ARTICLE | doi:10.20944/preprints202107.0581.v1
Subject: Life Sciences, Biochemistry Keywords: Sarcodia suieae; Acetyl-xylogalactan; RAW264.7; Heat stress; Apoptosis
Online: 26 July 2021 (14:25:38 CEST)
We aimed to evaluate the protective effects of acetyl-xylogalactan on the activity of RAW264.7 macrophages against heat stress. To this end, we assessed cell survival, phagocytic activity, in-tracellular Ca2+ level, mitochondria potential exchange, apoptotic assay findings, galactosidase activity and the RNA-seq by NGS and real-time polymerase chain reaction (PCR) expression. In our evaluation of macrophage morphology at 37°C and 41°C, the macrophages showed an oval shape at 41°C , unlike the spindle shape at 37°C. Therefore, 41°C was chosen as the heat stress condition. Subsequently, we designed an experiment to evaluate changes in the RAW264.7 macrophages after acetyl-xylogalactan treatment under heat stress. The survival of RAW264.7 macrophages treated with acetyl-xylogalactan was higher than that of controls that were not treated with acetyl-xylogalactan. Moreover, on the basis of the results of the annexin-V detection assay, the apoptotic activity of macrophages appeared to have reduced after treatment with ac-etyl-xylogalactan. Moreover, treatment with acetyl-xylogalactan resulted in a stronger recovery trend in the intracellular Ca2+ and mitochondrial membrane potential after heat stress. RNA sequencing and real-time polymerase chain reaction (PCR) illustrated that Sarcodia suieae acetyl-xylogalactan could upregulate the expression of the anti-apoptosis Cflar gene and down-regulate the expression of the apoptosis factors Ddit3, and Hyou1 to protect macrophages under heat stress.
ARTICLE | doi:10.20944/preprints201701.0098.v1
Online: 23 January 2017 (03:30:01 CET)
HER2/neu positive breast tumors predict a high mortality and comprise 25-30% of breast cancer. We have shown that Flavokawain A (FKA) preferentially reduces the viabilities of HER2 overexpressing breast cancer cell lines (i.e. SKBR3 and MCF7/HER2) versus those with less HER2 expression (i.e. MCF7 and MDA-MB-468). FKA at cytotoxic concentrations to breast cancer cell lines also has minimal effect on the growth of non-malignant breast epithelial MCF10 cells. FKA induces G2M arrest in cell cycle progression of HER2 overexpressing breast cancer cell lines through inhibition of Cdc2 and Cdc25C phosphorylation and down-regulation of Myt1 and Wee1expression leading to increased Cdc2 kinase activities. In addition, FKA induces apoptosis in SKBR3 cells by increasing the protein expression of Bim and BAX and decreasing expression of Bcl2, Bclx/L , XIAP and survivin. FKA also down-regulates the protein expression of HER-2 and inhibits AKT phosphorylation. Herceptin plus FKA treatment leads to enhanced growth inhibitory effect on HER-2 overexpressing breast cancer cell lines through down-regulation of Myt1, Wee1, Skp2, Survivin and XIAP. Our results suggest the promise of FKA as a novel apoptosis inducer and G2 checkpoint abrogating agent in combination with Herceptin for treatment of HER2 overexpressing breast cancer.
ARTICLE | doi:10.20944/preprints202212.0023.v1
Subject: Life Sciences, Molecular Biology Keywords: spinal cord injury; X-linked inhibitor of apoptosis protein; neuroprotection; apoptosis; transgenic mice
Online: 1 December 2022 (09:59:17 CET)
Trauma to the spinal cord causes extensive neuronal death contributing to the loss of sensory-motor and autonomic functions below the injury level. Apoptosis affects neurons after spinal cord injury (SCI) and is associated with increased caspase activity. Cleavage of X-linked inhibitor of apoptosis protein (XIAP) after SCI may contribute to this rise of caspase activity. Accordingly, we have shown that the elevation of XIAP resulted in increased neuronal survival after SCI and improved functional recovery. Therefore, we hypothesize that neuronal overexpression of XIAP can be neuroprotective after SCI with improved functional recovery. In line with this, studies of a transgenic mouse with overexpression of XIAP in neurons revealed that higher levels of XIAP after spinal cord trauma favours neuronal survival, tissue preservation, and motor recovery after the spinal cord trauma. Using the human SH-SY5Y cells overexpressing XIAP we show further that XIAP reduced caspase activity and apoptotic cell death after pro-apoptotic stimuli. In conclusion, this study shows that the levels of XIAP expression are an important factor for the outcome after spinal cord trauma and identifies XIAP as an important therapeutic target for alleviating the deleterious effects of SCI.
ARTICLE | doi:10.20944/preprints201911.0177.v1
Subject: Life Sciences, Molecular Biology Keywords: macrophage migration inhibitory factor; HNSCC cell lines; proliferation; cell cycle; apoptosis
Online: 15 November 2019 (08:48:50 CET)
Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that contributes to the progression of several cancers. MIF overexpression has been reported in head and neck squamous cell carcinoma (HNSCC) patients. However, the exact role of MIF in HNSCC is not fully understood. Our aim was to evaluate the amount of secreted MIF and the role of MIF in the proliferation, cell cycle, and apoptosis in HNSCC cell lines. The MIF levels in conditioned media from human primary (HN18 and HN30) and metastatic (HN17 and HN31) HNSCC cell lines were evaluated using ELISA. The HNSCC cell lines were treated with recombinant MIF and its effect on proliferation, cell cycle, and apoptotic status was determined by MTT and flow cytometry, respectively. The HNSCC-secreted MIF concentration ranged from 49.33‒860 pg/ml. Exogenous MIF (25 ng/ml) significantly increased HN18, HN30, and HN31 cell proliferation. Moreover, MIF induced cell cycle progression and inhibited apoptosis in these cells. However, MIF did not affect growth or apoptosis in HN17 cell. In conclusion, the HNSCC cell lines were evaluated secrete MIF. Exogenous MIF promotes various effects on proliferation, cell cycle, and apoptosis in HNSCC cells.
ARTICLE | doi:10.20944/preprints201611.0066.v2
Online: 14 November 2016 (04:05:38 CET)
Aquaporin-11 (AQP11) is an intracellular water channel expressed at the endoplasmic reticulum (ER) of the kidney proximal tubule. Its gene disruption in mice leads to intracellular vacuole formation at one week old and the subsequent development of polycystic kidneys at three week old. As the damaged proximal tubular cells with intracellular vacuoles later form cysts, autophagy may play a role in their survival. We examined the autophagy activity before and after the development of cysts in AQP11(-/-) kidneys. We first observed an enhanced expression of LC3 gene (Map1lc3b) as well as other autophagy-related genes in AQP11(-/-) mice by quantitative PCR analysis. We then examined the formation of autophagosomes visualized by a green fluorescent fusion protein, GFP–LC3 in its transgenic mice. The expression of GFP-LC3 puncta was increased in the proximal tubule of AQP11(-/-) mice before the cyst formation. Interestingly, they were also observed in the cyst-lining epithelial cell. Further PCR analyses revealed the enhanced expression of apoptosis- and ER stress-related caspase genes before and after the cyst formation suggesting that ER stress may have enhanced autophagy. We conclude that autophagy will play an important role in the development and the survival of the kidney cysts in AQP11(-/-) mice.
ARTICLE | doi:10.20944/preprints201608.0188.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: oleandrin; osteosarcoma; apoptosis; caspases
Online: 22 August 2016 (05:35:06 CEST)
Our previous study has found the anti-tumor activity of oleandrin in osteosarcoma cells in vitro, but the signal transduction process of cell apoptosis induced by oleandrin is uncertain, which is explored in this study. Fluorescence staining and flow cytometry (FCM) was performed to detect the cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Caspase-3 activity was detected using a commercial kit. The protein expression of cytoplasmic cytochrome c, mitochondrial cytochrome c, bcl-2, bax, caspase-9, Fas, FasL, caspase-8 and caspase-3 was detected using western blot. A pan-caspase inhibitor, z-VAD-fmk, was applied to block the apoptotic pathway and the apoptosis status were re-tested. We found that oleandrin significantly induced the increased apoptosis of U2OS cells. Meanwhile, the intracellular ROS was elevated, but the MMP decreased. The cytochrome c in mitochondria was notably decreased but increased in cytoplasm. The caspase-3 activity was also enhanced with the increase of drug concentration and treatment time. Oleandrin also down-regulated the level of bcl-2, but remarkably up-regulated the expression of bax, cleaved caspase-9, Fas, FasL, cleaved caspase-8 and cleaved caspase-3. Furthermore, the pre-treatment with z-VAD-fmk almost completely reverted the oleandrin-induced apoptosis. The results suggested that oleandrin induces the apoptosis of osteosarcoma cells via mitochondrial- and death receptor-dependent pathways.
ARTICLE | doi:10.20944/preprints202009.0634.v1
Subject: Life Sciences, Biochemistry Keywords: compound 3K; ovarian cancer; pyruvate kinase M2; autophagy; apoptosis
Online: 26 September 2020 (13:34:05 CEST)
Ovarian cancer is the common cause of death among gynecological cancers. Although ovarian cancer initially responds to chemotherapy, the frequent recurrence in patients remains a therapeutic challenge. Pyruvate kinase M2 (PKM2) plays a pivotal role in regulating cancer cell survival. However, its therapeutic roles remain unclear. Here, we investigated the anticancer effects of compound 3K, a specific PKM2 inhibitor, on autophagic and apoptotic pathway regulation in SK-OV-3 (PKM2-overexpressing human ovarian adenocarcinoma cell line). The anticancer effect of compound 3K was examined using the MTT and colony formation assay in SK-OV-3. The results of tissue microarray showed that PKM2 expression positively correlated with the severity of the tumor. Moreover, the expression of pro-apoptotic proteins increased in SK-OV-3 following compound 3K treatment. Compound 3K induced AMPK activation, which was accompanied by the inhibition of mTOR. Additionally, this compound inhibited glycolysis, resulting in reduced proliferation in SK-OV-3. Compound 3K treatment suppressed tumor progression in vivo xenograft model. Our findings suggest that the inhibition of PKM2 by compound 3K affected Warburg effects and induced autophagic cell death. Therefore, the use of specific PKM2 inhibitors to block the glycolytic pathway and target cancer cell metabolism represents a promising therapeutic approach for treating PKM2-overexpressing ovarian cancer.
ARTICLE | doi:10.20944/preprints201612.0107.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: apoptosis; gynecologic cancer; lipopolysaccharide; proliferation; Toll-like receptor
Online: 20 December 2016 (11:12:07 CET)
Toll-like receptor 4 (TLR4) is a member of the TLR family. Members of the TLR family play an important role in innate immune responses and are induced by recognition of pathogen-associated molecular patterns. They are also involved in cell proliferation and apoptosis in cancer. We investigated the role of TLR4 in apoptotic cell death in gynecological cancer cells; gynecological cancer is associated with infertility and spontaneous abortion. To examine the effect of TLR4 activation on apoptotic signaling in cancer cells, cultured primary cancer cells were treated with the TLR4 agonist lipopolysaccharide (LPS). The morphology of cancer cells was compared with normal myometrial cells. Enhanced growth rate and loss of contact inhibition with cellular overlap was observed in the cancer cells. The molecular mechanism analysis revealed differential expression of tumor suppressor genes in LPS-treated cancer cells. The expression of apoptosis-related caspase-3 was increased significantly in cancer cells with TLR4 activation after exposure to LPS. Taken together, these results suggest the pro-apoptotic activity of TLR4 as a potential therapeutic target for the treatment of gynecological cancers.
ARTICLE | doi:10.20944/preprints201608.0145.v1
Subject: Chemistry, Medicinal Chemistry Keywords: methyl 3,4-dihydroxybenzoate; oxidative stress; apoptosis; neuroprotection; nuclear factor erythroid 2-related factor 2
Online: 15 August 2016 (10:42:05 CEST)
This study investigated the neuroprotective effects of methyl 3,4-dihydroxybenzoate (MDHB) against t-butylhydroperoxide(TBHP) induced oxidative damage in SH-SY5Y (human neuroblastoma cells) and the underlying mechanisms. SH-SY5Y were cultured in DMEM+10% FBS for 24 hours and pretreated with different concentrations of MDHB or N-acetyl-L-cysteine (NAC) for 4 hours prior to the addition of 40 μM TBHP for 24 hours. Cell viability was analyzed using the methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays. An annexin V-FITC assay was used to detect cell apoptosis rate. The 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay was used to determine intracellular ROS levels. The activities of antioxidative enzymes (GSH-Px and SOD) were measured using commercially available kits. The oxidative DNA damage marker 8-OHdG was detected using ELISA. Western blotting was used to determine the expression of Bcl-2, Bax, caspase 3, p-Akt and Akt proteins in treated SH-SY5Y cells. Our results showed that MDHB is an effective neuroprotective compound that can mitigate oxidative stress and inhibit apoptosis in SH-SY5Y cells
ARTICLE | doi:10.20944/preprints201808.0146.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: histone deacetylase inhibitor, MHY2256, p53, apoptosis, autophagy, Ishikawa, endometrial cancer
Online: 7 August 2018 (14:37:46 CEST)
We previously found a novel a new sirtuin (SIRT) inhibitor MHY2256 that exerts anticancer activity through p53 acetylation in MCF-7 human breast cancer cells. Here, we investigated the anticancer activity of MHY2256 against hormone-related cancer, which is an endometrial cancer with poor prognosis. We found that MHY2256 markedly reduced cellular proliferation at low concentrations against Ishikawa endometrial cancer cells. The IC50 values of MHY2256 were much lower than that of salermide. Furthermore, MHY2256 significantly reduced the protein expression and activities of SIRT1, 2, and 3 with similar effects as salermide, a well-known SIRT inhibitor. Particularly, MHY2256 markedly inhibited tumor growth in a tumor xenograft mouse model of Ishikawa cancer cells. During the experimental period, there was no significant change in the body weight of mice treated with MHY2256. Detailed analysis of the sensitization mechanisms of Ishikawa cells revealed that late apoptosis was largely increased by MHY2256. Additionally, MHY2256 increased G1 arrest and reduced cell cyclic-related proteins, suggesting that apoptosis by MHY2256 was achieved by cellular arrest. Particularly, p21 was greatly increased by MHY225656, suggesting that cell cycle arrest by p21 is a major factor in MHY2256 sensitization in Ishikawa cells. We also detected a significant increase in acetylated p53, a target protein of SIRT1, in Ishikawa cells after MHY2256 treatment. In a mouse xenograft model, MHY2256 significantly reduced tumor growth and weight without apparent side effects. These results suggest that MHY2256 exerts its anticancer activity through p53 acetylation in endometrial cancer and can be used for targeting hormone-related cancers.
ARTICLE | doi:10.20944/preprints202012.0591.v1
Online: 23 December 2020 (14:09:09 CET)
: Plant-derived terpenes have aroused considerable interest as chemotherapeutic agents for a variety of diseases. This study aimed at the isolation and purification of the scarce terpenes psiadin, plectranthone and saudinolide from their respective plants, followed by the determination of antiproliferative activity, against hepatic cancer cell lines (HepG2, Hep3B), and the potential molecular mechanisms. Time- and dose-dependent cytotoxicity, evaluated using MTT and colony-forming assays, were exhibited by psiadin and plectranthone against the cancer cells. Flow cytometry showed that these two terpenes blocked cell cycle progression and induced mitochondrial-mediated apoptosis, particularly through increased cytochrome c and disruption of mitochondrial membrane potential. Additionally, they initiated the generation of reactive oxygen species as well as inhibiting NF-B. Psiadin lowered several essential cyclins and cyclin-dependent kinases and reduced RB activation. It was concluded that psiadin, in particular, has a significant therapeutic potential with the biggest advantage of differentiating between cancer and normal cells which is acutely lacking in current cytotoxic drugs. Its precise mode of action needs further investigation but appears predominantly to cause cell cycle arrest by interfering with cyclin production. It will be important to determine, in future studies, whether these terpenes will similarly inhibit other cancer cell lines and retain its activity against tumors in vivo.
ARTICLE | doi:10.20944/preprints202203.0153.v1
Subject: Medicine & Pharmacology, Behavioral Neuroscience Keywords: Apoptosis; Cerebellum; Methadone; Neuroinflammation
Online: 10 March 2022 (14:22:33 CET)
Background: Methadone is a centrally-acting synthetic opioid analgesic widely used in the methadone maintenance therapy (MMT) programs throughout the world. Considering its neurotoxic effects particularly on the cerebellum, this study aims to address the behavioral and histological alterations in the cerebellar cortex associated with methadone administration. Materials and Methods: Twenty-four adult male albino rats were randomized into two groups of control and methadone treatment. Methadone was subcutaneously administered (2.5–10 mg/kg) once a day for two consecutive weeks. The functional and structural changes in the cerebellum were compared to the control group. Results: Our data revealed that treating rats with methadone not only induced cerebellar atrophy, but also prompted the actuation of microgliosis, astrogliosis, and apoptotic biomarkers. We further demonstrated that treating rats with methadone increased complexity of astrocyte processes and decreased complexity of microglia processes. Our result showed that methadone impaired motor coordination and locomotor performance and neuromuscular activity. Additionally, relative gene expression of TNF-α, caspase-3 and RIPK3 increased significantly due to methadone. Conclusions: Our findings suggest that methadone administration has a neurodegenerative effect on the cerebellar cortex via dysregulation of microgliosis, astrogliosis, apoptosis, and neuro-inflammation.
REVIEW | doi:10.20944/preprints201808.0542.v1
Online: 31 August 2018 (05:56:58 CEST)
Neuronal cell death in the central nervous system has always been a challenging process to decipher. In physiological condition, neuronal cell death is restricted in the adult brain even as people ages. However, in pathological conditions of various neurodegenerative diseases, the cell death and shrinkage of a specific brain region represent a fundamental pathological feature across different neurodegenerative diseases. In this review, we will briefly go through the general pathways of cell death and describe the evidence of the cell deaths in the context of common neurodegenerative diseases individually, discussing our current understandings of cell death in connecting with the renowned pathogenic proteins, including tau, amyloid-beta, alpha-synuclein, huntingtin, and TDP-43.
REVIEW | doi:10.20944/preprints201804.0181.v2
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: p53, JMY, regulation, apoptosis, motility
Online: 18 April 2018 (13:46:19 CEST)
Following an event damaging the DNA, p53 levels increases inducing cell cycle arrest or apoptosis. JMY protein is a transcription co-factor involved in p53 regulation. After a DNA damage, also JMY levels increase and, as this protein accumulates in the nucleus, it forms a complex with P300 and Strap1 which increases the ability of p53 to induce transcription of proteins triggering apoptosis but not cell cycle. Therefore, Increase levels of JMY “direct” p53 activity toward triggering apoptosis. JMY expression is also linked to increased motility as it downregulates the expression of adhesion molecules of the Cadherin family and induces actin nucleation, making the cell less adhesive and more mobile. According to the scenario this gene can therefore have both a suppressive or a tumour promoting activity.
ARTICLE | doi:10.20944/preprints201910.0136.v1
Subject: Medicine & Pharmacology, Gastroenterology Keywords: apoptosis; cardiotrophin-1; colon; inflammation
Online: 12 October 2019 (03:38:00 CEST)
Ulcerative colitis (UC) is a relatively frequent, chronic disease that impacts significantly the patient’s quality of life. Although many therapeutic options are available, additional approaches are needed because many patients either do not respond to current therapies or show significant side effects. Cardiotrophin-1 (CT-1) is a cytokine with potent cytoprotective, anti-inflammatory, and antiapoptotic properties. The purpose of this study was to assess if the administration of CT-1 could reduce colon damage in mice with experimental UC. UC was induced with 5% dextran sulfate sodium (DSS) in the drinking water. Some mice received i.v. dose of CT-1 (200 µg/kg) 2 hours before and 2 and 4 days after DSS administration. Animals were followed during 7 days after DSS. The severity of UC was measured by standard scores. Colon damage was assessed by histology and immunohistochemistry. Inflammatory mediators were measured by Western blot and PCR. CT-1 administration to DSS-treated mice ameliorated both the clinical course (disease activity index), histological damage, inflammation (colon expression of TNF-α, IL-17, IL-10, INF-γ, and iNOS), and apoptosis. Our results suggest that CT-1 administration before UC induction improves the clinical course, tissue damage and inflammation degree in DSS-induced UC in mice.
ARTICLE | doi:10.20944/preprints202301.0247.v1
Subject: Life Sciences, Biochemistry Keywords: statin; natural compounds; Bcl2 family proteins; intrinsic apoptosis pathway; caspase dependent apoptosis
Online: 13 January 2023 (09:29:40 CET)
Glioblastoma multiforme (GBM) is one of the deadliest cancers. Temozolomide (TMZ) is the most common chemotherapy used for GBM patients. Recently, combination chemotherapy strategies have more effective antitumor effects and focus on slowing down the development of chemotherapy resistance. A combination of TMZ and cholesterol lowering medications (statins) is currently under investigation in in vivo and clinical trials. In our current investigation, we have used a triple combination therapy of TMZ, Simvastatin (Simva), and Acetylshikonin (ASH) and investigated its apoptotic mechanism in GBM cell lines (U87 and U251). We used viability, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), caspase-3/-7, acridine orange (AO) and immunoblotting autophagy assays. Our results showed that TMZ/Simva/ASH combination therapy significantly induced more apoptosis compared to TMZ, Simva, ASH, and TMZ/Simva treatments in GBM cells. Apoptosis via TMZ/Simva/ASH treatment induced mitochondrial damage (increase of ROS, decrease of MMP) and induced caspase-3/7 activation in both GBM cell lines. Compared to all single treatments and the TMZ/Simva treatment, TMZ/Simva/ASH significantly increased positive acidic vacuole organelles. We further confirmed that the increase of AVOs during the TMZ/Simva/ASH treatment was due to partial inhibition of autophagy flux (accumulation of LC3β-II and decrease in p62 degradation) in GBM cells. Our investigation also showed that TMZ/Simva/ASH-induced cell death was depended on autophagy flux as further inhibition of autophagy flux increased TMZ/Simva/ASH-induced cell death in GBM cells. Finally, our results showed that TMZ/Simva/ASH treatment potentially depends on an increase of Bax expression in GBM cells. Our current investigation might open new avenues for more effective treatment of GBM but further investigations are required for better identification of the mechanisms.
REVIEW | doi:10.20944/preprints202005.0158.v1
Subject: Life Sciences, Molecular Biology Keywords: apoptosis; cancer; phosphorylation; kinases; Bcl-2
Online: 9 May 2020 (08:40:40 CEST)
The regulation of apoptosis depends upon the Bcl-2 protein family. The process of cell death and survival is highly complicated and regulated by various types of extrinsic as well as intrinsic network of biological system. Several enzymes and regulators play crucial role in cell death and survival cycle not only in healthy but also in pathological state particularly in cancer. In cancerous cells, various proto-oncogenes and anti-apoptotic proteins are activated and responsible for the cell survival and longevity. The mechanism of activation and inactivation of various proteins in cell survival is regulated by the process of phosphorylation (kinases) and dephosphorylation (phosphatases). The current review will summarize the dynamics of Bcl-2 phosphorylation and its role in apoptosis and cell survival.
Subject: Biology, Anatomy & Morphology Keywords: spontaneous; racemization; apoptosis; post translational modification
Online: 18 November 2020 (11:09:30 CET)
Biochirality is evident in the hierarchical relation of molecular and cellular physiology during organism development and aging. Chirality influences the higher levels of biological processes, such as perception, memory and cognition, through intermolecular interactions between DNA, proteins, and lipids. At the molecular level, an organism's aging is the accumulation of macro-molecules with the aberrant composition, chirality, and folding. Cellular aging is driven by the non-physiological phase transitions (PhTs) withing membrane-bound and membrane-less compartments. Genomic instability and protein aging, as the interconnected root-causes of cell and organism aging, share two essential feature – spontaneous nature and accumulation over a lifetime. Consequently, we will analyze the interaction between the enzymatic (Enz) and spontaneous (Sp) post-translational modifications (PTMsEnz and PTMs Sp). Both forms of PTMs significantly contribute to the balance of L- and D-amino acids (L/D-AAs) in organisms, modulating the functions of nervous and immune systems. The most abundant form of PTM - enzymatic phosphorylation is bio-chemically associated with the spontaneous racemization (RzSp). The crass talk of enzymatic phosphorylation and spontaneous racemization, as an essential determinant of protein aging and aggregation, associated with the aberrant autophagy, apoptosis, and cell signaling, is discussed in this review.
ARTICLE | doi:10.20944/preprints201706.0126.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: 2,5-Dihydroxyacetophenone; multiple myeloma; MAPK; apoptosis
Online: 29 June 2017 (08:40:36 CEST)
2,5-Dihydroxyacetophenone (DHAP) is an active compound obtained from Radix Rehmanniae Preparata, which is widely used as a herbal medicine in many Asian countries. DHAP has been found to possess anti-inflammatory, anti-anxiety, and neuroprotective qualities. For the present study, we evaluated the anti-cancer effects of DHAP on multiple myeloma cells. It was discovered that DHAP downregulated the expression of oncogenic gene products like Bcl-xl, Bcl-2, Mcl-1, Survivin, Cyclin D1, IAP-1, Cyclin E, COX-2, and MMP-9, and upregulated the expression of Bax and p21 proteins, consistent with the induction of G2/M phase cell cycle arrest and apoptosis in U266 cells. DHAP inhibited cell proliferation and induced apoptosis, as characterized by the cleavage of PARP and the activation of caspase-3, caspase-8, and caspase-9. Mitogen-activated protein kinase (MAPK) pathways have been linked to the modulation of the angiogenesis, proliferation, metastasis, and invasion of tumors. We therefore attempted to determine the effect of DHAP on MAPK signaling pathways, and discovered that DHAP treatment induced a sustained activation of JNK, ERK1/2, and p38 MAPKs. DHAP also potentiated the pro-apoptotic and anti-proliferative effects of bortezomib in U266 cells. Our results suggest that DHAP can be an effective therapeutic agent to target multiple myeloma.
ARTICLE | doi:10.20944/preprints201807.0066.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: oridonin; radiation; sensitization; ROS; apoptosis
Online: 4 July 2018 (10:19:51 CEST)
Although many attempts have been made to improve the efficacy of radiotherapy to treat cancer, radiation resistance is still an obstacle in lung cancer treatment. Oridonin is a natural compound with promising antitumor efficacy that can trigger cancer cell death; however, its direct cellular targets, efficacy as a radiosensitizer, and underlying mechanisms of activity remain unclear. Herein, we report that oridonin exhibits additive cytotoxic and antitumor activity with radiation using H460 non-small cell lung cancer cell lines. We assessed the radiosensitizing effect of oridonin by MTT, clonogenic, reactive oxygen species (ROS) production, DNA damage, and apoptosis assays. In vitro, oridonin enhanced the radiation-induced inhibition of cell growth and clonogenic survival. Oridonin also facilitated radiation-induced ROS production and DNA damage and enhanced apoptotic cell death. In vivo, the combination of oridonin and radiation effectively inhibited H460 xenograft tumor growth, with higher caspase-3 activation and H2A histone family member X (H2AX) phosphorylation compared with that of radiation alone. Our findings suggest that oridonin possesses a novel mechanism to enhance radiation therapeutic responses by increasing DNA damage and apoptosis. In conclusion, oridonin may be a novel small molecule to improve radiotherapy in non-small cell lung cancer.
ARTICLE | doi:10.20944/preprints201906.0091.v1
Subject: Life Sciences, Biotechnology Keywords: apoptosis; cyclosaplin; molecular docking; protein kinases; Sandalwood
Online: 11 June 2019 (09:45:07 CEST)
Natural products from plants such as, chemopreventive agents attract huge attention because of their low toxicity and high specificity. The rational drug design in combination with structure based modeling and rapid screening methods offer significant potential for identifying and developing lead anticancer molecules. Thus, the molecular docking method plays an important role in screening a large set of molecules based on their free binding energies and proposes structural hypotheses of how the molecules can inhibit the target. Several peptide based therapeutics have been developed to combat several health disorders including cancers, metabolic disorders, heart-related, and infectious diseases. Despite the discovery of hundreds of such therapeutic peptides however, only few peptide-based drugs have made it to the market. Moreover, until date the activities of cyclic peptides towards molecular targets such as protein kinases, proteases, and apoptosis related proteins have never been explored. In this study we explore the in silico kinase and protease inhibitor potentials of cyclosaplin as well as study the interactions of cyclosaplin with other cancer-related proteins. Previously, the structure of cyclosaplin was elucidated by molecular modeling associated with dynamics that was used in the current study. Docking studies showed strong affinity of cyclosaplin towards cancer-related proteins. The binding affinity closer to 10 indicated efficient binding. Cyclosaplin showed strong binding affinities towards protein kinases such as EGFR, VEGFR2, PKB and p38 indicating its potential role in protein kinase inhibition. Moreover, it displayed strong binding affinity to apoptosis related proteins and revealed the possible role of cyclosaplin in apoptotic cell death. The protein-ligand interactions using LigPlot displayed some similar interactions between cyclosaplin and peptide-based ligands especially in case of protein kinases and a few apoptosis related proteins. Thus, the in silico analyses gave an insight of cyclosaplin as a potential apoptosis inducer and protein kinase inhibitor.
ARTICLE | doi:10.20944/preprints201911.0343.v1
Subject: Life Sciences, Endocrinology & Metabolomics Keywords: anti-apoptosis; anti-oxidant; curcumin; hyperglycemia; hyperlipidemia
Online: 27 November 2019 (09:57:14 CET)
Curcumin is the main secondary metabolites of Curcuma longa and other Curcuma spp, and has been reported to have some potential in preventing and treating some physiological disorders. This study investigated the effect curcumin in inhibiting high-fat diet and streptozotocin (STZ)-induced hyperglycemia and hyperlipidemia in rats. Twenty-six male Sprague-Dawley (SD) rats (170-190 g) were randomly divided into a standard food pellet diet group (Control group), a high-fat diet and streptozotocin group (HF+STZ group), and a high-fat diet combined with curcumin and STZ group (HF+ Cur +STZ group). Compared with the HF+STZ group, the HF+Cur+STZ group exhibited significantly reduced fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (AST) and aspartate transaminase (ALT) levels, and liver coefficients; in the livers of these rats, the expression of malondialdehyde (MDA) and Bax was downregulated, whereas that of superoxide dismutase (SOD) and Bcl-2 was upregulated. Moreover, the liver histology of these rats was improved and resembled that of the control rats. These results suggest that curcumin prevents high-fat diet and STZ-induced hyperglycemia and hyperlipidemia, mainly via anti-oxidant and anti-apoptotic mechanisms in the liver.
ARTICLE | doi:10.20944/preprints202203.0063.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: MPP+; neurotoxicity; autophagy; apoptosis; neuroprotection; Parkinson's disease
Online: 3 March 2022 (10:36:29 CET)
Autophagy is a cellular homeostatic process by which cells degrade and recycle their malfunctioned contents, and impairment in this process leads to Parkinson's disease (PD) pathogenesis. Dioscin, a steroidal saponin, has induced autophagy in several cell lines and animal models. The role of dioscin-mediated autophagy in PD remains to be investigated. Therefore, this study aims to investigate the hypothesis that dioscin-regulated autophagy and autophagy-related genes (ATG) could protect neuronal cells in PD via reducing apoptosis and enhancing neurogenesis. In this study, the 1-methyl-4-phenylpyridinium ion (MPP+) were used to induce neurotoxicity and impair autophagic flux in a human neuroblastoma cell line (SH-SY5Y). The result showed that dioscin pre-treatment counters MPP+-mediated autophagic flux impairment and alleviates MPP+-induced apoptosis by downregulating activated caspase-3 and Bax expression while increasing Bcl-2 expression. In addition, dioscin pre-treatment was found to increase neurotrophic factors and tyrosine hydroxylase expression, suggesting that dioscin could ameliorate MPP+-induced degeneration in dopaminergic neurons and benefit the PD model. Interestingly, the neuroprotective activities of dioscin were suppressed when co-treated with chloroquine (CQ), an autophagosome-lysosome inhibitor. In summary, we showed dioscin’s neuroprotective activity in neuronal SH-SY5Y cells might be partly related to its autophagy induction and suppression of the mitochondrial apoptosis pathway.
ARTICLE | doi:10.20944/preprints201805.0337.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: Periostracum cicadae; IgA nephropathy; inflammation; fibrosis; apoptosis
Online: 24 May 2018 (06:32:22 CEST)
Periostracum cicadae, the cast-off shell of the cicada Cryptotympana pustulata Fabricius, is used in traditional Chinese medicine for its diaphoretic, anticonvulsive, sedative, antipyretic, and antiallergic effects. However, the exact pathogenesis of immunoglobulin A nephropathy (IgAN) remains unclear, thereby hindering investigations to identify novel therapeutic agents. A rat IgAN model was established by administration of bovine serum albumin, lipopolysaccharide, and carbon tetrachloride, which simultaneously established blood stasis and a heat syndrome model. The animals were sacrificed to detect changes in protein levels in urine and blood. Immunofluorescence was performed to assess IgA deposition in the glomeruli. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6) levels were measured in bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assay. Hematology and eosin, periodic acid-Schiff, TUNEL, and immunohistochemical staining were performed to evaluate histopathological changes in kidney tissues. Additionally, target-related proteins were measured by western blotting. Periostracum cicadae resulted in a reduction in blood and urine protein levels. Serum TNF-α, IL-1β, and IL-6 levels significantly decreased in the periostracum cicadae-treated groups compared to the IgAN group. Furthermore, a reduction in MCP-1, TLR4, and IgA expression levels and a dose- dependent increase in caspace-3 expression were observed in response to periostracum cicadae treatment. TGF-β1 levels decreased, whereas that of Fas increased in the kidney tissues of the periostracum cicadae-treated groups. The findings of the present study indicate that periostracum cicadae induces apoptosis and improves kidney inflammation and fibrosis in IgA nephropathy rat models.
ARTICLE | doi:10.20944/preprints202212.0323.v2
Subject: Life Sciences, Virology Keywords: Apoptosis; Dengue Virus; microRNAs; Viral Nonstructural Proteins
Online: 26 January 2023 (10:47:59 CET)
The World Health Organization has estimated an annual occurrence of approximately 392 million Dengue virus (DENV) infections in more than 100 countries where the virus is endemic, and this represents a serious threat to humanity. DENV is a serologic group with four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) belonging to the genus Flavivirus, in the family Flaviviridae. Dengue is the most widespread mosquito-borne disease in the world. The ~10.7 kb DENV genome encodes three structural proteins (capsid [C], pre-membrane [prM], and envelope [E]) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS1 protein is a membrane-associated dimer and a secreted, lipid-associated hexamer. Dimeric NS1 is found on membranes both in cellular compartments and cell surfaces. Secreted NS1 (sNS1) is often present in patient serum at very high levels, which correlates with severe dengue symptoms. This study was conducted to discover how NS1 protein, microRNAs-15/16 (miRNAs-15/16), and apoptosis are related during DENV-4 infection in human liver cell lines. Huh 7.5 and HepG2 cells were infected with DENV-4, and miRNAs-15/16, viral load, NS1 protein, and caspases-3/7 were quantified after different times of infection. This study demonstrated that miRNAs-15/16 are overexpressed during infection of HepG2 and Huh 7.5 cells by DENV-4 and have a relationship with NS1 protein expression, viral load, and activity of caspases-3/7, thus making these miRNAs potential injury markers during DENV infection in human hepatocytes.
ARTICLE | doi:10.20944/preprints202012.0376.v1
Subject: Medicine & Pharmacology, Allergology Keywords: Desipramine; Autophagy; Apoptosis; Death receptor-5; TRAIL
Online: 15 December 2020 (12:09:09 CET)
Autophagy, an alternative cell death mechanism, is also termed programmed cell death type II. Autophagy in cancer treatment needs to be regulated. In our study, autophagy inhibition by desipramine or the autophagy inhibitor chloroquine (CQ) enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2 [death receptor (DR5)] expression and subsequently TRAIL-induced apoptosis in TRAIL-resistant A549 lung cancer cells. Genetic inhibition of DR5 substantially reduced desipramine-enhanced TRAIL-mediated apoptosis, proving that DR5 was required to increase TRAIL sensitivity in TRAIL-resistant cancer cells. Desipramine treatment upregulated p62 expression and promoted conversion of light chain 3 (LC3)-I to its lipid-conjugated form, LC3-II, indicating that autophagy inhibition occurred at the final stages of autophagic flux. Transmission electron microscopy analysis showed the presence of condensed autophagosomes, which resulted from the late stages of autophagy inhibition by desipramine. TRAIL, in combination with desipramine or CQ, augmented the expression of apoptosis-related proteins cleaved caspase-8 and cleaved caspase-3. Our results contributed to the understanding of the mechanism underlying the synergistic anti-cancer effect of desipramine and TRAIL and presented a novel mechanism of DR5 upregulation. These findings demonstrated that autophagic flux inhibition by desipramine potentiated TRAIL-induced apoptosis, suggesting that appropriate regulation of autophagy is required for sensitizing TRAIL-resistant cancer cells to TRAIL-mediated apoptosis.
ARTICLE | doi:10.20944/preprints202212.0011.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: MST4; EMT; AKT; Apoptosis; Breast cancer
Online: 1 December 2022 (04:46:12 CET)
The mammalian STE 20-like protein kinase 4 (MST4) gene is highly expressed in several cancer types, but little is known on the role of MST4 in breast cancer and the function of MST4 during epithelial-mesenchymal transition (EMT) has not been fully elucidated. Here, we report that overexpression of MST4 in breast cancer results in enhanced cell growth, migration, and invasion, whereas inhibition of MST4 expression significantly attenuates these properties. Further study shows that MST4 promotes EMT by activating Akt and its downstream signaling molecules such as E-cadherin/N-cadherin, Snail, and Slug. MST4 also activates AKT and its downstream pro-survival pathway. Furthermore, by analyzing breast cancer patient tissue microarray and silicon datasets, we found that MST4 expression is much higher in breast tumor tissue compared to normal tissue, and significantly correlates with cancer stage, lymph node metastasis and a poor overall survival rate (p<0.05). Taken together, our findings demonstrate the oncogenic potential of MST4 in breast cancer, highlighting its role in cancer cell proliferation, migration/invasion, survival, and EMT, suggesting a possibility that MST4 may serve as a novel therapeutic target for breast cancer.
REVIEW | doi:10.20944/preprints202112.0484.v1
Online: 30 December 2021 (12:32:40 CET)
Cell death by apoptosis is a major cellular response, in the control of tissue homeostasis and as a defense mechanism in case of cellular aggression like an infection. Cell self-destruction is part of antiviral responses, aimed at limiting the spread of a virus. Although it may contribute to the deleterious effects in infectious pathology, apoptosis remains a key mechanism for viral clearance and resolution of infection. The control mechanisms of cell death processes by viruses have been extensively studied. Apoptosis can be triggered by different viral determinants, through different pathways, as a result of virally induced cell stresses and innate immune responses. Zika virus (ZIKV) induces Zika disease in humans which has caused severe neurological forms, birth defects and microcephaly in newborns during the last epidemics. ZIKV also surprised by revealing an ability to persist in the genital tract and in semen, thus being sexually transmitted. Mechanisms of diverting antiviral responses such as the interferon response, the role of cytopathic effects and apoptosis in the etiology of the disease have been widely studied and debated. In this review, we examined the interplay between ZIKV infection of different cell types and apoptosis and how the virus deals with this cellular response. We illustrate a duality in the effects of ZIKV-controlled apoptosis, depending on whether it occurs too early or too late, respectively in neuropathogenesis, or in long-term viral persistence. We further discuss a prospective role for apoptosis in ZIKV-related therapies, and the use of ZIKV as an oncolytic agent.
ARTICLE | doi:10.20944/preprints201901.0315.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: glioblastoma; p53; apoptosis; doxorubicin; bortezomib; vorinostat.
Online: 30 January 2019 (13:03:22 CET)
In prostate cancer, p53 maximizes apoptosis in response to severe DNA damage, not DNA replication stress. Here, we examined the apoptotic response of two glioblastoma cells, p53-wild type U87 and a p53-mutated T98G cell, for the same stresses. We ascertained that p53 intensified apoptosis in response to severe DNA damage, not DNA replication stress in glioblastoma. We further asked if p53-mediated apoptosis can be induced by cellular stress other than severe DNA damage. We analyzed two compounds, bortezomib and vorinostat, respective inhibitors of 26S proteasome and histone deacetylase, to evaluate their capacity to activate p53-mediated apoptosis. The cellular stress incited by bortezomib, not vorinostat, activated p53-mediated apoptosis. Next, we asked if the cellular stress generated by combining the two compounds had a synergistic effect on apoptosis. Our results demonstrated that doxorubicin with bortezomib or CFS-1686, or bortezomib with vorinostat have a significant synergistic effect on apoptosis only in p53-wild type cell. Under high stress, p53 translocates from cytosol into the nucleus to cause apoptosis possibly. Together, p53 maximizes apoptosis for cellular stress caused by severe DNA damage, disruption of protein turnover, and for the stress induced by drug combination including doxorubicin with bortezomib or CFS-1686, and bortezomib with vorinostat.
ARTICLE | doi:10.20944/preprints201810.0034.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: T-2 toxin, toxicity, autophagy, apoptosis
Online: 2 October 2018 (16:51:09 CEST)
T-2 toxin produced by fungi of Fusarium genus is highly toxic to human and animals and has been shown to induce apoptosis in various organs/tissues. Apoptosis and autophagy are interconnected processes and these interactions are important for cellular homeostasis as well as pathogenesis. In this study, we report for the first time that T-2 toxin induced autophagy in human liver cells (L02). We showed that T-2 toxin induced the formation of acidic vesicular organelles, concordant with the time and dose-dependent alterations in LC3-phosphatidylethanolamine conjugate (LC3-II) LC3-I/II and p62/SQSTM1 suggesting an enhanced autophagic flux. The T-2 toxin-induced formation of autophagosome and lysosomal fusion was observed by expressing mRFP-GFP-LC3 in L02 cells by lentiviral transduction, and autophagosome was observed by transmission electron microcopy. We found that while T-2 toxin activated both apoptosis and autophagy, activation of autophagy appears to be a leading event reflecting the protective mechanism of cells against the insults by T-2 toxin. Activating autophagy by rapamycin (RAPA) inhibited the apoptosis while suppressing autophagy by chloroquine greatly enhanced the T-2 toxin-induced apoptosis suggesting the crosstalk of autophagy and apoptosis. In summary, our study showed that activation of autophagy protects liver cells from T-2 toxin-induced apoptosis suggesting autophagy may be targeted for prevention of the T-2 toxin-induced toxicity in human and animals.
ARTICLE | doi:10.20944/preprints202111.0526.v1
Subject: Biology, Animal Sciences & Zoology Keywords: VP28; WSSV; real-time PCR; viral load; apoptosis
Online: 29 November 2021 (11:55:17 CET)
White Spot Syndrome Virus (WSSV) has emerged as one of the most prevalent and lethal viruses globally, and infects both shrimps and crabs in the aquatic environment. This study aimed to investigate the occurrence of WSSV in different ghers of Bangladesh and the virulence of the circulating phylotypes. We collected 360 shrimp (Penaeus monodon) and 120 crab (Scylla sp.) samples from the South-East (Cox’s Bazar) and South-West (Satkhira) coastal regions of Bangladesh. The VP28 gene-specific PCR assays and sequencing revealed statistically significant (p < 0.05, Kruskal Wallis test) differences in the prevalence of WSSV in shrimps and crabs between the study areas (Cox’s Bazar and Satkhira), and over the study periods (2017-2019). The mean Log load of WSSV varied from 8.40 (Cox’s Bazar) to 10.48 (Satkhira) per gram of tissue. The mean values for salinity, dissolved oxygen, temperature and pH were 14.71±0.76 ppt, 3.7±0.1 ppm, 34.11±0.38˚C and 8.23±0.38, respectively in the WSSV-positive ghers. The VP28 gene-based phylogenetic analysis showed an amino-acid substitution (E→G) at 167th position in the isolates from Cox’s Bazar (referred to as phylotype BD2) compared to the globally circulating one (BD1). Shrimp PL artificially challenged with BD1 and BD2 phylotypes with filtrates of tissue containing 0.423 X 109 copies of WSSV per mL resulted a median LT50 value of 73 hrs and 75 hrs, respectively. The in-vivo trial showed higher mean Log WSSV copies (6.47±2.07 per mg tissue) in BD1 challenged shrimp PL compared to BD2 (4.75±0.35 per mg tissue). Crabs infected with BD1 and BD2 showed 100% mortality within 48 hrs and 62 hrs of challenge, respectively with mean Log WSSV copies of 12.06±0.48 and 9.95±0.37 per gram tissue, respectively. Moreover, shrimp antimicrobial peptides (AMPs) penaeidin and lysozyme expression was lower in BD1 challenged group compared to BD2 challenged shrimps. These results collectively demonstrated that relative virulence properties of WSSV based on mortality rate, viral load and expression of host immune genes in artificially infected shrimp PL could be affected by single aa substitution in VP28.
ARTICLE | doi:10.20944/preprints202009.0326.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: HepG2 cells; cytotoxicity; apoptosis; Hoechst staining; molecular dynamics
Online: 15 September 2020 (04:18:12 CEST)
The natural products and conventional chemotherapeutic drugs are believed to increase the cure rates of anti-cancer treatment while reducing their toxicity. The current study investigates the cytotoxic and apoptogenic effects of bioactive compounds from Monotheca buxifolia on Hep G2 cell lines. The effect on the viability of Hep G2 cells was evaluated by MTT assay; Morphological changes were studied, the apoptotic activity was demonstrated through Annexin-V-FITC/ PI, a molecular dynamics simulation study was conducted to explore the binding pattern of the compounds in the active site of the PPRAδ protein. The isolated compounds lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate inhibited the growth of hepatocellular cancer cells, as determined by MTT assay and annexin V-FITC/PI. The IC50 value for lauric acid was 56.4584 ± 1.20 µg/ml, that for oleanolic acid was 31.9421 ± 1.03 µg/ml, and that for bis(2-ethylhexyl) phthalate was 83.8019 ± 2.18 µg/ml. After 24 h of treatment, 29.5% of Hep G2 cells treated with lauric acid, 52.1% of those treated with oleanolic acid, and 22.4% of those treated with bis(2-ethylhexyl) phthalate were apoptotic. Morphological assay and Hoechst staining microscopy revealed the morphological alterations of cell membrane accompanied by nuclear condensation after treatment. The high fluctuation indicates the high potency and adopting various interactions, and vice versa, the oleanolic acid showed highly residues fluctuation, which remains stable in the active site of PPARδ protein and involved in various interactions while remaining locally fluctuated in the binding site the other two compounds. In conclusion, a significant apoptogenic effect was exhibited by lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate against HepG2 cells in inducing apoptosis. Our findings indicate that these bioactive compounds hold promise as potential therapeutic for hepatocellular carcinoma.
ARTICLE | doi:10.20944/preprints202107.0543.v1
Subject: Life Sciences, Biochemistry Keywords: Apoptosis; TUNEL; Caspase; image processing; thresholding; signal quantification; Drosophila
Online: 23 July 2021 (11:55:04 CEST)
Apoptosis is associated with numerous phenotypical characteristics, and is thus studied with many tools. In this study, we compared two broadly used apoptotic assays: TUNEL and staining with an antibody targeting the activated form of an effector caspase. To compare them, we developed a protocol based on commonly used tools such as filters, zprojection and thresholding. Even though it is commonly used in imageprocessing protocols, thresholding remains a recurring problem. Here we analyzed the impact of processing parameters and readout choice on the accuracy of apoptotic signal quantification. Our results show that TUNEL is quite robust, even if image processing parameters can allow or not to detect subtle differences of the apoptotic rate. On the contrary, images from anticleaved caspase staining are more sensitive to handle and proved to necessitate to be processed more carefully. We then developed an open source Fiji macro automatizing most steps of the image processing and quantification protocol. It is noteworthy that the field of application of this macro is wider than apoptosis as it can perfectly be used to treat and quantify other kind of images.
ARTICLE | doi:10.20944/preprints202209.0055.v1
Subject: Biology, Other Keywords: Adenocarcinoma HeLa; Anticancer; Apoptosis; Crude extract; Stomopneustes variolaris
Online: 5 September 2022 (09:31:44 CEST)
Cancer is one of the world's most serious health problems and the top cause of mortality today. S. variolaris is a valuable sea creature that has long been used as a folk medicine to cure and prevent ailments. A subcritical water extraction was used method to obtain crude extract from spikes of S. variolaris. The extract had shown antiproliferative effect in HeLa cells with an IC50 of 723.1 ± 9.73 µg/ml, but no toxicity in HEK293. Westernblot was used to detect protein expression; Bax, caspase-8, and IκBα were increased, whereas IKKα and p-NFκB-65(Ser 536) were downregulated. RNA/mRNA expression was revealed by RT-qPCR, and fold changes of caspase-3, cytochrome-c, Bax, Apaf-1, caspase-9, and Bak genes’ expression were raised. Using gel electrophoresis, the treatment groups showed more DNA fragmentation than the control group. GC-MS was used to identify the components in the crude extract; anticancer activity could be attributed to dodecanoic acid, hexadecanoic acid, and tetradecanoic acid content. In conclusion, these results showed the potential use of the crude extract of a spike from S. variolaris with anticancer activity against cervical cancer.
ARTICLE | doi:10.20944/preprints201712.0007.v1
Subject: Life Sciences, Molecular Biology Keywords: non-invasive apoptosis detection sensor; breast cancer; HDACi
Online: 1 December 2017 (12:08:13 CET)
Breast cancer is the most common malignancies in women and the second leading cause of cancer death in women. Triple negative breast cancer (TNBC) subtype is a breast cancer subset without ER, PR and HER2 expression, limiting treatment options and presenting a poorer survival rate. Thus, we investigated whether HDACi would be used as potential anti-cancer therapy on breast cancer cells. In this study, we found TNBC and HER2-enrich breast cancers are extremely sensitive to Panobinostat, Belinostat of HDACi via experiments of cell viability assay, apoptotic marker identification and flow cytometry measurement. On the other hand, we developed a bioluminescence based live cell non-invasive apoptosis detection sensor (IADS) detection system to evaluate the quantitative and kinetic analyses of apoptotic cell death by HDAC treatment on breast cancer cells. In addition, the use of HDACi may also be accompanied with chemotherapeutic agent such as doxorubicin to synergic drug sensitivity on TNBC cell (MDA-MB-231), but not in breast normal epithelia cells (MCF-10A), providing therapeutic benefits against breast tumor in clinic.
ARTICLE | doi:10.20944/preprints202011.0215.v1
Subject: Medicine & Pharmacology, Allergology Keywords: Pancreatic Cancer; PLGA-PAA copolymer; Hydroxytyrosol; Curcumin; Apoptosis
Online: 5 November 2020 (14:51:15 CET)
Background and Aim: Pancreatic cancer (PC) is a highly aggressive malignancy associated with low survival rates. Many chemotherapeutic regimens have been investigated for advanced unresectable and metastatic PC, but with only minimal improvement in survival and prognosis. The present study aimed to investigate the anti-cancer function of free and nano-encapsulated hydroxytyrosol (Hyd) and curcumin (Cur), and its combinations (Hyd-Cur) on the PANC-1 cell line.Methods: The poly lactide-co-glycolide-co-polyacrylic acid (PLGA-co-PAA) nano-encapsulated Hyd and Cur were synthesized, and MTT assay was performed to evaluate cytotoxic effects of free and nano-encapsulated Hyd, Cur, and Hyd-Cur. Moreover, effects of free and nano-encapsulated Hyd, Cur, and Hyd-Cur were evaluated on viability, migration, morphological alterations, colony formation, and apoptosis on PANC-1 cell line. The mRNA expression levels of MMP2, MMP9, BAX, BCL-2, and Cas9 genes were assessed after treated PANC-1 cells with free and nano-encapsulated Hyd, Cur, and Hyd-Cur.Results: The obtained results showed that free and nano-encapsulated Hyd, Cur, and Hyd-Cur treatments significantly decreased the viability, migration, and colony formation in the PANC-1 cells. Furthermore, apoptosis rates in PANC-1 cells were increased in a concentration and time dependent manner in all of the treatment groups. Moreover, anti-proliferative activity of nano-encapsulated Hyd-Cur was significantly more than other treatments.Conclusion: According to our results, Hyd-Cur combination and nano-encapsulation therapy exerts more profound apoptotic and anti-proliferative effects on PANC-1 cell line than free Hyd or Hyd monotherapy.
ARTICLE | doi:10.20944/preprints202111.0366.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: β-Apopicropodophyllin; Radiosensitizer; Topoisomerase inhibitor; ROS, Apoptosis; Colorectal cancer
Online: 19 November 2021 (14:54:40 CET)
β-apopicropodophyllin (APP), a derivative of podophyllotoxin (PPT), has been identified as a potential anti-cancer drug. This study tested whether APP acts as an an-ti-cancer drug and can sensitize colorectal cancer (CRC) cells to radiation treatment. APP had an anti-cancer effect against the CRC cell lines HCT116, and DLD-1, SW480 and COLO320DM with IC50 values of 7.88 nM, and 8.22 nM, 9.84 nM and 7.757 nM, respec-tively induction of DNA damage. Colonogenic and cell counting assays indicated that the combined treatment of APP and γ-ionizing radiation (IR) showed greater retardation of cell growth than either alone, suggesting that APP sensitizes CRC cells to IR. Annexin V-propidium iodide (PI) assays and immunoblot analysis showed that the combined treatment of APP and IR increased apoptosis in CRC cells compared with either APP or IR alone. Results obtained from the xenograft experiments also indicated that the combination of APP and IR enhanced apoptosis in in vivo animal model. Apoptosis induction by the combined treatment of APP and IR resulted from reactive oxygen species (ROS). Inhibition of ROS by N-acetylcysteine (NAC) restored cell viability and decreased the induction of apoptosis by APP and IR in CRC cells. Taken together, these results indicate that a combined treatment of APP and IR might promote apoptosis by inducing ROS in CRC cells.
ARTICLE | doi:10.20944/preprints202103.0242.v1
Subject: Life Sciences, Biochemistry Keywords: apoptosis; evading apoptosis; expression variability; cancer functional pathway; prostate cancer phenotype; immortality; proliferation; P53 signaling; transcriptomic network
Online: 9 March 2021 (08:44:15 CET)
Prostate cancer is a leading cause of death among men but its genomic characterization and best therapeutic strategy are still under debate. The Genomic Fabric Paradigm (GFP) considers the transcriptome as a multi-dimensional mathematical object subjected to a dynamic set of expression correlations among the genes. Here, GFP is applied to gene expression profiles of three (one primary, and two secondary) cancer nodules and the surrounding normal tissue from a surgically removed prostate tumor. GFP was used to determine the regulation and rewiring of the P53 signaling, apoptosis, prostate cancer and several other pathways involved in survival and proliferation of the cancer cells. Genes responsible for the block of differentiation, evading apoptosis, immortality, insensitivity to anti-growth signals, proliferation, resistance to chemotherapy and sustained angiogenesis were found as differently regulated in the three cancer nodules with respect to the normal tissue. The analysis indicates that even histo-pathologically equally graded cancer nodules from the same tumor have substantially different transcriptomic organizations, raising legitimate questions about the validity of meta-analyses comparing large populations of healthy and cancer humans. The study suggests that GFP may provide a personalized alternative to the biomarkers’ approach of cancer genomics.
ARTICLE | doi:10.20944/preprints201803.0184.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: colorectal cancer cells; metformin; apoptosis; oxidative stress
Online: 21 March 2018 (03:30:49 CET)
Accumulating evidence suggests that metformin, used as an antidiabetic drug, possesses anticancer properties. Metformin reduced the incidence and growth of experimental tumors in vivo. In a randomized clinical trial among nondiabetic patients, metformin treatment significantly decreased the number of aberrant crypt foci compared to the untreated group with a follow-up of 1 month. In our study, HT29 cells were treated with graded concentrations of metformin, 10 mM/25 mM/50 mM, for 24/48 hours. We performed immunofluorescence experiments by means of confocal microscopy and Western blot analysis to evaluate a panel of factors involved in apoptotic/autophagic processes and oxidative stress response. Moreover, HT29 cells treated with metformin were analyzed by flow cytometry assay to detect the cell apoptosis rate. The results demonstrate that metformin exerts growth inhibitory effects on cultured HT29 cells by increasing both apoptosis and autophagy; moreover, it affects the survival of cultured cells inhibiting the transcriptional activation of nuclear factor E2–related factor 2 (NRF-2) and nuclear factor–kappa B (NF-κB). The effects of metformin on HT29 cells were dose- and time-dependent. These results are very intriguing, since metformin is emerging as a multifaceted drug: it has a good safety profile and is associated with low cost, and it might be a promising candidate for the prevention or treatment of colorectal cancer.
Subject: Life Sciences, Microbiology Keywords: h. pylori; restriction modification system; virulence; il-8; apoptosis
Online: 1 November 2019 (12:52:39 CET)
Helicobacter pylori is a common human pathogen that causes gastroduodenal diseases. H. pylori genome consists of numerous restriction-modification (R-M) genes. It is established that N6-adenine methylation plays a crucial role in bacterial gene regulation and virulence, but not much is known about the role of C5-cytosine methylation. In this study, we examined the influence of an orphan cytosine methyltransferase, hpyAVIBM on gastric infection in mice and cultured cells. Histopathological staining showed that the deletion of hpyAVIBM in H. pylori strain SS1 had increased damaging hemorrhagic effects on the mice stomach. The gelatin-zymography result demonstrated that the mice infected with mutant SS1ΔhpyAVIBM had significantly up-regulated pro-MMP-9 than those infected with SS1. Additionally, ELISA results of pro-inflammatory cytokines proved that mutant strain caused significantly more inflammatory effect on mice stomach than its wild-type counterpart. The immunohistochemistry data showed that mutant strain caused attenuated epithelial cell damage. Co-culture studies of H. pylori with AGS (Human Gastric Adenocarcinoma cell line) cells revealed that SS1ΔhpyAVIBM instigated significantly more apoptotic death in the AGS cells compared to the wild-type strain. Our results indicated that DNA methylation by hpyAVIBM plays a crucial role in modulating virulence factors in bacterial cells and their interaction with the host cells.
REVIEW | doi:10.20944/preprints201811.0543.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: hydroxyurea; cerebellum; neuron; immunohistochemistry; electron microscopy; cell death; apoptosis
Online: 22 November 2018 (07:09:28 CET)
The cytostatic agent hydroxyurea (HU) has proven to be beneficial for a variety of conditions in the disciplines of oncology, hematology, infectious disease and dermatology. It disrupts the S-phase of the cell cycle by inhibiting the ribonucleotide reductase enzyme, thus blocking the transformation of ribonucleotides into deoxyribonucleotides, a rate limiting step in DNA synthesis. HU is listed as an essential medicine by the World Health Organization. Several studies have indicated that HU is well tolerated and safe in pregnant women and very young pediatric patients. To our knowledge, only a few controlled studies about the adverse effects of HU therapy have been done in humans. Despite this, the prevalence of central nervous system abnormalities, including ischemic lesions and stenosis have been reported. This review will summarize and present the effects of HU-exposure on the prenatal and perinatal development of the rat cerebellar cortex and deep cerebellar nuclei neurons. Our results call for the necessity to better understand HU effects and define the administration of this drug to gestating women and young pediatric patients.
ARTICLE | doi:10.20944/preprints201810.0342.v1
Subject: Biology, Physiology Keywords: 4-octylphenol; male sertoli cells; reproductive toxicity; apoptosis; mitochondria
Online: 16 October 2018 (08:35:47 CEST)
The toxic effects of 4-octylphenol (4-OP) have been studied in species such as mouse and fish; however, the toxic effects of 4-OP in male specific niche cells has not been researched. In this study, we investigated the molecular mechanism of toxicity of 4-OP in mouse TM4 Sertoli cells. TM4 cells were treated with four concentrations (0, 10, 30, and 50 µM/mL) of 4-OP at time points 24, 48, and 72 h. Cell viability and apoptosis assay was conducted following exposure. 4-OP significantly decreased cell viability in a concentration- and time-dependent manner, and increased apoptosis. Quantitative PCR analysis showed that Bad, Bax, and Bak mRNA expression levels were higher in exposed cells than in the control, but Bcl-2 expression was decreased. Western blotting revealed that 4-OP induced activities of caspase-3 and phosphorylation of Bad in a concentration- and time-dependent manner. Additionally, cytochrome C protein did not colocalize with mitochondria marker dye by 24 h. Cytochrome c protein expression increased in a time-dependent manner with 50 µM/mL. These results suggest that 4-OP induces mitochondria-mediated apoptosis by regulation of Bcl-2 family proteins and caspase-3 activation in male Sertoli cells.
ARTICLE | doi:10.20944/preprints202205.0097.v1
Subject: Chemistry, Medicinal Chemistry Keywords: KV1.3; potassium ion channels; antiproliferative activity; apoptosis; anticancer drugs
Online: 9 May 2022 (03:35:09 CEST)
The voltage-gated potassium channel KV1.3 has been recognized as a tumor marker and represents a promising new target for the discovery of new anticancer drugs. We designed a novel structural class of KV1.3 inhibitors through structural optimization of benzamide-based hit compounds and structure-activity relationship studies. The potency, and selectivity of the new KV1.3 inhibitors were investigated using whole-cell patch- and voltage-clamp experiments. 2D and 3D cell models were used to determine antiproliferative activity. Structural optimization resulted in the most potent and selective KV1.3 inhibitor 44 in the series with an IC50 value of 470 nM in oocytes and 950 nM in Ltk cells. Kv1.3 inhibitor 4 induced significant apoptosis in Colo-357 spheroids, while 14, 37, 43, and 44 significantly inhibited Panc-1 proliferation.
REVIEW | doi:10.20944/preprints201810.0033.v1
Subject: Life Sciences, Immunology Keywords: apoptosis; viral persistence, hepatitis C virus; immunity; chronic infection
Online: 2 October 2018 (16:34:19 CEST)
Hepatitis C virus (HCV) represents a challenging global health threat in ~200 million infected individuals. Clinical data suggests that only ~10-15% of acutely HCV-infected individuals will achieve spontaneous viral clearance despite exuberant virus-specific immune responses, which is largely attributed to difficulties in recognizing the pathognomonic symptoms during the initial stages of exposure to the virus. Given the paucity of a suitable small animal model, it is also equally challenging to study the early phases of viral establishment. Further, the host factors contributing to HCV chronicity in a vast majority of acutely HCV-infected individuals largely remain unexplored. The last few years have witnessed a surge in studies showing that HCV adopts a myriad mechanisms to disconcert virus-specific immune responses in the host to establish persistence that includes, but not limited to viral escape mutations, viral growth at privileged sites, and antagonism. Here, we discussed a few hitherto poorly explained mechanisms employed by HCV that are believed to lead to chronicity in infected individuals. A better understanding of these mechanisms would aid the design of improved therapeutic targets against viral establishment in susceptible individuals.
REVIEW | doi:10.20944/preprints201806.0161.v1
Subject: Life Sciences, Molecular Biology Keywords: miR-29a; cholestasis; apoptosis; ER stress; toll like receptors; epigenetics
Online: 11 June 2018 (14:03:48 CEST)
Both fibrosis and cirrhosis of the liver are the end results of most kinds of chronic liver damage and present a common but difficult clinical challenge throughout the world. The inhibition of the fibrogenic, proliferative, and migratory effects of hepatic stellate cells (HSCs) has become an experimental therapy for preventing and even reversing hepatic fibrosis. Furthermore, a complete understanding of the function of non-coding RNA-mediated epigenetic mechanisms in HSC activation may improve our perception of liver fibrosis pathogenesis. This review focuses on an evolving view of molecular mechanisms in which HSC activation by miR-29a signaling may moderate their profibrogenic phenotype, thus supporting the use of miR-29a agonists as a potential therapy for treating liver fibrosis in the future.
ARTICLE | doi:10.20944/preprints202104.0223.v1
Subject: Medicine & Pharmacology, Allergology Keywords: Apoptosis; PARP; Caspase 3; Neurodegeneration; GPR4 receptor; MPTP; Parkinson’s disease
Online: 8 April 2021 (09:12:06 CEST)
GPR4, a member of proton activated GPCRs group. Previously we have reported that GPR4 is constitutively active at physiological pH and knockout of GPR4 has shown to protect dopaminergic neuronal cells from caspase-dependent mitochondrial apoptotic cell death. In this study we have investigated the role of GPR4 in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) treated mice model of Parkinson’s disease. Subchronic administration of MPTP in mice produces oxidative stress induced apoptotic cell death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and motor deficits. Treatment with NE52-QQ57, a selective antagonist of GPR4 reduced dopaminergic neuronal loss MPTP-intoxicated C57BL6/J mice and improved motor deficit and memory impairment. Co-treatment with NE52-QQ57 significantly decreases the protein level of proapoptotic marker (Bax), and increases the antiapoptotic marker (Bcl-2) in the SNpc and striatum tissue collected from the brain of MPTP inflicted mice. Further, MPTP induced activation of caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP) was significantly decreased in the SNpc and striatum tissue of NE52-QQ57 cotreated mice. Further mice receiving both MPTP and NE52-QQ57 mice showed significantly higher TH positive cells in the SNpc and striatum than MPTP treated mice alone. Moreover, NE52-QQ57 cotreatment improved the motor activity in the rotarod test and pole test and also improved spatial memory in Y maze test. Our findings suggest GPR4 as a potential therapeutic target for PD whereas the activation GPR4 is involved in the caspase mediated apoptotic cell death in SNpc and striatum of MPTP-intoxicated mice.
ARTICLE | doi:10.20944/preprints201808.0043.v1
Subject: Life Sciences, Molecular Biology Keywords: cryptotanshinone; NSCLC; cell cycle arrest; apoptosis; PI3K/Akt/GSK3β
Online: 2 August 2018 (09:05:32 CEST)
Cryptotanshinone (CTT) is a natural product and a quinoid diterpene isolated from the root of the Asian medicinal plant, Salvia miltiorrhiza bunge. Notably, CTT has a variety of anti-cancer actions, including the activation of apoptosis, anti-proliferation, and a reduction in angiogenesis. We further investigated the anti-cancer effects of CTT in A549 and H460 which are NSCLC cell lines. CTT treatment in NSCLC cells reduced cell growth through PI3K/Akt/GSK3β pathway inhibition, G0 / G1 cell cycle arrest, and the activation of apoptosis. CTT induced increase of Bax and cleavage of apoptosis-related signaling such as caspase-3, caspase-9, poly-ADP-ribose polymerase (PARP), and Bax, as well as inhibition of anti-apoptosis related signaling such as Bcl-2, survivin, and cellular-inhibitor of apoptosis protein 1 and 2 (cIAP-1 and -2). It also induced G0/G1 phase cell cycle arrest by decreasing the expression of cyclin A, cyclin D, cyclin E, Cdk 2, and Cdk 4. In addition, CTT reduced the protein expression of the PI3K/Akt/GSK3β signaling pathway related to cell proliferation. These results highlight the latent potential of CTT as natural therapeutic agent for NSCLC.
ARTICLE | doi:10.20944/preprints201808.0432.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: Breast cancer; Moracin D; Apoptosis; FOXM1; β-catenin; GSK 3β
Online: 24 August 2018 (10:58:08 CEST)
Though Moracin D derived from Morus alba was known to have anti-inflammatory and antioxidant activities, the underlying antitumor mechanism of Moracin D was never unveiled so far. Thus, in the recent study, the apoptotic mechanism of Moracin D was elucidated in breast cancer cells. Herein, Moracin D exerted significant cytotoxicity in MDA-MB231 and MCF7 cells. Also, Moracin D increased sub G1 population, cleaved poly (ADP-ribose) polymerase (PARP) and attenuated the expression of pro-cysteine aspartyl-specific protease (procaspase 3), c-Myc, cyclin D1, B-cell lymphoma 2 (Bcl-2),and X-linked inhibitor of apoptosis protein (XIAP) in MDA-MB231 cells. Of note, Moracin D reduced expression of Forkhead box M1 (FOXM1), β-catenin, Wnt3a, and upregulated glycogen synthase kinase 3 beta (GSK 3β) on Tyr216 along with disturbed binding of FOXM1 with β-catenin in MDA-MB-231 cells. Conversely, GSK3β inhibitor SB216763 reversed the apoptotic ability of Moracin D to reduce expression of FOXM1, β-catenin, pro-caspase3 and pro-PARP in MDA-MB-231 cells. Overall, these findings provide novel insight that Moracin D inhibits proliferation and induces apoptosis via suppression of Wnt3a/FOXM1/β-catenin signaling and activation of caspase and GSK3β
ARTICLE | doi:10.20944/preprints201911.0239.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: edible marine alga; anticancer; hizikia fusiformis; saringosterol acetate; apoptosis
Online: 20 November 2019 (11:19:19 CET)
Hizikia fusiformis is a common, edible marine alga found in Asia. Although the anticancer activity of its extracts has previously been investigated, its active compounds have not been identified. In this study, saringosterol acetate (SA) was isolated from H. fusiformis extracts by centrifugal partition chromatography (CPC) system (two phase solvents condition: n-hexane:ethyl acetate/methanol:water = 5:3:7:1, v/v), exhibited anticancer effects in the human lung adenocarcinoma epithelial cell line, A549, by inducing apoptosis and sub-G1 phase cell cycle arrest. In addition, SA increased the expression of the pro-apoptotic proteins, Bax and cleaved caspase 3, and decreased that of the anti-apoptotic protein Bcl-xL. Although, SA did not affect the expression of p53, induces expression of Bid and caspase 8. In conclusion, we suggested that SA induces apoptosis against A549 cells via Bid and caspase 8 dependent pathway.
ARTICLE | doi:10.20944/preprints201712.0196.v1
Subject: Medicine & Pharmacology, Other Keywords: traumatic brain injury; fluid percussion injury; neurodegeneration; apoptosis; sensorimotor deficit
Online: 28 December 2017 (08:58:32 CET)
Traumatic brain injury (TBI) can result in persistent sensorimotor and cognitive deficits, which occur through a cascade of deleterious pathophysiological events over time. In this study, we investigated the hypothesis that neurodegeneration caused by TBI leads to impairments in sensorimotor function. TBI induces the activation of the caspase-3 enzyme, which triggers cell apoptosis in an in vivo model of fluid percussion injury (FPI). We analyzed caspase-3 mediated apoptosis by TUNEL staining and PARP and annexin V western blotting. We correlated the neurodegeneration with sensorimotor deficits by conducting the animal behavioral tests including grid walk, balance beam, inverted screen test, and climb test. Our study demonstrated that the excess cell death or neurodegeneration correlated with the neuronal dysfunction and sensorimotor impairments associated with TBI.
ARTICLE | doi:10.20944/preprints201809.0347.v1
Subject: Biology, Physiology Keywords: Aeluropus lagopoides; HepG2; HCT-116; MCF-7; apoptosis; chemical composition
Online: 18 September 2018 (11:40:12 CEST)
Natural product especially secondary metabolites that produced by plants under the stressed condition shown to have a different pharmacological impact. Aeluropus lagopoides is one of the typical halophyte plants survivals under stressed conditions. It has been used for wound healing and as a painkiller. The bioactivity and the chemical composition of this plant have poorly investigated. Consequently, chemical components of A. lagopoides leaves were extracted using hexane (nonpolar), ethyl acetate (semi-polar), n-butanol (polar) to extract the most extensive variety of metabolites. The cytotoxicity and anticancer impact of extracted secondary metabolites evaluated against breast (MCF-7), colon (HCT-116), and liver (HepG2) cancer cell lines using SRB test. The mechanism of action verified by observing the appearance of apoptotic bodies using the fluorescent microscope while their antiproliferative impact had been evaluated using flow cytometer. Results revealed that secondary metabolites extracted using hexane and ethyl acetate were having the highest cytotoxicity and thus anticancer activity effect on HepG2 with IC50 (24.29 ± 0.85, 11.22 ± 0.679 µg/mL) respectively. Where apoptotic bodies observed, flow cytometer results exhibited that secondary metabolites can inhibit cell cycle in G0/G1 phase. Accordingly, A. lagopoides hexane and ethyl acetate extracts may consider as a candidate anti-cancer drug.
REVIEW | doi:10.20944/preprints202107.0612.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: Gastric cancer; natural products; autophagy; apoptosis; angiogenesis; metastasis; chemo-resistance.
Online: 27 July 2021 (22:20:18 CEST)
Gastric cancer, also known as stomach cancer, is a cancer which develops from the lining of the stomach. Accumulated evidences and epidemiological studies have been indicated that natural products play an important role in gastric cancer prevention and treatment, although its mechanism of action did not elucidate yet. Particularly, experimental studies have been showed that natural products displayed a protective effect against gastric cancer via numerous molecular mechanisms such as suppression of cell metastasis, anti-angiogenesis, inhibition of cell proliferation, induction of apoptosis, and modulation of autophagy. Although chemotherapy remains the standard treatment for advanced gastric cancer along with surgery, radiation therapy, hormone therapy and immunotherapy, but its adverse side effects including neutropenia, stomatitis, mucositis, diarrhea, nausea, and emesis are well documented. Additionally, intake of naturally occurring phytochemicals could increase the efficacy of gastric chemotherapy and chemotherapeutics resistance. However, natural product structural stability and powerful bioactivity are important to develop novel treatments for gastric cancer that may minimize such adverse effects. Therefore, the purpose of this review is to summarize the potential therapeutic effects of natural products on prevention and treatment of gastric cancer with intensive molecular mechanisms of action, bioavailability, and safety efficacy.
ARTICLE | doi:10.20944/preprints202301.0021.v1
Subject: Life Sciences, Other Keywords: TBI; brain; injury; microglia; caspase; apoptosis; retina; degeneration
Online: 3 January 2023 (08:38:56 CET)
A Traumatic brain injury (TBI) is among the main causes of sudden death after head trauma. These injuries can result in severe degeneration and neuronal cell death in the CNS, including the retina which is a crucial part of the brain responsible for perceiving and transmitting visual information. The long-term effects of mild-repetitive TBI (rmTBI) are far less studied thus far, even though damages induced by repetitive injuries occurring in the brain are more common, especially amongst athletes. rmTBI can also have a detrimental effect on the retina and the pathophysiology of these injuries are likely to differ from the severe TBI (sTBI) retinal injury.Here we showed how rmTBI and sTBI can dissimilarly affect the retina. Our results indicate an increase in the number of activated microglial cells and Caspase3-positive cells in the retina in both traumatic models, suggesting a rise in the level of inflammation and cell death after TBI. The pattern of microglial activation appears evenly distributed and widespread but differs amongst the various retinal layers. sTBI induced microgial activation in both the superficial and deep retinal layers. In contrast to sTBI, no significant change occurred following the repetitive mild injury in the superficial layer, only the deep layer (spanning from the inner nuclear layer to the outer plexiform layer) shows microglial activation. This difference suggests that alternate response mechanisms play a role in the case of the different TBI incidents. The Caspase3 activation pattern showed a uniform increase in both the superficial and deep layers of the retina. This suggests a different action in the course of the disease in sTBI and rmTBI models and points to the need for new diagnostic procedures.Our present results suggest that the retina might serve as such a model of head injuries since the retinal tissue reacts to both forms of TBI and is the most accessible part of the human brain.
REVIEW | doi:10.20944/preprints202204.0093.v1
Subject: Life Sciences, Immunology Keywords: Electrophile signaling; drug mechanism; immunology; antioxidant response; apoptosis
Online: 11 April 2022 (10:39:31 CEST)
Here we draw insights from the latest serendipitous findings made on the opposing roles of a validated drug-target protein Keap1. We weigh up how natural reactive electrophiles and electrophilic small-molecule drugs in clinical use directly impinge on seemingly conflicting, yet both Keap1-electrophile-modification-dependent, cell-survival- vs. cell-death-promoting behaviors. In the process, we convey how understanding reactive chemical-signal regulation at a single-protein-specific level is an enabling necessity in deconstructing otherwise intricate reactive-small-molecule-responsive cellular pathways. We hope this opinion piece further spurs the broader interests of basic and pharmaceutical research communities toward better understanding of molecular mechanisms underpinning reactive small-molecule-regulated signaling subsystems.
REVIEW | doi:10.20944/preprints202111.0127.v1
Subject: Biology, Animal Sciences & Zoology Keywords: Apoptosis; preclinical research; diabetes type 2; HIPPO pathway
Online: 8 November 2021 (11:45:49 CET)
Diabetes mellitus is a heterogeneous disease of complex etiology and pathogenesis. Hyperglycemia leads to many serious complications, but also directly initiates the process of β cell apoptosis. A potential strategy for the preservation of pancreatic β cells in diabetes may be to inhibit the implementation of pro-apoptotic pathways or to enhance the action of pancreatic protective factors. The HIPPO signaling pathway is proposed and selected as a target to manipulate the activity of its core proteins in therapy - basic research. MST1 and LATS2 as major upstream signaling kinases of the Hippo pathway are considered as target candidates for pharmacologically induced tissue regeneration and inhibition of apoptosis. Manipulating the activity of components of the HiPPO pathway offers a wide range of possibilities, and thus is a potential tool in the treatment of diabetes and the regeneration of β cells. Therefore, it is important to fully understand the processes involved in apoptosis in diabetic states and to fully characterize the role of this pathway in diabetes. Therapy consisting in slowing down or stopping the mechanisms of apoptosis may be an important direction of diabetes treatment shortly.
REVIEW | doi:10.20944/preprints202106.0690.v1
Subject: Medicine & Pharmacology, Allergology Keywords: splenic micronodular B-cell lymphoma; corticosteroids; apoptosis; shrinkage
Online: 29 June 2021 (08:35:56 CEST)
Splenic micronodular T-cell/histiocyte-rich large B-cell lymphoma is possibly derived from nodal T-cell/histiocyte-rich large B-cell lymphoma; however, a transition between the nodal and splenic micronodular forms has not been described to date. Of note, the only lymph nodes to be involved in association with the splenic micronodular pattern of the disease are the splenic hilar lymph nodes, and that, with partial involvement only. Kan et al, in their series of articles, have raised the possibility that corticosteroids, when prescribed prior to splenectomy, cause histopathological and functional modulations (apoptosis, necrosis, tissue shrinkage), which modify or even obscure the diagnostic morphological features. The indications for glucocorticoid therapy are either related to the suspected lymphoma, or else to other comorbidities, like asthma and autoimmune disorders. We propose that patients with the splenic, rather than nodal subset of the disease are likely to have been prescribed corticosteroids prior to histopathologic examination of the involved tissue, causing disparate morphologies in the spleen. Apoptosis, as induced by corticosteroids, is hypothesized as the major mechanism initiating the histopa-thological and functional changes in the splenic micronodular variant of our patients.
Subject: Life Sciences, Biochemistry Keywords: chronic intermittent hypoxia; autophagy; apoptosis; cardiomyocyte damage; calcineurin
Online: 24 June 2021 (14:58:22 CEST)
Calcineurin plays a key role in cardiovascular pathogenesis by exerting pro-apoptotic effects in cardiomyocytes; however, its involvement in the regulation of cardiomyocyte autophagy under chronic intermittent hypoxia (CIH) remains largely unknown. Here we showed that CIH induced calcineurin activity in H9C2 cells, resulting in the attenuation of adenosine monophos-phate-activated protein kinase (AMPK) signaling and inhibition of H9C2 cell autophagy. Au-tophagy, LC3-II levels, and AMPK phosphorylation were significantly elevated in response to CIH in H9C2 cells by day 3; however, these effects were reversed, and calcineurin activity and apoptosis were significantly increased by day 5. The calcineurin inhibitor, FK506, restored AMPK activation and LC3 protein levels, and reduced CIH-induced H9C2 cell apoptosis, while calcineurin overexpression significantly attenuated the increase in LC3 levels and enhanced H9C2 cell apop-tosis. Calcineurin inhibition failed to induce autophagy or alleviate apoptosis in H9C2 cells ex-pressing a dominant negative K45R AMPK mutant. Autophagy downregulation abrogated the protective effects of FK506-mediated calcineurin inhibition. These results indicated that calcineurin suppressed adaptive autophagy during CIH by downregulating AMPK activation. Our findings showed the underlying mechanisms of calcineurin and autophagy regulation during H9C2 cell survival in response to CIH, and suggested a new strategy for preventing CIH-induced cardiomyocyte damage.
REVIEW | doi:10.20944/preprints202102.0289.v1
Subject: Life Sciences, Biochemistry Keywords: Cytokine, Depression, Ischemia, Stroke, Apoptosis, Excitotoxicity, Onecosis, Inflammation,
Online: 11 February 2021 (16:46:06 CET)
Ischemic Stroke precedes depression . Post Stroke Depression (PSD) is a major driver for poor recovery, negative quality of life, poor rehabilitation outcomes and poor functional ability. This systematic reviews confirmed the post stroke depression as the norm as complex ischemic cascade involve the bioenergetic failure, deranged iron homeostasis ( calcium influx, Na influx, potassium efflux etc) excitotoxicity, acidotoxicity,disruption of the blood brain barrier, cytokine mediated cytotoxicity, reactive oxygen mediated toxicity , activation of cyclooxygenase pathway and generation of toxic products, infiltration of immune mediated cells resulting the cell death and deranged neuronal networks in mood related brain regions. This review focus on the pathobiology of stroke in the context and make the argument that PSD is the norm after a stroke rather than the exception.
REVIEW | doi:10.20944/preprints202007.0344.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: UPR; NOXA; DR5; BCL2; apoptosis; necroptosis; ferroptosis; proteotoxic
Online: 16 July 2020 (07:41:31 CEST)
To maintain proteostasis, cells must integrate information and activities that supervise protein synthesis, protein folding, conformational stability, and also protein degradation. Extrinsic and intrinsic conditions can both impact on normal proteostasis, causing the appearance of proteotoxic stress. Initially, proteotoxic stress elicits adaptive responses aimed to restore proteostasis, allowing cells to survival the stress condition. However, if the proteostasis restoration fails, a permanent and sustained proteotoxic stress can be deleterious and cell death ensues. Many cancer cells convive with high levels of proteotoxic stress and this condition could be exploited in a therapeutic perspective. Understanding the cell death pathways engaged by proteotoxic stress is instrumental to better hijack the proliferative fate of cancer cells.
REVIEW | doi:10.20944/preprints201903.0116.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: beetroot; betanins; cancer chemoprevention; anti-oxidant; inflammation; apoptosis
Online: 11 March 2019 (08:00:34 CET)
Carcinogenesis is the process whereby a normal cell is transformed into a neoplastic cell. This action involves several steps starting with initiation and followed by promotion and progression. Driving these stages are oxidative stress and inflammation, which in turn encompasses a myriad of aberrant gene expressions, both within the transforming cell population and the cells within the surrounding lesion. Chemoprevention of cancer with bioreactive foods or their extracted/purified components occurs via normalizing these inappropriate gene activities. Various foods/agents have been shown to affect different gene expressions. In this review we discuss whereby the chemoprevention activities of the red beetroot itself may disrupt carcinogenesis and the activities of the water soluble betalains extracted from the plant.
REVIEW | doi:10.20944/preprints202110.0251.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: pluripotent; embryo; stem cells; genomic stability; cell cycle; apoptosis; differentiation; cancer
Online: 18 October 2021 (15:12:11 CEST)
Abstract: Remarkably, the p53 transcription factor, referred to as “the guardian of the genome”, is not essential for mammalian development. Moreover, efforts to identify p53‑dependent developmental events have produced contradictory conclusions. Given the importance of pluripotent stem cells as models of mammalian development, and their applications in regenerative medicine and disease, resolving these conflicts is essential. Here we attempt to reconcile disparate data into justifiable conclusions predicated on reports that p53‑dependent transcription is first detected in late mouse blastocysts, that p53 activity first becomes potentially lethal during gastrulation, and that apoptosis does not depend on p53. Furthermore, p53 does not regulate expression of genes required for pluripotency in embryonic stem cells (ESCs); it contributes to ESC genomic stability and differentiation. Depending on conditions, p53 accelerates initiation of apoptosis in ESCs in response to DNA damage, but cell cycle arrest as well as the rate and extent of apoptosis in ESCs are p53-independent. In embryonic fibroblasts, p53 induces cell cycle arrest to allow repair of DNA damage, and cell senescence to prevent proliferation of cells with extensive damage.
ARTICLE | doi:10.20944/preprints201804.0082.v2
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: Resveratrol; TRAIL; apoptosis; lung cancer cells; NF-κB; Cytochrome c
Online: 13 August 2018 (06:18:03 CEST)
Aims TRAIL is a promising anticancer agent that has the potential to sensitize a wide variety of cancer or transformed cells by inducing apoptosis. However, resistance to TRAIL is a growing concern. Current manuscript aimed to employing combination treatment to investigate resveratrol induced TRAIL sensitization in NSCLC. Method A549 and HCC-15 cells were used in experimental design. Cell viability was determined by morphological image, crystal violet staining and MTT assay. Apoptosis was evaluated by LDH assay, Annexin V and DAPI staining. Autophagy and apoptosis indicator protein were examined by western blotting. TEM and puncta assay were carried out to evaluate the autophagy. MTP and ROS activity were evaluated by JC-1 and H2DCFDA staining. Findings Resveratrol is a polyphenolic compound capable of activation of tumor suppressor p53 and its pro-apoptotic modulator PUMA. Herein, we showed the p53-independent apoptosis by decrease the expression of phosphorylated Akt-mediated suppression of NF-κB that is also substantiated with the downregulation of anti-apoptotic factors Bcl-2 and Bcl-xl in NSCLC, resulting in an attenuation of TRAIL resistance in combined treatment. Furthermore, apoptosis was induced in TRAIL-resistant lung cancer cells with a co-treatment of resveratrol and TRAIL assessed by the loss of MMP, ROS generations which resulting the translocation of cytochrome c from the mitochondria into the cytosol due to mitochondrial dysfunction. Moreover, autophagy flux was not affected by resveratrol-induced TRAIL-mediated apoptosis in NSCLC. Significance Overall, targeting the NF-κB (p65) pathway via resveratrol attenuates TRAIL resistance and induces TRAIL-mediated apoptosis which could be the effective TRAIL-based cancer therapy regimen.
ARTICLE | doi:10.20944/preprints202005.0212.v1
Subject: Medicine & Pharmacology, General Medical Research Keywords: Annona squamosa; phytomedicine; proliferation; apoptosis; migration; estrogen receptor; tumor size
Online: 12 May 2020 (12:37:43 CEST)
: Annona squamosa L. is an important medicinal plant used in traditional medicine for the treatment of various diseases. Different parts of A. squamosa L. have various therapeutic effects; however, the anticancer activity of the leaves has not yet been identified. In vitro, MTT, nuclear staining, and LDH assays were used to evaluate cell survival and proliferation in cells exposed to the extracts. The effect of the extracts on cell migration was investigated using a monolayer wound repair assay, and the apoptotic effects were evaluated using flow cytometry. A breast cancer model was used to study the effect of the extract on the tumor size, and the expression of different proliferative and apoptotic markers was evaluated by immunohistochemical analysis. At a concentration of 100 µg/mL, A. squamosa leaf extracts exerted strong antiproliferative and cytotoxic effects against various cell lines. The extracts reduced wound closure and strongly induced apoptosis. In vivo study, rats were sacrificed 24 h after the last injection, and tumor size, as well as the expression of proliferative and apoptotic markers, were observed to be greatly affected by treatment with the extracts. Therefore, A. squamosa leaf extract may be developed as a potential novel drug to treat breast cancer in the future
ARTICLE | doi:10.20944/preprints201903.0045.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: Thienopyrimidines, RP-010, Prostate cancer, Metastasis, Wnt/β-catenin, Apoptosis.
Online: 4 March 2019 (13:37:02 CET)
Thienopyrimidines are a versatile group of compounds that contain a biologically active pharmacophore and reported to have anticancer efficacy in vitro. Here, we report for the first time, that thieno[3,2-d]pyrimidine - based compounds, designated the RP series, have efficacy in prostate cancer cells. The lead compound, RP-010, was efficacious in PC3 and DU-145 prostate cancer (PC) cells (IC50< 1µM). The cytotoxicity of RP-010 was significantly lower in normal cells. RP-010 (0.5, 1, 2, and 4 µM) arrested prostate cancer cells in the G2 phase of the cell cycle, induced mitotic catastrophe and apoptotic signaling in both PC cell lines. Mechanistic studies suggested that RP-010 (1 and 2 µM) inhibits the wingless-type MMTV (Wnt)/β-catenin signaling pathway, mainly by inducing β-catenin fragmentation, while down regulating important proteins in the pathway, i.e. LRP-6, DVL3, and c-Myc. Interestingly, RP-010 (1 and 2 µM) induced the nuclear translocation of the negative feedback proteins, Naked 1 and Naked 2, in the signaling pathway. In addition, RP-010 (0.5, 1, 2, and 4 µM) significantly decreased the migration and invasiveness of PC cells in vitro. Finally, RP-010 did not produce significant toxic effects in zebrafish at concentrations up to 6 µM. In conclusion, RP-10 is a promising anticancer compound in metastatic prostate cancer and did not produce overt toxicity in an in vivo zebrafish model. Future mechanistic and efficacy studies are needed in-vivo to optimize the lead compound RP-010 for clinical use.
ARTICLE | doi:10.20944/preprints202001.0320.v1
Subject: Life Sciences, Other Keywords: conjugated linolenic acid; Caco-2 cell; lipid peroxidation; apoptosis; pyroptosis
Online: 27 January 2020 (02:03:33 CET)
The probiotic bacterial strain Lactobacillus plantarum ZS2058 has been proved to manifest comprehensive functions, which were due to ability to synthesise conjugated fatty acids (CFAs). To investigate the specific functions of CFAs produced by this probiotic bacterium, α-linolenic acid was isomerized by Lactobacillus plantarum strain ZS2058, and two different conjugated α-linolenic acid (CLNA) isomers were successfully isolated. These isoforms, CLNA1 (c9, t11, c15-CLNA, purity 97.48%) and CLNA2 (c9, t11, t15-CLNA, purity 99.00%), both showed the ability to inhibit the growth of three types of colon cancer cells in a time- and concentration-dependent manner. In addition, the expression of MDA in Caco-2 cells was increased by CLNA1 or CLNA2, which indicated lipid peroxidation was related to the antiproliferation activity of CLNAs. Examination of the key protein of pyroptosis showed that CLNA1 induced the cleavage of caspase-1 and gasdermin-D, while CLNA2 induced the cleavage of caspase-4, 5 and gasdermin-D. The addition of relative inhibitors could alleviate the pyroptosis by CLNAs. CLNA1 and CLNA2 showed no effect on caspase-3, 7, 9 and PARP-1, which were key proteins associated with apoptosis. And no sub-diploid apoptotic peak appeared in the result of PI single staining test. In conclusion, CLNA1 activated caspase-1 and induced Caco-2 cell pyroptosis, whereas CLNA2 induced pyroptosis through the caspase-4/5-mediated pathway. The inhibition of Caco-2 cells by the two isomers was not related to apoptosis. This is the first report showing the ability of CLNAs to activate antioxidant defenses resulting in pyroptosis.
Subject: Materials Science, Biomaterials Keywords: bismuth selenide nanoparticles; photothermal killing; apoptosis; autophagy; stress-related signaling pathway
Online: 10 May 2021 (10:52:30 CEST)
With a highly efficient optical absorption capability, bismuth selenide (Bi2Se3) nanomaterial can be used as an outstanding photothermal agent for anti-tumor treatment and shows promise in the field of nanotechnology-based biomedicine. However, little research has been done on the relevant mechanism underlying the photothermal killing effect of Bi2Se3 nanomaterial. Herein, the photothermal effects of Bi2Se3 nanoparticles on A549 cells were explored with emphasis put on autophagy. Firstly, we characterized the structure and physicochemical property of the synthesized Bi2Se3 and confirmed their excellent photothermal conversion efficiency (35.72%), photostability, biocompatibility and ability of photothermal killing on A549 cells. Enhanced autophagy was detected in Bi2Se3-exposed cells under an 808 nm laser. Consistently, an elevated expression ratio of LC3-II to LC3-I, a marker of autophagy occurrence, was induced in Bi2Se3-exposed cells upon NIR irradiation. Meanwhile, the expression of cleaved-PARP was increased in the irradiated cells dependently on the exposure concentrations of Bi2Se3 nanoparticles. Pharmacological inhibition of autophagy by 3-methyladenine (3-MA) further strengthened the photothermal killing effect of Bi2Se3. Meanwhile, stress-related signaling pathways including p38 and SAPK/JNK were activated coupled with the attenuated PI3K/Akt signaling. Our study figures out that autophagy and the activation of stress-related signaling pathways were involved in the photothermal killing of cancerous cells by Bi2Se3, which provides a more understanding of photothermal nanomaterials.
REVIEW | doi:10.20944/preprints201810.0428.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: amino acids, cancer, energy metabolism, autophagy, apoptosis, glutamine, diabetes type 2.
Online: 18 October 2018 (16:45:01 CEST)
Production of energy is a main task of cancer cells metabolism, since costs of duplicating are enormous. Although energy is derived in cells by dismantling carbon to carbon bonds of any macronutrient, cancer nutritional needs for energetic purposes have been studied primarily as dependent on glycolysis. Since the end of the last century, awareness of dependence of cancer metabolism on amino acids not only for protein syntheses but also for matching energy needs has grown. The roles of specific amino acids, like glutamine, glycine and serine have been explored in different experimental conditions and reviewed. Moreover, there are epidemiological evidences that some amino acids used as supplement for therapeutic reasons (the branched chain ones) may reduce incidence of liver cancer, and some molecular mechanism has been proposed as functional to their protective action. On the contrary, metabolic signature of some pathology connected with increased risk of cancer, like prolonged hyperinsulinemia in insulin resistant patients, is signed by plasma elevated levels of the same branched chain amino acids, posing puzzling questions to clinicians. Most recently, peculiar formulations of amino acids, deeply different if compared to amino acids compositions normally present in foods, have shown the power to master epigenetics slowing growth or driving cancer cells to apoptotic death, while being even beneficial for normal cells and for animals health and life span. In this review, we will analyze and try to disentangle some of the many knots dealing with complexities of amino acids biology and linked to cancer metabolism.
REVIEW | doi:10.20944/preprints201811.0592.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: caspase-3; cell death; apoptosis; cell proliferation; neurons; glia; cerebellum; development
Online: 26 November 2018 (11:48:00 CET)
Caspase-3, onto which there is a convergence of the intrinsic and extrinsic apoptotic pathways, is the main executioner of apoptosis. We here review the current literature on the intervention of the protease in the execution of naturally occurring neuronal death (NOND) during cerebellar development. We will consider data on the most common altricial species (rat, mouse and rabbit), as well as humans. Among the different types of neurons and glia in cerebellum, there is ample evidence for an intervention of caspase-3 in the regulation of NOND of the post-mitotic cerebellar granule cells (CGCs) and Purkinje neurons as a consequence of failure to establish proper synaptic contacts with target (secondary cell death). It seems possible that also the GABAergic interneurons undergo a similar type of secondary cell death, but the intervention of caspase-3 in this case still remains to be clarified in full. Remarkably, CGCs also undergo primary cell death at the precursor/pre-migratory stage of differentiation, in this case without the intervention of caspase-3. Glial cells as well undergo a process of regulated cell death, but it seems possible that expression of caspase-3, at least in the Bergmann glia, is related to differentiation rather than death.
REVIEW | doi:10.20944/preprints201809.0281.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: axon guidance; growth cone; cytoskeleton; caspases; apoptosis; signal integration; basal level of caspase activity; death associated inhibitor of apoptosis; axon branching; Netrin; DCC; frazzled; slit; robo; Drosophila
Online: 16 September 2018 (09:43:52 CEST)
Navigating growth cones are exposed to multiple signals simultaneously and have to integrate competing cues into a coherent navigational response. Integration of guidance cues is traditionally thought to occur at the level of cytoskeletal dynamics. Drosophila studies indicate that cells exhibit a low level of continuous caspase protease activation, and that axon guidance cues can activate or suppress caspase activity. We base a model for axon guidance on these observations. By analogy with other systems in which caspase signaling has non-apoptotic functions, we propose that caspase signaling can either reinforce repulsion or negate attraction in response to external guidance cues by cleaving cytoskeletal proteins. Over the course of an entire trajectory, incorrectly navigating axons may pass the threshold for apoptosis and be eliminated, whereas axons making correct decisions will survive. These observations would also explain why neurotrophic factors can act as axon guidance cues and why axon guidance systems such as Slit/Robo signaling may act as tumor suppressors in cancer.
ARTICLE | doi:10.20944/preprints202211.0509.v1
Subject: Chemistry, Medicinal Chemistry Keywords: isatin; indolin-2-one; acute myeloid leukemia; apoptosis; ERK1/2; MAPK
Online: 28 November 2022 (09:59:20 CET)
Searching for bioactive compounds within the huge chemical space is like trying to find a needle in a haystack. Isatin is a unique natural compound which is endowed with different biopertinent activities specially in cancer therapy. Herein, we envisaged that adopting a hybrid strategy of isatin and α,β-unsaturated ketone would afford new chemical entities with strong chemotherapeutic potential. Of interest, compounds 5b and 5g demonstrated significant antiproliferative activities against different cancer genotypes according to NCI assay. Concomitantly, their IC50 against HL-60 cells were 0.38 ± 0.08 and 0.57 ± 0.05, respectively, demonstrating remarkable apoptosis and mod-erate cell cycle arrest at G1 phase. Intriguingly, an impressive safety profile for 5b was reflected by a 37.2 times selectivity against HL-60 over PBMC from a healthy donor. This provoked us to further explore their mechanism of action by in vitro and in silico tools. Conclusively, 5b and 5g stand out as strong chemotherapeutic agents that hold a clinical promise against acute myeloid leukemia.
Subject: Life Sciences, Other Keywords: epigallocatechin-3-gallate; acetaminophen; cytochrome P-450; bioactivation; apoptosis; autophagy; hepatotoxicity
Online: 28 July 2019 (16:45:51 CEST)
Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea. In this study, the effects of dietary EGCG on oxidative stress and the metabolism and toxicity of acetaminophen in liver were investigated. Rats were fed the diets with (0.54 %) or without EGCG supplementation for four weeks and were then intraperitoneally injected with acetaminophen (1g/kg). Results showed EGCG lowered hepatic oxidative stress and cytochrome P450 (CYP) 1A2, 2E1, and 3A, and UDP-glucurosyltransferase activities prior to acetaminophen injection. After acetaminophen challenge, the elevations in plasma alanine aminotransferase activity and histological changes in liver were ameliorated by EGCG treatment. EGCG reduced acetaminophen-induced apoptosis by increasing the Bax/Bcl2 ratio in liver. EGCG mildly increased autophagy by increasing the LC3B II/I ratio. Lower hepatic acetaminophen-glutathione and acetaminophen-protein adducts contents were observed after EGCG treatment. EGCG increased glutathione peroxidase and NAD(P)H quinone 1 oxidoreductase activities and reduced organic anion-transporting polypeptides 1a1 expression in liver after acetaminophen treatment. Our results indicate that EGCG may lower oxidative stress and reduce the metabolism and toxicity of acetaminophen. The reductions in CYP-mediated acetaminophen bioactivation and uptake transporter, as well as enhanced antioxidant enzyme activity, may limit the accumulation of toxic products in liver and thus lower hepatotoxicity
ARTICLE | doi:10.20944/preprints201907.0279.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: beta adrenoblocker; anticancer; non-small cell lung cancer; clonogenic; apoptosis; necrosis
Online: 25 July 2019 (06:20:58 CEST)
Beta adrenoblockers is a large class of drugs mainly used to manage abnormal heart rhythms. Over the last couples of decades, the anticancer effects of these compounds has been extensively studied. There is much evidence about their activity in non-small cell lung, pancreatic, breast, colorectal, prostate and ovarian cancer. However, the mechanism of beta blockers anticancer activity is still not known, and more detailed studies are needed. The aim of our study was to evaluate the anticancer activity of beta adrenoblockers in non-small cell lung cancer cell lines A549 and H1299. In order to find the relationship with their selectivity to beta adrenoreceptors, in our study we used selective (atenolol, betaxolol, esmolol, metoprolol) and non-selective (pindolol, propranolol and timolol) beta blockers. The effect on cell viability was evaluated by MTT assay and the activity on cell ability to form colonies was tested by clonogenic assay. The type of cell death was evaluated by cell double staining with Hoechst 33342 and Propidium iodide. The most active adrenoblockers against both tested cancer cell lines were propranolol and betaxolol. They completely inhibited lung cancer cell colony formation at 90% of EC50 (half maximal effective concentration) value. Most tested compounds induced cell death through apoptosis and necrosis. In A549 cell lines apoptosis was mainly induced while in H1299 cell line compounds induced both apoptosis and necrosis. There was no correlation established between beta adrenoblocker anticancer activity and their selectivity to beta adrenoreceptors.
ARTICLE | doi:10.20944/preprints202107.0520.v1
Subject: Medicine & Pharmacology, Allergology Keywords: apoptosis; autophagy; bafilomycin A1; diffuse large B cell lymphoma
Online: 22 July 2021 (13:43:07 CEST)
Diffuse large B cell lymphoma (DLBCL), the most frequent type of non-Hodgkin lymphoma in adulthood, remains challenging clinical issues. Despite enhanced remission rates can be achieved, there are one-third of patients who will not respond to current treatment or will relapse with resistant disease, necessitating ongoing efforts on effective treatment strategies and agents. The vacuole H+-ATPase inhibitor bafilomycin A1 is broadly used to block late stage of autophagy flux at high concentration. In this study, we show that, to our surprise, bafilomycin A1 effectively inhibited and killed DLBCL cells at nanomolar concentrations (5nM). Bafilomycin A1 targeted cell cycle regulators cyclin D1 and cyclin E2 to induce cell cycle arrest in G0/G1 phase. Meanwhile, it induced caspase-dependent apoptosis with concomitant cleaved caspase-3 and Parp. Furthermore, we found that bafilomycin A1 inhibited autophagy flux at both early and late stages of the autophagy flux through activating ERK and mammalian target of rapamycin signaling, as well as by inhibiting the degradation of autolysosomes. We speculated that bafilomycin A1 as autophagy inhibitor might enhance the effect of DLBCL chemotherapeutic drug rituximab. Accordingly, our results provided evidence that the combination of bafilomycin A1 with rituximab enhanced the inhibition of DLBCL cells notably. Taken together, our data suggest that bafilomycin A1 may be a promising candidate drug in the therapy of diffuse large B cell lymphoma.
Subject: Biology, Anatomy & Morphology Keywords: Zika virus; ER stress; unfolded protein response; apoptosis; CHOP
Online: 16 February 2021 (13:48:02 CET)
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus considered as a threat to human health due to large epidemics and serious clinical outcomes such as microcephaly in newborns. Like all flaviviruses, ZIKV relies on the cellular machinery to complete its viral cycle, with the endoplasmic reticulum (ER) being the critical site of viral replication factories. The sudden high protein load in the ER induces an ER stress to which the cell responds with an appropriate unfolded protein response (UPR) in an attempt to restore its disturbed homeostasis. When the restoration fails, the cell signaling leads to a programmed cell death by apoptosis with the upregulation of the UPR-induced C/EBP homologous protein (CHOP) which acts as the main trigger for this fatal outcome. Our previous studies have shown the ability of ZIKV to manipulate various cellular responses in order to optimize virus production. ZIKV is able to delay apoptosis to its benefit and although ER stress is induced, the UPR is not complete. Here we discovered that ZIKV impairs the expression of CHOP/DDIT3, the main factor responsible of ER-stress driven apoptosis. Surprisingly, the mechanism does not take place at the transcriptional level but at the translational level.
ARTICLE | doi:10.20944/preprints202008.0389.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: ECN; neuropathic pain; oxidative stress; apoptosis; myelin sheath; spectroscopy
Online: 18 August 2020 (12:00:15 CEST)
7β-(3-ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), a sesquiterpenoid obtained from a natural origin (Tussilago farfara)has proved to be effective in minimizing various side effects associated with opioids and nonsteroidal anti-inflammatory drugs. The current study focused on investigating the effects of ECN on neuropathic pain induced by partial sciatic nerve ligation (PSNL) by mainly focusing on oxidative stress, inflammatory and apoptotic proteins expression in mice. Neuropathic pain was induced in mice by PSNL surgery performed on day 1 and ECN (1 and 10 mg/kg, i.p.), was administered once daily for 11 days, starting from the third day after surgery. ECN post-treatment was found to reduce hyperalgesia and allodynia in a dose dependent manner. ECN significantly reversed the severity of neuropathic pain by improving distress symptoms and survival rate. ECN remarkably reversed the histopathological abnormalities associated with oxidative stress, apoptosis and inflammation. Furthermore, ECN prevented the suppression of antioxidants (glutathione, glutathione-S-transferase, catalase, superoxide dismutase, NF-E2-related factor-2 (Nrf2), hemeoxygenase-1 and NAD(P)H: quinone oxidoreductase) by PSNL. Moreover, pro-inflammatory cytokines (tumor necrotic factor alpha, interleukin 1 beta, interleukin 6, cyclooxygenase-2 and inducible nitric oxide synthase) expression was reduced by ECN administration. Treatment with ECN was successful in reducing caspase-3 level consistent with the observed modulation of pro-apoptotic proteins. Additionally, ECN showed protective effect on the lipid content of myelin sheath as evident from FTIR spectroscopy which showed the shift of lipid component bands to higher values. Thus, anti-neuropathic potential of ECN might be due to inhibition of oxidative stress, inflammatory mediators and pro-apoptotic proteins.
ARTICLE | doi:10.20944/preprints202008.0054.v1
Subject: Life Sciences, Biotechnology Keywords: Amaryllidaceae; Boophone haemanthoides; Alkaloids; Terpenoids; Parkinson’s disease; Neuroprotection; Apoptosis
Online: 3 August 2020 (00:08:27 CEST)
Parkinson’s disease (PD) is a neurodegenerative disease that progresses with increasing age and some of its major symptoms include tremor, postural and movement related difficulties. Till date, the treatment of PD remains a challenge because available drugs only treat the symptoms of the disease or possess serious side effects. In light of this, new treatment options are needed, hence this study investigates the neuroprotective effects of an organic Boophone haemanthoides extract (BHE) and its bioactive compounds using an in vitro model of PD involving the toxin 1-methyl-4-phenylpyridinium (MPP+) and SH-SY5Y neuroblastoma cells. A total of seven compounds were isolated from BHE viz: distichamine (1), 1α,3α-diacetylnerbowdine (2), hippadine (3), stigmast-4-ene-3, 6-dione (4), cholest-4-en-3-one (5), tyrosol (6), and 3-hydroxy-1-(4`-hydroxyphenyl)-1-propanone (7). Six compounds (1, 2, 4, 5, 6, 7) were investigated and five showed neuroprotection alongside the BHE. This study gives insight into the bioactivity of the non-alkaloidal constituents of Amaryllidaceae since the isolated compounds and the BHE showed improved cell viability, increased ATP generation in the cells as well as inhibition of MPP+-induced apoptosis. Together, these findings support the claim that the Amaryllidaceae plant family could be a potential reserve of bioactive compounds for the discovery of neuroprotective agents.
ARTICLE | doi:10.20944/preprints201908.0167.v2
Subject: Life Sciences, Virology Keywords: Zika virus; apoptosis; viral replication; Bcl-2 protein family
Online: 18 September 2019 (03:05:26 CEST)
Zika virus (ZIKV) is an emerging human mosquito-transmitted pathogen of global concern, known to be associated with complications such as congenital defects and neurological disorders in adults. ZIKV infection is associated with induction of cell death. However, previous studies suggest that the virally-induced apoptosis occurs at a slower rate compared to the course of viral production. In this present study, we investigated the capacity of ZIKV to delay host cell apoptosis. We provide evidence that ZIKV has the ability to interfere with apoptosis whether it is intrinsically or extrinsically induced. In cells expressing viral replicon-type constructions, we show that this control is achieved through replication. Finally, our work highlights an important role for anti-apoptotic Bcl-2 family protein in the ability of ZIKV to control apoptotic pathways, avoiding premature cell death and thereby promoting virus replication in the host-cell.
ARTICLE | doi:10.20944/preprints201810.0768.v1
Subject: Chemistry, Medicinal Chemistry Keywords: silybin; prostate cancer; 2,3-dehydrosilybin; cell proliferation; cell apoptosis.
Online: 1 November 2018 (18:16:29 CET)
As part of our ongoing silybin project, this study aims to introduce a basic nitrogen-containing group to 7-OH of 3,5,20-O-trimethyl-2,3-dehydrosilybin or 3-OH of 5,7,20-O-trimethyl-2,3-dehydrosilybin via an appropriate linker for in vitro evaluation as potential anti-prostate cancer agents. The synthetic approaches to 7-O-substituted-3,5,20-O-trimethyl- 2,3-dehydrosilybins through a five-step procedure and to 3-O-substituted-5,7,20-O-trimethyl-2,3- dehydrosilybins via a four-step transformation have been developed. Thirty-two nitrogen-containing derivatives of silybin have been achieved through these synthetic methods for the evaluation of their antiproliferative activities towards both androgen-sensitive (LNCaP) and androgen-insensitive prostate cancer cell lines (PC-3 and DU145) using WST-1 cell proliferation assay. These derivatives exhibited greater in vitro antiproliferative potency than silybin. Among them, 11, 29, 31, 37, and 40 were identified as five optimal derivatives with IC50 values in the range of 1.40–3.06 µM, a 17- to 52-fold improvement in potency as compared with silybin. All these five optimal derivatives can arrest the PC-3 cell cycle in the G0/G1 phase and promote PC-3 cell apoptosis. Derivatives 11, 37, and 40 are more effective than 29 and 31 in activating PC-3 cell apoptosis.
REVIEW | doi:10.20944/preprints201705.0209.v2
Subject: Life Sciences, Immunology Keywords: influenza virus; apoptosis; antiviral agent; innate immunity; host response
Online: 14 August 2017 (04:41:22 CEST)
Human influenza A viruses (IAVs) cause global pandemics and epidemics, which remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral outbreaks, new treatments are urgently needed. Developing new virus control modalities requires better understanding of virus-host interactions. Here we describe how IAV infection triggers cellular apoptosis, and how this process can be exploited towards development of new therapeutics, which might be more effective than the currently available anti-influenza drugs.
ARTICLE | doi:10.20944/preprints201806.0141.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: Gambogenic acid; apoptosis; CNE-2Z cells; volume-sensitive outwardly rectifying chloride
Online: 8 June 2018 (16:00:24 CEST)
Nasopharyngeal carcinoma (NPC) has not been thoroughly studied, and the pathogenesis of NPC is unclear. Scientists have neither discovered effective therapies nor achieved a desirable prognosis. Some studies have found that the regulation of intra- and extracellular ion channels hinges directly on cell apoptosis, and treatment with Gambogenic acid (GNA) brings changes to the volume-sensitive outwardly rectifying chloride (VSOR Cl-) current of CNE-2Z cells recorded by the patch clamp method. Nevertheless, rarely have any researchers probed into the relevance between this variation and the anti-tumor mechanism of GNA. This paper is suggested that GNA activates the VSOR Cl- current on the CNE-2Z cell membrane, and the activation of VSOR Cl- currents by GNA in CNE-2Z cells is blocked by the chloride channel blockers DIDS and DCPIB. GNA induces the down-regulation of GRP78 and up-regulation of ATF4 as well as chop proteins, which is evidence for the induction of CNE-2Z cell apoptosis, and this correlates with ER stress. GNA can activate the VSOR Cl- channel and lead to the occurrence of ER stress, thus inducing the apoptosis of CNE-2Z cells and inhibiting the proliferation of CNE-2Z cells.
ARTICLE | doi:10.20944/preprints201804.0342.v1
Subject: Medicine & Pharmacology, Veterinary Medicine Keywords: apoptosis; breast cancer; E. coli O157:H7; necrosis; Shiga-like toxin
Online: 26 April 2018 (10:41:36 CEST)
Apoptosis and cell cycle arrest induction are targeted in the strategy of cancer therapy. Furthermore, bacterial toxin such as shiga-like toxin producing Escherichia coli has been suggested to be used as novel therapeutic agent against tumor malignancies either as independent anti-neoplastic agents or in combination treatment with chemo or radiotherapy. The aim of study was to investigate the potency of shiga-like toxin originated from local strains of E. coli O157:H7 as a new cancer therapy. As many as 10 culture cells T47D cell line were subjected by crude extract Shiga like toxin originated from five local isolates of E. coli O157:H7 i.e. KL-48(2), SM-25(1), SM-7(1), DS-21(4), and one isolate ATCC 43894 as a control with IC50 doses, respectively. The treatment was observed for 24 h, with two replications. An FITC-Annexin V and PI assay was used to observe apoptosis and necrosis effect, and simultaneously with cell cycle analysis using propidium iodide (PI) staining. Results of study showed T47D cell treated with Shiga-like toxin from local strain KL-48 (2) show the lowest viable cell, followed by SM 7(1), ATCC 43894, SM-25(1), DS-21(4) and contrary with control with each percentages as 15.20, 16.36, 22.17, 22.64, 33.86, and 94.36%, respectively. The results were also confirmed by the induction of the cell cycle arrest in phase G0-G1 as inactive phase, i.e. 66.41, 63.37, 61.52, 55.36 and 47.28% for T47D cell treated with toxin of KL-48(2), ATCC 43894, SM 25(1), SM 7(1), and DS 21(4), respectively. These results show tendency deleterious effect of Shiga-like toxin from local isolates on T47D cell, so that concluded they have potency as a good anticancer drug in Gb3-expressing breast cancer.
ARTICLE | doi:10.20944/preprints201804.0242.v1
Subject: Life Sciences, Biochemistry Keywords: Non-small cell lung cancer; Lambertianic acid; Apoptosis; TRAIL; XIAP; NF-B
Online: 18 April 2018 (15:25:10 CEST)
Lambertianic acid (LA) is a biologically active compound from the leaves of Pinus koraiensis. In the present study, apoptotic mechanisms of LA plus TNF-related apoptosis-inducing ligand (TRAIL) were elucidated in non-small cell lung cancer cells (NSCLCs). Cytotoxicity assay, flow cytometry, immunoprecipitation and Western blotting were performed. Here, combined treatment of LA and TRAIL increased cytotoxicity, sub-G1 population and cleaved poly (ADP-ribose) polymerase (PARP) and caspase3/8/9 in A549 and H1299 cells compared to LA or TRAIL alone. Furthermore, combined treatment of LA and TRAIL significantly decreased anti-apoptotic proteins such as B-cell lymphoma 2 (Bcl-2), Fas-like inhibitor protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) and enhanced the activation of pro-apoptotic proteins Bid compared to LA or TRAIL alone. In addition, combined treatment of LA and TRAIL upregulated the expression of Death receptor 4 (DR4) and downregulated phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-B), inhibitory protein of kB family (p-IB) and FLIP in A549 and H1299 cells along with disrupted binding of XIAP with caspase3 or NF-B. Overall, these findings suggest that lambertianic acid enhances TRAIL-induced apoptosis via inhibition of XIAP/NF-B in TRAIL resistant NSCLCs.
ARTICLE | doi:10.20944/preprints202102.0390.v1
Subject: Life Sciences, Biochemistry Keywords: Oroxylum indicum; Oroxyquinone; Traditional medicine; Bioassay Guided Fractionation; caspase-independent apoptosis; anti-metastatic
Online: 17 February 2021 (12:55:57 CET)
Leaf crude extract (aqueous) of Oroxylum indicum (L.) Kurz induces genomic DNA fragmentation, comet formation, and inhibition of cell proliferation in prostate cancer cell line, PC3 as assessed by agarose gel electrophoresis, comet assay, and MTT assay respectively. The bioactive compound was purified through bioassay-guided fractionation using preparative HPLC and MTT as-say. The brown and water-soluble compound was characterized using 1H and 13C nuclear magnetic resonance (NMR), fourier transform infrared (FT-IR) and electrospray ionization (ESI) mass spectrometry, and the compound was iden-tified as a glycosylated hydroquinone derivative, 2-[p-(2-Carboxyhydrazino)phenoxy]-6-(hydroxymethyl) tetrahy-dro-2H-pyran-3,4,5-triol (molecular formula, C13H18N2O8; molecular mass = 330). The identified phytocompound has not been reported earlier elsewhere. Therefore, the common name of the novel anticancer phytocompound isolated from oroxylum indicum in this current study is named as oroxyquinone. The half-maximal inhibitory concentration (IC50) of oroxyquinone on PC3 cells was 19.44 µg/ml (95% CI = 17.97 to 21.04). Oroxyquinone induced cell cycle arrest at S phases and inhibition of cell migration on PC3 as assessed by flow cytome-try and wound healing assay respectively. On investigating the molecular mechanism of inducing apoptosis, the results indicated that the oroxyquinone induced apoptosis through the p38 pathway and cell cycle arrest, however, not through caspase-3 and PARP pathways. The present study identifies a novel an-ticancer molecule and provides scientific evidence supporting the therapeutic potency of OI for ethnomedicinal uses.
REVIEW | doi:10.20944/preprints202004.0224.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: substance P,; NK-1R,; AML,; Aprepitant,; anti-leukemic,; apoptosis,; non-solid tumor
Online: 14 April 2020 (15:19:45 CEST)
Acute myeloid leukemia (AML) is an incurable hematological malignancy. To treat the disease successfully, new therapeutic strategies are urgently needed. One of these strategies can be the use of neurokinin-1 receptor (NK-1R) antagonists (e.g., aprepitant), because the substance P (SP)/NK-1R system is involved in cancer progression, including AML. AML patients show an up-regulation of the NK-1R mRNA expression; human AML cell lines show immunoreactivity for both SP and the NK-1R (it is overexpressed: the truncated isoform is more expressed than the full-length form) and, via this receptor, SP and NK-1R antagonists (aprepitant, in a concentration-dependent manner) respectively exert a proliferative action or an antileukemic effect (apoptotic mechanisms are triggered by promoting oxidative stress via mitochondrial Ca++ overload). Aprepitant inhibits the formation of AML cell colonies and, in combination with chemotherapeutic drugs, is more effective in inducing cytotoxic effects and AML cell growth blockade. NK-1R antagonists also exert an antinociceptive effect in myeloid leukemia-induced bone pain. The antitumor effect of aprepitant is diminished when the NF-κB pathway is overactivated and the damage induced by aprepitant in cancer cells is higher than that exerted in non-cancer cells. Thus, the SP/NK-1R system is involved in AML and aprepitant is a promising antitumor strategy against this hematological malignancy. In this review, the involvement of this system in solid and non-solid tumors (in particular in AML) is up-dated and the use of aprepitant as an anti-leukemic strategy for the treatment of AML is also mentioned (a dose of aprepitant (> 20 mg/kg/day) for a period of time according to the response to treatment is suggested).
ARTICLE | doi:10.20944/preprints202206.0317.v1
Subject: Life Sciences, Other Keywords: chick pea; chick pea water; apoptosis; cytotoxicity; colorectal cancer; polyphenols
Online: 22 June 2022 (11:50:20 CEST)
Chickpea is an essential legume, a staple food in many cultures and contains nutrients with potential health benefits. The chickpea water (CPW) leached out after cooking is usually discarded, which may potentially have significant anti-cancer and other health beneficial properties. This study compared the in-vitro bioactivity of CPW with chickpea polyphenol extract (CPPE) to evaluate its impact on pathways of colorectal cancer progression and development. Morphological observation by APOPercentage, cell viability detection using a cytotoxic assay and cell migration-scratch assay points to measure rate of metastasis were employed. Overall antioxidant activity of CPW and CPPE were measured using ABTS and DPPH free-radical assays. At 50 µg/mL concentration and above, both CPW and CPPE extracts significantly reduce cell viability in HT-29 colon cancer cell lines (p < 0.05). Moreover, a quantitative analysis of the extent of apoptosis demonstrated that at 250 and 500 μg/mL concentrations, both extracts induced significant apoptosis compared to the untreated control. Meanwhile, the cell migration scratch area decreases by 34.42% and 15.27% when treated with CPW and CPPE, respectively. In summary, CPW demonstrated comparable in vitro anti-cancer properties and antioxidant activity in colorectal cancer cells to CPPE. Further, in vivo studies are warranted to evaluate the physiological bioactivity of CPW and CPPE in targeting pathways of cancer development and progression
ARTICLE | doi:10.20944/preprints202109.0179.v1
Subject: Life Sciences, Cell & Developmental Biology Keywords: ovarian cancer; cannabis; phytocannabinoids; apoptosis; cytotoxicity; cell cycle; MAPK4; PARP1
Online: 9 September 2021 (11:57:38 CEST)
Ovarian cancer (OC) is the most lethal gynecologic malignancy. Cannabis sativa is being used to treat different medical conditions. We sought to examine the effectiveness of combinations of cannabis compounds against OC. Cytotoxic activity was determined by XTT assay on HTB75 and HTB161 cell lines. Apoptosis and cell cycle were determined by fluorescence-activated cell sorting (FACS). Gene expression was determined by quantitative PCR. The two most active fractions, F5 and F7, from a high Δ9–tetrahydrocannabinol (THC) cannabis strain extract and their standard mix (SM) showed cytotoxic activity against OC cells. The most effective phytocannabinoid combination was THC+cannabichromene (CBC)+cannabigerol (CBG). F5, F7 and SM affected cell cycle, led to cell apoptosis and to a marked reduction in cell migration. Moreover, these fractions act in synergy with niraparib, and were ~50 fold more cytotoxic to OC cells than to normal keratenocytes. Niraparib+F7 treatment was effective on OC patient's cells. F7 and the niraparin+fraction (F5 and F7) treatments reduced Mitogen-Activated Protein Kinase 4 (MAPK4) gene expression; this reduction may act in synergy with the niraparib inhibition of Poly (ADP-ribose) polymerase 1 (PARP1) activity. Combinations of cannabis compounds and niraparib should be examined for efficacy in pre-clinical studies and clinical trials.
ARTICLE | doi:10.20944/preprints202108.0167.v1
Subject: Biology, Physiology Keywords: LED; light; retina; microglia; caspase; apoptosis; Bcl-2; BAX; degeneration
Online: 6 August 2021 (14:04:11 CEST)
Vision is our primary sense as the human eye is the gateway for more than 65% of information reaching the human brain. Today’s increased exposure to different wavelengths and intensities of light from Light Emitting Diodes (LEDs) sources could induce retinal degeneration and accompanying neuronal cell death. Damage induced by chronic phototoxic reactions occurring in the retina accumulates over years and it has been suggested to be responsible for the etiology of many debilitating ocular conditions. In this work, we examined how LED stimulation affects vision by monitoring changes in the expression of death and survival factors as well as microglial activation in LED-induced damage (LID) of the retinal tissue. We found an LED exposure-induced increase in the mRNA levels of major apoptosis-related markers -BAX, Bcl-2, and Caspase-3 and an accompanying wide-spread microglial and Caspase-3 activation. Everyday LED light exposure was accounted for all the described changes in the retinal tissue of mice in this study, indicating that overuse of non-filtered direct LED light can have detrimental effects on the human retina as well.
ARTICLE | doi:10.20944/preprints202012.0682.v2
Subject: Life Sciences, Biochemistry Keywords: interleukin-33; gastric epithelium; proliferation; apoptosis; cell cycle; gastric cancer
Online: 9 March 2021 (13:12:02 CET)
Background: Interleukin (IL)-33 is a member of interleukin (IL)-1 family of cytokines which has been linked to the development of inflammatory conditions and cancer in the gastrointestinal tract. This study is designed to investigate whether IL-33 has direct effect on human gastric epithelial cells (GES-1) and on human gastric adenocarcinoma cell line (AGS) and gastric carcinoma cell line (NCI-N87), assessing its role in regulation of cell proliferation and cell cycle, apoptosis and necrosis. Cell cycle regulation was also determined in ex vivo gastric cancer samples obtained during endoscopy and surgical procedures. Methods: cell lines and tissue samples underwent stimulation with rhIL-33. Proliferation was assessed by XTT and CFSE assay, we also evaluated apoptosis by Caspase 3/8 Activity assay and Annexin V assays. Cell cycle were analyzed by means of Propidium Iodine assay and gene expression regulation was assessed by RT-PCR Profiling. Results: we found that IL-33 has an antiproliferative and proapoptotic effect on cancer cell line, while it can stimulate proliferation and reduce apoptosis in normal epithelial cell line. These effects are also confirmed by the analysis of cell cycle gene expression which showed a reduced expression of proproliferative genes in cancer cells, in particular genes involved in G0/G1 and G2/M checkpoint. These results are confirmed by the gene expression analysis on surgical and bioptic specimens. Conclusions: the aforementioned results indicate that IL-33 may be involved in cell proliferation in an environment- and cell type-dependent fashion.
ARTICLE | doi:10.20944/preprints202103.0160.v1
Subject: Medicine & Pharmacology, Allergology Keywords: 99mTc-Sestamibi; Breast Specific Gamma Imaging; Breast Cancer; Apoptosis; Theragnostic
Online: 4 March 2021 (14:00:05 CET)
The aim of this study was to investigate the possible role of 99mTc-sestamibi in the regulation of cancer cell proliferation and apoptosis. To this end, Te in vivo values of 99mTc-sestamibi uptake have been associated to the in-situ expression of both Ki67 and caspase-3. For in vitro investiga-tions BT-474 cells were incubated with three different concentration of 99mTc-sestamibi: 10µg/ml –1µg/ml – 0,1µg/ml. Expression of caspase-3 and Ki67, as well as the ultrastructure of cancer cells, were evaluated at T0 and after 24, 48, 72 and 120 hours after 99mTc-sestamibi incubation. Ex vivo data strengthened the known association between the sestamibi uptake and the Ki67 expression. Linear regression analysis showed a significant association between the sestamibi uptake and the number of apoptotic cells evaluated as caspase-3 positive breast cancer cells. As concern the in vitro data, a significant decrease of the proliferation index was observed in breast cancer cells incubated with high concentration of 99mTc-sestamibi (10µg/ml). Amazingly, a significant increase in caspase-3 positive cells in cultures incubated with 10µg/ml 99mTc-sestamibi was observed. This study suggested the possible role of sestamibi in the regulation of pathophysiological process involved in breast cancer.
ARTICLE | doi:10.20944/preprints201911.0206.v1
Subject: Medicine & Pharmacology, Pharmacology & Toxicology Keywords: leukemia; relapse; quiescence; dormancy; curcumin derivative; Hsp90; apoptosis; protein aggregation
Online: 17 November 2019 (14:06:46 CET)
Relapsed leukemia following initial therapeutic response and remission is difficult to treat and causes high patient mortality. Leukemia relapse is due to residual quiescent leukemia cells that escape conventional therapies and later reemerge. Eliminating not only growing but quiescent leukemia cells is critical to effectively treating leukemia and preventing its recurrence. Such therapeutic agents, however, are lacking in the clinic. Here we report that a 4-arylmethyl derivative of the natural anticancer compound curcumin demonstrates a dual effect in eliminating both growing and quiescent leukemia cells. This curcumin derivative, C212, on the one hand, inhibits growing leukemia cells at a higher efficacy than curcumin by inducing apoptosis and cell cycle arrest; it, on the other hand, kills quiescent leukemia cells that are resistant to conventional chemotherapy drugs. Furthermore, C212 drives leukemia cells into and kills them at deep quiescence, avoiding the potential risk associated with awaking therapy-resistant subpopulation of quiescent leukemia cells. Lastly, we show that C212 induces apoptosis and drives cells into deep dormancy at least partially by binding to and inhibiting Hsp90, leading to client protein degradation and protein aggregation. Further elucidating the molecular mechanisms underlying the dual function of C212 in eliminating both growing and quiescent leukemia cells will aid the development of novel therapies against leukemia relapse.
REVIEW | doi:10.20944/preprints201908.0038.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: solid cancers; cell cycle; apoptosis; inflammation; mitochondria; stemness; tumor suppression
Online: 5 August 2019 (03:43:33 CEST)
The Signal Transducer and Activator of Transcription (STAT)3 and 5 are activated by many cytokine receptors to regulate specific gene expression and mitochondrial functions. Their role in cancer is largely context dependent as they can both act as oncogenes and tumor suppressors. We review here the role of STAT3/5 activation in solid cancers and summarize their association to survival in cancer patients. The molecular mechanisms that underpins the oncogenic activity of STAT3/5 signaling includes the regulation of genes that control cell cycle, cell death, inflammation and stemness. In addition, STAT3 mitochondrial functions are required for transformation. On the other hand, several tumor suppressor pathways act on or are activated by STAT3/5 signaling including the p19ARF/p53 pathway, tyrosine phosphatases, suppressor of cytokine signaling 1 and 3, the sumo ligase PIAS3, the E3 ubiquitin ligase TMF/ARA160 and the miRNAs miR-124 and miR-1181. Cancer mutations and epigenetic alterations may alter the balance between pro-oncogenic and tumor suppressor activities associated to STAT3/5 signaling explaining their context dependent association to tumor progression both in human cancers and animal models.
ARTICLE | doi:10.20944/preprints201908.0006.v1
Subject: Biology, Ecology Keywords: chestnut shells; polyphenols; bioactive compounds; apoptosis; cytotoxicity; human cell lines
Online: 1 August 2019 (04:37:49 CEST)
In this study, bioactive compounds present in chestnut shells (CS) - derived from the industrial peeling processing - were extracted through different procedures. The aqueous extracts were characterized and the highest extraction yield and total phenolic content was obtained by Conventional Liquid Extraction (CLE). Gallic and protocatechuic acids were the main simple phenols in the extract with 86.97 and 11.20 mg/g chestnut shells dry extract (CSDE), respectively. Six tumor cell lines (DU 145, PC-3, LNCaP, MDA-MB-231, MCF-7 and Hep G2) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of CSDE (1-100 µg/mL) for 24 h and cell viability was evaluated using MTT assay. A reduced rate in cell viability was observed in DU 145, PC-3, LNCaP and MCF-7 cells while viability of MDA-MB-231 and Hep G2 cells was not affected except for PNT2 cells at a concentration of 100 µg/mL. Furthermore, CSDE-at concentrations of 55.5 and 100 µg/mL- lead to a significant increase of apoptotic cells in DU 145 cells of 28.2% and 61%, respectively. In conclusion, our findings showed that CS might be used for the extraction of several polyphenols that may represent good candidates for alternative therapies or in combination with current chemotherapeutics.
ARTICLE | doi:10.20944/preprints201805.0335.v2
Subject: Life Sciences, Other Keywords: Alzheimer’s Dementia; anaerobic neurotoxicity; inflammation; neuronal apoptosis; Non-REM Sleep
Online: 4 June 2018 (13:16:46 CEST)
Research into the causes of neurotoxicity in Alzheimer’s Dementia (AD) has focused on neurofibrillary tangles and beta amyloid (Aβ) plaques. This paper proposes the heterodox theory that these hallmarks of AD are the visible effects, not direct causes of neuronal necrosis. Rather AD results from a combination of age-induced, disproportional decline in physiological support for aerobic metabolism, and dysregulation of the sleep cycle processes. The hypothesis is that the decimation of neurons in AD results from a combination of neurotoxicity and increased apoptosis caused by: 1. direct damage from toxic waste products of anaerobic glycolysis due to a progressive decline in the capacity of neurons to perform oxidative phosphorylation (OXPHOS) and an increased reliance on anaerobic glycolysis to meet metabolic needs; 2. impaired cellular repair and effluent release due to dysregulation of non-rapid eye movement (NREM) sleep allowing damage to cell membranes and synaptic junctions to accumulate inducing a chronic inflammatory response; 3. indirect damage from products produced by inflammatory reaction to toxic metabolites; 4. neuronal apoptosis from the AβPP-mediated pathway due to the age-induced decline of growth hormone (GH), GH-releasing hormone (GHRH) and insulin-like growth factor (IGF).
ARTICLE | doi:10.20944/preprints201705.0218.v1
Subject: Chemistry, Food Chemistry Keywords: anticancer activity; apoptosis; oleanolic acid; cell cycle arrest; mapk signaling
Online: 31 May 2017 (11:28:30 CEST)
We evaluated oleanolic acid (OA)-induced anti-cancer activity, apoptotic mechanism, cell cycle status, and MAPK kinase signaling in DU145 (prostate cancer), MCF-7 (breast cancer), and U87 (human glioblastoma) cells. The IC50 values for OA-induced cytotoxicity were 112.57 in DU145, 132.29 in MCF-7, and 163.60 in U87 cells, respectively. OA (at 100 µg/mL) increased the number of apoptotic cells to 27.0% in DU145, 27.0% in MCF-7, and 15.7% in U87 cells, when compared to control cells. This enhanced apoptosis was due to increases in p53, cytochrome c, and Bax expression. OA-treated DU145 cells were arrested in G2 because of the activation of p-ERK and p21, and the decrease in cyclin B1 and cyclin E expression. Furthermore, OA-treated MCF-7 cells were arrested in G1 owing to the activation of p-JNK, p-ERK, p21, and p27, and the decrease in p-AKT, cyclin E, and CDK2. U87 cells also exhibited G1 phase arrest caused by the increase in p-ERK, p-JNK, p21, and p27, and the decrease in CDK2. Thus, OA arrests the cell cycle in different phases, and increases apoptosis in cancer cells. These results suggest that OA may alter the expression of cell cycle regulatory proteins in a cancer type-dependent manner.
ARTICLE | doi:10.20944/preprints201710.0157.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: docosahexaenoic acid; apoptosis; SIRT6; Hedgehog signaling; non-small cell lung cancer cells
Online: 24 October 2017 (05:43:16 CEST)
Omega-3 polyunsaturated fatty acids (ω3-PUFAs), including docosahexaenoic acid (DHA), have been shown to exert anticancer effects by inducing apoptotic cell death. However, the mechanism for DHA-induced cell death in lung cancer is not fully understood. Here, we show that DHA induces apoptosis in two human EGFR mutant non-small cell lung cancer (NSCLC) cell lines, and that DHA-induced cell death is accompanied by SIRT6 activation and attenuated Hedgehog (Hh) signaling. Knockdown of SIRT6 using siRNAs inhibited DHA-induced apoptosis, whereas SIRT6 overexpression increased apoptotic cell death. DHA-induced SIRT6 activation was associated with downregulation of Hh signaling, and knockdown of SIRT6 resulted in augmentation of Hh signaling. Pretreatment of NSCLC cells with a Smoothened agonist prevented DHA-induced decreases in the levels of Hh signaling proteins and increases in cleaved PARP levels. Moreover, endogenous production of ω3-PUFAs in PC9 cells via fat-1 expression resulted in elevated SIRT6 levels and reduced levels of Hh signaling molecules, including Gli, following DHA treatment. Overall, these results implicate that ω3-PUFAs induce apoptosis by downregulating Hh signaling via SIRT6 activation in human EGFR mutant NSCLC cells. These findings suggest that ω3-PUFAs potentially represent an effective therapy for the chemoprevention and treatment of NSCLC.
ARTICLE | doi:10.20944/preprints201805.0085.v1
Subject: Chemistry, Medicinal Chemistry Keywords: 7-acetamido-2-aryl-5-bromoindoles; trifluoroacetylation; cytotoxicity; apoptosis; tubulin polymerization; molecular docking
Online: 4 May 2018 (07:47:06 CEST)
Structurally related 7-acetyl-2-aryl-5-bromoindoles 2a–d and the 7-acetamido-2-aryl-5-bromoindoles 4a–d as well as their corresponding 3-trifluoroacetyl–substituted derivatives 5a–d and 5e–h were evaluated for potential antigrowth effect in vitro against the human lung cancer (A549) and cervical cancer (HeLa) cells. All of the 3-trifluoroacetyl–substituted 7-acetamido-2-aryl-5-bromoindoles 5e–h were found to be more active against both cell lines when compared to the chemotherapeutic drug, Melphalan. The most active compound 5g induced apoptosis in a caspase dependent manner for both cell lines. Compounds 5e–h were found to significantly inhibit tubulin polymerization. Molecular docking of 5g into the colchicine-binding site suggests that the compounds bind to tubulin by different type of interactions including pi-alkyl, amide-pi stacked and alkyl interactions as well as hydrogen bonding with the protein residues to elicit anticancer activity.
REVIEW | doi:10.20944/preprints202106.0227.v1
Subject: Medicine & Pharmacology, Allergology Keywords: endoplasmic reticulum; endoplasmic reticulum stress; apoptosis; homeostasis; unfolded protein response; type II diabetes
Online: 8 June 2021 (13:07:30 CEST)
The endoplasmic reticulum (ER) plays a multifunctional role in lipid biosynthesis, calcium storage, protein folding, and processing. Thus, maintaining ER homeostasis in insulin-secreting beta-cells is essential. Several pathophysiological conditions and pharmacological agents disrupt the ER homeostasis, thereby causing ER stress. The cells react to ER stress by initiating an adaptive signaling process called the unfolded protein response (UPR). However, the ER initiates death signaling pathways whenever the ER stress persists. ER stress has been linked to several diseases, such as cancers, obesity, and diabetes. Thus, the regulation of ER stress may provide possible therapeutic targets for many diseases. Current evidence suggests that chronic hyperglycemia and hyperlipidemia linked to type II diabetes disrupt ER homeostasis, resulting in irreversible UPR activation and cells death. Despite much progress in understanding the pathophysiology of UPR and ER stress, to date, the mechanisms of ER stress in relation to type II diabetes remain unclear. This review provided up-to-date information regarding the current status of UPR, ER stress mechanisms, insulin dysfunction, oxidative stress, and the therapeutic potential of targeting specific ER stress pathways.
REVIEW | doi:10.20944/preprints202103.0714.v1
Subject: Medicine & Pharmacology, Allergology Keywords: NAFLD; NASH; HCC; mitochondrial dynamics; hepatocytes; KCs, HSCs; apoptosis; metabolic reprogramming; Warburg effect
Online: 30 March 2021 (09:29:34 CEST)
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third-leading cause of cancer-related mortality. Currently, the global burden of nonalcoholic fatty liver disease (NAFLD) has dramatically overcome both viral and alcohol hepatitis thus becoming the main cause of HCC incidence. NAFLD pathogenesis is severely influenced by lifestyle and genetic predisposition. Mitochondria are highly dynamic organelles which may adapt in response to environment, genetics and epigenetics in the liver (“mitochondrial plasticity”). Mounting evidence highlighted that mitochondrial dysfunction due to loss of mitochondrial flexibility, may arise before overt NAFLD and since the early stages of liver injury. Mitochondrial failure not only promotes hepatocellular damage, but also release signals (mito-DAMPs) which trigger inflammation and fibrosis, generating an adverse microenvironment in which several hepatocytes select anti-apoptotic programs and mutations that may allow survival and proliferation. Furthermore, one of the key events in malignant hepatocytes is represented by remodeling of glucidic-lipidic metabolism combined to reprogramming of mitochondrial functions, optimized to deal with energy demand. In sum, this review will discuss how mitochondrial defects may be translated into causative explanations of NAFLD-driven HCC, emphasizing future directions for research purposes and for development of potential preventive or curative strategies.
ARTICLE | doi:10.20944/preprints202005.0098.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: breast cancer; snake venom; cytotoxin II; recombinant protein; apoptosis; cell cycle progression; cytotoxicity
Online: 6 May 2020 (15:12:05 CEST)
Breast cancer (BC) is among the leading causes of mortality from cancer in women. Many of the available anticancer drugs have various side effects. Therefore, researchers are seeking novel anticancer agents particularly from natural compounds and in this regard, snake venom is still one of the main sources of drug discovery. Previous studies showed potential anticancer effects of Cytotoxin II (CTII) from Naja naja oxiana against the different types of cancers. In this study, a pET-SUMO-CTII vector was transformed into SHuffle® T7 Express, an Escherichia coli strain, for recombinant protein expression (rCTII) and the cytotoxic effects of this protein was assessed in MCF-7 cells. The flow cytometry assay was applied to measure the apoptosis and cell cycle. Also, mRNA levels of the Bax, Bcl2, P53, caspase-3, caspase-8, caspase-9, caspase-10, matrix metalloproteinases (MMP)-3, and MMP-9 were analyzed by quantitative real-time PCR to determine the underlying cellular pathways affected by rCTII. The results of this study showed that treatment with 4 µg mL-1 of rCTII enhanced apoptosis through the intrinsic and extrinsic pathways. Also, the increase of the cells' proportion in the sub-G1 phase as well as a reduction in S phase was observed. In addition, the expression of MMP-3 and MMP-9 was decreased in the treated group in comparison to the control group that may contribute to the reduced migratory ability of tumor cells. These experimental results indicate that rCTII has anti-proliferative potential, and so this protein could be a potential drug for BC therapy in combination with other drugs.
ARTICLE | doi:10.20944/preprints202301.0269.v1
Subject: Life Sciences, Other Keywords: Intervertebral disc degeneration; nucleus pulposus; autophagy; apoptosis; external fixator; saline injection
Online: 16 January 2023 (04:44:40 CET)
Purpose: Intervertebral disc is a leading avascular organ in the body. Hence, it may utilize autophagy and apoptosis to alive in unfavorable condition like stress and mechanical force. But excessive force and stress is a key cause to intervertebral disc degeneration (IVDD). Purpose of this study is to identify, external fixator (EF) and saline injection (SI) may induce autopahgy and apoptosis mediated nucleus pulposus (NP) cells death in rat tail’s disc and which method is better for studying autophagy and apoptosis. Methods: Sixteen 9-week-old male Sprague–Dawley rat tails were treated with 0.9% saline and EF (two-cross kirschner wires) for 6 weeks and 12 weeks as well. Treated discs were dissected for checking out participation of autopahgy and apoptosis to intervertebral disc degradation (IVDD). For identifying them, we performed H&E staining, Masson’s trichrome staining, and Immunohistochemistry (IHC) for LC3, Beclin-1and P62 as well as MMP-2, MMP-3 and TIMP-1.Furthermore, we performed quantitative polymerase chain reaction (qPCR)to check out autophagy related gene expression (beclin-1, LC3 and P62) and apoptosis related gene expression (MMP-2, MMP-3 and TIMP-1) respectively. Results: More insidious nucleus pulposus (NP) cell degeneration was indentified in (EF) group than in (Ctrl) control group. And rate of degradation was elevated with increasing duration of compression, but SI group cannot distinguish the margin of AF and NP cells. Highest along with lateral expression was found for LC3, Beclin-1, and P62 of both groups. Up-regulated and central expression was observed for MMP-2, MMP-3 and TIMP-1 of both groups, although SI group cannot recognize the margin between NP and AF cells. Utmost autophagy related gene expression was identified in EF group than in SI group. Besides, maximum autophagy accumulating materials was found in EF group than in SI group. And it was amplified along with increasing the duration of compression. Highest, apoptotic gene expression was observed in SI group, whereas, EF group showed lowest expression. Conclusion: External fixator (EF) method was better to study autophagy and apoptosis because it enhanced IVDD after compression which is actively linked with autophagy and apoptosis. Degeneration process was conversely proportional along with duration of compression. On contrary, saline injection (SI) could not distinguish AF and NP cell border.