Genistein Mediated Molecular Pharmacology, Cell-Specific Anti-Breast Cancer Mechanism with Synergistic Effect and in silico Safety Measurement

Department of Biochemistry and Molecular Biology, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh. Department of Genetic Engineering and Biotechnology, Faculty of Biological Science and Technology, Jashore University of Science and Technology (JUST), Jashore-7408, Bangladesh. ABEx Bio-Research Center, East Azampur, Dhaka-1230, Bangladesh. Department of Biotechnology and Genetic Engineering, Faculty of life science, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh. Department of Biochemistry and Molecular Biology at Bangabandhu Sheikh Mujibur Rahman Science and Technology University, Gopalgonj-8100, Bangladesh. Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi, Bangladesh. Department of Pharmacy, Faculty of Life Science, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh. Global Biotechnology & Biomedical Research Network (GBBRN), Department of Biotechnology and Genetic Engineering, Faculty of Biological Sciences, Islamic University, Kushtia-7003, Bangladesh. Department of Pathology, College of Korean Medicine, Kyung Hee University, Seoul-02447, South Korea. Korean Medicine-Based Drug Repositioning Cancer Research Center, College of Korean Medicine, Kyung Hee University, Seoul-02447, Korea.


Introduction:
Breast cancer (BC) is considered a major public health concern among women in health science.
BC is responsible for 30% of all cancers in females and is the world's second leading cause of mortality [1]. Although they're diverse risk factors, which can increase the possibility of developing BC including aging, sex, gene mutations, family history, and unhealthy lifestyle, [2], abnormal hormones namely estrogen play an effective role in BC progression [3]. There are currently no established treatments are available, and having some important side effects including-reduction of blood cells, [4], sore throat, hair loss, ulcers, fatigue, nausea, change in taste, appetite loss, constipation, diarrhea, change in skin color, and changes of several hormonal levels [5] and some limitations of existing treatment are high cost, less effective, allergic reaction [6]. Multidrug-resistant (MDR) tumors are the major limitation of conventional treatment, leading to increased cancer-related deaths. Numerous drug molecules likely-anthracyclines (daunorubicin, doxorubicin, mitoxantrone, and epirubicin), taxanes (docetaxel, paclitaxel), and capecitabine were previously used successfully, but now these drugs are becoming resistant to cancer patients [7]. So, the modern medical emphasis is on preventing and treating breast cancer with natural nutritional components [8] [9] [10]. Under the pharmaceutical compoundsphytoestrogens are a natural dietary component with having potent anti-cancer activity against multiple cancer; most importantly, ovarian, prostate, as well as breast [11] [12].
One of the major soy phytoestrogens is Genistein (Gen), which has been described as a breast cancer preventive factor [13]. Soy-based foods, for example-soy cheese, and soy drinks i.e., soy milk with soy-based beverages, are the plentiful sources of Gen; of late, the amount of Gen content within mature soybeans has shown from 5.6 to 276 mg/100 g, but 81 mg/100 g is an average content [14], where regular daily intake of this isoflavones among adults in China, and Japan is about 25-50 mg which is several-folds higher than the consumption of these compounds by women in the western countries which accounted for less than 3 mg [15]. Gen has a structural similarity to 17β-estradiol and it binds to estrogen receptor ER-β compare to ER-α [16] [17].
In the case of estrogen receptor-α, Genistein acts as an antagonist. So, Genistein-mediated anticancer activity is involved in suppressing the expression and activity of estrogen receptor-α. E. J.
Choi summarised that Genistein regulates cell proliferation with apoptosis via ER-α dependent pathway in MCF-7 breast cancer cells through the underline mechanism of downregulating Cyclin D1 and up-regulating Bcl-2/Bax (full elaboration add here) ratio at the dose of 50 μM [37]. But in ER-β, Gen plays as an agonist activity and increases the receptor activity. Therefore, ER-β dependent anti-cancer activity of Genistein mediated by activating the receptor and potentiate chemotherapy efficacy to treat cancer treatment [38]. H. Jiang state that ER-β1 increased the anti-cancer efficacy of Gen in MDA-MB-231, MCF-7, cells, and in BALB/c mice by inhibiting cell proliferation through arresting cells in G2/M and G0/G1 phase, which led to cell cycle blockade at the dose of 10 −6 -10 −4 mol/l [39]. It has also been found to bind with the estrogen-responsive G protein-coupled receptor-30 (GPR-30) or G protein-coupled estrogen receptor-1 (GPER-1) [40], and inhibit cell proliferation [41]. HER-2 is an essential biomarker in breast cancer overexpressed in around 20-30% of breast cancer tumors [42]. So, regulating the HER-2 is a significant factor in BC treatment. M. S. Sakla et al. summarised that Genistein at > or =1 microM inhibited proto-oncogene of HER-2, and subsequently followed the HER-2 protein expression, phosphorylation, and promoter activity through an ER-independent mechanism in BC cells as delaying tumors onset in transgenic mice [43]. Therefore, GPR-30 is a potent alternate to ER that causes BC and can be a target in breast cancer cell treatment [44].
Kim GY et al. summarized that Genistein suppresses the GPR-30 activation in BRCA-1 mutated BC cells, resulting in G2/M phase arrest mediated by suppressing Akt phosphorylation [45].
Furthermore, Gen is a naturally occurring protein tyrosine kinase (PTK) inhibitor that is hypothesized to be responsible for the lower rate of BC observed in Asian women consuming soy. T. Akiyama et al. reported that Gen scarcely inhibited the enzyme activities of threonineand serine-specific protein kinases like cAMP-dependent protein kinase, Ca2+/phospholipiddependent enzyme protein kinase C, and phosphorylase kinase, and this mechanism are mediated through phosphorylation of the EGF receptor [46].

Cell-specific molecular mechanism of Genistein mediated anti-breast cancer activity in vitro
Cancerous cell lines derived from humans are critical models for cancer biology research in laboratories and determine the therapeutics advantage of anti-cancer agents [47]. Anti-cancer activity of phytochemicals is cell-specific, where one phytochemical is effective in only one or more than one cell line. This may be the difference in the cell components system. In this section, cell line-specific anti-cancer activity of Genistein is summarized in

Genistein effect on MCF-7 BC cell
According to R. F. Prietsch et al., Gen (0.01-100µm) promoted apoptosis via mediating the autophagy-dependent mechanism along with increasing the ratio of Bax/Bcl-2 and inhibit the oxidative stress of cancer progression through changing the expression of antioxidant enzymes.
For example, Gen down-regulates the thioredoxin reductase enzyme (TrxR) and up-regulates the expression of Glutathione peroxidase (GPx) [48]. X. Liu et al. summarized that Genistein (5-20µm) induces apoptosis through the mitochondrial-dependent pathway by decreasing the Bcl-2/Bax ratio, increasing tumor suppressor gene P 73 expression and ATM phosphorylation with G2/M phase arrest permanently [49]. Similarly, Genistein (50-200µm) hinders the cellular growth and induces the apoptosis mediated pathway by following the downregulation of Bcl-2 protein, as well as upregulation of the Bax, together with decreasing the cyclin D1 expression in MCF-7 breast cancer cell [37]. At low concentration Gen (1µM) stimulate cell proliferation but at higher concentration (25µM) induces apoptosis mechanism by up-regulating of the CDKN1A and p53 responsive gene and down-regulating the CCNG1 GADD45A, B NF-kappa B, BCL-2, and TNFR, ESR1, NCOA2, and NCOA3 [50]. Another research investigated that Gen (50µM) highlights apoptosis by up-regulating Poly-(ADP-ribose)-polymerase,p53, and down-regulating Bcl-2/Bax protein [26]. In vitro study by M. L. De Lemos investigated that Gen (10µM) induces apoptosis by breaking plasma membrane breakdown, nuclear membrane, and up-regulating pS2 expression [27]. A late study reported that Gen (100 µM) induces programmed cell death and suppresses cell growth by up-regulating Caspase 7, apoptosis signalling kinase-1, ADP ribose, and p38 dependent mitogen protein kinase [51].

Genistein effect on MDA-MB-231 BC cell
Recently an experimental conducted by X. Liu et al. Gen (5-20µm) induces apoptosis through the mitochondrial-dependent pathway by reducing the Bcl-2/Bax ratio and growth inhibition with decreasing expression of mutant p53 and increasing the expression of p73, leading to the activation of G2/M phase arrest and ATM/Cdc25C/Chk2/Cdc2 checkpoint pathway [49].
Genistein induces the apoptosis process and directly inhibits the growth of cells through the prevention of NF-κB activity by Notch-1 pathway and with downregulating cyclin B1, Bcl-xL, and Bcl-2 expression, resulting in the arrest of the cell cycle at G2/M phase at 5-20µm [66], while at 5-50µm, this phytochemicals induces apoptosis by targeting the endogenous copper ion, reducing Cu(II) to Cu(I) through the production of reactive oxygen species (ROS) [67]. Before that, in vitro study by K. Dampier et reported that Gen (10µM) induces apoptosis and inhibits the rapidly proliferating cells, cell cycle arrest at G2 phase with degrading proto-oncogene c-fos, prohibited protein-1 (AP-1), and also ERK activity [68]. Another research by X. Yang et al.
In the case of angiogenesis, Mukund et al. explained that Genistein (100µm) reduced angiogenesis by blocking the transactivation of downstream HIF-1α effectors, e.g., VEGF, leading to the reduction of hypoxia-inducible factor-1α expression in MDA-MB-231 BC cell [22]. Furthermore, 1-10 µg/ml of Gen suppressed angiogenesis and cell mutation by decreasing tyrosine kinase, Ribosomal S6 kinases, and DNA topoisomerases I, II [54], while at 50µM concentration decreased angiogenesis with inhibiting cell division through the underline mechanism of downregulating COX, Topoisomerase II enzyme, and TPA, EROD protein activity [53]. Followed by angiogenesis, Gen (15-30 µM) [21], (5-20 µM) [20]  activity, and glucose uptake rate leading to phagocytosis cancer cell [69]. Apart from this, inhibition of cell viability through decreasing the DNA methyltransferase activity, DNMT1 expression and affecting the expression of TSGs likely-APC, ATM, PTEN, SERPINB5 by at 60-100µm of Genistein [65]. Another recent study by D. G. Pons et al. summarized that Genistein (1µm) causes a considerable decrease in cell viability through the mitogen-dependent protein kinase pathway that can promote apoptosis [63].
In MDA-MB-231, BC cell growth controlling is a significant target for Genistein. Gong et al. state that Gen (5-50 µM) inhibits cell growth by partly inducing apoptosis via downregulation of the Akt, NF-κB cascade pathways in vitro study [70]. Another in vitro analysis displayed the cell growth inhibitory activity was evident by Genistein (2.5-400µm) through the up-regulation of two crucial TSGs e,g. p21WAF1 (p21), p16INK4a (p16), and downregulation of two tumorpromoting genes like-c-MYC, and, BMI1, ultimately inhibit cancer progression [71].Y. Fang et al. concluded that Genistein (40µm) inhibits cellular growth via following the activation of DNA-dependent damage response and ATR signaling pathway with activating the BRCA-1 complex, inhibiting cohesion complex and increasing phosphatide, which is distributed among CDK1, CDK2, and CDK3 [72]. Recently it was established that Genistein (1000ppm) suppressed the tumor growth by cell cycle regulating via maintaining the expression level of the cyclin D1 protein, leading to G0/G1 phase arrest, which causes cell cycle blockage [39]. Subsequently, T.
In the case of cell proliferation, low dose Genistein (10µm) slightly inhibited cell proliferation by reducing the level of P-STAT3/STAT-5 ratio [55]. In comparison, at a higher dose of 20-40µm, it

Effect of Genistein on T-47D breast cancer cell:
V. Mukund et al. summarize that Genistein (50µm) lowers angiogenesis by preventing the transactivation of downstream HIF-1α effectors such as VEGF, resulting in the lessening the expression of hypoxia-inducible factor-1α in T-47D type of breast cancer cell lines [22]. Cell proliferation efficacy was evident by Genistein (10nm) with apoptosis induction through the mitochondrial-dependent pathway via up-regulating the cytochrome c oxidase activity and downregulating the ATP synthase/cytochrome c oxidase ratio [55]. Genistein at the 1nm-100µm inhibits cell proliferation through ERK1/2 mediated signaling by the downregulation of phosphorylated p90RSK [75], while 10µM of Gen induces apoptosis and inhibits cell proliferation through degrading proto-oncogene c-fos levels and prohibited Protein 1 (AP-1) and ERK expression [68]. Another in vitro study by T. T. Rajah revealed that Gen (10-100 µM) inhibits Cell proliferation and Tumor growth by downregulating MEK5, pERK5, and NF-κB proteins [73].

The Actions of Genistein on HCC38 breast cancer cell:
M. G. Donovan stated that Genistein (4-10ppm) inhibits cell growth by increasing the BRCA1 protein level and reducing the CpG methylation, consequently decreases the AHR binding at BRCA1 [78].

The Actions of Genistein on DD-762 Cell and Sm-MT C breast cancer cell:
Nakagawa et al. appraised that Gen (7-274.2µM) inhibits cell proliferation by up-regulating caspase-3 protein activity [80].

The Actions of Genistein on BT-474 breast cancer cell
Genistein low concentration (1 µM) can promote cancer but at high concentration (50 µM) inhibits the cell division with downregulating Tyrosine kinase, HER2 activation, and MAPK pathway [43]. A similar study approached the same result on the BT-474 cell line. Gen (3.125-25 M) inhibits cell replication and arrests the cell cycle in the G2/M phase, as well as inhibiting the expression of EGFR, HER2, and ER-alpha [81].

The Actions of Genistein on BT20 breast cancer cell
Cappelletti et al. revealed that Gen (15-30 µM) inhibits metastasis through down lifting levels of CDKs, Tyrosine kinase, DNA topoisomerase II, and Paracrine stimulation BT20 Cell Line [21].

The Actions of Genistein on 184-B5/HER breast cancer cell
Katdare et al. showed that Gen (2.5-10 µM) impedes the cell cycle by inducing apoptosis by increasing the P16INK4a gene and decreasing HER-2/neu, Tyrosine kinase [82].

The Actions of Genistein on MCF-10A, MCF-ANeoT, MCF-T63B breast cancer cell
An early study showed that Gen (1-10 µg/ml) obstructs angiogenesis and cell mutation by decreasing the expression of DNA topoisomerases I and II, Ribosomal S6 kinases and Tyrosine kinase [54]. Overview of anti-cancer activities were tabulated in Table 01.

Breast Cancer Treatment breast cancer drugs
Except for the strong anti-cancer activity alone, Genistein possesses the synergestic property with many other anti-cancer drugs, which assist it in overcoming the chemopreventive resistance mechanism in breast cancer treatment.  [102]. According to another research study, genistein, in the form of phytoestrogens, can effectively suppress the breast cancer resistance protein, also known as ABCG2, which is responsible for multidrug resistance activity [103]. Moreover, others research studies have analyzed that the Genistein's ability to autophosphorylate protein tyrosine kinases (PTK), particularly EGF-R, by contesting ATP than other the protein substratum. The genistein antiproliferative effect was also attributable to interfering with the mitogen-activated tyrosine kinase cascade [46]. autophagic cell death only in T47D breast cancer cell [105]. Another early study reported that Tamoxifen, with Gen (1-10 µg/ml), impede angiogenesis and cell mutation by downregulating DNA topoisomerases I and II, Ribosomal S6 kinases, tyrosine kinase, and Cell cycle regulators [54]. Gen also shows a prohibitory effect with Tamoxifen which induces apoptosis by destroying nuclear membrane [27], arrest cell cycle by decreasing expression of HER2 in a dose-dependent manner [81]. changing p21, p53, and Bax protein expression [60]. The Table 02 contains a summary of the effects of phytoestrogens in combination with anti-cancer therapies that have been previously described. In vitro MCF-7

Clinical trials
Human clinical trials have confirmed the in vitro and in vivo research findings; in some cases, when consumed at a consistent dose, pure Genistein had no estrogenic effect on breast tissue [29], [30], although others dietary soy supplementation had proestrogenic properties on breast tissue [31]- [33]. Several secondary endpoints were evaluated in a recently published clinical study to determine whether purified Genistein has an effect on the endometrial thickness, vaginal cytology, and breast density (  [109], [110] have been preserved for the three years in the genistein-administered group, while levels of both BRCA-1, and 2 have decreased in the placebo group (Table 3) [29] [30] . Genistein also significantly reduced sister chromatid exchanges, implying that it may prevent genotoxicity and subsequent mutagenesis (Table 03) [30].
With a good bioavailability score of 0.55, Genistein maintained drug-like characteristics without any violating of Lipinski, Ghose Veber, Muegge, and Egan rules. In drug development, absorption is an important parameter. Our in silico predicted result showed that Genistein is highly soluble in water (-3.376) and absorbed by human intestine, with permeable to Caco-2 cell.
After absorption, consequentially comes distribution, another principal descriptor for drug development, which depends mumerous factors i.e., steady-state volume of distribution (VDss), blood-brain barrier (BBB) and central nervous system(CNS) permeability, binding to plasma protein, and many more [117].Our analysis suggested that Gen has the potentiality to have a poor VDss value, but is able to cross the BBB and central CNS properly. Human cytochrome P450 (CYP) isoforms are involved in drug metabolism in the liver. Its inhibition could lead to drug toxicity, drug-drug interactions, and other adverse effects. The predicted metabolic result reported that Genistein is the inhibitor of CYP1A2, CYP2C19, CYP2C9, and CYP3A4 but a non-inhibitor of CYP2D6. Excretion property based on total renal clearance parameter was predicted, where total clearance (logCLtot) of Genistein was 0.151 ml/min/kg.The toxicity profile of Genistein has been predicted based on eye corrosion, hepa-toxicity, Skin sanitization, OATP2B1 inhibitor, OATP1B3 inhibitor, OCT2 inhibitor, carcinogenicity, AMES toxicity, hERG I inhibitors, and BSEP inhibitors. Results outlined that Genistein showed toxicity only in the liver and OATP2B1 inhibitor, wherease positive to eye corrosion, hepatoxicity, AMES toxicity, hERG potassium channel inhibition, and carcinogenicity. Anti-cancer activities were determined based on P-glycoprotein and aromatase enzyme inhibition and estrogen receptors modulating affinity. Our analysis revealed that Genistein has a potent affinity to estrogen receptors, aromatase enzyme, but its non-inhibitor P-glycoprotein.
To achieve more information for better bioavailability and drug-likeness of Genistein, detailed information is summarized in Fig 02. The results of the bioavailability radar have been depicted by the lipophilicity: XLOGP3 between −0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å2, solubility: log S not higher than 6, saturation: fraction of carbons in the sp3 hybridization not less than 0.25 and flexibility: no more than 9 rotatable bonds with the colored zone defined the desired physicochemical space for good oral bioavailability indicating that they possess good drug-likeness properties. After the validation of bioavailability, potent drug likeness properties in cancer research models, it can be estimated that Genistein can be used as an effective anti-cancer natural agent with its synergistic use with other chemotherapeutics for the treatment of breast cancer.

Concluding remarks:
Nowadays, breast cancer is the major leading cause of death due to have diverse pernicious side effects of proposed synthetic drugs, multi drug resistance issues and many more. But natural food products are given more attention to the people in combating diseases such as-cancer insurgencies, mainly, breast cancer. For this reason, this current investigation was conducted to explore the better pharmacological activity of natural phytochemicals namely-Genistein that offer therapeutic activities in tissues of breast cancer patients, cell specific anti-breast cancer mechanism, and most importantly, possess potential synergistic activity with multidrug-resistant tumors. Here, the safety assessment of the Genistein conducted by the online-based server and predict their pharmacokinetics profiling as a safe therapeutic compound but more wet-lab based research is badly needed to discover the cell-specific breast cancer controlling, toxicological mechanism and their pharmacokinetics properties.