GAMBOGIC ACID AS ANTICANCER AGENT: A REVIEW

Gambogic acid, a common traditional Chinese medicine and widely distributed throughout South China, Vietnam, Cambodia, and Thailand. It is prenylated xanthone which is the significant bioactive compound of gamboge. Gambogic acid is known as a strong apoptotic inducer in cancer cells. It has been found as strong anticancer agent against various types of cancer cells lines such as breast cancer, pancreatic, and cervical cancer. It induces apoptosis, down regulates the antiapoptotic proteins (survivin and BCL2,) and down regulates the activities of P-glycoprotein in drug sensitive human breast MCF-7 and drug-resistant MCF-7/ADR cells. Similarly, it also exerts alteration in P13K, AKT, p21, MMP-2 &-9, and phosphorylated-AKT expressions. The current review highlights the anticancer and chemo-preventive perspectives of gambogic acid and its mechanistic role against human and animal cancers.


Introduction
Natural bioactive compounds from fruits and vegetables have been used as therapeutic agents in clinical and biological activity against various human maladies such as cancer, cardiovascular, and neurodegenerative disorders. Gambogic acid (GA), a naturally occurring compound which is derived from Garcinia hanburyi tree. It has been found to exert multiple intracellular and extracellular actions such as antimetastatic, anti-inflammatory autophagy, programmed cell death, anti-angiogenesis, and cell cycle arrest, respectively (Kashyap et al., 2016;Seo et al., 2019).

Anticancer perspectives
Breast cancer GA has been found to prevent from tumorigenesis in the MCF-7 subcutaneous xenograft tumor model through inhibiting proliferation and inducing apoptosis (Wang et al., 2018a;Sang et al., 2018). Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) has the ability to induce apoptosis in cancer cells that has developed significant interest in treating different types of cancers. In breast cancer cells, gambogic acid increased sensitivity of TRAIL and increased TRAIL-induced apoptosis in cancer cells. Wang et al (2018) discovered that GA with TRAIL cooperation decreased anti-apoptotic proteins level and activated Bid (BH3 interacting-domain death agonist) which promoted cross talk between extrinsic and intrinsic apoptotic signaling, instead of enhancing the TRAIL DR4 and DR5 receptors (Wang et al., 2018b). Different researchers and investigators determined that encapsulation of GA with polyethylenimine-poly (d,l-lactide-coglycolide) caused momentous augmentation in apoptotic cell death in an in vitro study on triplenegative breast cancer, and in vivo suppression of TNBC tumor growth . In another exploration by Doddapaneni et al. 2016, they found that encapsulation of GA as PEGylated liposomal formulation in MDA-MB-231 orthotopic xenograft significantly suppressed the growth of tumor, with 50% tumor-volume, and reduced tumor weight alongside inhibition of Bcl2 expression, apoptotic markers, cyclinD1, and micro-vessel density marker-CD31. Moreover, it also increased the levels of p53 and Bax (Doddapaneni et al., 2016).
GA increased cell toxicity, apoptosis, down regulated expressions of anti-apoptotic proteins survivin and Bcl2, and downregulated expression and activities of P-glycoprotein (P-gp) in drug sensitive human breast MCF-7 and drug-resistant MCF-7/ADR cells (Wang et al., 2015b).
Sensitization of doxorubicin (DOX) resistant breast cancer cells to DOX-mediated cell death and enhancement in the intra-cellular accumulation of DOX via inhibiting both P-gp expression and activity were reported after gambogic acid treatment. In addition, DOX in combination with GA led to the production of intra-cellular reactive oxygen species and inhibition of anti-apoptotic protein surviving. In DOX-induced apoptosis, over-expression of surviving blocked the sensitizing effects of GA. Moreover, ROS-mediated activation of p38 MAPK was revealed in GA -mediated suppression of Survivin expressions (Wang et al., 2015a).

Glioma cancer
A group of researchers studied the anticancer role of GA against human U251 glioma cells, they found that reduction in phosphorylation of P38, AKT, and mTOR, as well as decrement in the upstream binding factor (UBF), phosphorylation of ribosomal protein precursors (Pre) and insulinlike growth factor I (IGF-1) were reported after GA treatment (Luo et al., 2020;Sang et al., 2019).
In T98G glioblastoma cells, GA dose dependently showed potent anti-proliferative activity via apoptosis induction, enhancement in Bax and AIF expression, PARP and cleavage of caspase-3,-7 & -9 and down regulation of Bcl-2 expression (Thida et al., 2016). A peer of researchers, they investigated induction of up regulation of leucine-rich repeat and Ig-like domain-containing-1(LRIG1) which further exhibited downstream Akt/mTORC1 inhibition and epidermal growth factor receptor (EFGR) degradation after GA treatment in U87 glioma cells. Further, U87 cell apoptosis and growth inhibition, AMP-activated protein kinase activation mediated GA-induced LRIG1 upregulation, while AMPK inhibition by shRNA or compound C reduced GA -induced EGFR/Akt inhibition and cytotoxicity in U87 cells (He et al., 2013). Time and dose-dependent anti-cancer action of GA investigated by group of researchers against human glioma cell lines such as U251MG and U87MG showed decreased cell proliferation, induced apoptosis in cells, and inhibited colony formation. Development of monodansylcadaverine in autophagic vacuoles, up regulation of expressions of Beclin 1, LC3-II and Atg5, enhancement in punctate fluorescent signals of glioblastoma cells pre-transfected with GFP-tagged LC3 plasmid were reported (Luo et al., 2012). During an in vitro study on rat C6 glioma cells, GA dose and time dependently induced apoptotic cell death as it triggered intrinsic mitochondrial apoptotic pathways whereas during an in vivo trial, intravenous treatment of GA showed momentous reduction in tumor volumes through apoptotic induction (Qiang et al., 2008).

Skin cancer
In recent study reported by Li and their colleagues, they found that dose and time dependent GA in vitro study in melanoma A375, B16-F10 cells regulating the protein expressions, inhibiting the proliferation, migration, invasive and adhesive properties, suppressing the EMT and angiogenesis processes and reducing the enzymatic activities of MMP-2 and MMP-9. Furthermore, GA also suppressed the abnormal PI3K/Akt and ERK signaling pathways . In human malignant melanoma (MM) cells A375, GA (2.5-7.5 microg/mL) for 36 h suppressed the caspase-3 activity and Bax/ Bcl-2 ratio, respectively (Xu et al., 2009).

Mechanisms References Breast cancer
Inhibited proliferation and induced apoptotic cell death (Wang et al.,2018a;Sang et al., 2018). Increased the sensitivity of breast cancer cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) Enhanced TRAIL-induced apoptosis (Wang et al., 2018b).
Caused momentous augmentation in apoptotic cell death Suppressed TNBC tumor growth Xu et al., 2016) Significantly suppressed the tumor growth, tumor volume, and caused reduction in tumor weight Showed inhibition on expression of bcl2, apoptotic markers, surviving, cyclinD1, and microvessel density marker-CD31. Increased the levels of p53 and Bax (Doddapaneni et al., 2016)

Skin cancer
Suppressed the proliferation, migration, invasive and adhesive properties Inhibited the EMT and angiogenesis processes Lowered the enzymatic activities of MMP-2 and MMP-9 Suppressed the abnormal PI3K/Akt and ERK signaling pathways.  Colon cancer Induced cell proliferation, migration and invasion Altered expressions of AKT, p21, P13K, MMP-2 and -9 , and phosphorylated-AKT (Zhou and Ma, 2019).
Suppressed the cell viability and activated the SHH signaling .

Liver cancer
Enhanced the levels of p-AMPK

Up-regulated E-cadherin linked with LKB1
Induced E-cadherin, and down regulated the ZEB1 Suppressed the antioxidant enzyme TrxR1 reduction activity (Duan et al., 2014).

Lung cancer
Enhanced intracellular ROS level, C/EBP-homologous protein, expression levels of glucose-regulated protein (GRP)78 Activated transcription factor 6 and caspase 12 Activated the inositol-requiring enzyme 1alpha and phosphorylation levels of protein kinase R-like ER kinase (Zhu et al., 2019).
Inhibited cell growth, induced cells autophagy Caused upregulation of Beclin 1, and transformation of autophagosome markers such as LC31 to LC2II (Ye et al., 2018).
A study reported by Deschatrette et al. (2013) showed that gambogic acid i has been found to lower the thymidylate synthetase mRNA levels and level of thymidylate synthetase mRNA n a gastric carcinoma cell line. A contradictive relationship was observed in hepatoma cells between DHFR expression and resistance to GA with a dihydrofolate reductase (DHFR) gene amplification and cells transfected with an inducible DHFR transgene. In vitro, GA suppressed the DHFR activity, and lowered the affinity of the enzyme for dihydrofolate (Deschatrette et al., 2013). enhanced their sensitivity to GA and gambogenic acid (Wang et al., 2009).

Lung cancer
Different doses of GA as 0, 0.5, and 1.0 μmol/L were treated with non-small cell lung cancer A549 cells in a concentration-dependent manner and reduced the cell viability, enhancement intracellular ROS level, C/EBP-homologous protein, expression levels of glucose-regulated protein (GRP)78, activated transcription factor 6 and caspase 12, as well as the inositol-requiring enzyme 1 alpha and phosphorylation levels of protein kinase R-like ER kinase (Zhu et al., 2019). A study conducted by Ye and co-workers, they explored that gambogic acid in non-small cell lung cancer (NSCLC) NCI-H441 of xenografts inhibited cell growth, induced cells autophagy, caused upregulation of Beclin 1, and transformation of autophagosome markers such as LC31 to LC2II. Moreover, GA induced autophagy through an ROS-dependent pathway (Ye et al., 2018).
In human non-small cell lung cancer (NSCLC) cells (A549 and SPC-A1 cells), different doses of GA at 0, 0.5, 0.75, and 1.0 μmol/l showed momentous inhibition in cell viability, enhancement in cell apoptosis, reduction in the expression levels of Jagged1, Jagged2, DLL1, DLL3, DLL4, PK3K and Bcl2, increment in expression level of active caspase-3, and inhibited Akt phosphorylation, and NICD nuclear translocation (Zhu et al., 2018). Combined form of GA with cisplatin produced a significant inhibitory effect on A549 and NCI-H460 cells. They significantly increased autophagy, and inhibited the activation of S6, mTOR and Akt. In addition, gambogic acid combined with rapamycin induced more cell death as compare to GA anti-tumor action alone (Zhao et al., 2017).

Pancreatic cancer
In a recent study, pancreatic cancer cell lines treated with gambogic acid and showed induction of the expression of Beclin-1 and LC3-II proteins and enhancement in the formation of both acidic vesicular organelles and autophagosomes, as well as autophagic flux. Moreover, increased cytotoxicity of gambogic acid was linked with inhibition of autophagy by chloroquine. In addition, promotion of ROS production and destruction of mitochondrial membrane which is linked with autophagy activation were reported after GA treatment .
Pancreatic cancer cells treated with GA showed growth inhibition, down-regulation of TOP2A, ALDOA, and ATG4B, and up-regulation of DUSP1, DDIT3, and DUSP5, respectively (Youns et al., 2018). Similarly, there are multiple mechanisms through gambogic acid treatment against pancreatic cancer cell lines such as suppression of the growth, and induction of cell cycle arrest at S-phase and apoptosis. In PANC-1 and BxPC-3 cells, synergistic action of GA with gemcitabine was reported. GA activated apoptosis induced by gemcitabine via increasing expression of cleaved caspase-3 and 9, cleaved-PARP and Bax and reduced Bl-2 expressions. Reduction in the expression of the ribonucleotide reductase subunit-M2 (RRM2) protein and mRNA were reported after GA treatment which is linked with resistance to gemcitabine via extracellular signal regulated kinase (ERK)/E2F1 signaling pathway inhibition. Furthermore, both compounds in the xenograft cancer model significantly repressed tumor growth and down regulated the p-ERK, E2F1, and RRM2 (Xia et al., 2017;Saeed et al., 2014). In Panc-1 pancreatic cancer cells, gambogic acidloaded magnetic Fe3O4 nanoparticles significantly inhibited the ETS1-mediated proliferation and migration of cells, lowered the expression of ETS1, as well as it down-streamed target genes cyclin D1, u-PA and VEGF . In another study, encapsulation of GA with magnetic Fe3O4 nanoparticles improved anticancer potential via induction of apoptosis, increasing of Bax, caspase 3 and caspase 9, decreasing protein expressions of Bcl-2 .

Blood cancer
GA increased production of ROS in hematopoietic malignant cell lines (Ortiz-Sánchez et al., 2009). In acute myeloid leukemia cell lines of experimental subjects such as Jurket, HL-60 and MV4-11 cells, nano-emulsion encapsulated GA enhanced the efficacy rate in both in vivo and in vitro studies (Feng et al., 2018). Administration of gambogic acid more than 0.5 μM to human leukemia cell line K562 dose-dependently induced apoptosis, inhibited cell proliferation, and down regulated the levels of nuclear factor-κB (NF-κB), BCL-2, phosphatidylinositol3-kinase (PI3K), c-myc, and phosphorylation of serine-threonine kinase (p-AKT) .
Cancer cells lines such as U937 and HL-60 were treated with gambogic acid and caused suppression in cell growth, promotion in differentiation, and upregulation of p21waf1/cip1 expression, respectively (Chen et al., 2014). Gambogic acid also reported as significant anti-cancer agent against different chronic myelogenous leukemia cell lines i.e. KBM5-T315I, KBM5, and K562 of nude mice via inducing apoptosis, cell proliferation inhibition and suppression of the growth of imatinib-resistant Bcr-Abl-T315I (Shi et al., 2014). In another study reported by Li and colleagues, they found that down regulation in the expression of SRC-3 along with inhibition of Akt kinase and its down-streamed targets p70, S6 kinase 1(S6K1) and glycogen synthase kinase 3 beta (GSK3beta) were observed after gambogic acid treatment against myelogenous leukemia cell line K562 cells in humans. In addition, these changes also affected the expressions of apoptosis (gene Bcl-2) in K562 cells (Li et al., 2009).
In another study conducted by Tao and colleagues, they explored that N-(2-ethoxyethyl) gambogamide (NG-18), a derivative of gambogic acid in against leukemia (HL-60 cells) also effectively suppressed the culture human tumor cells proliferation, induced apoptosis whereas tumor apoptosis is linked with up-regulated pro-apoptotic Bcl-2 family member Bax, and downregulated anti-apoptotic protein Bcl-2 (Tao et al., 2007). In multidrugresistant lymphoma Raji/DNR cells, gambogic acid in a dose-dependent manner down-regulated expression of P-glycoprotein (Zhou et al., 2016). A study reported by Zhao and co-workers showed that gambogic acid exhibited mechanisms such as suppression of cell growth, induction of apoptosis, caused over expression of SRC-3, suppressed cyclin D3 and Bcl-2, Bcl-6 expressions, modulated down-stream gene expression, and induced the de-acetylation of histone H3 at lysine 9 and lysine 27 in B-cells non-Hodgkin lymphoma (NHL) (Zhao et al., 2016). Likewise, a group of researchers unveiled that GA in vitro and in vivo trials has potent anticancer effect on Bcell lymphoma (DLBCL) cells such as GCB-and ABC-DLBCL cells through inducing apoptosis, and inhibiting cells growth (Shi et al., 2015). In a study against Jeko-1 human mental cells lymphoma cell apoptosis, GA inhibited cell growth as time and dose-dependently. It is involved in suppressing ratio of Bax and Bcl-2 with arresting cell cycle and decreasing mitochondrial membrane potential and activating caspase-3,-8,-9 .

Conclusion
Researchers conducted extensive work on natural products to explore their role as an anticancer agent in last decades. Among these products, GA has attained popularity as a promising novel antitumor agent and has ability to inhibit proliferation and induce apoptosis. It inhibits the cell viability, enhances cell apoptosis, reduces the expression levels of PK3K, DLL1, DLL3, DLL4, Jagged1, Jagged2, and Bcl2, increases the active caspase-3 level, and inhibits the Akt phosphorylation in human malignancies. Extensive work is required to further investigate its toxicity and interaction with other chemotherapic agents in clinical setting.
Huang GM, Sun Y, Ge X, Wan X, Li CB. Gambogic acid induces apoptosis and inhibits colorectal tumor growth via mitochondrial pathways. World J Gastroenterol. 2015a