Gambogic Acid Treatment Research Articles

Gambogic acid inhibits tumor cell adhesion by suppressing integrin β1 and membrane lipid rafts-associated integrin signaling pathway

Cell adhesion plays an important role in the steps of cancer metastasis. Regulation of cell–cell (intercellular) and cell–matrix adhesion is a promising strategy for cancer progression. Gambogic acid is a xanthone derived from the resin of the Chinese plant Garciania hanburyi, with potent anti-metastasis activity on highly metastatic cells. The aim of this study was to investigate the function and mechanism of gambogic acid on tumor adhesion. We found that gambogic acid strongly inhibited the adhesion of human cancer cells to fibronectin. This inhibition was associated with the deformation of focal adhesion complex, which was mediated by suppressing the expression of integrin β1 and integrin signaling pathway. In vitro, cell lipid rafts clustering was inhibited following treatment of gambogic acid, which induced the suppression of integrin β1 and focal adhesion complex proteins colocalization within rafts. Moreover, gambogic acid significantly decreased cellular cholesterol content, whereas cholesterol replenishment lessened the inhibitory effect of gambogic acid on cell adhesion. Real-time PCR analysis showed that gambogic acid reduced mRNA levels of hydroxymethylglutaryl-CoA reductase and sterol regulatory element binding protein-2, while increased acetyl-CoA acetyltransferase-1/2. Taken together, these results demonstrate that gambogic acid inhibits cell adhesion via suppressing integrin β1 abundance and cholesterol content as well as the membrane lipid raft-associated integrin function, which provide new evidence for the anti-cancer activity of gambogic acid.

Gambogic acid inhibits the growth of osteosarcoma cells in vitro by inducing apoptosis and cell cycle arrest

The natural product gambogic acid (GA) has been demonstrated to be a promising chemotherapeutic drug for some cancers because of its ability to induce apoptosis and cell cycle arrest. Until now, no studies have looked at the role of GA in osteosarcoma. In this study, we observed the effects of GA on the growth and apoptosis of osteosarcoma cells invitro. We found that GA treatment inhibits the proliferation of osteosarcoma cells by inducing cell cycle arrest. Moreover, we found that GA induces apoptosis in MG63, HOS and U2OS cells. Furthermore, we showed that GA treatment elevates the Bax/Bcl-2 ratio. GA mediated the G0/G1 phase arrest in U2OS cells; this arrest was associated with a decrease in phospho-GSK3-β (Ser9) and the expression of cyclin D1. Similarly, in MG63 cells, GA mediated G2/M cell cycle arrest, which was associated with a decrease in phospho-cdc2 (Thr 161) and cdc25B. Overall, our findings suggest that GA may be an effective anti-osteosarcoma drug because of its capability to inhibit proliferation and induce apoptosis of osteosarcoma cells.

Gambogic acid triggers DNA damage signaling that induces p53/p21 Waf1/CIP1 activation through the ATR-Chk1 pathway

Gambogic acid (GA) has been wildly studied to show potent anti-tumor effects in vivo and in vitro. We have confirmed that GA stabilized and activated p53 through down-regulating the expression of MDM2 in variety of cancer cell lines. However, GA-induced p53 activation could be partially reversed by caffeine, a PI3k inhibitor. Therefore, questions of whether GA induces post-translational modifications of p53 and subsequent activation of p53; and if that is the case, which upstream signaling pathway(s) is (are) responsible for that are proposed. Here, the relationship between p53 activation and its post-translational modifications was investigated in the human cancer cell lines HepG2 and A549 in response to GA or adriamycin treatment. GA induces p53 phosphorylation at sites Ser15 and Ser20 in a concentration- or time-dependent way, which was a direct result of DNA damage, as γ-HA2X foci and ‘comet’ DNA fragments were detected. GA induces p53 phosphorylation through activation of an ATM- and Rad3-related pathway, and GA-induced phosphorylation of Chk1 is also involved. Upon treatment with GA, ATR activation is clearly associated with p53 phosphorylation, as well as activation of its target gene p21 Waf/CIP1. Furthermore, we found the dephosphorylation of Cdk1 at Thr161 induced by GA was abrogated, followed by a remarkable disruption of G2/M arrest when the cells were pre-incubated with caffeine. Interestingly, the sensitivity to caffeine enhanced the cytotoxicity of GA as well. Taken together, these data showed an important role of the DNA damage response mediated by ATR-Chk1 in p53/p21 Waf/CIP1 activation and downstream G2/M arrest during GA treatment.

Spectacular modification of Gambogic acid on microwave irradiation in methanol: Isolation and structure identification of two products with potent anti-tumor activity

Treatment of gambogic acid with methanol in acidic condition under microwave irradiation led to the formation of two new products bearing spectacular A ring systems which were different from reported Garcinia natural products. The chemical structures of these two compounds were elucidated by extensive NMR and MS spectroscopic analysis as well as crystallographic study. Both of the two products could significantly inhibit the growth of various tumor cell lines in vitro with IC(50) values at muM level through apoptotic pathway.

Gambogic Acid Induces G0/G1 Cell Cycle Arrest and Cell Migration Inhibition Via Suppressing PDGF Receptor β Tyrosine Phosphorylation and Rac1 Activity in Rat Aortic Smooth Muscle Cells

Gambogic acid (GA) is the major active compound of Gamboge, a brownish or orange resin exuded from Garcinia hanburryi tree in Southeast Asia. Previous studies have demonstrated that GA exhibits potent anticancer effects by inducing cell cycle arrest or apoptosis in many types of cancer cell lines and blocking angiogenesis via inhibition of vascular endothelial cell proliferation and migration. Proliferation and migration of vascular smooth muscle cells (VSMCs) are critical steps in the progress of atherosclerosis and restenosis after angioplasty. In the present study, we investigated whether GA has an inhibitory effect on the proliferation and migration of VSMCs and its possible mechanism. The inhibitory effect of GA on the proliferation induced by PDGF-BB and EGF was measured by using Cell number counting assay and [(3)H]-thymidine incorporation. The effects of GA on the cell cycle progression and viability stimulated by PDGF-BB and EGF were also analyzed by flow cytometry analysis. The inhibitory effect of GA on the migration stimulated by PDGF-BB was measured by transwell chamber assay. The effect of GA on the Cell cycle regulatory molecules (cyclinD1, cyclinE, CDK2, CDK4), PDGFR and its downstream signaling molecules including ERK1/2, PLCγ1, AKT and JNK was measured by western blotting. The effect of GA on the Rac1 activity was measured by using GST-pulldown assay. The effects of GA on the tyrosine phosphorylation stimulated by PDGF-BB and EGF and the capacity of GA binding with PDGF-BB and EGF were also measured. We found that GA significantly inhibited proliferation, migration and DNA synthesis in primary cultured rat aortic VSMCs at concentrations of 0.25, 0.5, 1.0 and 2.0 µmol/L after stimulation of 50 µg/L platelet-derived growth factor-BB (PDGF-BB). GA induced G0/G1 phase arrest in the cell cycle progression of VSMCs. No obvious necrosis or apoptosis was found with GA treatment. The expressions of CDK2, CDK4, cyclin D1 and cyclin E, cell cycle regulatory molecules, were significantly suppressed by GA treatment in a concentration-dependent manner. The phosphorylation of PDGF receptor β (PDGFR-β) and the activities of downstream intracellular signaling molecules including phospho-ERK, phospho-PLCγ1, phospho-AKT, phospho-JNK and GTP-Rac1 were also inhibited by GA pretreatment. GA inhibited PDGFR-β phosphorylation through inhibiting the activity of tyrosine directly, rather than indirectly via binding PDGF-BB. The results of this study provide preliminary evidence that the inhibitory effects of GA on VSMCs proliferation and migration may be mediated through multiple signal pathways controlled by PDGF-Rβ activation and its downstream intracelluar signaling.

Mechanisms of Gambogic Acid-Induced Apoptosis in Non-Small Cell Lung Cancer Cells in Relation to Transferrin Receptors

Gambogic acid (GA) is one of the important active ingredients of gamboge. Our study examined the expression of transferrin receptors (TFR) on the cell surface of human lung SPC-A1 and SK-MES-1 cells and measured their GA-induced apoptosis rate. The results showed that SPC-A1 cells with a higher TFR expression were more sensitive at the same GA concentrations. To examine its distribution in cultured cells and study the mechanisms of apoptosis, we labeled GA with a 125I tracer and examined the expression of apoptosis-related proteins. We found that GA uptake into SPC-A1 cells was higher than into SK-MES-1 cells; apoptosis-related proteins Caspase 2, Caspase 9, Caspase 10, Bax and p53 were involved in GA-induced apoptosis. We conclude that GA has an apoptosis-promoting effect on non small cell lung cancer cells. In clinical practice, the histopathological quantitation of TFR expression levels in tumor tissues may become a predictor of the sensitivity of patients' tumors to GA treatment.

Gambogic acid reduced bcl-2 expression via p53 in human breast MCF-7 cancer cells

In this study, we investigated the correlation between p53 and bcl-2 in gambogic acid (GA)-induced apoptosis. MTT assay was employed to evaluate MCF-7 cell viability after GA treatment. Cell morphological changes were observed follow-up under the inverted microscope after GA withdrawal. To observe the cell apoptosis, DAPI staining was used. Meanwhile, p53 small interfering RNA (si-RNA) was adopted to knock p53 down. All expressions of p53 and bcl-2 were evaluated by Western blot analysis. MTT assay showed that GA inhibited MCF-7 cell growth effectively in a time-dependent manner. With 0.5 h GA treatment, p53 was significantly increased, whereas bcl-2 was reduced potently with 6 h GA treatment. After GA withdrawal, p53 expressions were maintained in high levels. Furthermore, bcl-2 decreasing was attenuated after co-treatment with PFT alpha, a p53 transcription blocker. Same result was observed after p53 knock-down by p53 si-RNA. Gambogic acid induced human breast cancer cells MCF-7 apoptosis by reducing bcl-2 expression via p53.

Gambogic acid induces G0/G1 arrest and apoptosis involving inhibition of SRC-3 and inactivation of Akt pathway in K562 leukemia cells

Gambogic acid (GA), a major active component of gamboge, exhibits potent anticancer activity in many kinds of cancer cells. However, the anticancer mechanism of GA is not clearly understood. Here we showed that GA could cause growth inhibition, induce the G0/G1 phase cell cycle arrest and apoptosis in human chronic myelogenous leukemia cell line K562 cells. Since steroid receptor coactivator-3 (SRC-3), overexpressed in many human malignancies including leukemia, is a central target for cancer therapy, we also explored the effects of GA on SRC-3 and SRC-3-regulated gene products in K562. GA treatment downregulated the expression of SRC-3 and then inhibited the activity of Akt kinase and its downstream targets p70 S6 kinase 1 (S6K1) and glycogen synthase kinase 3β (GSK3β) without changes in total protein levels of these three proteins, which thus influenced the expression of the apoptosis related gene Bcl-2 in K562 cells. These results suggest that GA might exhibit its strong antitumor effects via the interruption of SRC-3.

Gambogic acid induced tumor cell apoptosis by T lymphocyte activation in H 22 transplanted mice

Multiple lines of evidence have demonstrated that gambogic acid (GA) is an efficient apoptosis inducing agent. However, the mechanisms of GA induced apoptosis have been controversial, despite the tremendous effort made during recent years. Here we report a novel mechanism through which GA induces cell apoptosis. Instead of dealing with tumor cells directly, GA first activates inactive T lymphocytes, which in turn triggers cancer cell apoptosis. This is supported by the observation that GA inhibited tumor growth and extended the survival time of mice bearing H 22 tumor. cDNA microarray analysis indicated that 22.92% of the 48 genes that were affected with GA treatment were immune related genes. RT-PCR assay revealed that GA up-regulated MHC-II and TCR transcriptions, implicating that GA activates T lymphocytes to induce tumor cell apoptosis in vivo. HE staining showed that T lymphocytes penetrated into tumor tissues after GA administration. Western blotting revealed that GA enhanced CD4 + and CD8 + expressions. Annexin-V/PI double-staining and DNA ladder assays confirmed that GA induced tumor cell apoptosis. In summary, this report demonstrated, for the first time, that GA mainly activates T lymphocytes to induce cancer cell apoptosis in H 22 transplanted mice.

Gambogic acid, a potent inhibitor of survivin, reverses docetaxel resistance in gastric cancer cells

Objective: Chemoresistance is a major obstacle to successful cancer chemotherapy. In this study, we examined the ability of gambogic acid (GA) to reverse docetaxel resistance in BGC-823/Doc gastric cancer cells. Methods: The cytotoxic and apoptotic effect of drugs were evaluated by MTT assay and double staining with both Annexin-V-FITC and PI. Cell cycle analysis was determined by PI-stained flow cytometry. Expression of survivin and bcl-2 were evaluated by real-time quantitative RT-PCR. Results: Treatment of BGC-823/Doc cells with gambogic acid at concentrations of 0.05 μM, 0.1 μM, and 0.2 μM, led to a dramatic increase in docetaxel-induced cytotoxicity without any cytotoxicity by itself. In parallel, gambogic acid treatment caused an increase in apoptotic cell death by docetaxel. Cell cycle analysis indicated that gambogic acid treatment potentiated docetaxel-induced G2/M arrest. Analysis of apoptotic associated gene revealed that gambogic acid singly or in combination with docetaxel significantly downregulate the mRNA expression of survivin, while with no effect on bcl-2. Conclusions: Our results describe the potential role of gambogic acid to reverse docetaxel resistance though downregulation of survivin, which may make it an attractive new agent for the chemosensitization of cancer cells.

Gambogic acid inhibits the catalytic activity of human topoisomerase IIα by binding to its ATPase domain

This study is intended to characterize the cellular target of gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hurburyi tree, which possesses potent in vitro and in vivo antitumor activities. The antiproliferative activity of GA was further confirmed here in a panel of human tumor cells and multidrug-resistant cells. We found that GA significantly inhibited the catalytic activity of topoisomerase (Topo) II and, to a comparatively less extent, of Topo I, without trapping and stabilizing covalent topoisomerase-DNA cleavage complexes. Down-regulation of Topo IIalpha but not Topo I and Topo IIbeta, reduced GA-induced apoptosis and the phosphorylation of c-Jun, and restored cell proliferation upon GA treatment. Moreover, GA antagonized etoposide-induced DNA damage and abrogated the antiproliferative activity of etoposide, whereas it did not affect camptothecin-induced DNA damage. By dissecting the actions of GA on the individual steps of Topo IIalpha catalytic cycle, we found that GA inhibited DNA cleavage and ATP hydrolysis. Moreover, GA directly bound to the ATPase domain of Topo IIalpha, and may share common binding sites with ATP. The results reported here show that GA exerts its antiproliferative effect by inhibiting the catalytic activity Topo IIalpha. They also indicate that GA inhibits Topo IIalpha-mediated DNA cleavage and modulate the activity of Topo II poisons, which provide rationale for further clinical evaluation of GA.

Repression of telomerase reverse transcriptase mRNA and hTERT promoter by gambogic acid in human gastric carcinoma cells

To investigate the effects and potential mechanisms of gambogic acid (GA), a naturally occurring anticancer agent, on the expression and regulation of telomerase in human gastric carcinoma cells. GA-induced inhibition of cell proliferation was evaluated by the commonly employed MTT assay on two human gastric carcinoma cell lines, MGC-803 and SGC-7901. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplication protocol-polymerase chain reaction and reverse transcription-polymerase chain reaction, respectively. The hTERT promoter activity was measured by luciferase assay. The expression of c-MYC, an apoptotic gene that modulates the expression of hTERT promoter, was quantified by Western blotting. The proliferation of human gastric carcinoma cell lines, MGC-803 and SGC-7901, was significantly inhibited with GA treatment. Both telomerase activity and hTERT mRNA expression were notably decreased in cells treated with GA. The activity of hTERT promoter and the expression of c-MYC were also remarkably decreased in GA-treated cells. This study demonstrated that GA treatment of human gastric carcinoma cell lines, MGC-803 and SGC-7901, significantly reduced the expression of c-MYC in a time- and concentration-dependent manner accompanied with the down-regulation of the hTERT transcription and the ultimate reduction in telomerase activity. Our results indicate that the hTERT is a target of c-MYC activity and identify a feasible mechanism of GA's potent anticancer activity.

Inhibition of human telomerase reverse transcriptase gene expression by gambogic acid in human hepatoma SMMC-7721 cells

The activation of human telomerase, a process regulated by the human telomerase reverse transcriptase (hTERT), is a crucial step during cellular immortalization and malignant transformation. We have reported that gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hanburyi tree, is an effective telomerase inhibitor and thus displays potent anticancer activity both in vitro and in vivo. Here we present the direct interaction of GA with oncogene c-MYC, a ubiquitous transcription factor involved in the control of cell proliferation and differentiation, as the molecular mechanism of GA's inhibitory effect on telomerase activity. Consistent with the recently reported association between c-MYC overexpression and induction of telomerase activity, we find here that GA treatment of a human hepatoma cell line SMMC-7721 significantly reduced the expression of c-MYC in a time- and concentration-dependent manner accompanied with the down-regulation of the hTERT transcription and the ultimate reduction in telomerase activity. Our results indicate that the hTERT is a target of c-MYC activity and identify a feasible mechanism of GA's potent anticancer activity.