Research On The Anti-tumor Effect And Mechanism Of Neogambogic Acid

Background and purposeGamboge is the dry resin secreted from Garcinia hanburyi Hook.F.G. In recent years, the anti-tumor effect of Gamboge has been gradually and greatly concerned. Studies have confirmed that neogambogic acid (NGA) is one of the main active ingredients of the anti-tumor effect of Gamboge and that it is expected to be developed into a new anti-tumor drug. But the research on its anti-tumor spectrum has not yet completed. It has not been found that the information of distribution of NGA in the organs and tissues. In addition, the research on its mechanism was limited to NGA impacting on cell cycle of leukemia. Much more important information concerning the application of NGA was far from perfect. In view of this, the dissertation aims to further elaborate the anti-tumor activity of NGA in vitro and in vivo. And on this basis, from apoptosis, cell cycle, cell signal transduction, telomerase activity and angiogenesis, we tried to explore the anti-tumor mechanism of NGA and its molecular basis, which would establish the theoretical basis for the development and clinical application of NGA.Methods and results1 The anti-tumor effect of NGA in vitroMTT and CCK-8 were used to assay the inhibition rate of tumor cells treated with NGA in different concentration. According to the inhibition rate, the IC50 (50 percent inhibitory concentration) of each tumor cell line was calculated, which was used to evaluate the anti-proliferative effect of NGA on tumor cells in vitro. The results showed that NGA could play anti-proliferative effect on 16 tumor cell lines. Their IC50 was between 1.14μg/mL and 4.25μg/mL, indicating that NGA has exact anti-tumor activity in vitro (IC50<10μg/mL). The experimental results of the normal human liver cell line HL-7702 treated with NGA showed that the IC50 of NGA on HL-7702 cell line was 5.23±0.04μg/mL, and the IC50 of NGA on human hepatoblastoma Hep G2 cell line was 1.22±0.12μg/mL. In addiction, the results also showed that in a certain concentration range (between 1.25μg/mL and 2.50μg/mL) NGA could selectively play strong anti-proliferative effect on tumor cells while play weaker anti-proliferative effect on normal liver cells.2 The anti-tumor effect of NGA in vivoAfter acute toxicity test, we calculated the LD50 (median lethal dose) of NGA on mice by statistical software SPSS11.5. The result showed that the LD50 was 36.66mg/kg. On this basis, we determined that the high, medium and low doses of the mice experiments were 8.0mg/kg,4.0mg/kg and 2.0mg/kg respectively. The anti-tumor effect of NGA on mice bearing S180 ascitic tumor was evaluated by survival time of tumor-bearing mice. The results showed that NGA at medium dose (4.0 mg/kg) has the best effect on prolonging survival time of tumor-bearing mice, which was significantly higher than the efficacy of the positive control drug 5-FU (fluorouracil)(10.0 mg/kg)(P<0.05). But the efficacies of the high dose (8.0 mg/kg) and low dose (2.0 mg/kg) were lower than the efficacy of 5-FU (10.0 mg/kg) (P<0.05). And the average survival time of low dose group and negative control group had no significant difference (P>0.05). These suggested that NGA played therapeutic effect in a narrow dosage range, and that the efficacy of high dose group was worse than the efficacy of medium dose group may be caused by the toxic side effects of NGA.After establishing the high performance liquid chromatography (HPLC) determination method of NGA in blood and the tissues of organs, the bearing S180 solid tumor model was established to study the distribution of NGA in mice. The results showed that the peak concentration of NGA in heart, liver, lung, spleen, kidney and tumor tissues were 9.58±2.79,16.49±4.17,14.51±3.68.8.23±1.88,8.85±2.37,10.28±2.75μg/g respectively. But in the experimental conditions, NGA failed to be detected in brain tissue, whose reason may be that NGA failed to pass the blood-brain barrier or its concentration level was lower than the detection limit. According to the results of distribution experiment and the results of the experiments in vitro, human hepatoblastoma cell line Hep G2 was determined to be the cell line of the tumor xenograft model in nude mice. Then human hepatoblastoma Hep G2 cell xenograft model in nude mice was established. And the anti-tumor effect of NGA on human tumor in vivo was evaluated by the relative tumor volume (RTV) and tumor growth inhibition rate (TGI). The results showed that the NGA could significantly inhibit the growth of the human hepatoblastoma xenograft in nude mice. In the experimental time frame, after 8 days of treatment, it could be observed that RTV decreased along with the increase of dose. After 14 days of treatment, RTV of the negative control group,5-FU group, NGA high dose (8.0mg/kg) group, medium dose (4.0mg/kg) group and low dose (2.0mg/kg) group were 10.72±4.83,7.57±2.24,1.34±0.36,2.52±1.37 and 7.02±1.94 respectively. TGI of 5-FU group, NGA high dose (8.0mg/kg) group, medium dose (4.0mg/kg) group and low dose (2.0mg/kg) group were 46.54%,83.75%,68.09% and 22.14% respectively. These suggested that in a certain dosage range, NGA had exact anti-tumor effect in vivo.3 The anti-tumor mechanism of NGAAfter determining NGA having the exact anti-tumor effects in vivo and in vitro, we exerted further research on the anti-tumor mechanism of NGA from apoptosis, cell cycle, cell signal transduction, telomerase activity and angiogenesis.After cells stained by Annexin V-FITC/PI, flow cytometry analysis showed that the maximum percentage of apoptosis appeared and reach to (49.44±3.12)% at 24 hours after Hep G2 cells were exposed to 2.0μg/mL NGA, indicating that NGA could induce early apoptosis of Hep G2 cells in vitro. Analysis by agarose gel electrophoresis showed that NGA could induce the DNA in nucleus of Hep G2 evidently to be degraded, but no DNA ladder was observed. Its reason might be that after cells occurring early apoptosis, DNA was further degraded into fragments of about 200 base pairs (bp).To further research on the mechanism of apoptosis, the levels of apoptosis-related proteins Bax and Bcl-2 in Hep G2 cells exposed to NGA in vitro were detected by Western blot. The results showed that Bax increased along with the increase of drug concentration while Bcl-2 decreased along with the increase of drug concentration. The Bax/Bcl-2 ratios of the control group, NGA (1.0μg/mL) group, NGA (2.0μg/mL) group and NGA (3.0μg/mL) group were 0.06,0.29,2.25 and 21.14 respectively, indicating that NGA in a certain concentration range could increase the Bax/Bcl-2 ratio of Hep G2 cells in vitro.The levels of Bax and Bcl-2 in human hepatoblastoma Hep G2 cell xenograft in nude mice was assayed by immunohistochemistry. The results showed that the levels of Bax and Bcl-2 of NGA low dose (2.0mg/kg) group and that of negative control group had no significant difference (P>0.05). For NGA medium dose (4.0mg/kg) group and NGA high dose (8.0mg/kg) group, the levels of Bax were significantly higher than the negative control group, while the levels of Bcl-2 were significantly lower than the negative control group. The Bax/Bcl-2 ratios of the negative control group, NGA (2.0mg/kg) group, NGA (4.0mg/kg) group and NGA (8.0mg/kg) group were 0.729,0.808,1.679 and 1.688 respectively, indicating that NGA in a certain dosage range could increase Bax/Bcl-2 ratio of human hepatoblastoma Hep G2 cell xenograft in nude mice. Evidently, both the results in vitro and in vivo supported the hypothesis that NGA could increase the level of Bax and (or) reduced the level of Bcl-2, which increased Bax/Bcl-2 ratio to induce apoptosis.In terms of the progression of the cell cycle, Hep G2 cells treated with 2.0μg/mL NGA for different time were collected and assayed by flow cytometry. Cell phase analysis showed that compared with the control group, the indexes of the cells arrested in S phase significantly increased during the first 0-36 hours and the indexes of the cells arrested in G0/G1 phase significantly increased when treated with NGA for 48 hours, indicating that NGA could induce cells to be arrested in S phase and to be further arrested in G0/G1 phase with the time passing by.In terms of the MAPK signal transduction pathway, the levels p-ERK1/2 and p-MEK1/2, the activated forms of MEK and ERK of Ras\Raf\MEK\ERK cascade pathway, were assayed by immunohistochemistry. The results showed that NGA could down-regulate the levels of p-ERK1/2 and p-MEKl/2, indicating that NGA could play anti-proliferative effect by reducing the signals of ERK signaling pathway to arrest cell cycle. But the adjustment point may be in the upstream of MEK in Ras/Raf/MEK/ERK cascade pathway, as the level of p-MEK1/2, the MEK activation form, had also been reduced.In terms of telomerase activity, the level of hTERT (human telomerase reverse transcriptase) mRNA was assayed by FQ-PCR (fluorescence quantitative polymerase chain reaction) to reflect telomerase activity. The results showed that the levels of hTERT mRNA of each group of Hep G2 cells treated with NGA was significantly lower than the control group (P<0.05). The level of hTERT mRNA of each group of Hep G2 cells treated with NGA decreased along with the increase of drug concentration, and there is significantly difference among each group (P<0.05). These indicated that NGA could inhibit telomerase activity in drug concentration dependently. Consequently, we inferred that NGA could play anti-tumor effect by inhibiting telomerase activity.In terms of the angiogenesis of tumor, the level of CD31 (platelet endothelial cell adhesion molecule) in Hep G2 tumor tissue in nude mice was assayed by immunohistochemistry. The results showed that CD31-positive expression IOD (integrated option density) values of three groups treated with NGA were significantly lower then that of the negative control group, and the differences were statistically significant (P<0.05). These indicated that NGA could reduce the levels of CD31 in solid tumor tissue in a certain dosage range, suggesting that NGA could inhabit angiogenesis in solid tumors to some extent.ConclusionsNGA has a broad spectrum anti-tumor effect in vitro, In a certain concentration range, NGA can selectively play strong anti-proliferative effect on tumor cells and relatively weaker anti-proliferative effect on normal cells.NGA can be widely distributed in mice. The maximum concentration was found in liver followed by that of the lung. NGA can significantly prolong the life span of the S180 ascites tumor bearing mice, indicating that NGA has an exact anti-tumor activity on animal tumors in vivo. NGA can significantly reduce the RTV of human hepatoblastoma Hep G2 xenografts in nude mice and increase TGI to 83.75%, indicating that NGA has an exact anti-tumor activity on human hepatoblastoma Hep G2 xenografts in nude mice in vivo.NGA can play an anti-tumor effect by inducing apoptosis, arresting cell cycle, inhibiting telomerase activity and inhibiting angiogenesis. NGA inducing tumor cells apoptosis is by increasing the ratio of Bax/Bcl-2. In addition, NGA can blocks MAPK cascade by reducing the activity of key enzymes in MAPK/ERK signaling pathway, causing cell cycle arrest and resulting in anti-proliferative effect. Meanwhile, cell cycle arrest can still induce tumor cell programmed cell death.