Studies On The Synergistic Effects Of Trail With Mitomycin C And Ginseng Polysaccharides On Human Colon Cancer Cells

Colorectal cancer (CRC) is the human malignant tumor. Globally greater than1million people get colorectal cancer yearly resulting in about0.5million deaths-with it being the fourth most common cause of cancer death after lung, stomach, and liver cancer. Colorectal cancer is a disease involved a series of complex biological, chemical changes in the cells. Based on the research of the mechanism of the tumorigenesis of colorectal cancer, it is important to explore effective drugs and combination methods for therapy of this disease.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of TNF family, is a potent cancer cell-specific apoptosis-inducing agent with little to no effect on normal tissues. So do the TRAIL agonistic antibodies targeting TRAIL receptors (TRAIL-Rs)(mapatumumab and lexatumumab). This advantage makes them attractive candidate anticancer drugs. TRAIL induces apoptosis through binding to proapoptotic receptors DR4and DR5. However, acquired tumor resistance of cancer cells to TRAIL is a roadblock, allowing them to evade the proapoptotic effects of TRAIL. The effectiveness of TRAIL and TRAIL-R agonistic antibodies as monotherapies may be limited by resistance, as it is for other anticancer agents.Drugs are commonly combined to augment treatment efficacy and suppress the emergence of resistant clones. Thus agents that can modulate some of these mechanisms of resistance to TRAIL have a potential in improving the cytokine therapy. It has been reported that DNA-damaging agents and small-molecule inhibitors effectively sensitize cancer cells to TRAIL.Mitomycin C is an antibiotic that has antitumor activity, and it has received the greatest amount of preclinical and clinical work and already been widely used to treat various cancers. MMC is known to act synergistically with a series of clinical drugs.The polysaccharides contain the active components of ginseng and hanve been reported to possess various antitumor activities. Ginseng polysaccharides also have synergistic effects with various clinical drugs.In this thesis, we tested the synergistic effects of TRAIL with Mitomycin C (MMC) and ginseng polysaccharides respectively. We also further investigated the mechanism accounting for the synergistic effect.The cell viability assay, flow cytomentry assay and Western-blot assay were applied to investigate whether MMC potentiated TRAIL-induced cell apoptosis in HCT116(p53-/-) human colorectal cancer cells. The results indicated that the combination treatment with MMC and TRAIL decreased cell viability and enhanced cell apoptosis significantly. MMC can also enhance TRAIL-induced apoptosis in TRAIL-resistant HT-29cells. Our results showed that pretreatment with MMC further enhanced the sensitivity to lexatumumab and mapatumumab in HCT116(p53-/-) cells and HT-29cells respectively.To identify the mechanism by which MMC sensitizes colon cancer cells to TRAIL and TRAIL agonistic antibodies, we quantified multiple extrinsic and intrinsic cell death pathway components that could be affected by MMC to lead to more efficient apoptotic signaling. We found that MMC down-regualted Bcl-2, Bcl-XL and Mcl-1expression. By contrast, MMC increased the content of proapoptotic proteins such as Bax and Bim. MMC also down-regulated caspase inhibitor family members c-IAP-1and XIAP. Furthermore, death receptors DR4and DR5were increased by MMC in a dose denpendent manner in protein content, cell surface receptor density and mRNA level. And up-regulation of DR4and DR5expression by MMC is not specific to cell type.We used a specific cancer cells stably transfected with shRNA against DR4(HCT116shDR4), DR5(HCT116shDR5), DR4and DR5(HCT116shDR4/shDR5) and then examined the impact on suppression of cell viability by the combination of MMC and TRAIL. We found that the silencing of DR5or both receptors increased the cell viability. These results suggest that DR5plays an important role in MMC on TRAIL-induced cytotoxicity.Given the induction of DR4and DR5by MMC and the important role of DR5in the synergistic effects as demonstrated above, we were particularly interested in the mechanisms by which MMC induces expression of DR4and DR5. MMC induced expression of DR5and DR4in HCT116, HCT116(p53-/-), Bax-knockout, Bax parental cells, and Bim-knockdown HCT116(p53-/-) cells, indicating that induction of TRAIL receptors are independent of p53, Bax and Bim expression. But Bim knockdown and Bax knockout suppressed the synergistic effect of MMC and TRAIL.We found that MMC increased the levels of phosphorylation of JNK and its downstream substrate, phosphorylation of c-Jun, indicating that MMC activates JNK pathways. The JNK-specific inhibitors SP600125abrogates up-regulation of DR4and DR5, indicating that MMC induces DR4and DR5expression through a JNK-dependent mechanism. We also transfected JNK-specific siRNA to inhibit JNK activation through silencing JNK expression and then examined its impact on DR4and DR5expression MMC induced DR4expression in control and control siRNA-transfected cells, but not in JNK1and JNK2siRNA-transfected cells. MMC did not induce DR5expression only in JNK2siRNA-transfected cells. We also tested the combination effect of MMC and TRAIL on JNK1and JNK2knockdown cells. We found that the synergistic effect of MMC and TRAIL decreased in the JNK1knockdowen cells. Collectively, we conclude that MMC induces DR4and DR5expression through a JNK-dependent mechanism.We next tested the therapeutic combination of MMC and TRAIL in vivo. Mice bearing xenografted HCT116(p53-/-) colon tumors and HT-29colon tumors were treated with intraperitoneal dose MMC (1mg/kg) and intravenous dose of TRAIL (100μg) every other day. Animals were treated with ten consecutive cycles of the combination therapy regimen. The combination therapy suppressed tumor growth and the ki67expression significantly, indicating that the therapeutic combination of MMC and TRAIL has activity in vivo.The other aim of this thesis is investigating the synergistic effects of ginseng polysaccharides and TRAIL. First we did research on the anticancer activity of ginseng polysaccharides alone on human colon cancer cells. Five representive ginseng polysaccharides and their temperature-modified products were chosen to be assayed for their effects on HT-29cell proliferation by MTT assay, on cell cycle progression by flow cytometry, and on caspase-3activation by western-blot analysis. The HG:rich ginseng pectin inhibited cell proliferation and induced cell cycle arrest in the G2/M phase. The temperature-modified HG-rich pectin had dramatically increased antiproliferative effect and induced apoptosis accompanied by the activation of caspase-3. Starch-like glucan and arabinogalactan of ginseng exhibited no antiproliferative effects. Even after temperature modification, their inhibitory effects either remained unchanged or increased slightly. The HG-rich pectin exerts its antiproliferative effect via cell cycle arrest and the temperature modification markedly increased the antiproliferative effect.We next did some research on the synergistic effects of different ginseng polysaccharides and TRAIL. The results showed that WGPA-3-RG and WGPA-4-RG, which have both RGI domain and HG domain, can not only suppress HT-29cells proliferation alone, but also synergize with TRAIL in suppressing the cell viability of HT-29cells. We also found that the temperature-modified ginseng polysaccharide MWGPA-3-HG has significant synergistic effect with TRAIL in suppressing cell viability of HT-29cells and HCT116cells. The Western-blot was applied to test the DR4and DR5expression of the cells treated with different concentrations of MWGPA-3-HG. The results showed that MWGPA-3-HG increased DR4and DR5expression in a dose-dependent manner. We speculated that increasing expression of DR4and DR5might account for the synergistic effect of MWGPA-3-HG and TRAIL.In conclusion, we found that the Mitomycin C and ginseng polysaccharides have synergistic effects with TRAIL in suppressing human colon cancer. All these results will provide the theory basis for the clinical trials of TRAIL.