Activation Of Critical Proteinum During Apoptosis Of Human Colorectal Cancer Cells Induced By Mcp

BackgroundColon cancer is one of the most common malignant tumour in the world,Its morbidity has had the tendency to rise obviously The morbidity is rising year by year in china. Chemotherapy is the major means for the treatment of advanced cancer patients.The main principle of chemotherapeutic is the use of cytotoxic drugs to destroy cancer cells and prevent cancer cells from further multiplying. Although the outcomes of cancer treatment have been significantly improved by chemotherapy in the last three decades, the clinical success of cancer chemotherapy has been severely hindered by de novo and acquired chemoresistance and the severe non-specific toxicity od high dose chemotherapy to vital tissues and organs.Therefore,searching for low toxicity or non-toxic chemosensitizers to enhance the anticancer effect of conventional chemotherapeutic agent and reverse chemoresistance is an area of significant interest in cancer research and oncology.In the search of naturally occurring substances that could be useful in controlling and treating cancer metastasis, modified citrus pectin (MCP), a complex water soluble indigestiblepolysaccharide obtained from the peel and pulp of citrus fruits and modified by means of high pH and temperature treatment , has emerged as one of the most promising anti-metastatic drugs. Earlier work by our research demonstrated that MCP can increase the effect of inhibiting tumor growth, liver metastasis and angiogenesis. The recognized potent anti-metastatic activity of MCP,the exact targrnts and molecular mechanism of MCP are well known.But,its unclear the potent antitumour activity of MCP. In the most recent study,MCP induces apoptosis in multiple myeloma cells without significantly altering normal cell viability ,MCP-triggered apoptosis is associated with activation of caspase-8 and caspase-3. From the last research we infer that MCP have antitumour activity in colon cancer.Objective:1,To estimate the effect of MCP on colon cancer cell proliferation and apoptosis .2,To infer the possible mechanism of MCP in vitro. .Methods:1. Cell lines :human colon cancer cell lines SW116,HCT116,HT-29,SW620,SW480.2. Methyl thiazolyl tetrazolium(MTT) assay was used to measure the proliferation of human colon cancer cell lines( SW116,HCT116,HT-29,SW620,SW480 )cultured with different concentrations (0mg/ml,1 m g/ml,2.5mg/ml,5mg/ml)of MCP for 48 h in vitro..3. Morphology change of colon cancer cells after 24 hours exposed to MCP.4. Flow cytometric analysis of the effect of MCP on apoptosis after 24 hours.5. Western blotting analysis of the effect of MCP on caspase-8, caspase-9, caspase-3,PARP protein expression levels after 48 hours.Results:1. Significant cell death were induced by MCP in all testing colon cancer cell lines . because of the solubility of MCP,we could not measure IC50 of MCP. 2. Cells treated by concentration of MCP(2.5mg/ml)did not show any visible apoptosis characters after 24 hours because of the solubility of MCP.3. Cells were treated with 2.5mg/ml MCP for 24 hours in comparison with the untreated cells in vitro. MCP(2.5mg/ml) enhanced apoptisis after 24 hours treatment on all tested colon tumor lines significantly.4. Wstern blotting results showed that the treatment of cell line HCT116 for 48 hours with MCP (2.5mg/ml,5mg/ml) resulted in the activation of caspase-9 ,caspase-3 followed by proteolytic cleavage of poly(ADP-ribose) polymerase enzyme and the caspase-8 cleavage to a less degree.Conclusions:1.MCP ,a novel carbohydrate-based agent, induces apoptosis in various multiple colon cancer cell lines .2.MCP triggered apoptosis is associated with activation of caspase-9 and caspase-3 followed by proteolytic cleavage of poly(ADP-ribose) polymerase enzyme.