Research On The Relationship And The Mechanism Of Autophagy And Apoptosis Induced By5-Fu In HCT-116Cell Stem

Colorectal cancer is one of the common malignant tumor for people. Although surgery resection currently remain the only curative treatment for colorectal cancer, it is always unsatisfactionary. Many people have no effects or only minor ones for the adjuvant drugs. During the process of colorectal cancer cell apoptosis induced by chemotherapy, the tolerance of the cells often appears and escapes from the apoptosis. Now the relationship between autophagy and apoptosis induced by5-Fu is becoming a new hot spot, which will be resulted in an increased response rate in advanced colorectal cancer, and the new therapeutic strategies and a new adjuvant drugs can be explored.1. Detection of autophagy and apoptosis in HCT-116cells induced by5-Fu.Objective:Research on the autophagy and apoptosis induced by5-Fu in HCT-116cells.Methods:Culture the cells and take into control group and induced by5-Fu group. To evaluate autophagy induced by5-Fu, the methods include dyeing with MDC and Western blot. To evaluate apoptosis induced by5-Fu, the methods include dyeing with DAPI, FlowCytometry and Western blot.Results:By dyed with MDC, The pointlike distribution of autophagy body were observed by fluorescence microscope. By dyed with DAPI, it observed the DNA of chromatin changed as apoptosis, and the apoptosis rate induced by5-Fu group significantly increased than the control group by FlowCytometry. Also the protein level of beclinl, LC3B and p53increased than the control group with Western blot.Conclusion:The result shows that5-Fu can induce the autophagy and apoptosis of HCT-116cells.2. The role of autophagy in apoptosis induced by5-Fu in HCT-116cellsObjective:Effects of proliferation, apoptosis and gene expression by inducers or inhibitors for autophagy and apoptosis on HCT-116cells.Methods:After the HCT-116cells treated with5-Fu in combination with autophagy inducer(rapamycin), autophagy inhibitor(3-MA) or apoptosis inhibitor(Z-VAD-FMK), respectively, cell proliferation observed by MTT, apoptosis rate detected by FlowCytometry and the gene level expression by RT-PCR.Results:The cell absorbency value reduced significantly, and the inhibit of cell proliferation significantly increased, as compared with in the control. The cell proliferation rates6,12and24h after induced by5-Fu were significantly lower than those at Oh. As compared with that in the controls, the apoptosis percentage of cells treated by using5-Fu and autophagy inducer increased significangtly. The apoptosis percentage of cells treated with5-Fu in combination with autophagy inducer, autophagy inhibitor or apoptosis inhibitor increased as compared with that in the controls.The apoptosis percentage of cells in5-Fu+rapamycin group increased significantly as compared with that in the5-Fu+3-MA and5-Fu+Z-VAD-FMK groups. The relative expressions of beclinl, LC3B and p53mRNA increased in HCT-116cells treated by5-Fu jointing with rapamycin, as compared with5-Fu group. On the contrary, The relative expressions of beclin1, LC3B and p53mRNA decreased in HCT-116cells treated by5-Fu jointing with autophagy inhibitor or apoptosis inhibitor. The relative expressions of p53mRNA increased in HCT-116cells treated by5-Fu and that jointing with rapamycin, as compared with5-Fu jointing with autophagy inhibitor or apoptosis inhibitor.Conclusion:The result indicate that5-Fu combined with autophagy inducer can inhibit HCT-116cells proliferation, and it combinated with autophagy inhibitor can promote the cell proliferation. And5-Fu combined with autophagy inducer can promote the apoptosis of HCT-116cells. The cell autophagy and apoptosis are two different programmed cell death ways, that have overlap.5-Fu jointing with autophagy inducer affects not only the autophagy pathway but also the apoptosis pathway in the cells.3.The role of beclinl gene in autophagy and apoptosis in HCT-116cells induced by5-FuObjective:Constructing beclinl gene interference and over-expression models, and research on the proliferation, apoptosis, protein level and gene expression effected by5-Fu on beclinl gene interference and over-expression models.Methods:Constructed pcDNA3.1-beclinland pGenesill.3-beclin1Ri recombinated vector, and transfected into the HCT-116cells by liposome method. To observed cell proliferation by MTT on beclinl gene interference and over expression models in HCT-116cells. And the effects of5-Fu on apoptosis in HCT-116, HCT-116-beclinl and HCT-116-beclinlRi cells by FlowCytometry. Also the methods of RT-PCR and western blot detect the gene expression and protein level respectly.Results:Cell prolifetation in the5-Fu and HCT-116-beclinl group decreased significantly as compared with the other groups. The apoptosis percentages of the5-Fu group, HCT-116-beclinl and HCT-116-beclinlRi6h and24h increased significantly as the control group. The apoptosis percentage between the HCT-116-beclinl, HCT-116-beclinlRi5-Fu and5-Fu is statistical different. It is also significantly between HCT-116-beclinl and HCT-116-beclinlRi. The cell apoptosis percentage in the HCT-116-beclinl was the highest. The expression of beclinl protein in HCT-116cells increased6h after5-Fu induced, which reached to the greatest12h later, and decreased24,48h, still higher than that in the control. The expression of p53rise6h. which increased gradually with the extension of time. In the HCT-116-beclinlRi cells are lower at6h after induced by5-Fu. which begin to rise12h, and reach to the max in24h. the expression of p53protein begin to rise6h, and reach to the max in24h. The expression of beclinl protein in HCT-116-beclinl increased gradually with time. The relative expression of LC3B/GAPDH gene of HCT-116cells, HCT-116-beclinl and HCT-116-beclinlRi were significantly different. The relative expression of p53/GAPDH in5-Fu group, HCT-116-beclinl and HCT-116-beclinlRi group was significantly higher than that in the HCT-116group. And it was significantly different in expression of p53mRNA between all of the groups.Conclusion:The over expression of beclinl gene restrain the cell prolification of HCT-116cells. The expression of beclinl in HCT-116cells induced by gene promoted the increased of p53protein expression, and the autophagy induced by5-Fu can increase cell apoptosis. The beclinl gene in the HCT-116cells can regulated the cell autophagy and apoptosis.