The Histone Deacetylase Inhibitor (Trichostatin A, TSA)Blocking The Growth Mechanism Of Colorectal Cancer

Background:Colorectal cancer is a common malignant tumor. In Western countries, its incidence (50/100000) and mortality ranks among the first three of the malignant tumors. In China, it runs after gastric cancer, lung cancer and esophageal cancer. It will become the common malignant tumors with high incidence, increasing morbidity and mortality. Therefore, currently, an important and urgent task for anti-cancer researchers is to further study the pathogenesis of colorectal cancer and find effective chemotherapeutic drugs.Up to now, it is widely accepted that the pathogenesis of colorectal cancer is viewed as a complex process involving multi-genes and multi-dimensions, for example, muti-oncogene activation and tumor suppressor gene inactivation are involved. P53is an important tumor suppressor gene; it works through the regulation of downstream genes. P53protein can promote the expression of p21and Bax genes, which results in cell cycle arrest and apoptosis. A number of anti-tumor drugs work through a p53-dependent way. But years of clinical studies have reported that nearly50%of the tumor sufferers are related to p53mutation and loss of its function. Trichostatin A (TSA), a potent specific and reversible histone deacetylase inhibitor, can inhibit cell growth, induce cell cycle arrest and promote cell differentiation and apoptosis.Though the anti-tumor effect of TSA has been carried out in some domestic and foreign researches, the range of its anti-tumor mechanism remains uncovered. On studying the mechanism of cell cycle arrest and cell apoptosis induction relative to TSA, the thesis provides an observation of how TSA has an effect upon the HCT116p53(+/+) and HCT116p53(-/-) colorectal cancer and upon HT29cell cycle and cell apoptosis resulted from p53mutation, and an analysis of TSA in terms of the regulation of the molecules closely related to apoptosis:PAPP, caspase3, Bax, Bcl-2, Bcl-xL, MCL1and Ku70d. This thesis provides more dimensions of TSA anti-tumor mechanism and theoretical evidence on clinical prevention and colon cancer treatment with histone deacetylase inhibitor.Objectives:First, observe what effect TSA has on cell cycle and cell apoptosis; explore what role p53dependent or non-dependent ways play in cell cycle arrest and cell apoptosis caused by TSA;Second, explore the apoptosis mechanism of colorectal cancer cell induced by TSA; detect how TSA regulates Bax promoter activity and what effects TSA has on the expression of apoptosis-related molecules, PAPP, caspase3, Bax, Bcl-2, Bcl-xL, MCL1and the mitochondrial pathway.Third, define the role of Ku70acetylation in TSA-induced apoptosis of colorectal cancer cells through detecting the effects of TSA on Ku70acetylation and Bax translocation.Experiments:Experiment OneAfter24h different concentrations (0,0.1,1,5μM) of TSA involving HCT116of p53(+/+) cells, HCT116p53(-/-) cells and mutant p53or HT29cells, measure cell cycle distribution and apoptosis by flow cytometry; assess what effects TSA has on the activity of p21by the promoter experiment; analyze what effects TSA has on the working of acetylated p53and p21protein by Western blotting.Experiment TwoAfter different concentrations (0,0.1,1,5μM) of TSA involving HCT116cells (with an expression of wild type p53gene) and HT29cells (with an expression of mutant p53), measure apoptosis by flow cytometry and Western blotting; observe mitochondrial membrane potential after mitotracker dye staining of living cells; detect the expressions of such molecules closely related with cell apoptosis as PARP, caspase-3, Bax, Bcl-2, Bcl-xL, MUL1by Western blotting; detect what effects TSA has on Bax activity with promoter expriment; detect the expression pattern of Bax and the shear situation of PARP by Bax of siRNA technology.Experiment threeAfter different concentrations (0,0.1,1,5μM) of TSA involving HCT116cells (with an expression of wild type p53gene) and HT29cells (with an expression of mutant p53), observe the variation of Ku70distributed at different time points (Oh,1h,4h,18h) by immunofluorescence staining; detect what effects TSA has on Ku70acetylation by IP-Western blotting; observe the variation of Bax expression amount in the cytoplasm and mitochondria by Western blotting, after6h and18h of TSA treatment respectively; detect Bax working and PARP shear situation by Ku70siRNA.Results:Experiment One1. G2/M cycle arrest emerged in HCT116P53(+/+) cells, HCT116p53(-/-) cells and HT29cells (with an expression of mutant p53), which were induced through TSA concentration dependence.2. Concentration-dependent TSA increased the expression of acetylated p53and p21protein in p53-positive HCT116cells, while promoting the activity of the p21promoter. In contrast, TSA had no effect both on the expression of acetylated p53and p21protein in p53-negative HCT116cells and on the activation of p21promoter.3. Concentration-dependent TSA was responsible for the induction of apoptosis of colorectal cancer cells, but the rate of p53-positive cells’apoptosis was significantly greater than that of the p53-negative cells.Experiment Two1. Concentration-dependent TSA was responsible for the induction of apoptosis of HCT116and HT29cells, and apoptosis of HCT116cells rated significantly higher than that of HT29cells.2. TSA, in a concentration dependent way, reduced mitochondrial membrane potential in the two groups of cells was found as well as a dose dependent activation of caspase-3.3. With the treatment of TSA in the two groups of cells, the expression of Bax protein was significantly increasing, while that of Bcl-2and Bcl-xL was significantly decreasing and relative to concentration-dependence as well. In contrast to this, no significant effects were showed upon the expression of MCL1protein in both groups of cells. 4. After p53-positive and p53-negative HCT116cells being treated by TSA, the Bax transcription activity of these cells increased in a concentration-dependent way.5. The expression amount of Bax and PARP shear fragment significantly increased after p53wild type and mutant cells being treated by TSA, while PARP shear fragment significantly decreased after using Bax siRNA to reduce the expression of Bax, which revealed the decreasing of cell apoptosis.Experiment three1. After HCT116cells and HT29cells being treated by TSA within1h, part of ku70trans-located from nucleus to cytoplasm. After4h it began to revert to the nucleus. Until18h, there remained a small amount of Ku70in the cytoplasma. The small amount of Ku70in HCT116cells cytoplasm became granular.2. After the TSA treatment of the two groups of cells, the expression of acetylated Ku70significantly increased.3. After6h TSA treatment of the two groups of cells, the expression of Bax in cytoplasm significantly decreased, while significantly increased in mitochondrial. After18h, this trend became much obvious; the expression of cytochrome C was opposite with Bax.4. With Ku70siRNA decreasing the expression of Ku70in the two groups of cells, and then with TSA treatment, the expression amount of Ku70and Bax and PARP shear fragment significantly decreased.Conclusions:1. TSA, in a concentration-dependent way, induced the arrest of G2/M phase in colon cancer cells. This effect of arrest was viewed as not being dependent on p53-p21pathway.2. TSA induced apoptosis in colon cancer cells by p53-dependent and non-dependent pathway. The normal function of p53was found to promote the TSA-induced apoptosis of colon cancer.3. The mitochondrial pathway was activated and apoptosis in colon cancer cells was induced through Bax-dependent pathway involved in TSA.4. Bax-dependent apoptosis was induced by TSA, mediated by acetylated Ku70.