| BackgroundColon cancer is one of the high incidence of human malignant tumor, its incidence has been on the rise. According to statistics, the incidence of colon cancer has risen to the third of the world, the mortality rate in malignant tumor is the second leading cause of death. In China, the mortality of colon cancer has been located in the fifth malignant tumor mortality, seriously harmful to human health. Currently, the main method in the treatment of colon cancer is: surgical treatment of main and auxiliary 5-fluorouracil based chemotherapy treatments. However, at present, colon cancer patients are mostly found in middle-late, in our country, and simple surgery cannot effectively remove all the tumor cells in the body; But if the surgery, on the basis of secondary to systemic chemotherapy, is not only beneficial to remove residual tumor cells, but can prevent tumor recurrence and metastasis, prolong patient survival. However, clinical datas showed that for the patients after surgical removal of the tumor with high recurrence rate, and higher resistance to chemotherapy, especially 5-fluorouracil, many patients because of drug resistance and eventually lead to death. However, in the present various kinds of chemotherapy regimens in the treatmen of tcolon cancer, 5-fluorouracil is indispensable key drug. Therefore, to reduce the resistance of 5-fluorouracil based chemotherapy drug, and to improve the therapeutic effect of middle-late colon cancer has important significance, is an important part of the colon cancer currently.Src is the first to be found in human history of proto-oncogenes, its coding the Src protein is a kind of close to the cell membrane of receptor tyrosine protein kinase(nrPTKs) a member of the family, has close relationship with the occurrence of tumor development. It is reported that the src showed high expression in 80% of colon cancer. In recent years, the src kinase inhibitors used in tumor treatment become the study hotspot. For it is a kind of the second generation of dasatinib src kinase inhibitor, in vitro experiments and animal experimental study and oxaliplatin into union with synergy for colon cancer cells. There are a number of 1st and 2nd stage with paclitaxel for clinical trials testing of dasatinib, capecitabine or oxaliplatin into for breast cancer, prostate cancer and colon cancer and other solid tumors therapeutic effect. Araujo JC a new three phase of clinical trial results show that the combination of dasatinib and taxol does not improve the survival of patients with metastatic castration therapy against prostate cancer. Therefore, it is now accepted that: SRC kinase inhibitors with traditional chemotherapy drug combination for the treatment of tumors. Carrato, however, do another similar phase 3 trials have found the opposite: Receptor tyrosine kinase inhibitor chougny for his combination FOLFIRI scheme does not improve survival of patients with metastatic colon cancer, and combinations lead to more serious security problems. De Bouards and others for chougny reports, also can inhibit the Src kinase activity at a cellular level. Therefore, in the process of colon cancer patients and postoperative chemotherapy src kinase inhibitors and the curative effect of traditional chemotherapy drug combination is unclear, needs further researches.Decreasing apoptosis is the important cause leading to the chemotherapy drug resistance. In this paper, based on this question, through the cell experiment, MTS, western bloting methods for detection dasatinib and 5-fluorouracil for src protein and activity in colon cancer cells; Flow cytometry instrument, Western blot methods such as study of 5-fluorouracil for comparison, combination dasatinib with 5-fluorouracil use impact on colon cancer celsl apoptosis; To further research on src kinase inhibitors and the curative effect of traditional chemotherapy drug combination and its effects. ? To detect the influence of the expression of the src in the group of the Dasatinib combines with 5-fluorouracil in the colon cancer cells Methods1.To detect the influence of the activity of the colon cancer cells with dasatinib and fluorouracil by MTS.2. Western blotting test the effect of 5-fluorouracil on src activity.3. Western blotting for detection of the influence of dasatinib to the activity of src in colon cancer cells.4. Western blotting for detection of dasatinib and 5-fluorouracil combined influence on Src activity. Results1. Dasatinib and 5-fluorouracil can suppress the activity of the colon cancer cells.MTS results show: SW480 and HT29 cells treated with 20 nm dasatinib for 48 hours, enzyme standard to test results, the celsl survival rate is 80%; SW480 and HT29 cells treated with 5-fluorouracil 50 um for 48 hours, the superiorities of the testing results of the standard instrument, the cells survival rate was 50%(SW480 and HT29 cell with 5-fluorouracil treatment conditions of IC50 of 50 um 48 hours).2. 5-fluorouracil regulation of colon cancer cells the activity of srcWestern blotting results showed that using the 50 um concentration of 5-fluorouracil in SW480 and HT29 cells at different times, on behalf of the src kinase activity of the expression of p-src increased significantly, and presents the time gradient according to the patient.3. Dasatinib suppress the activity of src in the colon cancer cellsWestern blotting results showed that different concentrations of dasatinib for 24 hours, deal with SW480 and HT29 cells, on behalf of the src kinase activity of the expression of p-src significantly reduced, and present a concentration gradient in patience; With 20 nm concentration of dasatinib processing SW480 and HT29 cells at different times, on behalf of the src kinase activity of the expression of p-src increased significantly, and presents the time gradient according to the patient.4. Dasatinib combines with 5-fluorouracil suppress the activity of src in the colon cancer cells.For Western blotting showed: Dasatinib combines with 5-fluorouracil reduce the expression of p-src in colon cancer cells.? Dasatinb combines with 5-fluorouracil induce apoptosis of the colon cancer cells. Methods1. Flow cytometry instrument to detect the influence of dasatinib to the early colon cancer cell apoptosis induced by 5- fluorouracil.2. Western blotting test the influence of dasatinib to 5-fluorouracil induced apoptosis of colon cancer cells apoptosis signal PARP, caspase 3, cleaved caspase 3, caspase- 7, cleaved the expression of caspase- 7. Results1. Dasatinb combines with 5-fluorouracil can induce apoptosis of the colon cancer cells.Flow cytometry instrument results show that the use of dasatinib for 20 nm, with 5- fluorouracil 50 um combination processing HT29 colon cancer cells, HT29 cell early apoptosis rate was 7.8%;Use 5- fluorouracil, HT29 cell early apoptosis rate was 14.3%;Compared the two groups: combination group of apoptosis rate than but with 5- fluorouracil group significantly decreased(p < 0.01).2. Dasatinib combines with 5-fluorouracil can reduce the expression of apoptosis signal of the colon cancer cells.Western blotting showed: used dasatinib for 20 nm and 5-fluorouracil 50 um combined with colon cancer cells SW480 and HT29 cell after 48 hours, the cell apoptosis signal PARP, cleaved caspase 3, cleaved the expression of caspase- 7 combination than 5- fluorouracil is decreased obviously, the results hint of sand for with 5- fluorouracil reduce colon cancer cell apoptosis rate. ? The expression of src in recurrence or metastatic and primary of the colon cancer tissues MethodsImmunohistochemical Envision method to detect the SRC in the colon cancer of the organization. ResultsThe expression of src in recurrence of colon cancer tissues is lower than primary colon cancer tissues.Immunohistochemical results show: the immunohistochemical study found that the same overall than the Src protein expression of patients with primary cancer recurrence or metastatic carcinoma. Suggesting that possible recurrence of cancers which lower expression of src is not sensitive to chemotherapy. The existence of the Src may also play a positive role in the process of the chemotherapy. Conclusion1. 5-fluorouracil combines with Dasatinib can reduce the expression of src protein in colon cancer cells;2. 5-fluorouracil combines with Dasatinib can reduce apoptosis of colon cancer cells;3. The src is a proto-oncogene, its src protein plays a positive role in the process of colon cancer chemotherapy. |
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