The Establishment Of Common Colorectal Cancer Cell Lines With Mitochondrial Dna-depleted And The Obserasion Of Their Biological Behaviors

The incidence of colorectal cancer(CRC) ranked fourth in tumors, the mortality rate ranked second, in developed countries. The incidence and mortality rates of CRC are increasing 3%-5% annually, and the patient age obviously getting younger and younger, in our country. The early symptoms of colorectal cancer are not typical, the diagnosis rate is very low, especially the recurrence and metastasis of colorectal cancer has become the main cause of the prognosis of patients is not ideal or even death. Therefore, exploring the mechanism of the development, invasion and metastasis of colorectal cancer, looking for more effective detection of indicators for early diagnosis, as well as to explore new and effective methods of treatment have a very important significance. ObjectiveThe change of mitochondrial DNA(mt DNA) content was significantly associated the prognosis of colorectal cancer cell. However, its effectson cell behavior and the mechanisms are unclear due to lack of appropriate cellular models. Therefore, we establishedcolorectal cancer rho 0(ρ0) cell lines(mitochondrial DNA-depleted) and, preliminary compared the the biological behaviors of ρ0 cell lines with their parental cells and investigated the potential mechanisms, which could provide a platform for research to find new treatments in colorectal cancer. Materials and Methods(1) Colorectal cancer cell lines SW480, HCT 116, HCT-8 were cultured in 1640 medium with 10 % FBS, pyruvate, uridine and different concentrations of ethidium bromide. The changes of mt DNA content were detected by real-time quantitative PCR.(2) Cellular morphologies of CRC ρ0 cells were examined under Optical microscope.(3) Numbers of mitochodira were observed under laser scanning confocal microscope after ρ0 and their parental cells staining with mito-tracker.(4) The growth curves of ρ0 cells and their parental cells were drew by cell counting method.(5) Using flow cytometry to detect cell cycle of the three pairs of cells above.(6) The invasive ability of these three pairs cells were detected by transwell invasion assay to.(7) The ROS levels of these three pairs cells were assessed by flow cytometry. Results(1) SW480ρ0 cell line can be obtained by treating SW480 with 50ng/m L EB for 25 passages and then with 100 ng/m L EB for 14 passages;HCT 116 ρ0 cell line can be gotten by treating HCT 116 cells with 100 ng/m L EB for 32 passages,200 ng/m L EB for 10 passages and 500 ng/m L EB for 3 passages in orderly; HCT-8ρ0 cells can be harvested by treating HCT-8 cells with 200 ng/m L EB for 24 passages.(2) ρ0 cells become larger and longer compared to their parental cells under Optical microscope.(3) The mitochondria mass in ρ0 cells increases under laser scanning confocal microscope after staining with mito-tracker.(4) SW480ρ0、HCT 116ρ0、HCT-8ρ0 cells grew ignificantly slower, furthermore, the growth curves were more gentle than their parental cells.(5) The proportions of cells in different stages of the cell cycle were similar in ρ0 cells and their parental cells of the three cell lines, the differences were not statistically significant(P > 0.05).(6) Transwell invasion assay results show that the numbers of invaded cells of three ρ0 cells were less than their parent cells, the differences were statistically significant(P <0.01).(7) Flow cytometric analysis revealed that the production of ROS in the threeρ0 cells were significantly reduced compared to their parental cells(P <0.01). Conclusionsρ0 cells can be successfully obtained by treated with EB. But there are still differences between different colorectal cancer cell lines. The ρ0 cells can steadily grow and divide in medium supplemented with sufficient uridine, pyruvate and 10% FBS. Furthermore reducing mt DNA copy numbers can significantly influence the shape of colorectal cancer cells. Loss of a functional mitochondrial electron transport chain in CRC cells inhibits cell-cycle progression, proliferation and invasion significantly decreased, but did not affect the percentage of all stages of the cell cycle, which may be caused by the decreased concentration of reactive oxygen species,...