Study On Correlation Between APC Gene Methylation And Proliferation, Apoptosis In Human Cervical Cancer Cells

Objective To detect the methylation status of 5'CpG island in the promotor of APC (adenomatous polyposis coli) gene in HeLa(HPV18 type), CaSki and SiHa(HPV16 type) cell lines of human cervical cancer, and to investigate the correlation of the silenced expression of APC gene and the methylation status of 5'CpG island in the promotor of APC gene, the third aim is to study if Hydralazine can re-express APC gene as a demethylating agent and to investigate the effect of Hydralazine on the growth of human cervical cancer cell lines, thus offering a new experimental proof for diagnosis and a demethylated treatment of cervical cancer.Methods The methylation status of 5'CpG island in the promotor of APC gene in HeLa, CaSki and SiHa cell lines of human cervical cancer were analyzed using methylated specific PCR (MSP) method. The three cervical cancer cell lines and a normal cell HECV were treated by different concentration of Hydralazine. MTT assay was used to observe the changes of proliferation activity of the cells after Hydralazine treatment. The methylation status of 5'CpG island in the promotor of APC gene in HeLa and CaSki cell lines were analyzed using MSP method. The expression of APC mRNA was studied by RT-PCR and FQ-PCR methods. FCM assay was used to observe the changes of cell cycle and apoptosis of the cells after Hydralazine treatment. The effect of Hydralazine on expression ofβ-catenin protein which correlates closely with APC protein was detected by SP method.Results (1)APC gene was methylated and did not express in HeLa cell, APC gene in CaSki cell was hemimethylated and expressed less, at the same time APC gene was not methylated and expressed normally in SiHa cell. (2) The three cervical cancer cell lines and a normal cell HECV were treated by 5, 10, 20, 40, 80, 160 and 320μmol/L Hydralazine, the growth of the three cervical cancer cell lines were all inhibited after having been treated by 20μmol/L Hydralazine for 72 h, the growth inhibitory ratio was increased gradually as the training time was lengthened and the training concentration of Hydralazine was increased. Growth inhibitory ratios of HeLa, CaSki and SiHa cell lines were (52.12±3.78) %, (44.31±2.59) % and (47.73±4.73) % respectively after having been treated by 40μmol/L Hydralazine for 72 h, on the contrary normal cell HECV's growth inhibitory ratio was only (27.18±0.79) %. (3) MSP assay showed that the promotor of APC gene in HeLa and CaSki cell lines which were treated by 40μmol/L Hydralazine for 72 h was demethylated. RT-PCR and FQ-PCR assays indicated that the expression of APC mRNA was up-regμlated in these two cell lines after treatment. (4) HeLa and CaSki cells were arrested in S phase and G2/M phase by Hydralazine, the apoptosis rate of these two cells treated with Hydralazine was increased significantly comparing with control groups(P<0.01).β-catenin protein can be expressed in cell membrane after the treatment with Hydralazine.Conclusion (1)The 5'CpG island in the promotor of APC gene is methylated or hemimethylated in HeLa(HPV18 type) and CaSki(HPV16 type) cell lines, APC methylation plays an important role in the carcinogenesis of cervical cells possibly. At the same time the 5'CpG island in the promotor of APC gene is unmethylated in SiHa cell whose HPV type is similar with CaSki cell line, that indicates there is no dependability between the HPV type of human cervical cancer cell and the methylation status of 5'CpG island in the promotor of APC gene. (2) APC mRNA can re-express or express increasingly in HeLa and CaSki cell lines treated with Hydralazine by demethylating. (3)The mechanism of inhibiting proliferation of cervical cancer cells by Hydralazine is not only through demethylating APC gene. (4)The main cause that Hydralazine inhibits proliferation of cervical cancer cells is arresting cells in S phase and G2/M phase and inducing apoptosis of these cells. (5) Wnt signaling pathway is prohibited by Hydralazine and the targeting genes of the pathway can not be transcribed, which may be one of the causes that Hydralazine inhibits proliferation of cervical cancer cells.