| ObjectivetThe mechanism of Estrogen receptor(ER) alpha gene expression silencing is not known in ER alpha negative breast cancer.Present study showed that it's mostly not associated with the genetic changes such as gene mutatiom loss of heterozygosity (LOH) and so on,but associated with the CpG island methylation in the core promotor of ER alpha gene.The aims of this study are to detect the methylation status of 5'CpG island in the core promotor of ER alpha gene in ER alpha negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines and to investigate if hydralazine can re-express ER alpha gene as a demethylating agent,sensitiving these two ER alpha negative cell lines to tamoxifen(TAM), the third aim is to study the synergistic effect of hydralazine combined with TAM on the growth of ER alpha negative breast cancer cells ,thus offering a new way for the treatment of ER alpha negative breast cancer.Methods: The status of 5'CpG island methylation of ER alpha gene in ER alpha negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines was analyzed by using methylation specific polymerase chain reaction(MSP).The expression of ER alpha and maspin gene mRNA was inspected by using RT-PCR. ER alpha negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines were treated with hydralazine or TAM alone,or in combination in vitro.Cell proliferation was evaluated by MTT assay,distribution of cell cylcle and rate of apoptosis were determined by flow cytometry, and cell ultrastructure was observed by transmission electron microscope.Results: (l)The 5'CpG island was methylated in the core promotor of ER alpha gene in ER alpha negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines;(2)The expression of ER alpha mRNA up-regulated in these two cell lines after treated with hydralazine,so was maspin mRNA;(3) MTT assay showed the proliferation activity of these two cell lines obviously reduced in hydralazine group with the concentration of hydralazine more than 10礛,but no significant difference was found among those groupsin which the concentration of hydralazine is 10,20,40,80 n M. The inhibitory effect on proliferation of these two cell lines was raised as training time increased compared with control group.the inhibitory effect on proliferation of these two cell lines was enhanced when hydralazine combined with TAM compared with control group and hydralazine group,the cells were arrested in GO/G1 phase by hydralazine or by hydralazine combined with TAM,the apoptosis rate was 8.71% and 11.2% in MDA-MB-231 and MDA-MB-435 breast cancer cell lines treated with hydralazine respectively,while the rate of apoptosis is 48.8% and 53.1% when these two cell lines were treaied by hydralazine combined with TAM.Under the observation of light microscope and ele;trical microscope,cells degenerate seriously in hydralazine group,the amount of mitochondrion was significantly decreased,the mitochondria were swollen,some of them turned into vacuoles and myelin figures were found, in combination group,cell morphology became irregular,cell debris and abnormal cytoplasmic blebs were easily found and cell membrane was broken down,apoptotic bodies were not observed,which was seldom found in control group.Conclusionsr(l) The 5'CpG island methylation in :he core promoter of ER alpha gene is probably responsible for ER alpha expression silencing in ER alpha negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines;(2)Hydralazine can re-express ER alpha and maspin by demethylating and sensitive ER alpha negative human breast cancer cell lines to TAM, and could be widely used as a demethylating agent in the clinic.Hydralazine and TAM synergistically inhibit proliferation and induce apoptosis of ER alpha negative human breast cancer cell lines .The synergism of hydralazine and TAM may be of important significance in the treatment of breast cancer. |
