Effects Of GPER Activation On The Prolifreation Of Breast Cancer Cells

Recently,a novel membrane-associated estrogen receptor: G protein-coupled estrogen receptor(GPER;formerly known as GPR30)was identified.GPER is different from the classical estrogen receptor ER?,ER?,the membrane localization characteristics gives it a new ability to mediate the rapid non-genomic effect of estrogen,thus aroused widespread concern.As a 7-fold transmembrane GPCRs,GPER indirectly participates in gene transcriptional regulation by mediating estrogen rapid non-genomic effects,affecting downstream function molecules in the corresponding target tissues such as affecting cell cycle progression or proliferation.There are more and more studies on the effect of G-1-activated GPER on cell proliferation,most of them focused on the field of tumors and reproductive systems,and the mechanisms of action reported in different cancer cell lines are also different,more cases and in-depth researches are needed to fully elucidate the mechanisms involved.Therefore,the effect of G-1 on the proliferation of breast cancer cells was studied by using E2 and TSA treatment as a control.MCF-7 and MDA-MB-231 were used as materials.The results were as follows:By using E2 and TSA treatment as a control,the growth curves of G-1 treated MCF-7 and MDA-MB-231 were calculated.The results showed that E2 treatment significantly promoted the growth of both breast cancer cell lines as expected;both G-1 and TSA treatment significantly inhibited the growth of two cell lines,more significant inhibition effect was detected by G-1 and TSA co-treatment groups.These results indicate that G-1 and TSA treatment has similar inhibition effects on the proliferation of breast cancer cells,and the effects of G-1 and TSA on the proliferation of breast cancer cells are synergistic.In order to understand the effect of the above treatment on the cell cycle progression of breast cancer cells,G-1 treated MCF-7 was detected by flow analysis with E2 treatment as a control.The results showed that the proportion of S phase and G2 / M phase cells in E2,G-1 treated group was significantly higher than that in control group.Especially in the treatment of G-1 for 24 h and 48 h,the proportion of G2 / M phase cells was as high as 38.7% and 64.5%,respectively.Together with the results of growth curve showed that E2 promotes the proliferation of breast cancer cells by increasing the proportion of S and G2 / M cells;G-1 activated GPER inhibits its proliferation by block the cells at G2 / M phase.In order to further study the molecular mechanism of G-1 treatment to inhibit the proliferation of breast cancer cells,the levels of cell cycle and apoptosis-related proteins,pERK,pAKT and acetylated histone H3 in MCF-7 and MDA-MB-231 were detected by Western blot and immunofluorescence respectively.The results showed that apoptosis-related proteins caspase6 and P53 were significantly increased,MCL-1 was significantly decreased;the relative expression of cell cycle-related proteins cyclin B1 and P21 was significantly increased,PCNA was significantly decreased;the level of pERK was increased and pAKT decreased.As a known histone deacetylase inhibitor,TSA can participate in the regulation of the cycle and apoptosis by improving the acetylation of histones in the promoter region of the relevant genes.We found that G-1 treatment can also improve the intracellular histone acetylation level,inhibit the proliferation and promote the apoptosis of breast cancer cells in a similar manner to TSA.The results of immunofluorescence also support the above conclusions.The effect of G-1-activated GPER on the migration of two breast cancer lines was detected by scratches.The results showed that G-1-activated GPER significantly inhibited the migration of two cell lines.In conclusion,G-1-activated GPER induces the apoptosis and cell cycle arrest,inhibits the proliferation and migration of breast cancer cells by inhibiting the phosphorylation of AKT and sustained activation of ERK to regulate the expression of apoptosis and cell cycle-related proteins.G-1 treatment can increase the acetylated histone H3 level,and inhibit the proliferation and promote the apoptosis of breast cancer cells in a similar manner to TSA.These results support that G-1 is likely to be a new type of anti-tumor drug as TSA,while GPER may be another potential therapeutic target for cancer.