Effect Of G Protein-coupled Receptor Inhibitor On17β-estradiol-induced Proliferation And Apoptosis In Ishikawa And HEC-1A Cells

ObjectiveEndometrial cancer is one of the three major malignant tumors of the female reproductive system.Under Long-term high levels of estrogen stimulation and lack of progesterone antagonist, the body can increase a chance of developing endometrial cancer. The mechanism incluses classic theory "nuclear effects" and non-gene transcription effects. G protein-coupled estrogen receptor(GPER) is a new estrogen receptor, also called G protein-coupled receptor30(GPR30).Kanda etc found that estrogen acting on GPER can cause to cells proliferation by increasing expression of apoptosis proteins Bcl-2and cell cycle protein D, inhibiting oxidative stress-induced apoptosis. Vivacqua etc proved that estrogen combining GPER in endometrial cancer cells and thyroid carcinoma cell induces proliferation and reduces apoptosis and is directly related to cancer. Previous studies of our research group found that expression of GPER and AKT in endometrial cancer cells is consistent after17β-estrogen activated Ishikawa and HEC-1A cells, suggesting that GPER is very likely mediated the PI3K/Akt pathway and promoted the occurrence of endometrial cancer. Aboving studies have shown that GPER may induce the program of cell growth and multiplication, while the effect of cell proliferation and apoptosis by inhibiting GPER-induced signaling pathway hasn’t reported so far. This experiment observe the effect of G protein-coupled receptor inhibitor on17β-estradiol-induced proliferation and apoptosis in Ishikawa and HEC-1A cells and preliminary discuss possibility of blocking GPER-induced signal path curing endometrial cancer.Methods1. Culturing Ishkawa and HEC-1A cells outside the body. 2. Determining Ishkawa and HEC-1A cells growth by MTT method.3. Determining endometrial cancer cell cycle with Flow cytometry instrument.4. The Annexin V-FITC flow cytometry to detect cell apoptosis.Resultsexperiment results Determined by MTT method:E2has effect on promoting proliferation of Ishikawa cells showing the time dependence (F=212.132, P<0.001) and the concentration dependence (F=128.954, P<0.001), while the role in HEC-1A cell proliferation was not significant (F=0.955, P=0.393),and there was no statistical significance (F=1.978, P=0.137)between the differences among the concentration. Under the action of10-6mol/L E2, the effect of PTX on Ishikawa and HEC-1A cell proliferation inhibition has a time-dependence (F=47.428, P<0.001; F=123.395, P<0.001) and a concentration-dependence (F=495.437, P<0.001; F=424.561, P<0.001).The result of cell cycle analysis:1. Effects of E2to endometrial cancer cell cycle distribution:In Ishikawa cells,the effect of E2to G0～G1phase and S phase of cell cycle distribution reveals concentration dependence.As E2concentration increased, the percentage of G0～G1phase decreased (F=34.078, P=0.001)and increased in S phase (F=20.568, P=0.002), while there was no statistically significant difference (F=0.245, P=0.790) in G2to M period; In HEC-1A cell, the distribution of cell cycle among different concentration of E2were no statistically significant differences (P>0.05).2. Effects of PTX on endometrial cancer cell cycle distribution.Under the action of10-6mol/L E2and PTX of different concentration, in Ishikawa cells, cell cycle distribution at G0～G1phase (F=21.028, P=0.002), G2to M (F=35.533, P<0.001) revealed concentration dependence, while there were no significant differences in S phase (F=1.665, P=0.266); in HEC-1A cell, cell cycle distribution at G0～G1phase (F=6.809, P=0.029)and S phase (F=22.266, P=0.002) are both concentration dependent, whereas at G2to M phase there didn’t show significant alteration(F=0.142; P=0.870).3. Action of PTX promoting cell apoptosis for endometrial carcinoma Ishikawa and HEC-1A cell both are showing a concentration dependence (F=27.294, P<0.001; F=82.069, P<0.001), with the increase of concentration of PTX, apoptosis both of Ishikawa and HEC-1A cells is increased gradually.ConclusionThe difference on cell proferliration and cell cycle distribution between Ishikawa and HEC-IA cell may be due to two different ER levels in cells.For transcription effect of E2is ER dependent and non-transcription effect is ER dependent in Ishikawa cell while is not ER dependent in HEC-IA cell.Action of PTX promoting cell apoptosis for endometrial carcinoma Ishikawa and HEC-IA cell both are showing a concentration dependence (F=27.294, P<0.001; F=82.069, P<0.001), with the increase of concentration of PTX, apoptosis both of Ishikawa and HEC-1A cells is increased gradually.