| Objective:1) To explore the effect of metformin to HER2 positive gastric cancer cell proliferation. colony forming, cell cycle arrest, apoptosis.2) To explore possible mechanisms that metformin inhibited HER2 positive gastric cancer cell proliferation.3) To explore the coordination effect of metformin and targeted drug trastuzumab.Methods:1) Western blot. qRT-PCR detect the expression levels of HER2 positive in common gastric cancer cell lines.2) Logarithmic growth phase HER2 positive gastric cancer cell NCI-N87 and OE19 were incubated in culture plates, and were divided into the experimental group [HER2 positive gastric cancer cell NCI-N87 and OE19 were intervened by metformin at different concentrations (10.20.30.40.50 mmol/L) for 24,48.72 hours] and the control group (HER2 positive gastric cancer cell NCI-N87 and OE19 were cultured in the DMEM medium). The effect of metformin to the proliferation of HER2 positive gastric cancer cell NCI-N87 and OE19 was detected by CCK-8. By crystal violet staining method to detect the influence of metformin to colony forming ability in the HER2 positive gastric cancer cell NC1-N87 and OE19. Flow cytometry(FCM) was used to detect the cell cycle arrest and apoptosis after the HER2 positive gastric cancer cell NCI-N87 and OE19 were treated with metformin. The expression levels of HER2 positive and PI3K/Akt/mTOR signaling pathways after dealing with metformin of the HER2 positive gastric cancer cell NCI-N87 and OE19 were detected by Western blot.3) The effect of trastuzumab combined with metformin to the proliferation of HER2 positive gastric cancer cell NCI-N87 and OE19 was detected bv CCK-8.Results:(1) The effect of metformin on HER2-positive gastric cancer cells.1) Western blot. qRT-PCR detection of HER2 protein expression levels in seven kinds of common human gastric cancer results show that HER2 protein is highly expressed in BNCI-N87 and OE19. low expressed in MGC-803. MKN-45 and lowest in AGS. BGC-823. SGC-7901.2) The relative proliferation rates of HER2 positive gastric cancer cell NCI-N87 were 1.391±0.070,1.289±0.044,1.199±0.017,0.978±0.052,0.952±0.041 after intervened by metformin at different concentrations (10,20,30,40,50 mmol/L) for 24 hours,1.779±0.069, 1.593±0.236,1.385±0.158,1.142±0.082,0.789±0.055 after intervened by metformin at the same concentration gradient for 48 hours, and 2.511±0.118,1.882±0.090,1.528±0.067, 0.898±0.041,0.499±0.019 after intervened by metformin at the same concentration gradient for 72 hours. There were significant differences in the relative proliferation rates in compared with the blank group(p<0.05). The relative proliferation rates of HER2 positive human gastric cancer cell line OE19 were 1.824±0.027,1.738±0.010,1.443±0.025,1.237±0.041, 0.932±0.011 after intervened by metformin at different concentrations (10,20,30,40,50 mmol/L) for 24 hours,2.359±0.041,2.010±0.033,1.462±0.029,1.103±0.018,0.723± 0.021 after intervened by metformin at the same concentration gradient for 48 hours, and 3.704±0.083,3.033±0.037,1.658±0.027,0.860±0.032,0.316±0.009 after intervened by metformin at the same concentration gradient for 72 hours.10,20 mmol/L metformin treatment group showed no significant difference in the 24 hours time point(P> 0.05). The rest of the time points of each drug group decreased gastric cancer cell proliferation rate was statistically significan(p<0.05).3) The crystal violet staining method was used to detect different concentrations of metformin (10,20,30,40,50 mmol/L) intervention NCI-N87 clone forming ability.10,20,30 group mmol/L clone formation rate were 72.500%+3.546%,25%+7.071%,6.500%+ 2.121% which were significant differences in the relative proliferation rates in compared with the blank group.There were no obvious colony formation in another two groups, which concentrations were 40 and 50 mmol/L. The crystal violet staining method was used to detect the same concentration gradient of metformin intervention OE19 clone forming ability.10,20 group mmol/L clone formation rate were 57.500%±10.607%、17.500%±3.536% which were significant differences in the relative proliferation rates in compared with the blank group.There were no obvious colony formation in another there groups. which concentrations were 30.40 and 50 mmol/L.4) The apoptosis rates of NCI-N87 were 5.450%±0.574%,9.250%±1.630%,9.850% ±0.759%.17.900%±4.028%,20.400%±1.512% after intervened by metformin at different concentrations (10,20,30,40,50 mmol/L) for 48 hours. There were significant differences in the apoptosis rates in compared with the blank group(p<0.05). The apoptosis rates of OE19 were 3.800%±0.572%.7.450% ± 1.303%.8.350%±1.439%.17.475%±5.773%.22.825% ±4.971% after intervened by metformin at the same concentration gradient for 48 hours. The difference was statistically significant between the 20.30,40.50 mmol/L group and the blank group in the apoptosis rates(p<0.05).5) The Gl% of NC1-N87 were 67.833%±0.702%,72.667%±1.060%,73.633%± 0.850%,77.267%±0.306%,81.867%±0.416% after intervened by metformin at different concentrations (10.20.30,40,50 mmol/L) for 48 hours. The difference was statistically significant between the 20.30,40,50 mmol/L group and the blank group in the apoptosis rates(p<0.05). The cell cycle arrest of OE19 were 66.800%±0.458%、68.800%±0.400%、 70.067%±1.102%、76.567%±0.709%、79.600%±0.900% after intervened by metformin at the same concentration gradient for 48 hours. The difference was statistically significant between the 20.30.40.50 mmol/L group and the blank group in the apoptosis rates(p<0.05).(2) The mechanism of metformin inhibition HER2-positive gastric cancer cell proliferationon.Western blot show that metformin can inhibit expression of HER2 protein, down regulate the expression of its downstream PI3K. reduce the phosphorylation of Akt and mTOR. thereby inhibiting the activation of downstream 4E-BP1 and p70S6K.(3) The coordination effect of metformin and targeted drug trastuzumab.1) CCK-8 detection of a simple target to drug trastuzumab (100ug/ml) at different time points on HER2 positive gastric cancer cell NCI-N87 and OE19 proliferation were no inhibitory effect.2) The relative proliferation rates of NCI-N87 were 1.314 ± 0.047.1.156 ± 0.069, 1.052 ± 0.011.1.002±0.020.0.900±0.012 after intervened by metformin at different concentrations (10,20.30.40.50 mmol/L) with trastuzumab for 24 hours.1.728±0.115.1.427 ± 0.036.1.243 ± 0.033.1.076 ± 0.018.0.761 ± 0.022 after intervened by metformin at the same concentration gradient with trastuzumab for 48 hours, and 2.457±0.110.2.176±0.074, 1.367 ± 0.070.0.974±0.034.0.602±0.033 after intervened by metformin at the same concentration gradient with trastuzumab for 72 hours. Compared with the same concentration and single drug of metformin group, the difference was statistically significant (p<0.05). The proliferation rates of OE19 were 1.596±0.050.1.740±0.086.1.307±0.023.1.086±0.035. 0.866 ± 0.025 after intervened by metformin at different concentrations (10,20,30,40,50 mmol/L) with trastuzumab for 24 hours,1.999±0.047,1.872±0.044,1.435±0.085,1.073 ±0.019,0.527±0.055 after intervened by metformin at the same concentration gradient with trastuzumab for 48 hours, and 2.748±0.120,2.260±0.027,1.497±0.058,0.796±0.025, 0.291±0.038 after intervened by metformin at the same concentration gradient with trastuzumab for 72 hours. Compared with The blank control group, single drug trastuzumab group and single drug metformin group with same concentration, the difference was statistically significant (p<0.05).Conclusion:1) Metformin inhibited HER2 positive gastric cancer cell proliferation and colony forming ability, promote cell apoptosis, cell cycle arrest cell growth arrest in the G1 phase; 2) Metformin can downregulate HER2 protein expression level, the inhibition of the pathway of PI3K/Akt/mT0R; 3) Trastuzumab alone had no growth inhibitory effect on HER2 positive gastric cancer cell NCI-N87 and OE19 in vitro, but could obviously inhibit the grouth of HER2 positive gastric cancer cell NCI-N87 and OE19 in combination with metformin. Trastuzumab in combination with metformin had synergistic effect. |
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