| Objectives：1To investigate the effects of Herceptin in combination with Rapamycinon proliferation,apoptosis and cell cycle on human gastric cancer cells NCI-N87in vitro,and to evaluate that whether Herceptin in combination with Rapa-mycin had synergistic effect.2To study the effects of Herceptin in combination with Rapamycin onthe expression of the Her2protein,P-Akt,P-mTOR and P-4E-BP1on humangastric cancer cells NCI-N87,and to explore the preliminary mechanism of thedown regulation of related protein and inducing apoptosis.Methodes:1Culture human gastric cancer cells NCI-N87routinely.2Detect the expression of Her2protein on human gastric cancer cellsNCI-N87and SGC-7901by flow cytometry.3MTT assay was used to determine the influence of the two drugs onproliferation of human gastric cells NCI-N87.4Flow cytometry was used to investigate the influence of Herceptin andRapamycin alone or in combination to cell cycle,apoptosis index as well as tothe expression of Her2protein in human gastric cells NCI-N87.5Detect the influence of Herceptin and Rapamycin alone or in combinat-ion to the expression of P-Akt,P-mTOR,P-4E-BP1in human gastric cells NCI-N87by Western blot method.Results:1Human gastric cancer cells NCI-N87showed over-expreesion of Her2protein,but SGC-7901showed opposited.2Herceptin alone could not inhibit the proliferation of Human gastric cancer cells NCI-N87.After human gastric cancer cells NCI-N87were treatedby different concentrations of Rapamycin (0.1nmol/l,1nmol/l,10nmol/l,50nmol/l,100n mol/l),the inhibitation ratios of24hours,48hours and72hourswere7.77±1.12%,14.83±1.38%,25.03±1.79%,24.83±2.94%,24.03±2.38%;15.90±1.66%,25.73±1.67%,32.7±1.75%,33.06±1.36%,32.97±2.15%;22.83±1.33%,35.53±2.65%,45.23±2.01%,44.03±3.35%,44.27±2.31%. From the testdata, we could see that different concentrations of Rapamycin can inhibit pro-liferation of NCI-N87cells.In certain concentration range(0-10nmol/l),withincreasing concentrations of Rapamycin and extending time of treatment,theinhibitory effects were enhanced, and there was a significant different betweenthe Rapamycin group and the control group(P<0.01). It showed that inhibitati-on was dose and time-dependent in certain concentration range.When the con-centrations of Rapamycin were10nmol/l,50nmol/l and100nmol/l,the inhibit-ation had no statistical difference (P>0.05).The inhibitation ratio of Herceptinin combinantion with Rapamycin was57.78±2.75%,and compared with thealone groups, the difference had statistical significance (P<0.01). In accordan-ce with the Jin’s formula,q was equal to1.72which was greater than1.15,andit showed that combination of the two drugs had a synergistic effect on NCI-N87cells．3After human gastric cancer cells NCI-N87were treated by Herceptin(100ug/ml) and Rapamycin(10nmol/l) alone or in combination for48hours,the percents of G1phase were49.87±1.14%,59.60±1.05%,72.83±0.87%; Sphase were34.27±0.67%,23.63±1.11%,11.6±1.25%;and G2phase were15.86±1.29%,16.77±1.06%,15.53±0.42%.The percents of G1, and G2phase of thecontrol group were43.03±0.68%,41.60±0.82%,15.37±0.42%.From the testdata,we could know that G1phase rate gradually increased,S phase rate gradu-ally decreased,cell cycle was arrested at G1phase in human gastric cancercells NCI-N87treated by Herceptin(100ug/ml) and Rapamycin (10nmol/l)alone or in combination for48hours,and the difference had statistical siginifi-cance compared the control group(P<0.01).G2phase rate did not change inNCI-N87cells,and there had no statistical difference compared with the control group (P>0.05). Compared with the alone groups, Herceptin in combi-nation with Rapamycin could distinctly increase G1phase rate and reduce Sphase rate, and the difference had statistical significance (P<0.01).4Apoptosis rates were3.38±0.45%,7.71±1.44,33.71±2.05%after Herce-ptin and Rapamycin alone or in combination acting on the NCI-N87cells for48hours. Apoptosis rate of the control group was0.73±0.06%.Herceptin alonecould not induce apoptosis of NCI-N87cells(P>0.05). Compared with thecontrol group.Rapamycin alone or in combination with Herceptin couldinduce apoptosis of NCI-N87cells,and the difference had statistical significan-ce(P<0.01).Compared with the alone groups,Herceptin in combination withRapamycin could obviously induce apoptosis of NCI-N87cells,and the differ-ence had statistical significance (P<0.01).5The expression of Her2protein were0.632±0.004,0.870±0.021,0.506±0.015by flow cytometry and the expression of P-Akt,P-mTOR and P-4E-BP1were0.430±0.045,0.623±0.031,0.110±0.046;0.570±0.053,0.320±0.046,0.120±0.026;0.387±0.086,0.370±0.662,0.117±0.050; the control group were0.943±0.042,0.887±0.025,0.683±0.031by western blot after Herceptin and Rapam-ycin alone or in combination acting on the NCI-N87cells for48hours.Theyshowed that Herceptin and Rapamycin alone or in combination acting on theNCI-N87cells for48hours could decrease the expression of Her2protein,P-Akt,P-mTOR and P-4E-BP1.Compared with the control group,and the diffe-rence had statistical siginificance (P<0.01).Compared with the alone groups,Herceptin in combination with Rapamycin could down-Regulate the express-ion of Her2protein, P-Akt,P-mTOR,P-4E-BP1obviously,and they had notablestatistical difference (P<0.01).Conclusions:1Herceptin alone had no growth inhibitory effect on human gastriccancer cells NCI-N87in vitro,but could obviously inhibit the grouth of NCI-N87cells, arrest cell cycle at G1phase and induce apoptosis in combinationwith Rapamycin.Herceptin in combination with Rapamycin had synergisticeffect. 2Herceptin in combination with Rapamycin could obviously down-regulate the expression of Her-2protein,P-Akt,P-mTOR,P-4E-BP1.3Apoptosis induced by Herceptin in combination with Rapamycin maybe related with arresting cell cycle at G1phase and decreasing the expressionof Her2protein,P-Akt,P-mTOR and P-4E-BP1in human gastric cancer cellsNCI-N87. |
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