Study On Growth Inhibitory Effects Of Icotinib In SNU-1Human Gastric Cancer Cells

Objective: Gastric cancer is harm to the health of human being seriously,being the fourth most common cancer and the second leading cause of cancerdeath worldwide.From a number of cases,patients with gastric cancer in Chinaaccount about47%of the total number of global incidence. Although surgeryremains the gold standard for the treatment of stomach cancer,combination ofpreoperative or postoperative adjuvant therapy. Despite chemotherapeuticregimens improved the survival of patients,the prognosis of patients withadvanced gastric cancer is still poor,with a median overall survival ofapproximately10to11months.The medical treatment of gastric cancer is stillvery difficult,current treatment remains unsatisfactory.The epidermal growthfactor receptor(EGFR) belongs to the super-family of receptor tyrosine kinaseand is overexpressed in many malignancies.Epidermal growth factor receptoris closely related to adhesion,invasion, metastasis,angiogenesis and inhibitionof apoptosis.Recently,the study showed:EGFR has low expression or noexpression in normal gastric mucosa,which has overexpression, being closelyrelated to the occurrence,development of gastric carcinoma.With the progressof molecular biology, targeted therapy especially epidermal growth factorreceptor-tyrosine kinase inhibitors (EGFR-TKIs) become an importantapproach in the treatment of gastric cancer.Icotinib is one kind of smallmolecular EGFR tyrosine kinase inhibitor,experiments had confirmed thatIcotinib could inhibite the proliferation of carcinoma of A431,BGC-823andTca8113.This study was designed to investigate the effect of Icotinib on theproliferation inhibitory of SNU-1human gastric cancer cells and to detect thedownstream signaling pathways,to explore the internal mechanism, then toprovide a new way for target therapy of gastric cancer. Methods:1Cell culture: Refrigerated SNU-1hunman gastric cancer cells were g-rown in100ml culture flasks with RPMI-1640including10%fetal bovineserum (FBS),100U/ml streptomycin and penicillin, at37℃,5%CO2humidiedincubator,and the cells were observed by inverted microscope.2MTS method was used to measure cell proliferation after SNU-1hu-man gastric cancer cells were treated with Icotinib, and got the cell inhibitoryrate curve of each group.3Flow cytometry was used to investigate the ratio of cell apoptosis andcell cycle after SNU-1hunman gastric cancer cells were treated with Icotinib.SNU-1hunman gastric cancer cells were suspended in100ml culture flasks ata density of2.5×10~5/ml.After24h,each group was added with various concen-trations（0,5,10,20,40,80μmol/L）of Icotinib. In control group, the sameamount of culture medium was added. After48h, the cells were harvested andfixed by cold ethanol (70%).The cells were stained with propidium iodide(PI)later, then were measured by flow cytometry (Beckman Coulter Company,America).4Western blot was used to measure the protein in the EGFR downstre-m signaling pathway including p-Akt,CyclinD1.5Statistical analysis was performed using SPSS13.0software package,and the results were described as x±s. Comparison of many groups wasperformed by the One-Way ANOVA, and multiple comparisons betweengroups used SNK. P<0.05was considered significant.Results:1MTS method was used to evaluate cell proliferative activity:The prol-iferation of SNU-1hunman gastric cancer cells could be inhibited by Icotinibat different concentration (5,10,20,40,80μmol/L) in a dose-dependent manner,and the inhibition rate of various concentrations of Icotinib showed significantdifference(P<0.05).The inhibitory rate of cell proliferation at the sameconceration of Icotinib has statistical in different time(24h,48h,72h).In a word, Icotinib could inhibit the SNU-1hunman gastric cancer cells growth by dose-and time-dependment manners.2Effects of Icotinib on cell apoptosis and distribution of cell cycle wer-e observed by flow cytometry: After SNU-1hunman gastric cancer cells weretreated with various concentrations of Icotinib（0,5,10,20,40,80μmol/L）for48h, FCM showed that the ratio of cell apoptosis and cell cycle were changed.The ratio of cell apoptosis detected by FCM were（1.900±0.361）%，（8.467±0.961）%，（14.267±3.702）%，（20.813±3.260）%，（26.533±3.102）%，（32.200±4.900）%, so the ratio of cell apoptosis was increased with theconcentration. So Icotinib could promote the apoptosis of SNU-1cells in adose-dependent way. The ratio of cell in G0/G1phase were（11.667±2.173）%，（20.200±2.955）%，（28.467±5.380）%，（37.567±4.219）%，（46.000±4.321）%，（56.367±6.526）%, the ratio of cell in S phase were（57.467±7.358）%，（48.900±4.590）%，（40.267±3.201）%，（31.700±2.052）%，（23.200±5.112）%，（14.567±2.581）%, the ratio of cell in G2/M phase were（23.067±3.553）%，（21.767±2.669）%，（23.567±3.478）%（，14.167±2.793）%（，17.900±1.609）%，（10.833±1.550）%.Compared with the control group, cell cycle phaseanalysis revealed that the proportion of SNU-1cells in G0/G1phase increased,while the proportion of SNU-1cells in S phase decreased,and showedsignificant differences (P<0.05).There was not statistically significantdifference in G2/M phase(P>0.05).3Compared with the control group,Icotinib signifieantly down-regulat-ed the expression of p-Akt,CyclinD1.Conclusion:1Icotinib can inhibit the proliferation of SNU-1hunman gastric cancercells in time-and dose-dependment manners.2Icotinib can change distribution of SNU-1cell cycle.It has evident ef-fect that indicated G0/G1phase arrest and the percent of S phase cell weredecreased.It can induce apoptosis of SNU-1cell.3Icotinib inhibits the EGFR/PI3K/Akt signal transduction pathway. Ic-otinib may be can effectively downregulated downstream protein expression i- nhibit proliferation of SNU-1cell and increase apoptosis.4The activation of EGFR/PI3K/Akt signaling transduction pathway m-ay be involved in the etiopathogenesis of gastric cancer.Icotinib might becomea new target therapy drug for gastric carcinoma.