The Inhibitory Effect Of Combination Of Cyclopamine And Oxaliplatin On The Growth Of Gastric Cancer Cells

Objective:(1)To investigate the expesstion of Hedgehog signal transduction pathway in normal gastric epithelial cells and gastric cancer cells. (2)To explore the effect of cyclopamine,a Hedgehog specific target inhibitor, alone or in combination with oxaliplatin on the growth,cell cycles,appotosis and Hedgehog signalling transduction pathway in gastric cancer cell lines.Methods:(1)The expression of Hedgehog signal transduction pathway was assayed by Reverse-transcript PCR in gastric epithelial cell GES-1 and gastric cancer cell BGC-823,SGC-7901 and MKN-45.(2)The expression of GLI protein was detected by Immunnocytochemistry in MKN-45 cells.(3)CCK-8 kit was applied to explore the effect of cyclopamine and oxaliplatin to MKN-45 cell proliferation,to see whether combination of cyclopamine could enhance the antiproliferative effect of oxaliplatin.(4)Cell cycle distribution was assessed by flow cytometry to analysis the effect of cyclopamine alone or in combination with oxaliplatin,and an annexin V/propidium iodide assay was used to identify induction of apoptosis.(5)The expression of Hedgehog signal transduction pathway proteins,SHH and GLI influenced by cyclopamine and oxaliplatin was detected by western-blotting.Results:(1)Hedgehog signal transduction pathway was expressed in gastric epithelial cell GES-1 and gastric cancer cells BGC-823,SGC-7901 and MKN-45.SHH and GLI was upregulated in two gastric cancer cells but not GES-1 and SGC-7901.(2)GLI protein was expressed in cytoplasm and nuclear of MKN-45 cells.(3)The antiproliferation effect of different concentrations of cyclopamine and oxaliplatin to MKN-45 gastric cancer cell was dose and time-dependent. The combined effect of the two agents was more effective than singal agent(P<0.05).When 15uM cyclopamine was addicted to 10ug/ml oxaliplatin,the growth inhibition rate could be more effective than the single agent 15ug/ml oxaliplatin or 15uM cyclopamine.(4) Apoptosis of MKN-45 gastric cancer cell induced by cyclopamine and oxaliplatin was related to the concentration of the two agents(P<0.05,P< 0.05),and the percentage of apoptosis of MKN-45 gastric cancer cells treated by combination therapy significantly increased(P<0.05).As the concentration of cyclopamine increased,the percentage of the G0/G1 phase of MKN-45 gastric cancer cell increased;the percentage of the S phase also decreased. The effect of oxliplatin to MKN-45 gastric cancer cell cycle distribution was similar to that of cyclopamine.The effect of cyclopamine in combination with oxaliplatin was more significant than either of the two agents.(5) Western-blotting assay showed that cyclopamine can downregulate the expression of SHH and GLI,while oxaliplatin had no influence on the expression of SHH and GLI proteins in MKN-45 cell.Conclusions:(1)Normal gastric epithelial cell haboured the Hedgehog pathway,but lacking the transcript factor GLI,while SHH and GLI were both expressed in gastric cancer cells,which indicated the ligand dependent activation of SHH might be responsible for the mechanism of Hedgehog pathway actvation.It seemed that the growth of SGC-7901 was independent of Hedgehog pathway.(2)GLI was positive in MKN-45 cells which located in cytoplasm and nuclear.(3)Cyclopamine and oxaliplatin can inhibit the growth of MKN-45 gastric cancer cell line,the effect of the two agents was more effective when in combination.(4)Cyclopamine in combination with oxaliplatin can increase the percentage of G0/G1 phase,and decrease the synethsis phase of S.It indicated that cyclopamine could enhance the antiproliferative effect of oxaliplatin in gastric cancer cells.(5)Cyclopamine but not oxaliplatin can downregulate the expression of SHH and GLI protein in Hedgehog pathway.