Interaction Between CD133 And PI3K-p85 Promote The Multidrug Chemoresistance Of Gastric Cancer Cells

ObjectivesTo study the molecule mechanism of CD133 expression promoting the multidrug chemoresistance of gastric cancer cells and find out whether CD133 makes it by the interaction between its intracellular tyrosine residues and PI3K-p85.Methods1.By Lentiviral packaging technologies,we built a CD133 stable overexpression MKN45 gastric cancer cell line and a CD133 stable interference KATOIII gastric cancer cell line.Then the Lentiviral transfection efficiency was observed under a fluorescence microscope,and the CD133 overexpression and knockdown efficiency was estimated by Western blot and q PCR.CCK-8 was used to evaluate the sensitivity of cells in each group to 5-FU and DDP;Western blot and q PCR were used to detect the protein and m RNA levels of CD133,P-gp,Bcl-2,Bax.2.Western blot was used to detect the p-Akt and Akt protein level;ELISA was used to measure the enzymatic activity of PI3 K.Then KATOIII and MKN45 were treated with PI3K/Akt pathway inhibitor LY294002 and activator EGF.CCK-8 was used to evaluate the sensitivity of cells in each group to 5-FU and DDP;Western blot was used to detect the protein level of P-gp,Bcl-2,Bax,p-Akt and Akt;ELISA was used to measure the enzymatic activity of PI3 K.3.At last,we built five mutant CD133 plasmid by site-directed mutagenesis(located in 818,819,828,846,852 tyrosine),then CCK-8 was used to evaluate the sensitivity of cells in each group to 5-FU and DDP,ELISA and Western blot was used to detect the activity of PI3 K,and GST-pull down was used to identify their interaction with p85.Results1.With the fluorescence microscope,Western blot and qPCR assay,the CD133 overexpression and knockdown cell lines were successfully established.CD133 knockdown in KATO III cells significantly increase its inhibitory rate to 5-FU and DDP,while CD133 overexpression in MKN45 cells significantly reduce its inhibitory rate to 5-FU and DDP.The protein and m RNA levels of P-gp and Bcl-2 in CD133 knockdown group were significantly lower than the control group,while the Bax level was higher;on the contrary,CD133 overexpression significantly increased the expression of P-gp and Bcl-2 and reduced the Bax level,all statistically different.2.CD133 knockdown and overexpression could respectively reduce and increase the p-Akt protein levels and the enzymatic activity of PI3 K.Then,PI3K/Akt inhibitor LY294002 significantly increased the inhibitory rate of KATO III cells to 5-FU and DDP,reduced the protein levels of p-Akt,P-gp,Bcl-2 and PI3 K activity,and increased the Bax expression.While activator EGF remarkably reduced the inhibitory rate of MKN45 cells to 5-FU and DDP,increased the protein levels of p-Akt,P-gp,Bcl-2 and PI3 K activity,and reduced the Bax expression.3.At last,site-directed mutagenesis was used to built five CD133 mutant plasmid(located in 818,819,828,846 852 tyrosine)and the seven plasmids(vector,wide type,818,819,828,846,852)were separately transfected into MKN45 cells.The inhibitory rate of CD133 wild type,818,819,828 and 846 groups to5-FU and DDP were significantly higher than the control group,while the 853 group showed no significant difference.The p-Akt protein level and PI3 K enzymatic activity were also remarkably increased except the 852 group.Then GST-p85 fusion protein was built and the GSTpull down assay demonstrated that the binding between CD133 and p85 in the 852 group was significantly lower than the other groups.ConclusionsCD133 could regulate the PI3K/Akt signal pathway by the interaction between its tyrosine852 with PI3Kp85,and thus contribute to the multidrug resistance of gastric cancer cells.