Destruction Of Membrane Microdomains(Lipid Rafts) Reverses Drug Resistance In Colorectal Cancer And Its Possible Molecular Mechanisms

Objective:I.By knocking down the structural proteins Caveolin-1,Flotillin-1,and Flotillin-2 on the lipid raft in connective and rectal cancer resected cells(HCT-15/ADM),the changes in drug resistance of tumor cells and the relation between lipid rafts and tumor resistance were determined,to evaluate the significance of changing the structure of lipid rafts as a potential target for reversing tumor resistance and provide new method for reversing tumor resistance.2.Analyzing the changes of key molecules P38/p-P38,JNK/p-JNK,Akt/p-Akt,P-catenin and Notch1 of the signaling pathways MAPK,PI3K/Akt,Wnt and Notch in HCT-15/ADM cells that have been knocked down by Caveolin-1,Flotillin-1 and Flotillin-2 proteins,separately.Signal transduction pathways related to tumor drug resistance reversal were determined to evaluate its significance in tumor cell drug resistance reversal,and preliminary explore the signal transduction mechanism of tumor drug resistance reversion.Methods:1.The HCT-15/ADM cell line was used to induce its recovery of drug resistance by Adriamycin(ADM).MTS method was used to detect cell viability under different ADM concentrations,and the half inhibitory concentration(IC50)and drug resistance index(RI)of ADM were calculated.2.Design three pairs of interference primers and one pair of negative control shRNA primer(shRNA1,2 and 3).shRNA-Cav1 interference plasmid was constructed and co-transfected 293ft cells with helper plasmid to infect HCT-15/ADM cells.Puromycin was then used to screen stable transfected cell lines.qRT-PCR and Western Blotting were performed to detect the effect of knock down,and the cell lines with the most obvious knockdown effect were selected.3.MTS method was used to determine the cell viability of HCT-15/ADM cell line after Caveolin-1 was knocked out to calculate its proliferation inhibition rate and reversal multiple,and then judge the change of HCT-15/ADM drug resistance.4.Immunodetection of P38/p-P38,JNK/p-JNK,Akt/p-Akt and β-catenin were conducted by Western-blotting in cells with knocking-down of Caveolin-1,Flotillin-1 and Flotillin-2(Flotillin-1 and Flotillin-2 stable transfected cell lines have been previously constructed).Results:1.HCT-15/ADM cell line recovers resistance after induction by ADM with the drug resistance index at 11.39 and the drug resistance index RI>5,which is consistent with moderate drug resistance.2.The shRNA-Cavl-3 plasmid was constructed and used for gene knock down.The qRT-PCR and Western Blotting results indicated that,compared to control,the three plasmids for knocking down Caveolin-1 were all effective,and shRNA-2 performed best.3.After knocking down of Caveolin-1,the IC50 of HCT-15/ADM cells decreased from 2.884 ± 0.038 μg/ml to 1.341 ± 0.049 μg/ml,with the reversal factor at 2.15,indicating that the sensitivity of HCT-15/ADM to doxorubicin increased by the deficient in Caveolin-1.4.The knockdown of Caveolin-1 led to decrease of p-JNK and Notch1 expression and increased p-Akt expression.The difference was statistically significant.However,there were no significant differences in the expression levels of P38,p-P38,JNK,Akt and β-catenin.5.The knockdown of Flotillin-1 and Flotillin-2 led to decrease of p-JNK and p-Akt expression and increased p-P38,β-catenin,Notch1 expression.The difference was statistically significant.However,there were no significant differences in the expression levels of P38,JNK and Akt.Conclusions:1.The knockdown of Caveolin-1 proteins can reverse the drug resistance of HCT-15/ADM cells.2.The p-JNK expression level in the JNK signal pathway was decreased after knocking down Caveolin-1,Flotillin-1 and Flotilin-2.The reversal of drug resistance after lipid raft destruction may be related to the JNK signaling pathway in the MAPK signaling pathway.3.After Caveolin-1 was reduced,the expression level of Notch1 was decreased,and the expression level of p-Akt was increased.there were no significant differences in the expression levels of P38,p-P38,JNK,Akt and β-catenin.After the reduction of Flotillin-1 and Flotillin-2,the expression levels of p-P38,β-catenin and Notch1 were up-regulated.The expression level of p-Akt was down-regulated,and there were no statistical differences in the expression levels of P38,JNK and Akt.Lipid rafts can affect the changes of different signaling pathways,which may provide a theoretical basis and direction for the study of molecular targeted therapy for lipid rafts to reverse drug resistance in colorectal cancer.