To Investigate The Reversal Effect And Mechanism Of All-transretinoic Acid On Drug Resistance In Colon Cancer

Objectives To investigate the reversal effect and mechanism of total trans-retinoic acid combined with oxaliplatin on drug resistance of HCT116/l-ohp cells.Methods At the initial stage,small dose oxaliplatin combined with large dose shock was used to culture HCT116 colon cancer oxaliplatin-resistant cells(HCT116/l-ohp).The OD value of HCT116/l-ohp at different concentrations was determined by MTT assay,and IC50 was calculated according to the OD value,so as to obtain the drug resistance multiple of HCT116/l-ohp cells.IC50 of HCT116/l-ohp cells treated with oxaliplatin plus ATRA at different concentrations was obtained by MTT assay.The gene expression levels of LC-3,Beclin1 and Bcl-2 in HCT116 colon cancer cell group,HCT116/l-ohp cell group and treatment group were detected by fluorescence quantitative PCR.Western Blot was used to detect the expression of LC-3,Beclin1,Bax,P62 and Bcl-2 proteins in HCT116 colon cancer cell group,HCT116/l-ohp cell group and treatment group.Results 1 This study after high-dose low-dose continuous joint impact resistance method to cultivate colon oxaliplatin into cells(HCT116/L-OHP),determined by MTT experiment test of oxaliplatin into the HCT116/L-OHP cell activity and HCT116/L-resistant stability of the OHP cells,preliminary experimental results show that L-OHP action HCT116 cell half inhibitory concentration(IC50)value of 6.3 mg/L,HCT116/L-OHP cells IC50 alue concentration of 67.73 mg/L,Resistance fold(RF)=10;It was found that the activity of HCT116/l-ohp cells cultured with different concentrations of oxaliplatin decreased significantly,indicating that oxaliplatin could inhibit the activity of HCT116/l-ohp cells in a dose-dependent manner.2.MTT assay was used to determine the optimal concentration of total trans-retinoic acid.1umol/L(0.901 0.026),2 umol/L(0.7410.029),5 umol/L(0.505 0.037),10 umol/L(0.295 0.019(3)by 2~-??CT??CT analysis method to calculate the corresponding processing three groups of cells after 24 h of LC-3,Beclin1,P62 m RNA relative expression,LC-3,Beclin1,P62 m RNA in HCT116 cells,HCT116/L-OHP,after all trans retinoic acid effect of drug-resistant cell relative to express respectively in three groups:Lc-3(1.000 0.000,11.310 0.856,3.557 0.985*),Beclin1(1.000 0.000,12.589 0.863,3.865 0.904*),P62(1.000 0.000,0.308 0.068,0.972 0.057*)suggest that total trans-retinoic acid in combination with oxaliplatin can reverse the resistance of HCT116/l-ohp.It may be related to the down-regulation of lc-3 and Beclin1mRNA and up-regulation of P62mRNA in drug-resistant cells(P<0.05).4.Western blotting results suggested that the expressions of LC-3,Beclin1 and Bcl-2 proteins in HCT116 cells,HCT116/l-ohp cells and drug-resistant cells treated with total trans-retinoic acid were down-regulated(P<0.05),and the expressions of Bax and P62proteins were up-regulated(P<0.05).Conclusions 1 All-transretinoic acid can inhibit the activity of HCT116/l-ohp cells in a dose-dependent manner;2 All-transretinoic acid can reverse the drug resistance of HCT116/l-ohp cells and induce its apoptosis by down-regulating its autophagy;3 In summary,it can be speculated that the regulation of autophagy of human colon cancer HCT116/l-ohp cells resistant to oxaliplatin may be a new method to enhance the sensitivity of cancer cells to chemotherapy drugs.Figure 8;Table 4;Reference 163...