Reversal effects of short hairpin RNA interference and tetrandrine on multidrug resistance of human colorectal cancer cell line LoVo/5-fluorouracil

Abstract

Objective To compare the reversal effects of short hairpin RNA (shRNA) interference and tetrandrine on multidrug resistance (MDR) of human colorectal cancer cell line LoVo/5-fluorouracil(5-FU ).Methods An eukaryotic expression plasmid of shRNA targeting MDR1 was constructed and transfected into human colorectal cancer cell line LoVo/5-FU (transfection group).LoVo/5-FU was also pretreated with tetrandrine (tetrandrine group).Drug sensitivity was detected by methyl thiazolyltetrazolium colorimetric method.Cell cycle,apoptosis of cells and positive expression rate of P-glycoprotein (P-gp) were determined by flow cytometry assay.The expressions of MDR1 mRNA and P-gp were detected by real-time polymerase chain reaction and Western blot,respectively.All data were analyzed by analysis of variance and SNK-q test.Results (1)Drug sensitivity:the 50％ concentration of inhibition(IC50)of the control group,tetrandrine group and transfection group were (7.3 ± 0.3),(4.4 ±0.7) and (2.4 ±0.4) mmol/L,respectively,a significant difference between the 3 groups was found(F =65.27,P ＜ 0.05 ).There was a significant difference in the IC50 between the tetrandrine group and the transfection group (q =6.67,P ＜ 0.05 ).(2) Changes of cell cycle:the proportion of cells in the G1 phase and S phase of the control group,tetrandrine group and transfection group were 38.13％ ± 3.75％,51.36％ ± 2.76％,59.24％±4.31％ and 20.46％±2.23％,14.32％± 1.91％,9.40％± 1.65％,respectively,a significant difference between the 3 groups was found(F =25.23,24.37,P ＜ 0.05 ).There were significant differences in the proportion of cells in the G1 phase and S phase between the tetrandrine group and the transfection group(q =3.67,4.35,P ＜ 0.05 ).(3) Cell apoptosis:the cell apoptotic rates of the control group,tetrandrine group and transfection group were 1.32％ ± 0.47％,3.24％ ± 0.26％,5.88％ ±- 0.44％,respectively,a significant difference between the 3 groups was found(F =99.26,P ＜ 0.05 ).There was a significant difference in the cell apoptotic rate between the tetrandrine group and transfection group(q =11.48,P ＜ 0.05 ).(4)The expression of P-gp:the positive expression rates of P-gp of the control group,tetrandrine group and transfection group were 96.9％ ± 2.3％,61.6％ ± 4.9％,76.6％ ± 3.6％,respectively,a significant difference between the 3 groups was found(F =67.83,P ＜ 0.05 ).There was a significant difference in the positive expression rate of P-gp between the tetrandrine group and transfection group (q =6.97,P ＜ 0.05 ).(5)The mRNA expression of MDR1:the mRNA expressions of MDR1 of the control group,tetrandrine group and transfection group were 1462 ±161,570 ±85,233 ± 81,respectively,a significant difference between the 3 groups was found(F =90.59,P ＜ 0.05 ).There was a significant difference in the mRNA expression of MDR1 between the tetrandrine group and transfection group (q =5.12,P ＜ 0.05 ).Conclusions MDR1 shRNA and tetrandrine could reverse M DR1 gene-mediated m.ultidrug resistance in human colon cancer cell line LoVo/5-FU,but the effect of MDR1 shRNA is better than that of tetrandrine.MDR1 shRNA and tetrandrine might take effect by inhibiting P-gp expression and down-regulating mRNA expression of MDR1. Key words: Colorectal neoplasms;  Short hairpin RNA;  Tetrandrine;  Multidrug resistance gene;  P-glycoprotein