Association Between RRM1 Level And Gemcitabine Sensitivity In Cervical Cancer Cell Lines

Purpose Cervical cancer is the second most common malignancy of women.Chemotherapy is one of the main treatments for cervical cancer,next to surgery and radiotherapy.Gemcitabine as a new chemotherapy agent has been used in the treatment of cervical cancer and ribonucleotide reductase subunit M1(RRM1) is its pivotal intercellular target.Our study is to investigate the association between RRM1 level and gemcitabine sensitivity in cervical cancer cell lines, and the methods of reversing gemcitabine chemoresistance are further explored.Materials and Methods Three cervical cancer cell lines(C33a,Hela,CaSki) were cultured in vitro.Cell chemotherapy sensitivity to gemcitabine,cisplatin,and gemcitabine plus cisplatin was determined by MTT assay.IC₅₀ is defined as the concentration of drug that results in 50%cell survival compared with untreated cells.RRM1 expression level was measured by RT-PCR and western blot.The association between gemcitabine sensitivity and RRM1 expression in cervical cancer cell lines was further analyzed.Gemcitabine-resistance cells were developed by continuous low concentration combind intermittent high concentration exposure to gemcitabine for 2 months,then its IC₅₀ and resistance index were measured by MTT,as well as its RRM1 expression by RT-PCR and western blot.Inhibition of RRM1 expression by RNAi technique was applied and its influence on gemcitabine-sensitivity was further evaluated.The expression level of RRM1 was assessed after exposure to cisplatin.Cell cycle distribution and apoptosis were analyzed by flow cytometry.Results The gemcitabine-sensitivity among cervical cancer cell lines varied in a wide range.The IC₅₀ values of gemcitabine for C33a,Hela,CaSki were 0.58μM,1.43M,and14.39μM,respectively,while their difference of cisplatin-sensitivity was not significant.An inverse correlation was observed between the expression level of RRM1 and the cell sensitivity to gemcitabine.The resistance index of gemcitabine-resistance cells was 3.6.Compared with the parental cells,gemcitabine-resistance cells had an increase in levels of RRM1 mRNA and protein expression.RRM1-siRNA could effectively knock down the expression of RRM1 and markedly increase the cell sensitivity to gemcitabine in a dosage-dependant manner.When treated with gemcitabine,cervical cancer cells were markedly blocked into S-phase.The proportion of S-phase cells was elevated with the increase of exposure duration.Meanwhile,compared with the single-agent treatment,combination of gemcitabine and cisplatin increased the number of apoptotic cells(P=0.035). Conclusions There is a negative correlation between RRM1 level and gemcitabine sensitivity in cervical cancer cell lines. Gemcitabine-resistant inducement can results in increased expression of RRM1.RRM1-siRNA interference is a functional pathway with down-regulation of RRM1 expression and reversion of gemcitabine-resistance.The combination of gemcitabine and cisplatin is synergistic in vitro.The mechanism of synergism may be explained by the increase of apoptosis of cells.