Effects Of Cyclosporine A Or Tacrolimus Combined With Epirubicin On The Proliferation And Apoptosis Of Human Hepatocellular Carcinoma Cell Lines

ObjectiveThe current studies were designed to investigate the effects of immunosuppressive agent—cyclosporine A and tacrolimus on the growth and epirubicin sensitivity of human hepatocellular carcinoma cell lines in vitro. After observing the antiproliferative effect of cyclosporine A or tacrolimus alone or in combination with epirubicin at various concentrations on these cells, we discussed the effect for hepatocarcinoma recurrent while immunosuppressive agent cooperate with chemotherapeutic drug , and explored the mechanism involved in favor of clinical practice primarily.Methods1 Through counting method to set out growth curve of SMMC-7721 and HepG2.2.15 cells , to understand theirs basic characteristic.2 Human hepatocellular carcinoma cell lines SMMC7721 and HepG2. 2. 15 were cultured in vitro, MTT assay was used to examine the antiproliferativeeffect of cyclosporine A or tacrolimus with or without epirubicin on these cells, and the results are expressed as a percentage of the control value. The survival curves were obtained by plotting the cell viability of the treated cells versus the logarithm of the drug concentration. The sensitivities of the anticancer drugs were obtained by determining the 1C50 of the drugs (the concentration of the drugs that reduced the cell viability to 50% of the control) from the survival curves. The drug sensitivity assays were repeated more than three times for each drug. The chemosensitizing effects of cyclosporine A or tacrolimus are expressed as a sensitivity index (SI), which was determined by dividing the IC50 ofepirubicin only by the IC50 of epirubicin in combination with cyclosporineA or tacrolimus, respectively.3 Flow cytometry (FCM) was used to examine the effects of epirubicin alone or in combination with cyclosporine A or tacrolimus on the apoptosis of HepG2.2.15 cells.4. The mRNA expression level of MDR1 gene was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Result1. Cyclosporine A alone have no effect on the proliferation of human hepatocellular carcinoma cells, Chemo-sensitivity to epirubicin combined with 5μg/ml cyclosporine A was increased to 1.92 and 4.03 folds, respectively for SMMC7721 and HepG2.2.15 cells, with the value of IC₅0 being 0.52μg/ml and 0. 62μg/ml respectively;As for tacrolimus , inhibitory effect on cell proliferation was observed only after 72h of incubation ,but tacrolimus didn't affect the chemo-sensitivity to epirubicin in human hepatocellular carcinoma cells.2. FCM results indicated that more HepG2.2.15 cells showed apoptoticphenotype when treated with lUg/ml epirubicin and 5Mg/ml cyclosporine A, than those treated with epirubicin alone(ratio 64.79% and 27.22% respectively);But when treated with lUg/ml epirubicin and 0. 5Mg/ml tacrolimus, HepG2.2.15 cells apoptotic phenotype was 29.37% , compare with those treated with epirubicin alone, there were no significantly different (p>0.05)3. The mRNA expression of MDRl gene was down-regulated in HepG2.2.15 cells treated with cyclosporine A. But tacrolimus didn' t affect the mRNA expression of MDRl gene in human hepatocellular carcinoma cells. Conclusions1. Cyclosporine A significantly improved the sensitivity of human hepatocellular carcinoma cells to epirubicin, especially for HepG2.2.15 cells. Down-regulated expression of MDRl mRNA might be a possible mechanism to overcoming multidrug resistance in human hepatocellular carcinoma cells.2. As for tacrolimus , inhibitory effect on cell proliferation was observed only after 72h of incubation ,but tacrolimus didn t affect the chemo-sensitivity to epirubicinin human hepatocellular carcinoma cells.