| Objective:To study the effect of arsenic trioxide(As2O3) combining with cisplatin(CDDP) on apoptosis of liver cancer cell HepG2,and explore its mechanism indepthly.Then to provide the firm practical base and theoretical warranty for arsenic trioxide(As2O3) combining with cisplatin (CDDP) in the clinical therapeutic use for hepatocellular carcinoma,and explore a new chemotherapy way for liver cancer.Methods:(1) We used MTT assay to detect the OD and calculate the cell inhibitatory rates of human HepG2 hepatocellular carcinoma cell lines treated with As2O3 and CDDP at different concentrations,then protracted the proliferation curve with drug concentrations changes.Then we evaluated the mutual effect nature of the two chemotherapeutant by calculating CDI(coefficient of drug in interation,CDI).(2) HepG2 cells were cultured in vitro at 37℃in humandified 5%CO2 in air.Human HepG2 hepatocellular carcinoma cell lines were treated with As2O3 and CDDP at different concentrations in vitro,then the liver cancer cell apoptosis and morphologic changes with time and concentrations changes were observed by acridine orange(AO)/ ethidiumbromide(EB) fluorescence microscopy,and apoptosis rate of HepG2 cell induced by As2O3 and CDDP were calculated.(3) Human HepG2 hepatocellular carcinoma cell lines were treated with As2O3 solely and combining with CDDP,and immunohistochemical staining ElivisionTM plus method incorporated with computator image assay were used to investigate the expression of Bax,Bcl-2 and Fas proteins in human HepG2 hepatocellular carcinoma cell lines.(4) Flow cytometry(FCM) was used to observe and assay cell cycle changes and apoptosis rate induced by As2O3 and CDDP at different doses and time.(5) By means of telomerase repeat amplification protocol(TRAP) combined with PAGE silver staining,we detected the changing of telomerase activity of human HepG2 hepatocellular carcinoma cell lines with As2O3 and CDDP in different doses and different periods.Results:(1) As2O3 at different concentrations(1μg/ml,2μg/ml,4μg/ml) solely and combining with CDDP(0.25μg/ml,0.5μg/ml,1μg/ml,2μg/ml) both could inhibit the proliferation of HepG2 cell lines and had obvious time-and concentration-dependent manner.It was found that As2O3 combining with CDDP obviously increased the inhibitatory rates of HepG2 cell lines than those of the corresponding individual drug groups and the increment of inhibitatory rates at middle and low combinative groups was even apparent:the inhibitatory rate of proliferation of HepG2 cell lines with As2O3(2μg/ml) combining with CDDP(0.25μg/ml,0.5μg/ml,1μg/ml) at 24 hours was 35.54%,41.77%,44.34%;at 48 hours was 62.14%,66.43%,69.43%;at 72 hours was 67.84%,77.93%,84.48%,respectively(P＜0.05).(2) The control group cells displayed dispersed and homogeneous fluorescence and human HepG2 hepatocellular carcinoma cell lines treated with As2O3 and CDDP displayed the typically morphological features of apoptosis.There were apparent nucleic fragmentation and particulate fluorescence.The apoptosis rate of 200 cells showed that the cell apoptosis had a time-and concentration-dependent manner;The difference of experimental group solely by As2O3 low concentration(1μg/ml) between the antithetical group had no notable significance(P=0.071), and the difference of experimental group treated solely with CDDP of middle and low concentrations between the antithetical group had notable significance;In the group treated with As2O3 and CDDP the proportion of apoptosis cell was quite high in the low drug concentration.(3) The immunohistochemical staining showed that the expression of the three genes had ostensible concerns with the drugs concentrations and times.As2O3(1.0μg/ml) and CDDP(0.25μg/ml) could change the expression of three genes early yet not so obviously.The expression of genes took place apparent changes with drugs concentrations rose and time prolonged:the expression of Bax and Fas increased and the expression of Bcl-2 decreased obviously in the experimental group,and the expression of Bcl-2 increased and Bax and Fas decreased in the control group.It was also found that As2O3 combining with CDDP obviously increased the expression of three genes than those of the corresponding individual drug groups and the expressions difference had prominent statistical significance(P＜0.05).(4) Flow cytometry study showed a typical subdiploid peak before G1 phase(Sub—G1, AP—apoptotic peak) and the apoptosis amounts with combing drug groups were higher than those of the corresponding individual drug groups.It could concluded that As2O3 and CDDP both could induce the liver cancer cells apoptosis and the combing drug groups obviously increased the apoptosis rate.It also showed that the portion of S phase cell decreased following treatment with As2O3 combing with CDDP and showed an arrestment at G2/M phase.(5) The temolerase activity of the human hepatocarcinoma cells line HepG2 was positive with As2O3 combing with CDDP and the corresponding individual drug groups at 48 hours and 72 hours,and the temolerase activity of the human hepatocarcinoma cells line HepG2 was partly negative with As2O3 combing with CDDP at 48 hours,yet the detection results showed wholely negative following treatment with As2O3 combing with CDDP at 72 hours.It could concluded that combing drug groups could not block temolerase activity at 24 hours because of the lower drug concentration and shorter time.It showed that the suppression of the expression of telomerase have a time-and concentration-dependent manner so that the inhibition increased following with concentration elevated and times prolonged.Conclusion:(1) My research proved that arsenic trioxide combing with cisplatin may obviously increase the anti—hepatocarcinoma effect in vitro.It may inhibit the proliferation of human hepatocarcinoma cells line HepG2in a time—and concentration—dependent manner.(2) The mechanism of inhibition of the proliferation of hepatoma cells was mainly inducing apoptosis of hepatoma cells selectively and the apoptosis rate of combing drug groups was more higher than the corresponding individual groups.It was demonstrated that arsenic trioxide combing with cisplatin may saliently induce apoptosis of human hepatoma cells synergeticly.It need deep-going probing for whether the lower drug concentration of combing drug groups may obtain more satisfied effect than the corresponding individual groups.(3) The induced—apoptosis effect of arsenic trioxide and cisplatin was relative to the expression level changes of Bax,Bcl-2 and Fas.It amplified the expression of Bax and Fas in the meantime laid down the expression of Bcl-2.The ratio of Bcl-2/Bax was decreased.In conclusion this was supposed to an important molecular mechanism of inducing apoptosis of hepatoma cells.(4)Arsenic trioxide combing with cisplatin could change hepatoma cell cycle and the portion of S phase cell decreased and showed an arrestment at G2/M phase.It can prohibit the cells mitosis so that prolong cell cycle time.It was supposed to another important mechanism of inducing apoptosis of human hepatocarcinoma cells line HepG2.(5) Arsenic trioxide combing with cisplatin may increase the anti—telomerase effect so that inhibit the proliferation of hepatoma cells.The inhibition of expression of telomerase activity might turn up before apoptosis yet was not the consequence of apoptosis so the relation of inhibition of telomerase activity and apoptosis of hepatoma cells was not yet explicit.It need deep—going probing for whether the inhibition of telomerase activity following with the treatment of As2O3 and CDDP started up the course of apoptosis of hepatoma cells.(6) As2O3 and CDDP may inhibit the proliferation of hepatoma cells and induce apoptosis of hepatoma cells in vitro.The combing use of the two medicines may embody the feature and preponderance of Chinese traditional medicine connected with Western medicine.So it need to continue to put up animals'endoexperimentation and clinical experiment so that provide credible experimental foundation and academic gist for ultimately using these two medicines diffusely to treat liver cancer in clinic.It also may explore a new expansive applied foreground way for chemotherapy of liver cancer.
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