| Objective:To study the effects of recombinant mutant human tumor necrosis factorαon proliferation of human hepatocellular cancer cell SMMC-7721 and its mechanisms. Meanwhile, to examine the effects of rmhTNF-αcombined with anti-cancer agents cisplatin on human hepatocellular cancer cell SMMC-7721 to investigate possible mechanism of inhibiting cell proliferation and inducing apoptosis by rmhTNF-αcombined with cisplatin.Methods:1 SMMC-7721 cells were incubated in culture medium in vitro. Effect of rmhTNF-α,DDP and rmhTNF-αcombined with DDP on the proliferation of SMMC-7721 cells was measured by MTT colorimetric method. 2 Apoptosis and distribution of cell cycle were examined by flow cytometry. 3 According to the result of MTT, establishing control and experimental group, extracting total RNA of each group, assessing the integrality and content of RNA, the level of survivin mRNA expression was examined by RT-PCR technique in the SMMC-7721 cells treated before and after with rmhTNF-αand DDP. 4 The expression of HIF-1 protein was analyzed by flow cytometric indirect immunofluorescent technigue.5 To determine whether the combination of rmhTNF-αwith DDP results in a synergistic cytostatic effect, Isobolanalysis formula was performed. In the analysis, there is synergy when then interaction index is less than 1; additivity when the interaction index is equal to 1; and antagonism when the interaction index is more than 1.Results : 1 MTT colorimetric method showed that rmhTNF-α,DDP and rmhTNF-αcombined with DDP could inhibit the proliferation of SMMC-7721 cells. After treated with rmhTNF-α,DDP for 48h,compared with control group, the OD values of treated groups decreased, and there was statistically significant difference between control group and every treatment group(P<0.01).Among every treatment group, there was also statistically significant difference(P<0.01).Furthermore, with the increasing concentration of rmhTNF-α,DDP, the inhibited rate increased gradually, that was rmhTNF-α,DDP inhibited the proliferation of SMMC-7721 significantly in a dose-dependent manner. MTT colcrimetric method was performed to evaluate the potential cytostatic effect of combining rmhTNF-αwith anti-cancer agents DDP in SMMC-7721 cells. The results showed: rmhTNF-α(400,1600iu/ml) excerted a synergistic effect in SMMC-7721 cells when combined with DDP .In the range of concentration from 400iu/ml to 1600iu/ml ,rmhTNF-αhad the enhancing synergistic effect combined with DDP with the increasing concentration. The IC50 of DDP in SMMC-7721 cells alone was 4.018μg/ml. When combined with 400iu/ml rmhTNF-α, the IC50 decreased to 2.658μg/ml; when combined with 1600iu/ml rmhTNF-α, the IC50 decreased to 1.621μg/ml; Thus, there was synergistic interaction between rmhTNF-αand DDP in SMMC-7721 cells.2 When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry , the results were: After treated with rmhTNF-αand rmhTNF-αcombined with DDP for 48h, the number of cells in G0/G1 phase were not changed obviously, while the number of cells in S phase decreased gradually, and G2/M phase increased gradually. That was, rmhTNF-αand rmhTNF-αcombined with DDP could induce an arrest of cell cycle in G2 phase . In addition, after treated with rmhTNF-α,DDP,rmhTNF-αcombined with DDP for 48h, the typical apoptotic peak which enhanced gradually with the difference group, the control group,rmhTNF-α(400iu/ml),rmhTNF-α(1600iu/ml),DDP(2.0μg/ml),rmhTNF-α(400iu/ml)+ DDP(2.0μg/ml),rmhTNF-α(1600iu/ml)+ DDP(2.0μg/ml), the apoptotic percentage was 2.32%,6.59%,9.25%,8.13%,16.47%,27.94% separately.Compared with control group, the apoptotic percentage of treated groups decreased, except rmhTNF-α(400iu/ml) group , there was statistically significant difference between control group and every treatment group(P<0.01).3 RT-PCR detection results: There was different depression of the survivin mRNA expression in different concentration groups of SMMC-7721 cells after being treated by rmhTNF-αalone,DDP alone,rmhTNF-αcombined with DDP, rmhTNF-α(1600iu/ml) combined with DDP group obviously repressed the surviving mRNA expression in SMMC-7721 cells after treating 48h.4 Analysis on expression of HIF-1 by FCM showed that: Treating SMMC-7721 cells with 0,rmhTNF-α(400iu/ml),rmhTNF-α(1600iu/ml),2.0μg/ml DDP,rmhTNF-α(400iu/ml)+2.0μg/ml DDP,rmhTNF-α(1600iu/ml)+2.0μg/ml DDP for 48h resulted in the FI values of HIF-1 decreasing. To the FI values of HIF-1 protein, there was statistically significant difference between every treated group and control group,combined group and cisplatin alone group(P<0.05), except the expression of HIF-1 between rmhTNF-α(400iu/ml) alone group,DDP alone group and control group.5 The analysis of isobolanalysis showed that the interaction index for rmhTNF-αcombined with DDP used in SMMC-7721 cells was 0.74 as the inhibition ratio was 50% respectively. It proved that rmhTNF-αindeed had a synergistic effect on inhibiting the proliferation of SMMC-7721 cells combined with DDP.Conclusions:1 We comfirmed that recombinant mutant human tumor necrosis factorαhad the capability of inhibiting the proliferation of SMMC-7721 cells in vitro by a dose-dependent manner. 2 With MTT colorimetric method, we confirmed that rmhTNF-αcombined with cisplatin had a synergistic effect on inhibiting the proliferation of SMMC-7721 cells.3 rmhTNF-α,rmhTNF-αcombined with cisplatin induced an arrest of cells cycle in G2 phase as well as apoptosis in SMMC-7721 cells. 4 rmhTNF-α's effects of inhibiting proliferation and inducing apoptosis in SMMC-7721 cells is due to down-regulating the expression of survivin,HIF-1. 5 By FCM, we confirmed that rmhTNF-αcombined with DDP had a synergistic effect on inducing the apoptosis of SMMC-7721 cells, thus, we assumed that rmhTNF could have increased the sensitivity to anti-cancer agents of SMMC-7721 cells.
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