Study On Anti-growth Activity Of Hepatoma Cancer Cells And Its Mechanism By Taxoids

As in recent years to paclitaxel and their precursors of rising demand,the search for new biological resources,protection of the ecological environment has been imminent.In recent years researchers isolated by extracting a great deal of paclitaxel analogs,but so far no second-generation drug Taxol listed. So the new taxanes drug study was to find a more low toxic side effects,a higher anti-tumor selective and a variety of tumor cells in particular,to have drug-resistant human tumor cells have a higher biological activity of taxanes compounds. The present experiment investigated the effects of 23 taxanes compounds extracted from the renewable branchlets and needles of Taxus cuspidate and Taxus canadensis on the proliferation of the human hepatoma tumor cells.We screened out three compounds with obvious inhibitory activity and the mechanism of anti-proliferation of Hep G2 cells was investigated in order to provide experimental basis and theoretical foundation for developing the high-efficiency and low-toxin drugs. Part one 1 The inhibitory effects of taxanes compounds on proliferationof human hepatoma tumor cellsObjective: As in recent years to paclitaxel and their precursors of rising demand,alleviate Taxol in short supply,the present experiment investigated the effects of 23 taxanes compounds extracted from the renewable branchlets and needles of Taxus cuspidate and Taxus canadensi on the proliferation of the human hepatoma tumor cells, and to screen out the compounds with significantly inhibitory effects on proliferation of tumor cells.Methods: The proliferation of Human hepatoma cancer cells which treated with 23 taxanes compounds was measured by monotetrazolium(MTT) colorimetric assay. The concentration effect curve of experimental compounds on the tumor cells was fit by Hill mathematical model to calculate the IC50 of medicineResults: 10 μmol/L cisplatin and Taxol inhibited the proliferation of human hepatoma cancer cells(Hep G2) strongly. The inhibition rates of cell proliferation were 71.02% and 78.93%;23 taxanes compounds acted on human hepatoma cancer cells(Hep G2), compounds 11,20,22 displayed inhibitory activities as the same or better effect than cisplatin or Taxol. Other compounds demonstrated weak anti-proliferative activity of human hepatoma tumor cell. Through logarithm regression equation of proliferative rate of tumor cells on the concentration of compounds 11,20,22,we got the IC50 values of the three compounds on the human hepatoma cancer cells, respectively 0.15、3.75 and 4.91μmol/L.Conclusions: Compounds 11,20 and 22 from renewable branchlets and needles of Taxus cuspidate and Taxus canadensi presented strong anticancer activity on human lung cancer cells;the anti-tumor effect on hepatoma cancer cells of taxanes compounds is not only closed related with its skeleton structure,but also with C-5 cinnamoyl side chain. Part two 2 The mechanisms of inhibitory effects of Taxinine onproliferation of human hepatoma cancer cellsObjective: A lot of experiments in vivo and in vitro have confirmed that many anticancer drugs can induce different types of sensitive tumor cells apoptosis. Traditional Chinese medicinal herbs contained many anticancer compounds which could induce apoptosis. The first part of our study had confirmed that three taxanes compounds inhibited the proliferation of human hepatoma cancer cells significantly. In this part,we explored the antitumor mechanism of the three taxanes compounds,and identified whether taxanes compound Taxinine display its antihepatoma cancer effects through the activities of inducing human hepatoma cancer cells(Hep G2) apoptosis.Methods:1 We detected the reporter genes by luciferase reporter gene assay. The reporter genes were p G13-Luc,p21-Luc,Bax-Luc, h Noxa-Luc, SRE-Luc, Ig K-Luc, NFAT-Luc and CRE-Luc.2 We examined cell apoptosis by FCM. Hep G2 cells were incubated 24 h by Taxinine at final concentration of 5 μmol/L.3 We examined the expressions of p53, Bax an Caspase-3 protein by Western blot.4 We observed the reversal effects of Z-VAD-FMK on Taxinine’s inhibitory activities on proliferation of Hep G2 cells.Results:1 The expressions of reporter gene in Hep G2 cells which were treated with compounds 11,20 and 22(at final concentration of 5μmol/L):The results showed that Taxinine can induce the expressions of reporter genes p G13-Luc, p21-Luc, Bax-Luc,h Noxa-Luc which were associated with the p53-dependent signaling pathways of Hep G2 cells significantly. But there were no effects to the expressions of reporter gene SRE-Luc,Ig K-Luc, NFAT-Luc and CRE-Luc, which were associated with other signaling pathways of Hep G2 cells. And the other two taxanes compounds showed no activities to the expressions of the reporter genes.2 Effects of Taxinine on apoptosis of Hep G2 cells: After treated for 24 h with Taxinine at final concentration of 5μmol/L, Hep G2 cells’ apoptosis rate 13.7%, had significant difference compared with that 5.8% of blank group(P<0.05).Taxinine increased apoptosis rate of Hep G2 cells.3 Taxinine could increase the expressions of p53, Bax and Caspase-3 of Hep G2 cells significantly.4 The reversal effects of Z-VAD-FMK inhibitor on Taxinine’s inhibition on proliferation of Hep G2 cells: After treated with 0.1,1 and 10 μmol/L cisplatin and Taxinine, the cell survival rates of Hep G2 cells were 95.10%、66.37%、21.27% and 79.82%、31.87%、7.38%; After treated with 20 μmol/L Z-VAD-FMK and cisplatin or Taxinine, the Hep G2 cells’ survival rates were 100.00%、70.90%、36.34% and 84.79%、45.67%、19.05%.Conclusions: The apotosis of human hepatoma cancer cells(Hep G2 cells) could be induced by taxanes compound Taxinine significantly; the expression of p53 was increasely stimulated in Hep G2 cells with Taxinine,and the expression of Bax and Caspase-3 increased as followed. And Z-VAD-FMK had the reversal effects of Taxinine ’s inhibitory activities on proliferation of Hep G2 cells.