Study On Anti-growth Activity Of Hepatoma Cancer Cells And The Mechanism Of Taxanes Isolated From The Taxus Canadensis

With rising demand for Taxol and their precursors, finding new biological resources, protecting of the environmental biodiversity has been generally accepted by scientific field. In past few decades chemists have isolated a great numbers of taxol analogs, but so far no one second generation of Taxol was marketed. So many chemists try to find more new taxanes analogues with hope to find a less toxic side effects and higher anti-tumor activity more selective and effective to more kinds of tumor cells, or have a higher biological activity to drug-resistant human tumor cells. The present experiment investigated the effects of 59 taxanes compounds extracted from the renewable branches and needles of Taxus canadensis on the proliferation of the human hepatoma tumor cells. We screened out 4 taxanes with potencial inhibitory effect and the anti-proliferation mechanism of these taxanes to BEL-7402 cells was studied with hope to develop the high-efficiency and low-toxicity drugs. Part One The Inhibitory Effecties of Nature Taxanes On Proliferation ofHuman Hepatoma Tumor CellsObjective: Due to rising demand for taxol and its precursors in recent years, Taxol was in short supply in clinic. Present experiment investigated the effects of 59 taxanes compounds extracted from the renewable branches and needles of Taxus canadensis on the proliferation of the human hepatoma tumor cells, and to explore the natural taxanes with significantly inhibitory activities on proliferation of selected tumor cells.Methods: The proliferation activity to human hepatoma cancer cells which were treated with 59 isolated taxanes form Canadian yew tree was measured by monotetrazolium(MTT) colorimetric assay method. The concentration effect curve of experimental taxanes on the tumor cells was fit by Hill mathematical model to calculate the IC50 of tested taxanes.Results: 10 μmol/L cisplatin and Taxol as references inhibited the proliferation of human hepatoma cancer cells(BEL-7402) strongly. 59 Taxanes compounds acted on human hepatoma cancer cells(BEL-7402), compounds 5, 19, 29 and 30 displayed inhibitory activities as the same or better effect than cisplatin or Taxol. Other compounds exhibited weak anti-proliferative activity to human hepatoma tumor cell. By logarithm regression equation of proliferative rate of tumor cells on the concentration of taxanes 5, 19, 29, and 30, we got the IC50 values of the tested taxanes on the human hepatoma cancer cells were 1.20, 1.24, 1.78 and 4.91 μmol/L, respectively.Conclusions: Compounds 5, 19, 29, and 30 from renewable branches and needles of Taxus canadensis exhibited potencial anticancer activity on human lung cancer cells. Part Two Study On the Relationship Between Structure and ActivityObjective: Early activity screening results found that the resistance of human liver with different mother nucleus structure of 4 kinds of taxanes have stronger inhibiting the activity of liver cancer. In this experiment to investigate these four taxanes for other inhibitory activity and structure-activity relationship of liver cancer cells. This work will further explore the basis of the activities from the viewpoint of chemical structure.Methods: The proliferation effect of Human hepatoma cancer cells were treated with compounds 5, 19, 29, and 30 was tested by monotetrazolium(MTT) colorimetric assay. The specific method is the same as the first part. Use Chemoffice software molecular mechanics model, to study the threedimensional of compound 19.Results: No side chain taxane compounds showed weaker or don’t show the activity of inhibiting tumor cell proliferation, 13 carbon atoms connected phenyl different serine side chain can led antitumor activity increases; A fat-soluble cinnamon acyl side chain at C-5 was associated with antitumor activity. A fat-soluble cinnamon acyl side chain at C-5 of the three-ring compound 19 and taxol quantify three-dimensional conformation is very similar. 10 μmol/L compounds 5, 19, 29, and 30 inhibited the proliferation of human hepatoma cancer cells(BEL-7402、SMMC-7221、Hep G2 and HLE cells) strongly. By logarithm regression equation of proliferative rate of tumor cells on the concentration of taxanes 5, 19, 29, and 30, we caculated the IC50 values of the four taxane compounds on the human hepatoma cancer cells, respectively 1.20, 1.24, 1.78 and 4.91 μmol/L(BEL-7402); 6.27, 30.91, 0.81 and 55.34 μmol/L(SMMC-7221); 0.52, 6.27, 4.91 and 20.09 μmol/L(Hep G2); 29.32, 42.51, 18.06 and 61.24 μmol/L(HLE).Conclusions: The anti-tumor effect on hepatoma cancer cells of taxanes compounds is not only closed related with their skeleton structures, but also with C-5 cinnamoyl side chain. The taxanes 5, 19, 29, and 30 were broadspectrum antitumor activity. Part Three The Mechanism of Isoalantolanctone Anticancer Activity onthe Liver CancerObjective: As many experimental results in vivo and in vitro have found that many drugs with anticancer activities can induce different types of sensitive tumor cells apoptosis. Traditional medicinal herbs contain a lot of anticancer components with induce apoptosis activities. In the first part of our study we have demonstrated that four taxanes can exhibite significantly the proliferation of human hepatoma cancer cells. As a continuation of our work, we explored the antitumor mechanism of the four taxane compounds in this part, and further identified whether taxanes compound taxinine display its antihepatoma cancer activities by inducing human hepatoma cancer cells(BEL-7402) apoptosis.Methods:(1) We investigated the reporter genes by luciferase reporter gene assay. The reporter genes were p21-Luc, p G13-Luc, h Noxa-Luc, Bax-Luc, Ig K-Luc, SRE-Luc, NFAT-Luc and CRE-Luc.(2) TUNEL apoptosis detection statutory 10 μmol/L compound 19 on BEL-7402 cells.(3) We studied cell apoptosis by FCM.BEL-7402 cells which were incubated 24 h by 9?,10?-deacetyltaxinine at final concentration of 10 μmol/L.(4) We studied the expressions of Bax Bcl-2, p53 and Caspase-3 protein by Western blot.(5) We observed the reversal effects of Z-VAD-FMK on Taxinine’s inhibitory activities on proliferation of BEL-7402 cells.Results:1 The expressions of reporter gene in BEL-7402 cells which were treated with natural taxanes 5, 19, 29, and 30(the final concentration was 10 μmol/L): The exprmental results indicated that 9?,10?-deacetyl- taxinine can induce the expressions of reporter genes p21-Luc, p G13-Luc, h Noxa-Luc, Bax-Luc, which were associated with the p53-dependent signaling pathways of BEL-7402 cells significantly. However, there were no effects was observed for the expressions of reporter gene Ig K-Luc, SRE-Luc, NFAT-Luc and CRE-Luc, this observation is in agreement with which was associated with other signaling pathways of BEL-7402 cells. And the other three taxanes exhibied no activities in this exprement to the expressions of the reporter genes.2 Effects of 9?,10?-deacetyltaxinine on apoptosis of BEL-7402 cells: After treated for 24 h with 9?,10?-deacetyltaxinine at concentration of 10 μmol/L at final, BEL-7402 cells’ apoptosis rate 12.23%, had showed significant difference to that of blank group 4.10%(P<0.05). 9?,10?-deacetyltaxinine increased apoptosis rate of BEL-7402 cells.(3) 9?,10?-deacetyltaxinine could increase the expressions of Bax, Bcl-2, p53 and Caspase-3 of BEL-7402 cells significantly.3 TUNEL experiment showed the result of 9?,10? deacetyltaxinine taxanes was positive.4 The reversal effects of Z-VAD-FMK inhibitor on 9?,10?-deacetyltaxinine’s inhibition to proliferation of BEL-7402 cells: After treatement with 0.1, 1 and 10 μmol/L cisplatin and 9?,10?-deacetyltaxinine, the cell survival rates of BEL-7402 cells were 94.59%, 64.39%, 33.56% and 65.82%, 35.41%, 1.87%. After treated with 20 μmol/L Z-VAD-FMK and cisplatin or 9?,10?-deacetyltaxinine, the BEL-7402 cells’ survival rates were 100.00%, 72. 19%, 40.63% and 85.97%, 40.15%, 6.53%.Conclusions:1 The apotosis effect of human hepatoma cancer cells(BEL-7402 cells) could be significantly induced by taxanes compounds 5, 19, 29 and 30;2 the expression of p53 was increasely stimulated in BEL-7402 cells with 9?,10?-deacetyltaxinine, and the expression of Bax and Caspase-3 was increased as indicated. And Z-VAD-FMK had the reversal effects of 9?,10?-deacetyltaxinine’s inhibitory activities on proliferation of BEL-7402 cells.3 Resistance to human liver cell activity not only with the skeleton structure of the compound taxanes close relations, and the compound resistance to endometrial cancer cells activity may also with C-5 of cinnamon acyl side chain and the number of hydroxyl side chain has certain related 9?,10?-deacetyltaxinine through the induction of human liver cancer BEL-7402 cells apoptosis and antineoplastic activity.(4) HSP90? in its antineoplastic played a role in the process.