Study On Growth Inhibition Of Human Cancer Cell Lines And Mechanism By Taxane Compound From Taxus Cuspidata

Taxol was discovered by Dr. Monroe E. Wall and Dr. Mansukh C. Wani at Research Triangle Institute (RTI) of North Carolina, USA, in 1967. Later, they isolated the compound from the Pacific yew tree, Taxus brevifolia, and noted its antitumor activity in a broad range of solid tumors. Taxol, currently the best-known drug approved for use in the treatment of ovarian and breast cancer, is very expensive, about 4800 dollars for one gram. And the supply of the drug from nature resources, yew trees and African fern pine (Podocarpus gracilior Pilger), which are grow very slowly and contain only a small amount of taxol, is very limited. Taxus cuspidata (Dongbei or Japanese Yew): evergreen tree, growing in the forest of 500-1000 m. Distributed in southeast Heilongjiang, east Jilin, Liaoning, Shaanxi of China and Japan, Korea, east Russia (Kurile Islands, Primorye, Sakhalin).Objective: 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. Currently taxanes components only paclitaxel and BaccatinⅢor 10-DeacetylbaccatinⅢprecursors for the semi-synthetic drug docetaxel (Taxotere) applied to clinical. 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 purpose of this study is from the northeast Taxus in the separation and purification are taxanes compounds anti-tumor cell proliferation activity of screening, for the development of new anticancer drugs provide a theoretical basis.Methods:1 Apply by MTT assay to select the Taxus in the Northeast taxanes compounds for anti-tumor cell proliferation activity screening. First study of different nuclear structure of mother taxanes compounds of cervical cancer HeLa cells; and then study the nuclear structure with the same mother, different connection taxanes chain compounds of cervical cancer HeLa cell proliferation; final choice of nuclear structure and the mother have the same side-chain taxanes compounds to study the different nucleus of Substituents on taxanes anti-cancer compounds HeLa cell proliferation. 2 Choice of the mother of nuclear structure, side-chain activity, replace the activity of the taxanes compounds, they inspected the five proliferation of tumor cells. 3 Mechanism research: The proliferation inhibition test, Taxol as a positive control, Hela cells in the selected compounds Compound(22) of the effective role 48, 96, 144 h, the cells were counted concentration, rendering cell proliferation curve to comparison. Flow cytometry and AnnexinⅤ-FITC Kit analysis the situation of apoptosis. WesternBlot detected to Bax , Bcl-2,Caspase-3 and Caspase-9 expression of HeLa cells deal with 2α,10β-Diacetyl-5-cinnamoyl-phototaxicin II.Results: 1 6/10/6 mother compounds core structure 7-Deacetyl-taxine E (17) that the use of 100μmol/L concentration to deal with HeLa cells, their survival rate was 97%, of HeLa cells do not show any inhibitory activity; 6/8/6 parent core structure compounds Decinnamoytaxinine E (12), 6/5/5/6 mother core structure compounds Taxinine L (23) and 6/12 mother of the nuclear structure of compounds Taxachitriene A (24) three compounds 100μmol/L concentration to deal with HeLa cells, their proliferation showed weak inhibitory activity, survival rates were 74%, 73% and 71%, compared with blank control were significantly different (P <0.05).2①6/8/6 the nuclear structure of mother five carbon atoms connected with water-soluble sugar side chain taxanes (10), with 1μmol/L, 10μmol/L and 100μmol/L to deal with HeLa cells, their survival rates were 98%, 86% and 79%; 6/8/6 home in the nuclear structure of five carbon atoms connected with the fat-soluble Cinnamoyl side chain taxanes(9) and connect with alkali side chain of Winterstein taxanes(2), with 1μmol/L, 10μmol/L and 100μmol/L to deal with HeLa cells, their survival rates were: 84%, 46%, 21% and 89 %, 86%, 34% of HeLa cells showed strong inhibitory activity showed a good dose-dependent relationship.②6/12 mother only nuclear structure, not connected to any side-chain compounds(24) with 1μmol/L, 10μmol/L and 100μmol/L to deal with HeLa cells, their survival rates were 100%, 97% and 71% of HeLa cells showed weak inhibitory activity, when the five carbon atoms in the introduction of fat-soluble compounds Cinnamoyl side chain(25), using the same concentration of (24) compounds to deal with HeLa cells, their survival rates were 100%, 100% and 65% did not show an obvious anti-tumor cell proliferation activity improved.③6/5/5/6 mother only nuclear structure, not connected to any side-chain compounds(23) with 1μmol/L, 10μmol/L and 100μmol/L to deal with HeLa cells, their survival rates were 100%, 93% and 73% of HeLa cells showed weak inhibitory activity, when the five carbon atoms in the introduction of fat-soluble compounds Cinnamoyl side chain(22), using the same concentration of (23) compounds to deal with HeLa cells, their survival rates were 100%, 64% and 32%, 10μmol/L and 100μmol/L group compared with blank control group was significant (P<0.05) and very significant differences (P<0.01), significantly improved the taxanes such compounds in anti-tumor cell proliferation activity.④6/10/6 mother only nuclear structure, not connected to any side-chain compounds(17) with 1μmol/L, 10μmol/L and 100μmol/L to deal with HeLa cells, their survival rates were 100%, 100 % and 97% of HeLa cells do not show any inhibitory activity; when five carbon atoms in the introduction of fat-soluble compounds Cinnamoyl side chain of (18) and Winterstein side chain alkaline compounds(19), using the same compound(17) the concentration of processing HeLa cells, their survival rates were 100%, 100%, 95% and 98%, 97%, 73%, did not show an obvious anti-tumor cell proliferation activity improved.3 When the mother of nuclear substituents on the compounds are acetyl-(8), with 100μmol/L to deal with HeLa cells, the survival rate dropped to 66%; when the mother has a nuclear hydroxy substituent taxanes(8) with 100μmol/L to deal with HeLa cells, the survival rate dropped to 51%; when the mother has two nuclear hydroxy substituent taxanes(9) deal with the same concentration of HeLa cells, the survival rate continued to decline 21%.4 The compounds(7) pairs of HEC-1 cells, T-98 cells and HMV-1 cell proliferation with inhibitory activity, IC50 were 32μmol/L, 37μmol/L and 23μmol/L, compounds(9) only HEC-1 cell proliferation with inhibitory activity, IC50 for 33μmol/L, compounds(22) pairs of HEC-1 cells, HOC-21 cells, T-98 cells and HMV-1 cell proliferation with inhibitory activity, IC50 were 58μmol/L, 15μmol/L, 11μmol/L and 36μmol/L.5 HeLa cell proliferation experiment results showed that: Taxanes 2α,10β-Diacetyl-5-cinnamoyl-phototaxicin II (22) interference with the role of tumor cell proliferation trends and very similar to Taxol (Figure 20), so that its to play the role of anti-tumor cell proliferation may be related to interfere with cell cycle-related.6 Confirmed by the experimental study of apoptosis, the compound(22) can inhibit tumor cell proliferation. Through apoptosis of tumor cells so as to reduce the number of tumor cells, caused by activation of caspase pathway of apoptosis Hela cells. And Bcl-2 family proteins are also involved in this apoptotic response, an increase of pro-apoptotic Bax protein expression, and inhibition of apoptosis inhibitory protein Bcl-2, in order to achieve its induced HeLa cell apoptosis.Conclusions: 1. With tricyclic 6/8/6 and 6/5/5/6 tetracyclic nucleus structure TAXANES with anti-tumor cell proliferation activity.2. Cinnamoyl side chains and Winterstein side chain, can increase tricyclic 6/8/6 and 6/5/5/6 tetracyclic nuclear structure of mother TAXANES anti-tumor cell proliferation activity.3. Mother's nuclear substituent hydroxy compounds for activity is higher than that for acetyl substituent of the compounds, and its activity is directly proportional to the amount of hydroxyl.4. Taxanes 22 through active of caspase pathway to apoptosis in Hela cells. By increasing the pro-apoptotic Bax protein expression, and inhibition of apoptosis inhibitory protein Bcl-2, to achieve its induced HeLa cell apoptosis.