The Study Of Anti-cancer Activity And Its Mechanism By Taxus Compounds From Taxus Cuspidata And Taxus Canadensis

Taxus cuspidata and Taxus canadensis are natural plants belonging to Taxus family.Paclitaxel（Taxol）isolated from Taxus is two terpenoids taxane composition because of its in clinically significant antitumor activity and the unique mechanism of action.However,as drug resistance,side effects,and many other problems remain on clinical application,therefore,it is so important to find stronger structure more simple analogue or alternative molecules.Currently researches around the compound taxanes structure-activity relationship and structural transformation are discussed increasingly.The present experiment investigated the effects of 16 purified taxanes compounds extracted from the Taxus plants on the proliferation of the human lung cancer cells.We screened out three kinds of compounds with obvious inhibitory activity and the mechanism of anti-proliferation of lung cancer cells was investigated in order to provide experimental basis and theoretical foundation for developing the high-efficiency and low-toxin drugs.Part one The inhibitory effects of 16 taxanes compounds on proliferation of human tumor cellsObjective:To observe the inhibitory effects of 16 taxanes--（1）7-deacetyltaxine B,（2）taxinine L,（3）5a,9a,10b,13a-tetrahydroxy-4（20）,11-taxadiene,（4）taxinine M,（5）taxuspine X,（6）2a,9a,10b-triacetyl-5-cinnamoyltaxicin I,（7）taxinine A,（8）2a,10b-diacetyl-5-cinnamoyl-phototaxicin II,（9）dicyclotaxane-5α,9α,13β-triol,（10）4a,10β,13a-triacetoxy-15-benzoyloxy-2a,20β-epoxy-11（15→1）abeotax-11-ene-5a,7β,9a-triol,（11）4a,7β,9a,10β,15-pentaacetoxy-2a,20β-epoxy-11（15→1）abeo-taxa-11-ene-5a,13a-diol,（12）7,9,10,13-tetraace-toxy-5-[3’-（N-formyl-N-methyl-amino）-3’-phenylpropanoyl]oxytaxa-4（20）,12-diene,（13）7,9,10,13-tetraace-toxy-5-[3’-（N-formyl-N-methyl-amino）-3’-phenylpropanoyl]oxytaxa-4（20）,12-diene、（14）2α,7β,9α,10β,13-pentaacetoxy-11β-hydroxy-5α-（2’-hydroxy-3’-N,N-dimethyl-amino-3’-phenyl）-propionyloxytaxa-4（20）,12-diene,（15）7β,9α,10β-triaceto-xytaxa-4（20）,12-diene-2α,5α,11β-triol and（16）4α,10β,13α-acetoxy-2α-benzoyloxy-7β,9α-epidioxy-5β,20-epoxytax-11-en-1β-ol on proliferation of human lung cancer cells;and to screen out the compounds with significantly inhibitory effects on its proliferation.Methods:The proliferation of human lung cancer cells which treated with 16 taxanes were monitored by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）assay.Cell survival was calculated from the percentage of surviving cells.The concentration effect curve of experimental compounds on the tumor cells was used to calculate the half inhibitory concentration(IC₅₀)of medicine.Results:（1）10mmol/L 16 kinds of compounds with the same concentration of taxanes in five kinds of human lung cancer cells（QG-90,PC-6,A549,RERF-LC-kj and QG-56）,compound 6,8 and 16 for PC-6,QG-90 and RERF-LC-kj cells all showed the inhibition of proliferation activity stronger than the positive control drug,cell survival rates were9.77%,10.37%,18.08%,7.97%and 16.38%,7.97%,7.97%,10.52%10.52%;,The proliferation inhibition activity of A549 and QG-56 cells was the same or lower than that of positive control paclitaxel,and the cell survival rate was 19.42%,28.80%,38.36%and 21.72%,30.28%and 2.96%,respectively.Other taxane compounds 1⁵,7 and 9¹5 showed lower or no inhibitory activity on the proliferation of five tumor cells.Conclusions:The first part of the experimental results show that the northeast yew and Canada branchlets and needles in the fields of separation and purification of 16 kinds of compounds,6,8 and 16 have significantly inhibit lung cancer cell proliferation activity,and its anticancer activity may have greater tension and its structure of C4,5,20 epoxy propane,and connecting with the C-five carbon atoms cinnamoyl side chain also has a strong correlation.Part two The mechanisms of inhibitory effects of taxanes compound on proliferation of human lung cancer cellsObjective:A large number of experiments in vivo and in vitro found that many ingredients in Chinese herbal medicine have obvious antitumor activity,and the effective components is accomplished by inducing tumor cell apoptosis.The first part of our study had confirmed that taxanes compounds inhibited the proliferation of human lung cancer QG-56 cells significantly.In this part,we explored the antitumor mechanism and identified whether taxanes exert its antitumor activity of cell proliferation through apoptosis.Methods:（1）Flow cytometry was used to detect the apoptosis of QG-56 cells after the treatment of compound 16 for 12 or 24 h at final concentration of 1μmol/L,which was determined by the method of AnnexinⅤ-FITC/PI stain.（2）Luciferase reporter gene detection system was used to detect the expression of tumor cell related genes after the detection of compound 16 in 24 h of QG-56 cells.（3）The expression of apoptosis protein p53 Bax bcl-2 and caspase-3 was detected by Western Blot method.Results:（1）Compound 16 apoptosis effect on QG-56 cells:1mmol/L compounds 16treated QG-56 cells after 12 h and 24 h,the cell apoptosis rate respectively,（10.97±1.89）%and（33.63±2.56）%,compared with the blank control group（7.41±7.41）%and（8.48±1.83）%,（P<0.05）;（2）1mmol/L three compounds taxanes 6 of 8 and 16 treated QG-56 cells after 24 h,16 compounds show obviously induced QG-56 cells with p53-dependent apoptosis signaling pathways related report gene pG13-Luc p21-Luc Bax-Luc and h Noxa-Luc expression,for other signaling pathways related report gene Ig K-Luc NFAT-Luc SRE-Luc and CRE-Luc did not show induction activity,and the other two compounds,genes of other report does not display the entrainment;（3）The expression of p53,Bax and caspase-3 protein was significantly increased after the treatment of QG-56 cells by 1mmol/L compound 16,and the p53 was increased to 1.58 times of the blank control.In addition,the number of Caspase-3 was increased to 1.98 times of Caspase-3,which caused the increase of Bax,decreased Bcl-2 to 4.17 times.（4）The cell survival rate increased from 81.65%,27.47%,2.96%to 84.79%,39.67%7.05%,respectively,after the use of caspase inhibitor with a final concentration of 20mmol/L and different concentrations of compound 16 in the lung tumor QG-56 cells.Conclusions:This part research results show that compound 16 on human lung cancer cells induced with QG-56 apoptosis and exert antitumor activity;Compound 16 induced QG-56 lung cancer cells apoptosis by raising the p53 expression,which further increases the expression of Bax,mitochondrial apoptosis raised by caspase-3 protein expression,this may be one of the apoptosis mechanism by taxane compounds;Caspase inhibitor can reverse the inhibitory effect of compound 16 on the proliferation of QG-56cells,and further elucidate that compound 16 induced apoptosis may be achieved through the caspase-3 signaling pathway.