Antitumor Activty Of PAB-D1 And Its Mechanism

Department of Pharmacy of Medical College of Chinese People's Armed Police Forces found the PAB-D1 by screening from eleven compounds which were the derivatives of Pseudolaric acid B extracted from the traditional herbs, Pseudolarixamabilis (Nelson) Regd [p.kaempferiGord].The experiment results showed that PAB-D1 possessed obvious anticancer activity both in vivo and in vitro in a dose-dependent manner. Further more, we have studyded the cytotoxic activity on kinds of cultured human cancer cell lines in vitro, inhibition on the growth of transplantable tumors in mice. Meanwhile we have investigated the mechanism and characteristic of the cytotoxic activity of the PAB-D1. The results of MTT, SRB, cell growth curve and colony formation of cancer cells assay in vitro demonstrated that PAB-D1 showed potent cytotoxic effect on human galactophore cancer Hela cells line, human ovarian cancer cells Skov-3 line and human epithelium nonnasality cancer KB cells line,human former gastric cancer BGC-823 cells line,with IC50 of 1.26μmol/L and 0.78μmol/L, 3.98μmol/L, 0.84μmol/L. We could see from the results of SRB assay that PAB-D1 showed anticancer activity by the cytostatic effect when it was used in the low concentration, the GI₅₀ of PAB-D1 to Hela,Skov-3,and KB,BGC-823 was 0.59171 μmol/L,1.062μmol/L and 1.091μmol/L,1.254μmol/L. meanwhile,PAB-D1 showed anti-cancer effect through the cytotoxic effect when it was used in the high concentration, the LC₅₀ of PAB-D1 to the above four cell lines was 1.42789μmol/L,2.348μmol/L,1.176μmol/L and 1.281μmol/L. Furethermore, the results of cell growth curve and colony formation of cancer cells matched with the above results.Judging from the above experience results, we could indicate that PAB-D1 possessed the obvious anticancer effect in vitro. The studies in vivo revealed that PAB-D1 significantly inhibited the growth of transplantable tumors H22 hepatoma in mice. The inhibitory rate to H22 by continuative injection for 8 days through abdomen was 27.62% and 69.21% respectively at the dosages for 40 mg/kg and 80 mg/kg. It displayed that PAB-D1 also showed obvious anticancer activity in vivo. In order to investigate the mechanism of the anticancer activity of PAB-D1, we studied the change of the cell cycle of cancer cells, and the effects of PAB-D1 on the biosynthesis of the apoptosis-related DNA and protein expression , and so on. The apoptosis induced by PAB-D1 in Hela,Skov-3,and KB,BGC-823 cells was also reserached. The four cells were treated with PAB-D1(0.5⁴μmol/L) for 24 hours, with Hoechst 33342 labeling cells, we observed obviously morphological features such as nuclear chromatin segmentation and condensation as well as apoptosis bodies under the fluorescentmicroscopes to Hela,Skov-3,and KB,BGC-823 cells. a significant amount of intranucleosomal DNA fragmentation was detected by genomic DNA gel electrophoresis from the DNA extracted from Hela, Skov-3,and BGC-823 cells which were treated with PAB-D1 for 24 hours , which was featured as "DNA ladder". the typical "Sub-G1 peak"were also detected through flow cytometry in Hela,Skov-3 cells, after they were treated with PAB-D1 for 24 hours in different concentration (0.625¹0μmol/L). The height of "Sub-G1 peak"augmented following the increase of the concentration of PAB-D1 in a dose-dependent manner. The above results demonstrated that PAB-D1 might induce apoptosis of tumor cells significantly. The effect of PAB-D1 on the tumor cell cycle was also studied. After Hela,Skov-3 were treated with PAB-D1 (0.625¹0μmol/L) for 24 hours, the cells in G1 phase reduced significantly while the cells in S phase increased lightly, the number of cells in G2+M seemed raise obviously,so we could draw the conclusion that PAB-D1 blocked the tumor cells at G2+M phase and arrestd followed cell circle . The change of apoptosis-related genes was the prime event for tumor cells apoptosis generally. We explored whether the effects on apoptosis by PAB-D1 are dependent on caspase-3 and/or bcl-2 and/or p53 function.After Hela cells we...