Antitumor Activity Of PB-LY And Its Mechanism

Objective: Pseudorlaric acid-B(PLAB) is Pseudolarix'main active component. People find that the PLAB and its derivatives have anticancer activity. We got a lot of its derivatives by substituting PLAB with different group. PB-LY is one of derivatives of PLAB. We select PB-LY to study the antitumor activity on kinds of cu1tured human cancer cell lines in vitro, inhibition on the growth of transplantable tumors in Mouse. The experiment resu1ts showed that PB-LY possessed obvious anticancer activity both in vivo and in vitro in a dose-dependent manner. Meanwhile we have investigated the mechanism and characteristic of the anticancer activity of the PB-LY.Methods and Results: 1 PB-LY effects the proliferation of cancer cellsIn a series of experiments, seven tumorigenic human cells, including human cervical cancer cell (Hela), human ovarian carcinoma cell (SKOV3), human lung adenocacinoma cell (A549), human stomach carcinoma cell (BGC-823), human leukemia cell(K562), human breast adenocarcinoma cell (MCF-7) and human osteosarcoma cell (HOS) were chosen to determine the cytotoxic activity of PB-LY. After incubating for 96h, PB-LY led to a gradual decrease of viable cells fraction with increasing concentrations. MTT assay showed that IC50 was 1.35μmol/L (Hela) to 5.98μmol/L (K562). Resu1ts of SRB assay showed anticancer activity by the cytostatic effect when it was incubated in the low concentration, the GI50 of PB-LY to Hela was 0.94μmol/L, when it was incubated in the high concentration, the LC50 of PB-LY to the Hela cell lines was 10.21μmol/L . Further more, the resu1ts of cell growth curve matched with the above resu1ts.2 PB-LY inhibited tumor growth in vivoThe effect of PB-LY on tumor growth was studied in KunMing Mouse using transplanted models with sarcoma S180. Intraperitoneal injection of PB-LY (40mg/kg and 80mg/kg) was performed every day, followed with tumor cells inocu1ation. Mouse S180 sarcoma was sensitive to PB-LY, 40mg/kg and 80mg/kg PB-LY treatment resu1ted in 13.5% and 36.5% (P<0.05) inhibition of tumor growth compared with untreated control. Curative doses of PB-LY were well tolerated and little systemic toxicity as indicated by an increase in body weight.3 PB-LY caused cell cycle arrestTo evaluate the possible role of cell cycle arrest in PB-LY caused growth inhibition, Hela cells were treated with PB-LY. Cell cycle distribution was evaluated by flow cytometric analysis after staining of cellu1ar DNA with propidium iodide at the different concentrations, which indicated that PB-LY induced an accumu1ation in G₂+M phase of cell cycle. After treatment with 0.5⁴.0μmol/L of PB-LY for 24h, the number of cells in G2+M phase was higher than that in untreated cells. The percentage of cells arrested in G1 phase was obviously reduction.The percentage of cells arrested in G2+M phase increased with the increasing of concentration.4 PB-LY induced tumor cells apoptosisPB-LY induced morphological changes by Hoechest staining, which were characteristics of apoptosis. Contrast cells displayed excellent growth characteristics -polygonal shape with round large nucleus featuring prominent mu1tiple nucleoli, and well spread on the growth surface. PB-LY evoked typical apoptotic features such as membrane blebbing, cell shrinkage and detachment, and chromosome condensation and fragmentation.Agarose gel electrophoresis showed typical DNA fragmentation pattern and confirmed the apoptosis induced by PB-LY. DNA fragmentation caused by PB-LY was dose- dependent. The intensity of DNA fragments increased as increasing amounts of PB-LY (0.5⁴.0μmol/L) was added to the cells. As a positive control, cells were treated with paclitaxel.Finally, flow cytometric analysis of Hela cells exposed to PB-LY confirmed the morphological observations above. The DNA fluorescence histograms of PI-stained cells showed the low DNA stainability of the PB-LY-treated apoptotic cells, which resu1ted in a distinct, quantifiable region A0 peak. In contrast, the G1 peak predominated in control cells. Quantification of dose-dependency was done by monitoring the amount of nuclei with subdiploid DNA content with flow cytometry. The apoptotic Hela cells increased up to 34.5% after incubated in PB-LY for 24h. The effect was also time-dependent. The proportions of apoptotic Hela cells incubated in PB-LY for 24h were higher than in the same concentration for 12h, and lower than that for 36h.5 PB-LY regu1ated expression of P53 mRNA, P21 mRNA, Caspase-3 mRNA, Bax mRNA and Bcl-2 mRNAExpression of P53 mRNA, P21 mRNA, Caspase-3 mRNA, Bax mRNA and Bcl-2 mRNA in Hela cells exposed to PB-LY was investigated by RT-PCR. The resu1ts showed that P53 mRNA , Bax mRNA and Caspase-3 mRNA levels increased,meanwhile the expression of P21 mRNA, Bcl-2 mRNA decreased for 24h cu1turing Hela cell in the present of PB-LY.Conclusion: PB-LY showed obvious anticancer activity by inducing apoptosis and causing cell cycle arrest in the dose-dependent manner. PB-LY also caused cell cycle arrest to G₂+M phase in the dose-dependent manner. It influenced the apoptosis-related gene, which were accompanied by upregu1ating P53, Caspase-3 and Bax,downregu1ating P21 and Bcl-2.Thus PB-LY can induced the apoptosis of cancer cells. It wou1d be a prospective anticancer compound, which were derivated from the natural product.