Antitumor Activity Of YB-B1S And Its Mechanism

D-24851 is a novel synthetic compound that was identified in a cell-based screening assay to discover cytotoxic drugs. And it has undergoing phaseâ… clinical trial. D-24851 destabilizes microtubules and blocks cell cycle transition specifically at G2/M phase. In vitro, D-24851 has potent cytotoxic activity toward a panel of established human tumor cell lines. In vivo, oral D-24851 treatment induced complete tumor regressions (cures) in rats.Of importance is that the administration of curative doses of D-24851 to the animals revealed no systemic toxicity and neurotoxicity. A serious of novel derivatives of D-24851 were synthesized by Department of Pharmacy of Medical College of Chinese People's Armed Police Forces. MTT assay showed many compounds had notable cytotoxic activities against human cervica cancer cell line in vitro. Further more, we select one of these compounds YB-B1S to study its cytotoxic activity on kinds of cultured human cancer cell lines in vitro and inhibition on the growth of transplantable tumors in mice. Meanwhile we have investigated the mechanism and characteristic of the cytotoxic activity of the YB-B1S.1 Effects of YB-B1S on the proliferation of cancer cellsIn a series of experiments, five tumorigenic human cells, including human osteosarcoma cell (HOS),human lung adenocacinoma cell (A549), human cervical cancer cell (Hela), human ovarian carcinoma cell (SKOV3), and human breast carcinoma cell (MCF-7) were chosen to determine the cytotoxic activity of YB-B1S. After incubating for 96h, YB-B1S led to a gradual decrease of viable cells fraction with increasing concentrations. MTT assay showed that IC₅₀ was 2.0μmol/L (MCF-7) to 2.8μmol/L (HOS). Also, the results of cell growth curve and colony formation of cancer cells matched with the above results.2 Inhibition of tumor growth in vivo by YB-B1S The effect of YB-B1S on tumor growth was studied in KunMing mice using transplanted models with sarcoma S180. Intraperitoneal injection of YB-B1S (50mg/kg and 100mg/kg) was performed every day, followed with tumor cells inoculation. Mouse S180 sarcoma was sensitive to YB-B1S, 50mg/kg and 100mg/kg YB-B1S treatment resulted in 26.39% and 60.55% (P<0.05) inhibition of tumor growth compared with untreated control. Curative doses of YB-B1S were well tolerated with low or no systemic toxicity in terms of body weight loss in contrast to the administration of cyclophosphamide.3 Causing cell cycle arrest by YB-B1STo evaluate the possible role of cell cycle arrest in YB-B1S caused growth inhibition, Hela cells were treated with YB-B1S. Cell cycle distribution was evaluated by flow cytometric analysis after staining of cellular DNA with propidium iodide at the different concentrations, which indicated that YB-B1S induced an accumulation in G₂/M phase of cell cycle. After treatment with 0.5⁴.0μmol/L of YB-B1S for 24h, the number of cells in G₂/M phase was higher than that in untreated cells. The percentage of cells arrested in G2/M phase increased with the increasing of concentration.4 Inducing apoptosis by YB-B1SThe morphological changes induced by YB-B1S can be detected by Hoechest staining, which were characteristics of apoptosis. Contrast cells displayed excellent growth characteristics -polygonal shape with round large nucleus featuring prominent multiple nucleoli, and well spread on the growth surface. YB-B1S evoked typical apoptotic features such as membrane blebbing, cell shrinkage and detachment, and nuclear condensation and fragmentation.Flow cytometric analysis of Hela cells exposed to YB-B1S confirmed the morphological observations above. The DNA fluorescence histograms of PI-stained cells showed the low DNA stainability of the YB-B1S-treated apoptotic cells, which resulted in a distinct, quantifiable region below the G₁ 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 51.9%, after incubated in YB-B1S for 24h. The effect was also time-dependent. The proportions of apoptotic Hela cells incubated in YB-B1S for 24h were higher than in the same concentration for 12h.Finally, agarose gel electrophoresis showed typical DNA fragmentation pattern and confirmed the apoptosis induced by YB-B1S. DNA fragmentation caused by YB-B1S was dose-dependent. The intensity of DNA fragments increased as increasing amounts of YB-B1S (1.0⁴.0μmol/L) was added to the cells. As a positive control, cells were treated with D-24851 (0.1μmol/L, 24 h).5 Regulating expression of p53 mRNA,p21 mRNA,caspase-3 mRNA and Bcl-2 mRNA by YB-B1SExpression of p53 mRNA,p21 mRNA,caspase-3 mRNA and Bcl-2 mRNA in Hela cells exposed to YB-B1S was investigated by RT-PCR. Data showed that p53 mRNA,p21 mRNA and caspase-3 mRNA levels increased,meanwhile the expression of Bcl-2 mRNA decreased for 24h culturing in the present of YB-B1S.6 Effect of YB-B1S on the microtubular network.To determine whether YB-B1S could affect microtubule, the effects of YB-B1S on the microtubule cytoskeleton of Hela cells were investigated by fluorescence microscopy, because of their extensive and thinly spreading microtubule cytoskeleton. In untreated Hela cells, microtubules formed a fine extensive network throughout the cytoplasm that was generally aligned with the cell axis. By contrast, microtubules in 0.25μmol/L YB-B1S -treated Hela cells began to appear disrupted, in 1.0μmol/L, microtubules no longer formed organized bundles, and in 2.0μmol/L, microtubules were detected in only a diffuse punctuated pattern.From all above, we conclude that YB-B1S showed obvious anticancer activity by inducing apoptosis and causing cell cycle arrest in the dose-dependent manner, which were accompanied by upregulating P53,P21 and caspase-3 mRNA, downregulating Bcl-2 mRNA. YB-B1S also caused cell cycle arrest to G2/M phase in the dose-dependent manner, interfering with microtubule polymerization and disrupting cytoskeleton. Thus YB-B1S is a prospective novel anticancer drug.