Antitumor Activity Of YB-L12 And Its Mechanism

D-24851, as one of the derivatives of indol-3-glyoxylic acid amide, is a novel synthetic anti-mitotic agent that destabilizes microtubules polymerization and arrests tumor cell growth at the G2/M phase, whose binding site does not overlap with the tubulin-binding sites of the other conventional microtubule-destabilizing agents vincristine or colchicines. D-24851 is orally active in a panel of human, murine and rat tumor cell lines in vitro and in xenografts. And it also exhibits cytotoxicity towards multidrug-resistant tumor cells. Of great importance is that D-24851 does not show any significant hematological or neurotoxic effects at efficacious doses in animals and in Phase I clinical trials. According to these above, a series of novel derivatives of indol-3-glyoxylic acid amide were synthesized, with the introduction of several active chemical groups isolated from natural products, by Department of Pharmacognosy of Medical College of Chinese People's Armed Police Forces. MTT assay showed many derivatives had notable cytotoxic activities against human cervix cancer Hela cells and ovarian cancer SKOV3 cells in vitro. Further more, we selected one of these derivatives YB-L12 which contains the benzyl group of pepper amine to study its cytotoxic activity against a variety of cultured human cancer cell lines in vitro and inhibition effect on the growth of transplanted tumors in mice. Meanwhile we investigated the mechanisms and characteristics underlying the cytotoxic activity of YB-L12. Main results would be summarized as follows.1 Effects of YB-L12 on the proliferation of cancer cellsIn a series of experiments, nine tumorigenic human cells, including human cervical cancer cells (Hela), human ovarian carcinoma cells (SKOV3), human stomach carcinoma cells(BGC-823), human colon cancer cells(LoVo),human breast carcinoma cells(MCF-7), human osteosarcoma cells(HOS), human lung adenocacinoma cells (A549), human erythroleukaemia cells(K562) and multidrug-resistant human erythroleukaemia cells induced by Doxorubicin (K562/AO2) and two normal human cells, including human vascular endothelial cells(VEC) and human hypertrophic scar fibroblast(fb) were chosen to determine the cytotoxic activity of YB-L12. Results of MTT assay showed that IC50 ranged from 0.10μM (BGC-823) to 9.41μM (A549). Because of these variations, the antitumoral efficacy of YB-L12 was determined by tumor cells of different origins. The cytotoxicity was also evaluated by the sulforhodamine B (SRB) assay and the GI50 of YB-L12 to K562 and K562/AO2 was 1.70μM and 0.94μM respectively. The results showed YB-L12 exhibited comparative cytotoxicity towards multidrug-resistant tumor cells(K562/AO2) to K562. Moreover, the results of cell growth curve of cancer cells matched with the above results. 2 Inhibition on tumor growth in vivo by YB-L12The effect of YB-L12 on tumor growth was studied in KunMing mice using transplanted models with sarcoma S180. Intragastric administration of YB-L12 (75mg/kg and 150mg/kg) was performed every day after tumor cells inoculation, and last for 6 days. Mouse S180 sarcoma was sensitive to YB-L12, as 75mg/kg and 150mg/kg YB-L12 treatment resulted in 34.89% and 47.60% (P<0.01) inhibition of tumor growth respectively, compared with untreated control. Curative doses of YB-L12 were well tolerated with low or no systematic toxicity in terms of body weight loss in contrast to the administration of cyclophosphamide. Effects of YB-L12 in vivo showed that daily administration of YB-L12 at less than toxic doses reduced the tumor weight.3 Causing cell cycle arrest by YB-L12To evaluate the possible role of cell cycle arrest in YB-L12-caused growth inhibition, Hela cells were treated with YB-L12. Cell cycle distribution was evaluated by flow cytometric analysis after staining of cellular DNA with propidium iodide at different concentrations, which indicated that YB-L12 induced an accumulation in G2/M of cell cycle. After treatment with 0.2~0.8μM YB-L12 for 12h and 24h, the percentage of cells in G2/M phase was higher than that of untreated cells, and it increased with the increasing concentration.4 Inducing apoptosis by YB-L12 The morphological changes in apoptotic cell nuclei induced by YB-L12 were detected by Giemsa and Hoechest 33342 nuclear staining in Hela and SKOV3 cells and visualized using light or fluorescence microscopy. YB-L12 evoked typical apoptotic features such as nuclear condensation and fragmentation, membrane blebbing, cell shrinkage and detachment. While contrast cells exhibited excellent growth characteristics-polygonal shape with round large nuclei or multiple nuclei. Flow cytometric analysis of Hela cells exposed to YB-L12 confirmed the morphological observations above. The DNA fluorescence histograms of PI-stained cells showed the low DNA stainability of the apoptotic cells treated with YB-L12, which resulted in a distinct, quantifiable region in front of 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 via flow cytometry. The proportion of apoptotic Hela cells was 8.02% after incubated in YB-L12 with low concentration(0.2μM) for 12h and increased up to 11.6% with high concentration(0.8μM), which were higher than the proportion of control cells. After incubated in YB-L12 with the same concentrations above for 24h, the proportion of apoptotic cells also increased as the concentration increasing. In addition to the concentration-dependent manner, YB-L12 induced cell apoptosis also in a time-dependent manner. The proportion of apoptotic Hela cells incubated in YB-L12 for 24h was higher than that for 12h with the same concentration.Finally, the results of agarose gel electrophoresis showed typical DNA fragmentation pattern and confirmed again the apoptosis induced by YB-L12. And DNA fragmentation caused by YB-L12 was dose-dependent. The intensity of DNA fragments increased as increasing concentration of YB-L12 (0.2~0.8μM) applied to the Hela and SKOV3 cells. As a positive control, cells were treated with D-24851 (0.1μM, 24 h).5 Regulating the levels of p53 mRNA,caspase-3 mRNA,bax mRNA and bcl-2 mRNA by YB-L12The levels of p53 mRNA,caspase-3 mRNA,bax mRNA and bcl-2 mRNA in Hela cells exposed to YB-L12 were investigated by semiquantitative RT-PCR. Data showed that levels of p53 mRNA,caspase-3 mRNA and bax mRNA increased,meanwhile the level of bcl-2 mRNA decreased in a dose-dependent manner after 24h culturing in the presence of YB-L12.6 The effect of YB-L12 on the microtubule organization in mitotic spindles.To test whether YB-L12 affects the microtubule organization in mitotic spindles, we treated Hela cells with YB-L12 for 12h. Microtubules were then visualized by laser scanning confocal microscope using an anibody againstα-tubulin. In solvent-treated Hela cells, microtubules formed a fine extensive network throughout the cytoplasm that was generally aligned with the cell axis. In contrast, after treated with 0.2μM YB-L12, microtubules in Hela cells were mainly organized in mitotic spindle with abnormal structures which had extremely long astral microtubules. While with 0.8μM, fragmented mitotic spindles were observed, which were similar to the effects on Hela cells by exposure to D-24851(0.1μM) but different from those by exposure to paclitaxel (0.01μM). Hela cells treated with paclitaxel (0.01μM) were found to have at least three regions of accumulated microtubules but not fragmentation of microtubules, which is consistent with the stabilizing effect of paclitaxel on microtubules. These data indicate that YB-L12 destabilizes the microtubules of mitotic spindles in a concentration-dependent manner similar to D-24851.From all above, we conclude that YB-L12 showed obvious anticancer activity by inducing cell apoptosis and causing cell cycle arrest at G2/M phase in a dose-dependent manner, which were accompanied by upregulating the levels of p53,caspase-3 and bax mRNA and downregulating the level of bcl-2 mRNA and destabilizing the microtubules of mitotic spindles and interfering with mitoses of tumor cells. Thus YB-L12 is a prospective novel anticancer agent.