Anti-MDR Tumor Cells Activities Of ZM-16 And Its Mechanism Of Action

Objective: This paper studied ZM-16 which was one of the novel derivatives of podophyllotoxin and its cytotoxic activity against a variety of cultured human cancer cell lines in vitro. Mechanism of anti-multidrug resistance of ZM-16 was studied preliminary for development of novel anti-tumor drug.Methods:1 The suppressive effect of ZM-16 on the proliferation of Hela, Skov3, Lovo, KB, KBv200, K562 and K562/A02 tumor cells were evaluated in vitro with MTT and SRB assays.2 The apoptosis of multidrug resistance cell K562/A02 and KBv200 induced by ZM-16 was studied by Gimsa staining, Hoechst33342/PI staining, DNA agrose gel electrophoresis and flow-cytometry (FCM) technique.3 The mRNA expression of apoptosis associated genes such as: p53, p21, caspase, bcl-2, bax and multidrug resistance related genes such as: mdr-1, topoⅡα, topoⅡβwere investigated by using semi-quantity RT-PCR technique.4 The expression of P-gp in K562/A02 cells was quantified by Western-blot and localized by immunohistochemistry technique, then the mechanism of anti-multidrug resistance of ZM-16 was analysised. 55 The influence of ZM-16 on tubulin of KBv200 cells was observed by laser confocal microscopy.Results:1 Results of MTT assay showed that ZM-16 significantly inhibits the growth of cancer cells. The IC50 value of ZM-16 ranged from 0.43μM (Skov3) to 20.95μM (Lovo). Human erythroleukaemia cells(K562) and multidrug-resistance human erythroleukaemia cells induced by Doxorubicin (K562/A02) were chosen to determine the cytotoxic activity of ZM-16. The cytotoxicity was evaluated by the sulforhodamineB (SRB) assay and the GI50 of ZM-16 to K562 and K562/A02 was 4.39μM and 2.28μM respectively. Moreover, the results of cell growth curve of K562/A02 cells matched with the above results.2 After treated with ZM-16, the morphological changes in apoptotic cell nuclei were detected by Giemsa staining and Hoechest 33342/PI nuclear staining in K562/A02 cells. ZM-16 evoked typical apoptotic features such as nuclear condensation and fragmentation, cell shrinkage and detachment. While control cells exhibited excellent growth characteristics round shape with round large nuclei. The results of agarose gel electrophoresis showed typical DNA fragmentation pattern and confirmed again the apoptosis in KBv200 cells induced by ZM-16. Flow cytometric analysis of K562/A02 cells exposed to ZM-16 confirmed the morphological observations above. The DNA fluorescence histograms of PI-stained cells showed the low DNA stainability of the apoptotic cells treated with ZM-16. Cells in the sub-G1 population, an indication of the number of apoptotic cells present, were detected by cell cycle analysis with flow cytometry. Quantification of dose-dependency was done by monitoring the amount of nuclei with subdiploid DNA content via flow cytometry. After incubated in ZM-16 with the different concentrations, the proportion of apoptotic K562/A02 cells also increased as the concentration increasing. These results indicated that ZM-16 induced K562/A02 cell apoptosis.3 To evaluate the possible role of cell cycle arrest in ZM-16 caused growth inhibition, K562/A02 cells were treated with ZM-16 for 6h and 24h. Cell cycle distribution was evaluated by flow cytometric analysis after staining of cellular DNA with propidium iodide at different concentrations, which indicated that ZM-16 caused G2/M arrest.4 The data of semiquantitative RT-PCR showed that levels of p53, p21 and caspase-3 mRNA increased, meanwhile the level of bcl-2 and mdr-1 mRNA decreased in a dose-dependent manner after culturing in the presence of ZM-16. The compound did not have obviously effect transcription of bax, topoⅡαand topoⅡβmRNA.5 Expression of P-gp in K562/A02 cells which were exposed to different concentration of ZM-16 was investigated by western-blot and immunocytochemistry assays. Data showed that P-gp protein levels decreased(P<0.01, compare with control group) for 48h culture in the present of ZM-16 in a concentration-dependent manner. 6 After KBv200 cells treated with ZM-16 for 6h, microtubules were visualized by laser scanning confocal microscope. In solvent-treated KBv200 cells, microtubules formed a fine extensive network throughout the cytoplasm that was generally aligned with the cell axis. In contrast, after treated with 1μM ZM-16, microtubules in KBv200 cells were mainly organized in mitotic spindle with abnormal structures which had extremely long astral microtubules. While with 4μM ZM-16, fragmented mitotic spindles were observed, which were similar to the effects on KBv200 cells by exposure to podophyllotoxin (1μM).Conclusions: ZM-16 showed obvious anticancer activity and anti-multidrug resistant cancer activity by inducing cell apoptosis and causing cell cycle arrest at G2/M phase in a dose-dependent manner, which were accompanied with upregulating the levels of p53, p21, caspase-3 mRNA and downregulating the level of bcl-2, mdr-1 mRNA and destabilizing the microtubules of mitotic spindles and interfering with mitoses of tumor cells.Thus ZM-16 is a prospective novel anti-multidrug resistant cancer agent.