Antitumor Effects And Mechanism Of Dicitrinone B On Human Malignant Melanoma (A375)

Natural products has always been an important resource of anticancer drugs, marine microorganisms might activate some silent genes and induce some unique biosynthetic pathways to produce a lot of natural products with new structure and good biological activity to adapt to high salt, high pressure, oligotrophic, cold and other unique physiological environment, So natural products from ocean has become the hotspot in recent years. In this study, we fermented a marine fungus called Penicillium citrinum which collected from Fujian Langqi Island in large scale. From its secondary metabolites, we isolated and purified Dicitririone B, a rare carbon-bridged citrinin dimer which has reported to have antitumor effects on tumor cells previously. Using extraction、column chromatograph、high performance liquid chromatography combined with a variety of means of spectroscopic methods including NMR spectroscopy, mass spectrometry, we purified and identified Dicitrinone B from its secondary metabolites, a rare carbon-bridged citrinin dimer which has reported to have antitumor effects on tumor cells previously.To evaluate the antimumor activity of Dicitrinone B comprehensively in vitro, twenty tumor cell lines derived from different types of tumors were used for evaluating cell growth inhibition, the results shows that different tumor cell lines had different levels of proliferation inhibition after treated with Dicitrinone B. The IC50 values of the three most sensitive cell lines including malignant melanoma cell lines A375, breast cancer cell lines SK-BR-3 and MCF-7 were 13.38 μM,14.28 μM and 15.70 μM, respectively. so the most sensitive cell A375, was chosed as target cell line for further study while the first-line clinical anticancer drugs 5-fluorouracil (5-Fu) was used as a positive control drug. WST-1 method showed Dictrinone B treatment inhibits A375 cells growth in a dose-and time-dependent manner; Hoechst 33258 staining and AO/EB staining showed typical chromatin condensation after Dicitrinone B treatment, and appeared typical apoptotic nuclear morphology; PI staining suggested that Dicitrinone B might inhibit A375 cells growth by blocking cells in the G2/M phase and the proportion of sub-G0/G1 cells, an important hallmark of apoptotic cells, significantly increased to 57.29% when the concentration of Dictrinone B reached to 20μM. Annexin-V/PI double staining further showed that inducing apoptosis was the way of A375 cells death and the induction of apoptosis presented in a dose-dependent manner while the apoptosis rate resented a little change than blank control group under the treatment by 5-Fu.Base on these study, we researthed the mechanism of the Dicitrinone B-inducing apoptosis in-depth. Using ROS-sensitive fluorescence dye DCFH-DA showed that treatment of A375 cells with Dicitrinone B led to increased ROS accumulation in a dose-dependent manner. JC-1 assay was applied to test the effects of Dicitrinone B on mitochondrial membrane potential and results revealed that Dicitrinone B concentration-dependently reduces mitochondrial membrane potential, western blot assay showed that Dicitrinone B pretreatment could up-regulating the expression of Bax, down-regulating the expression of Bcl-2 and activated caspase-9, caspase-8, caspase-3 and cleaved-PARP, indicated that the apoptosis was associated with both intrinsic and extrinsic apoptosis pathways, And the observation that apoptosis could be reversed by pan-caspase inhibitor Z-VAD-FMK allow us to conclude the involvement of caspase-dependent pathways. Furthermore, the results from real-time quantitative PCR were consistent with the results from western blot in the expression of Bax and Bcl-2. All the study indicated that dicitrinone B induced apoptosis, at least in part, mediated by two apoptosis pathways. The first one was through the ROS-related intrinsic mitochondrial pathway, and the second one was through the extrinsic apoptotic pathway.In summary, the present study indicate that Dicitrinone B which derived from Penicillium citrinum has better antitumor activity in vitro and could induce A375 cells apoptosis through different signaling pathways. so might serve as a potential antitumor agent.