The Anti-tumor Effect And Underlying Mechanisms Of Deacetyl-mycoepoxydiene:Both In Vitro And In Vivo Studies

BackgroundsCancer remains a serious threat of human health and life in the world. Chemotherapy is one of the most effective methods for cancer treatment. Natural products have become an important source of anticancer drugs. Deacety-mycoepoxydiene, a novel secondary metabolite produced by plant endophytic fungi Phomosis sp.,was confirmed to possess potential cytotoxic activities to various cancer cell lines.In our research, we investigate the anti-tumor mechanisms of deacetyl-mycoepoxydiene by in vitro and in vivo studies. Moreover, we also investigate the effect and mechanism of deacetyl-mycoepoxydiene on reversing resistance to taxol in tumor cells.Methods1. MTT assay was performed to evaluate the anti-tumor effect of deacetyl-mycoepoxydiene on a series of cancer cells. We chose human breast cancer cells MCF for further studies. The inhibitory effects of deacetyl-mycoepoxydiene on MCF-7 cells was estimated by the MTT method. DNA Ladder and flow cytometry assays were performed to determine the apoptosis cells and cell cycle arrest induced by deacetyl-mycoepoxydiene. Western blotting assay was carried out to analyze apoptosis related proteinscaspase-3、9,parp,p21, Bcl-2 and Bax. The efficacy of deacetyl-mycoepoxydiene in vivo was evaluated in MCF-7 xenografts mouse model. To examine the effect of deacetyl-mycoepoxydiene on microtubules by the methods of tubulin polymerization assay and immunofluorescence microscopy evaluation2. MTT colorimetric assay was employed to evaluate the cytotoxicity of DM to sensitive tumor cells A549 and drug resistant cells A549/T. The influence of DM on cellular taxol sensitivity was analyzed as well. The reversal fold was approximated by MTT, and the apoptotic percentage was estimated by flow cell cytometry (FCM). The accumulation of Rh123 in drug-resistance cells was observed in AnnexinV method. Additionally, the content of taxol and the expression of β-tubulin protein were respectively quantified by high performance liquid chromatography (HPLC) and Western blotting.Results1. We investigated the pro-apoptotic effect of deacetyl-mycoepoxydiene employing human cancer cells. The results of vitro cell culture experiments and human tumor xenografts showed that deacetyl-mycoepoxydiene can inhibit the growth of various human cancer cells both in vivo and in vitro. The anti-tumor mechanisms of deacetyl-mycoepoxydiene can be divided into the following aspects. (1) Deacetyl-mycoepoxydiene blocked cell mitosis and induced cell cycle arrest at G2/M phase in tumor cells, thus preventing cell growth. (2) We can also confirm that deacetyl-mycoepoxydiene promote microtubule polymerization both in cultured cells and vitro by the methods of immunofluorescence microscopy observation and tubulin polymerization assay. (3) Deacetyl-mycoepoxydiene reduced the expression levels of actin and interfered the function of cell cytoskeleton. (4) Deacetyl-mycoepoxydiene can modulate the expression levels of multiple related proteins(Caspase3、Caspase9、 P21、Bcl-2、Bax、Prp、Bcl-2-P)and induce the apoptosis of tumor cells, finally achieving anti-tumor effect.2.We investigated the effect and mechanism of deacetyl-mycoepoxydiene on reversing resistance to taxol in tumor cells.The results demonstrated that deacetyl-mycoepoxydiene enhanced the cellular taxol sensitivity and thus increased the apoptotic percentage of taxol-resitsant tumor cells.The content of taxol and Rh123 was greatly increased in deacetyl-mycoepoxydiene-treated A549/T cells. Moreover, Western blotting results showed that deacetyl-mycoepoxydiene inhibited the expression of β-tubulin protein in A549/T cells.ConclusionDeacetyl-mycoepoxydiene possesses strong anti-tumor effect against MCF-7 cells both in vitro and in vivo. Further study showed that the anti-tumor effect of deacetyl-mycoepoxydiene was associated with inhibiting microtubule and inducing cancer cell apoptosis via mitochondrial signaling pathway. Deacetyl-mycoepoxydiene could also induce cell cycle arrest at G2/M phase. Deacetyl-mycoepoxydiene can effectively reverse the multidrug resistance and increase the drug sensitivity of tumor cells. Therefore, deacetyl-mycoepoxydiene was a promising anti-cancer agent for cancer treatment.