| Epipolythiodioxopiperazines (ETPs), a group of important secondary metabolites from fungi, characterized by the bridged disulfide or polysulfide dioxopiperazine six membered ring, have been found to have wide range of bioactivities, such as antitumor, antimicrobial, antiviral immunosuppressive and enzyme inhibitory activities. It has been well recognized that disulfide or polysulfide functionalities are the key moiety for their bioactivities, and removal/cleavage of the sulfur bridge or addition of the reducing agent, e.g. dithiothreitol, usually will lead to complete loss of bioactivities. Due to its various types of novel structure and unique stereochemistry or skeleton, ETPs have drawn wide attention in recent years, especially for the antitumor activity.The present study focused on evaluating the antitumor activity of new chemical structure of eight kinds of fungi secondary metabolites (C80, C86, C87, 662, DR56-1, DR56-52, zw-2, DS56-10) in vitro, evaluating the antitumor activity of compound with more activity in vitro and in vivo, and investigating its mechanisms.METHODS:(1) Tumor cells were exposed to fungi secondary metabolites, cell viability was determined by sulforhodamine B (SRB) assay and the half growth inhibition (GI50) were calculated. (2) The anti-tumor efficacy of C87 in vivo was also measured in HeLa xenografts model in nude mice. (3) RTCA (Real Time Cellular Analysis) system was used to establish a real-time monitoring of tumor cell growth approach to evaluate the effect of compound C87 on cell proliferation. (4) After treatment with C87, the generation of ROS, cell cycle, the mitochondrial membrane potential of cells and apoptosis were measured by flow cytometry analysis. (5) HeLa cells were treated with C87 and DNA damage was measured by the number of AP sites. (6) Western blot was used to analyze the expression levels of the apoptotic associated molecules. Results:(1) SRB assay was employed to evaluate the anti-tumor efficacy of eight kinds of fungi secondary metabolites (C80, C86, C87,662, DR56-1, DR56-52, zw-2, DS56-10) against a panel of various tumor cell lines (including HCT116, HeLa, A549, SMMC7721, H460, H157, MDA-MB-231, H1993 cancer cell lines). The cell viability was significantly reduced following exposure to ETP compounds C80, C86, C87, which were stronger than others. (2) The cell viability was significantly reduced following exposure to C87 in a concentration-dependent and time-dependentmanner in tumor cells. C87 also caused potent inhibition of tumor growth in nude mice, yielding 33.22% of tumor growth inhibition. (3) The generation of ROS was increased after exposure to C87 in a concentration-dependent and time-dependent manner in tumor cells. Both of ROS generation and cell death induced by C87 could be blocked by NAC in tumor cells. (4) C87 could arrest cell cycle in G0-G1 phase in a concentration-dependent manner and up-regulate the expression level of p53 protein. (5) C87 could induce a concentration-dependent decrease in mitochondrial membrane potential. (6) C87 could induce apoptosis in a concentration-dependent and time-dependent manner and the apoptosis could be blocked by NAC. (7) C87 could induce cytochrome C release, stimulate activation of caspase-8、caspase-9, downstream caspase-3, and PARP cleavage in tumor cells.Conclusion:C87 screened from eight compounds inhibits the growth of various tumor cells in a concentration-dependent and time-dependent manner. The antitumor mechanisms may include:(1) C87 incurs cell growth inhibition or apoptosis by inducing a burst of ROS and intracellular oxidative stress and DNA damage; (2) C87 could arrest cell cycle in G0-G1 phase by up-regulating the expression level of p53 protein, leading to cell growth suppression. (3) By through destroying mitochondrial membrane potential and increasing membrane permeability, C87 induces release of cytochrome C, activation of Caspase-9, Caspase-8 and Caspase-3, PARP cleavage in tumor cells leading to tumor cell apoptosis. |
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