The Anti-tumor Mechanism Of CPD As A Mcl-1Protein Inhibitor

Over-expression of Bcl-2pro-survival family proteins (Bcl-2、Mcl-1, Bcl-2-like proteins) can confer resistance to apoptosis in tumor cells. Previous studies revealed that the specificity and broad-spectrum against Bcl-2proteins of small molecules determine their efficient, broad and low resistant anti-tumor effect. Mcl-1, a short half-life protein, is highly expressed in human malignancies and play important anti-apoptotic function. So inhibition of Mcl-1expression can potently make Mcl-1-dependent cells more susceptible to apoptosis.In this thesis, experiments were carried out to study a small molecular compound (2E,4E)-2-cyano-5-phenylpenta-2,4-dienamide (CPD) synthesized by our group.In the experiments of tumor suppressoion, microscope observations showed that CPD can kill human breast adenocarcinoma cell line MCF-7effectively, without obvious harm to normal human epidermal cells. MTT and AV/PI assays indicated that CPD can induce apoptosis in MCF-7.Results of MTT and western blot suggested that the tumor suppression induced by CPD may be related with Mcl-1. In vitro FP and ELISA experiments showed that CPD can bind with Mcl-l(Kj=6nM) competitively compared to BH3peptide. Protein Nuclear Magnetic Resonance (NMR) experiments by our group indicated that CPD binds in the BH3-binding groove of Mcl-1protein; Isothermal Titration Calorimetry (ITC) data revealed more information of combination. CPD was identified as a Mcl-1inhibitor (nM).Results of western blot and co-ip assays revealed that CPD can induce disruption of Mcl-1/Bak interactions, releasing pro-apoptotic protein Bak and translocating pro-apoptotic protein Bax at1.5hours, which finally triggered cytochrome C release to execute apoptosis at2hours. It suggested that CPD-induced apoptotic signaling pathway is endogenous apoptotic pathway, which is mitochondrial apoptotic pathway. shRNA experiments testified that the apoptosis induced by CPD was dependent on Bax/Bak.Anti-tumor effects In vivo by H22xenograft mouse model assay was that CPD could suppress the H22solid tumors and extend a longer life time of mouse with H22tumor. ABT-737can not kill MCF-7cells with highly expressed Mcl-1effectively, but CPD synerized with ABT-737promoted apoptosis of MCF-7cells, and suggested a new method that can break the resistence of MCF-7to ABT-737caused by highly expressed Mcl-1. This study not only reveals the antitumor mechanism of a small molecular apoptosis-inducing agent CPD, but also provides an inhibitor antagonizing Mcl-1for the study of Bcl-2targeted combination therapy in the future.