The Apoptotic Role Of The Mcl-1 Inhibitor UMI-77 On Gasric Cancer Cells

Background & Objective:Gastric cancer is one of the most common gastrointestinal malignancies with high morbidity and mortality in China. Chemotherapy is one of routine therapies for advanced and postoperative patients. The cure rate is still low, mainly because of the resistance of gastric cancer cells to apoptosis induced by chemotherapeutic drugs. Apoptosis happens in many physiologic conditions under strict control via multiple genes and signal pathways. Uncontroled apoptosis is closely implicated in the occurrence and development of tumors. Bcl-2 family proteins are key molecules that regulate endogenous apoptosis. One of these proteins, Mcl-1, is an important regulator with anti-apoptotic effect, and many chemicals has been developed to target this molecule for the treatment of tumor occurrence. In the present study, we investigated the pro-apoptotic role of UMI-77, a small molecule. Mcl-1 inhibitor, on gastric cancer cells in vitro, and hope that our results will provide more ideas for future clinical application of the small molecule inhibitor.Method:1. Pro-apoptotic role of UMI-77 on gastric cells:Two cell lines of gastric cancer MGC-803 and HGC-27, and the gastric epithelial cells GES-1 were treated with UMI-77 in different concentrations. MTS was used to detect cell survival and proliferation, and crystal violet staining for colony formation. The apoptotic rates were determined by flow cytometry using Annexin V/PI staining.2. Mechanistic Analyse of UMI-77’s effect on apoptosis in MGC-803:The activation of caspase-9, caspase-3 and cleavage of PARP, the protein level of Bcl-2, Bcl-XL and Mcl-1, and the release of Cytochrome C and Smac from motichondria were detected by Western blot. Mitochondrial membrane potential was determined by JC-1 staining with a flow cytometer. The effect of the caspase inhibitor z-VAD-fmk on UMI-77-induced apoptosis was assessed with MTS.3. Effect of Mcl-1 silencing on UMI-77-induced apoptosis:McL-1 silencing was conducted using small interfering RNA technique. Chemically synthesized Mcl-1 siRNA was transfected into MGC-803 cells using Lipofectamine 2000 Reagent, and the efficacy of gene silencing was confirmed by Western blot analysis. The expressed mRNA of Mcl-1, Bcl-2 and Bcl-XL were detected by qRT-PCR. Apoptotic rates of UMI-77 treated MGC-803 cells after Mcl-1 silencing were assessed with MTS.Results:1. UMI-77 treatment inhibited the cell survival, proliferation and clone formation in all the 3 cell lines, with higer inhibition rate in MGC-803 cells than in the other 2 cell lines. Flow cytometry anlysis showed that the UMI-77 obviously induced characteristic changes of apoptosis in MGC-803 cells, while only weak changes were observed in HGC-27 cells. Apoptosis induced by UMI-77 is time-and dose-dependent.2. Nomal mitochondrial membrane potential was observed to be destroyed after UMI-77 treatment. Moreover, releases of Cyt C and Smac from mitochondria into the cytoplasm were increased, and activation of caspase-9, caspase-3 and cleavage of PARP occurred after 24 h post the treatment. Furthermore, the caspase inhibitor z-VAD-fmk partially suppressed UMI-77-induced cell death. The expression levels of Bcl-2 and Bcl-XL were not altered when exposure to UMI-77, while Mcl-1 was down-regulated after UMI-77 after 24 h post the administration.3. Transfection with Mcl-1 siRNA successfully decreased the expression level of Mcl-1 in MGC-803 cells as shown by Western blot. Silencing Mcl-1 partially blocked the apoptosis induced by UMI-77.Conclusions:The examined 3 gastric cell lines displayed different sensitivity to UMI-77, with higher apoptotic rate in MGC-803 than in HGC-27 and GES-1. Changes in mitochondrial membrane potential and increased releases of Cyt C and Smac demonstrated that UMI-77 promoted apoptosis via the endogenous pathway. Higher expression of Mcl-1 in MGC-803 than in HGC-27 and GES-1 might explain the discrepancy in the UMI-77 sensitivity. Mcl-1 silencing partially abrogated apoptosis induced by UMI-77, indicating that mechanisms other than Mcl-1 are also involved in UMI-77-induced apoptosis of gastric cells.