AIM: To investigate the role of TR3 in induction of apoptosis in gastric cancer cells. METHODS: Human gastric cancer cell line, MGC80-3, was used. Expression of TR3 mRNA and its protein was detected by Northern blot and Western blot. Localization of TR3 protein was showed by immunofluorescence analysis under laser-scanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among 1000 cells randomly. Stable transfection assay was carried out by Lipofectamine. RESULTS: Treatment of MGC80-3 cells with TPA and VP-16 resulted in apoptosis, accompanied by the repression of Bcl-2 protein in a time-dependent manner. At the same time, TPA and VP-16 also up-regulated expression level of TR3 mRNA in MGC80-3 cells that expressed TR3 mRNA. When antisense-TR3 expression vector was transfected into the cells, expression of TR3 protein was repressed. In this case, TPA and VP-16 did not induce apoptosis. In addition, TPA and VP-16-induced apoptosis involved in translocation of TR3. In MGC80-3 cells, TR3 localized concentrative in nucleus, after treatment of cells with TPA and VP-16, TR3 translocated from nucleus to cytosol obviously. However, when this nuclear translocation was blocked by LMB, apoptosis was not occurred in MGC80-3 cells even in the presence of TPA and VP-16. CONCLUSION: Induction of apoptosis by TPA and VP-16 is through induction of TR3 expression and translocation of TR3 from nucleus to cytosol, which may be a novel signal pathway for TR3, and represent the new biological function of TR3 to exert its effect on apoptosis in gastric cancer cells.
PKCs have been implicated in the regulation of cellular differentiation, proliferation, apoptosis and signal transduction. It was demonstrated in this study that PKC? was located both at mitochondria and in cytosol in gastric cancer cell line BGC-823. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the translocation of PKCα from both mitochondria and cytosol to nucleus as clearly shown by laser-scanning-confocal microscopy, while the protein level of PKCα was not changed by TPA treatment as detected by Western blot. The results also revealed that TPA-induced translocation of PKCα was in close association with apoptosis induction, and such association was further affirmed by other experiments where various apoptotic stimuli and specific inhibitors of PKC were used. Taken together, these findings indicate that translocation of PKCα from both mitochondria and cytosol to nucleus in gastric cancer cell is accompanied by induction of apoptosis, and may imply a new mechanism of the potential linking between cell apoptosis and PKCα translocation.
AIM: To investigate the roles of PLCγ2 and PKCα in TPA-induced apoptosis of gastric cancer cells.METHODS: Human gastric cancer cell line MGC80-3 was used. Protein expression levels of PLCγ2 and PKCα were detected by Western blot. Protein localization of PLCγ2 and PKCα was shown by immunofluoscence analysis under laserscanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among 1 000 cells randomly.RESULTS: Treatment of gastric cancer cells MGC80-3 with TPA not only up-regulated expression of PLC-γ2 protein,but also induced PLC-γ2 translocation from the cytoplasm to the nucleus. However, this process was not directly associated with apoptosis induction. Further investigation showed that PKCa translocation from the cytoplasm to the nucleus was correlated with initiation of apoptosis. To explore the inevitable linkage between PLC-γ2 and PKCα during apoptosis induction,PLC inhibitor U73122 was used to block PLC-γ2 translocation,in which neither stimulating PKCα translocation nor inducing apoptosis occurred in MGC80-3 cells. However, when U73122treated cells were exposed to TPA, not only PLC-γ2, but also PKCα was redistributed. On the other hand, when cells were treated with PKC inhibitor alone, PLC-γ2 protein was still located in the cytoplasm. However, redistribution of PLC-γ2 protein occurred in the presence of TPA, no matter whether PKC inhibitor existed or not.CONCLUSION: PLC-γ2 translocation is critical in transmitting TPA signal to its downstream molecule PKCα. As an effector,PKCα directly promotes apoptosis of MGC80-3 cells.Therefore, protein translocation of PLCγ2 and PKCα is critical event in the process of apoptosis induction.
Bing Zhang Qiao Wu Xiao-Feng Ye Su Liu Xiao-Feng Lin Mu-Chuan Chen, Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China Bing Zhang, Medical school, Xiamen University, Xiamen 361005, Fujian Province. China
