Aim: To investigate and determine the mechanism and signal pathway of tetradecanoylphorbol-1, 3-acetate (TPA) in degradation of RXRalpha.
Methods: Gastric cancer cell line, BGC-823 was used in the experiments. The expression level of RXRalpha protein was detected by Western blot. Nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation. Localization and translocation of RXRalpha were observed under laser-scanning confocal microscope through labeling specific anti-RXRalpha antibody and corresponding immunofluorescent antibody as secondary antibody. Different inhibitors were used as required.
Results: In BGC-823 cells, RXRalpha was expressed in the nucleus. When cells were treated with TPA, expression of RXRalpha was repressed in a time-dependent and TPA-concentration-dependent manner. Meanwhile, translocation of RXRalpha from the nucleus to the cytoplasm occurred, also in a time-dependent manner. When cells were pre-incubated with proteasome inhibitor MG132 for 3 hrs, followed by TPA for another 12 hrs, TPA-induced RXRalpha degradation was inhibited. Further observation of RXRalpha translocation in the presence of MG132 showed that MG-132 could block TPA-induced RXRalpha redistribution. Conversely, when RXRalpha translocation was inhibited by LMB, an inhibitor for blocking protein export from the nucleus, TPA could not repress expression of RXRalpha.
Conclusion: TPA could induce the degradation of RXRalpha protein in BGC-823 cells, and this degradation is time- and TPA-concentration-dependent. Furthermore, the degradation of RXRalpha by TPA is via a proteasome pathway and associated with RXRalphatranslocation from the nucleus to the cytoplasm.