| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 95 === Bufalin and cinobufagin, the Chan su extracts, are administered to treat patients with congestive heart failure. Bufalin and cinobufagin have been known to induce cell apoptosis in several cancers. The present study evaluated the direct effects of bufalin and cinobufagin on aldosterone secretion, cell proliferation, apoptosis and the mechanisms in human adrenocortical carcinoma cells (NCI-H295 cells). The H295 cells were incubated with bufalin and cinobufagin. The concentrations of aldosterone in medium samples were measured by radioimmunoassay. The protein expressions of cholesterol side-chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein in H295 cells were analyzed by the method of Western blot. The cell proliferation or apoptotic effects of bufalin and cinobufagin on H295 cells were studied by WST-1 assay or protein expression of poly- (ADP-ribose) polymerase ([PARP]). The role of extracellular signal-regulated kinases (ERK1/2) in the effects of bufalin and cinobufagin on aldosterone secretion and apoptosis was studied by the inhibitor of MEK (U0126). The binding of transcription factors (SF-1) to regions of the StAR promoter was analyzed by electrophoretic mobility shift assay (EMSA). The data was expressed as mean±SEM, and analyzed by the analysis of variance and then multiple-range test. Our results demonstrated that bufalin and cinobufagin (10-7 M, 24 h) markedly inhibited aldosterone secretion, StAR protein and SF-1 protein expression in H295 cells. Therefore, we suggested that bufalin and cinobufagin reduced aldosterone secretion via the inhibitions of SF-1 and StAR protein expression. Treatment of bufalin and cinobufagin produced a similar effect on cell proliferation as compared with the effect of bufalin and cinobufagin on aldosterone release. The protein expression of cleaved PARP, a substrate of caspase-3, was increased by bufalin or cinobufagin treatment. Bufalin treatment increased the protein expression of phospho-ERK1/2, and U0126 (a MEK inhibitor) inhibited this bufalin-induced increase in phospho-ERK1/2 in H295 cells. Furthermore, the bufalin-decreased StAR protein expression and SF-1 binding as well as increased PARP cleavage were reversed by the treatment of U0126. These results showed that the effects of bufalin on StAR protein expression and apoptosis were associated with the activation of ERK1/2 cascade. In other words, ERK cascade might play an inhibitory role in aldosterone steroidogenesis and cell survival of H295 cells. These effects of bufalin or cinobufagin on H295 cells also provide a new idea in the treatment of human adrenocortical carcinoma in the future.
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