RESUMO
Previous studies have demonstrated that calycosin is a natural phytoestrogen with a similar structure to estrogen, which can inhibit cell proliferation and induce apoptosis in a variety of tumors. Calycosin exerts potential pharmacological effects on osteosarcoma cells by inducing apoptosis. The aim of the present study was to elucidate the specific molecular mechanism of calycosininduced apoptosis in osteosarcoma cells. Cell proliferation was determined by an MTT assay. Annexin V/PI and JC1 staining were used to detect apoptosis and mitochondrial dysfunction, respectively, by flow cytometry. Western blot analysis was used to detect the expression of caspases or mitochondrial proteins. The results revealed that calycosin reduced the cell viability of human osteosarcoma 143B cells, induced apoptosis and increased the loss of mitochondrial membrane potential (MMP). In addition, calycosin increased the expression of the proapoptotic antiapoptotic proteins cleaved caspase3, cleaved caspase9, cleaved poly(ADPribose) polymerase and Bcl2associated X protein (Bax), and decreased the expression of the antiapoptotic proapoptotic protein Bcell lymphoma2 (Bcl2), thus altering the Bax/Bcl2 ratio. In addition, the expression levels of cytochrome c were markedly decreased in the mitochondria and increased in the cytoplasm following calycosin treatment. Furthermore, calycosin treatment induced p38mitogenactivated protein kinase (MAPK) phosphorylation, whereas the p38MAPK inhibitor BIRB 796 markedly reversed cell viability, apoptosis and loss of MMP in 143B cells. These results suggested that calycosin inhibited osteosarcoma 143B cell growth via p38MAPK regulation of mitochondrialdependent intrinsic apoptotic pathways.
Assuntos
Neoplasias Ósseas/metabolismo , Isoflavonas/farmacologia , Mitocôndrias/metabolismo , Osteossarcoma/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Previous studies have shown that calycosin, a natural phytoestrogen which is structurally similar to estrogen, inhibits proliferation and induces apoptosis in estrogendependent cancer types via the estrogen receptor (ER)ßinduced inhibition of PI3K/Akt. Therefore, the aims of the present study were to investigate the effects of calycosin on human osteosarcoma (OS), and to examine the molecular mechanisms associated with ERß. Human OS MG63 cells were treated with various concentrations of calycosin, and MTT and flow cytometry assays were used to assess the effects of calycosin on cellular proliferation and apoptosis. In addition, protein expression levels of ERß, phosphorylated (p)PI3K, pAkt, cleaved poly (ADPribose) polymerase 1 (PARP) and cleaved caspase3 were evaluated by western blot analysis. The present results suggested that calycosin inhibited proliferation and induced apoptosis in MG63 cells. Furthermore, increased ERß expression was detected in OS MG63 cells treated with calycosin, and an ERß inhibitor (PHTPP) reversed calycosininduced cytotoxicity and apoptosis. Moreover, phosphorylation levels of PI3K and Akt were significantly downregulated after calycosin treatment, whereas PHTPP reversed their phosphorylation. ERßmediated PI3K/Akt downstream signaling pathways were found to influence the activity of poly (ADPribose) polymerase 1 and caspase3. Thus, the present results indicated that calycosin inhibited proliferation and induced apoptosis in OS MG63 cells, and that these effects were mediated by ERßdependent inhibition of the PI3K/Akt pathways.
Assuntos
Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Receptor beta de Estrogênio/metabolismo , Osteossarcoma/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/uso terapêutico , Receptor beta de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/genética , Humanos , Isoflavonas , Osteossarcoma/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Pirazóis , PirimidinasRESUMO
Rapamycin is known to inhibit the mammalian target of rapamycin complex (mTORC)1 signaling pathway, but it is unable to effectively inhibit mTORC2, resulting in activation of protein kinase B in multiple myeloma (MM) cell lines. Additionally, certain studies have suggested that resveratrol has an effect on human MM cells, and that rapamycin in combination with resveratrol may be useful in cancer therapy. The present study aimed to investigate the combined treatment effect of resveratrol and rapamycin on the MM MM1.S cell line. The results demonstrated that combined treatment with rapamycin and resveratrol effectively inhibited cell viability in the MM1.S cell line through inhibition of the mTORC1 and mTORC2 signaling pathways, compared with resveratrol or rapamycin monotherapy. In addition, cyclin D1 levels were decreased and the activation of caspase-3 and poly (ADP-ribose) polymerase was increased. These results suggested that downregulation of the mTOR signaling cascades is likely to be a crucial mediator in the impairment of viability and the induction of apoptosis resulting from combined therapy with resveratrol and rapamycin in MM1.S cells.