RESUMO
Dietary phytochemicals as adjuvants have been suggested to play important roles in enhancing chemotherapeutic potential owing to multitargeted chemopreventive properties and lack of substantial toxicity. Here, we investigated the efficacy of the combined treatment of various phytochemicals with the anticancer drug clofarabine in malignant mesothelioma MSTO-211H cells and normal mesothelial MeT-5A cells. The combined treatment of resveratrol and clofarabine produced a synergistic antiproliferative effect in MSTO-211H cells, but not in MeT-5A cells. In MSTO-211H cells, the nuclear accumulation of Sp1 and the levels of p-Akt, Sp1, c-Met, cyclin D1, and p21 were effectively decreased by the combined treatment of them. In combination with clofarabine, the ability of resveratrol to reduce the contents of Sp1 and its target gene products was also evident in a time- and dose-dependent experiment. The inhibition of phosphoinositide 3-kinase using Ly294002 augmented a decrease in the p21 level induced by their combination, but it showed no significant effects on expression of Sp1 and cyclin D1. Taken together, the data provide evidence that the synergistic antiproliferative effect of resveratrol and clofarabine is linked to the inhibition of Akt and Sp1 activities, and suggest that this combination may have therapeutic value in treatment of malignant mesothelioma.
Assuntos
Nucleotídeos de Adenina/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Arabinonucleosídeos/farmacologia , Mesotelioma/tratamento farmacológico , Estilbenos/farmacologia , Nucleotídeos de Adenina/administração & dosagem , Arabinonucleosídeos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Clofarabina , Ciclina D1/metabolismo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Mesotelioma/metabolismo , Mesotelioma/patologia , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Resveratrol , Fator de Transcrição Sp1/metabolismo , Estilbenos/administração & dosagemRESUMO
We previously reported that MSTO-211H cells have a higher capacity to regulate Nrf2 activation in response to changes in the cellular redox environment. To further characterize its biological significance, the response of Nrf2, a transcription factor that regulates ARE-containing genes, on the synergistic cytotoxic effect of clofarabine and resveratrol was investigated in mesothelioma cells. The combination treatment showed a marked growth-inhibitory effect, which was accompanied by suppression of Nrf2 activation and decreased expression of heme oxygenase-1 (HO-1). While transient overexpression of Nrf2 conferred protection against the cytotoxicity caused by their combination, knockdown of Nrf2 expression using siRNA enhanced their cytotoxic effect. Pretreatment with Ly294002, a PI3K inhibitor, augmented the decrease in HO-1 level by their combination, whereas no obvious changes were observed in Nrf2 levels. Altogether, these results suggest that the synergistic cytotoxic effect of clofarabine and resveratrol was mediated, at least in part, through suppression of Nrf2 signaling.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proliferação de Células/efeitos dos fármacos , Mesotelioma/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Nucleotídeos de Adenina/administração & dosagem , Apoptose/efeitos dos fármacos , Arabinonucleosídeos/administração & dosagem , Western Blotting , Clofarabina , Regulação para Baixo , Sinergismo Farmacológico , Heme Oxigenase-1/metabolismo , Humanos , Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Resveratrol , Estilbenos/administração & dosagem , Células Tumorais CultivadasRESUMO
The nuclear factor erythroid-derived 2 related factor 2 (Nrf2)/heme oxygenase (HO)-1 induction plays cytoprotective roles against oxidative injury, apoptosis, and anticancer therapy; however, little is known about its regulation in human mesothelioma MSTO-211H cells. In this study, we investigated Nrf2/HO-1 induction in response to sulforaphane and determined the signaling pathways involved in this process. Sulforaphane treatment decreased cell viability and triggered a rapid and transient increase in the intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) prevented sulforaphane-induced cytotoxicity. Erk1/2 was activated within 1h of sulforaphane addition, whereas Akt phosphorylation was suppressed until the first 8h, and was then maintained at an elevated level until 72h, displaying a biphasic regulatory feature. Nrf2 protein levels in both nuclear and whole cell lysates were increased after sulforaphane treatment and were decreased by pretreatment with NAC, actinomycin D and cycloheximide. Activation of the Nrf2/HO-1 system after sulforaphane treatment was suppressed by pretreatment with NAC or Ly294002, a PI3K inhibitor. Knockdown of Nrf2 with siRNA decreased cell viability and attenuated sulforaphane-induced HO-1 up-regulation. Overall, our results indicate that ROS generation and/or activation of PI3K/Akt signaling regulate cell survival and Nrf2-driven HO-1 expression in sulforaphane-treated MSTO-211H cells.
Assuntos
Mesotelioma/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Isotiocianatos , Fator 2 Relacionado a NF-E2/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Sulfóxidos , Tiocianatos/administração & dosagem , Tiocianatos/toxicidadeRESUMO
N-Acetylcysteine (NAC), sulfidryl-containing thiol antioxidant, has been heralded as chemopreventive agent, generally because of its ability to scavenge free radicals. It also suppresses the proliferation of many cancer cells; however, the antiproliferative mechanism(s) remain to be fully elucidated. In this study, we investigated a growth-suppressive mechanism of NAC action in androgen-independent prostate carcinoma PC-3 cells. NAC (≥ 1mM) inhibited the proliferation of PC-3 cells in a dose- and time-dependent manner. Moreover, NAC treatment suppressed the activation of NF-κB induced by IKK-ß as detected by the NF-κB reporter gene assay. NAC exerted a biphasic effect on the intracellular ROS levels depending on incubation time; the antioxidant effect was seen within 2h after NAC treatment, however, a pro-oxidant effect was evident after 48 h treatment. In addition to these effects, NAC treatment elicited a dose- and time-dependent increase in the Cyr61 expression that was accompanied by an increase in its mRNA and blocked by cycloheximide pretreatment. Importantly, NAC treatment caused an early but transient activation of Akt and Erk1/2. The NAC-induced increase in Cyr61 protein levels was suppressed by the PI3K inhibitor (Ly294002) and, to a lesser extent, MEK/Erk1/2 inhibitor (PD98059). Taken together, our data suggest that the antiproliferative effect of NAC is partially mediated by intracellular ROS production, the inhibition of NF-κB activity, and the activation of PI3K- and/or MEK/Erk-related intracellular signaling pathways, which lead to up-regulation of Cyr61 expression.
Assuntos
Acetilcisteína/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteína Rica em Cisteína 61/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Regulação para Cima/efeitos dos fármacos , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/prevenção & controle , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteína Rica em Cisteína 61/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Masculino , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/prevenção & controle , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Cadmium is a well-known toxic metal and occupational exposure to it is associated with lung cancer. In probing the possible non-genotoxic molecular targets of cadmium-induced nasal toxicity, we performed an mRNA differential display analysis for cadmium-treated human nasal septum carcinoma RPMI-2650 cells. Cadmium (≥ 0.5 µM) inhibited the cell proliferation. The intracellular ROS levels were induced by cadmium treatment. In addition, cadmium elicited the AKR1C3 expression. The cadmium-induced increase in AKR1C3 protein levels was suppressed by N-acetylcysteine (NAC) and, to a lesser extent, PI3K inhibitor (Ly294002). Cells pretreated with Ly294002 were more resistant to cadmium toxicity than control. The increase in AKR1C3 protein level was accompanied by an increase in the nuclear transcription factor Nrf2. Overall, our data suggest that cadmium-induced ROS cause up-regulation of AKR1C3 expression, at least partially via the activation of PI3K-related intracellular signaling pathways, and Nrf2 activation, thereby contributing to an adaptive intracellular response to cadmium toxicity.