RESUMO
Inhibitor-of-apoptosis protein (IAP) inhibitors have been reported to synergistically reduce cell viability in combination with a variety of chemotherapeutic drugs via targeted cellular IAP (cIAP) depletion. Here, we found that cIAP silencing sensitised colorectal cancer (CRC) cells to selenite-induced apoptosis. Upon selenite treatment, the K63-linked ubiquitin chains on receptor-interacting protein 1 (RIP1) were removed, leading to the formation of the death-inducing complex and subsequent caspase-8 activation. Although the ubiquitinases cIAP1 and cIAP2 were significantly downregulated after a 24-h selenite treatment, cylindromatosis (CYLD) deubiquitinase protein levels were marginally upregulated. Chromatin immunoprecipitation assays revealed that lymphoid enhancer factor-1 (LEF1) dissociated from the CYLD promoter upon selenite treatment, thus abolishing suppression of CYLD gene expression. We corroborated these findings in a CRC xenograft animal model using immunohistochemistry. Collectively, our findings demonstrate that selenite caused CYLD upregulation via LEF1 and cIAP downregulation, both of which contribute to the degradation of ubiquitin chains on RIP1 and subsequent caspase-8 activation and apoptosis. Importantly, our results identify a LEF1-binding site in the CYLD promoter as a potential target for combinational therapy as an alternative to cIAPs.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Proteínas Inibidoras de Apoptose/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ácido Selenioso/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteína 3 com Repetições IAP de Baculovírus , Sítios de Ligação , Caspase 8/genética , Caspase 8/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Enzima Desubiquitinante CYLD , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Proteínas Inibidoras de Apoptose/genética , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
It has previously been shown that selenite can act as an antitumor agent and inhibit cancer cell growth, although the mechanism responsible for this effect is not well understood. In this study, we have shown that selenite can induce cell cycle arrest and apoptosis in NB4 cells. Selenite treatment of these cells also inhibited the JNK/ATF2 axis. Further experiments demonstrated that selenite-induced production of reactive oxygen species (ROS) worked as an upstream of the JNK/ATF2 axis, cell cycle arrest and apoptosis. Inactivation of ATF2 resulted in decreased affinity of this transcription factor for the promoters of cyclin A, cyclin D3 and CDK4, which led to the arrest of the NB4 cells in the G0/G1 phase. Finally, in vivo experiments confirmed the antitumor activity of selenite and the mechanisms that were described in vitro. Taken together, our results indicate that selenite-induced ROS arrest NB4 cells at G0/G1 phase through inhibiting the JNK/ATF2 axis in vitro and in vivo.
