RESUMO
The transcription factor Atonal homolog 1 (Atoh1) plays crucial roles in the differentiation of intestinal epithelium cells. Although we have reported that the Atoh1 protein was degraded in colon cancer by aberrant Wnt signaling, a recent study has indicated that the Atoh1 protein is expressed in mucinous colon cancer (MC) and signet ring cell carcinoma (SRCC). However, the roles of the Atoh1 protein in MC are unknown. To mimic MC, a mutated Atoh1 protein was stably expressed in undifferentiated colon cancer cells. Microarray analysis revealed the acquisition of not only the differentiated cell form, but also malignant potential by Atoh1 protein stabilization. In particular, Atoh1 enhanced Wnt signaling, resulting in the induction of Lgr5 as a representative stem cell marker with the enrichment of cancer stem cells. Moreover, the fluorescent ubiquitination-based cell cycle indicator system with time-lapse live imaging demonstrated cell cycle arrest in the G0/G1 phase by Atoh1 protein stabilization. In conclusion, the Atoh1 protein regulates malignant potential rather than the differentiation phenotype of MC, suggesting the mechanism by which MC and SRCC are more malignant than non-mucinous adenocarcinoma.
Assuntos
Adenocarcinoma Mucinoso/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias do Colo/patologia , Via de Sinalização Wnt , Adenocarcinoma Mucinoso/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Pontos de Checagem do Ciclo Celular , Movimento Celular , Neoplasias do Colo/genética , Resistencia a Medicamentos Antineoplásicos , Fase G1 , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Estabilidade Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fase de Repouso do Ciclo CelularRESUMO
P-glycoprotein (P-gp) is an efflux transporter that regulates bioavailability of orally administered drugs at the intestinal epithelium. To develop an in vitro experimental model that mimics P-gp-mediated intestinal drug transport in vivo, we employed normal intestinal epithelium three-dimensionally cultured. Physiological expression of P-gp mRNA and the expression of its protein at the apical membrane were observed in the small intestinal epithelium grown as cystic organoids. Rhodamine123 (Rh123), a substrate for P-gp, was actively transported in the basoapical direction and accumulated in the luminal space, while the epithelial integrity was kept intact. Furthermore, we were able to monitor the whole process of Rh123 transport and its inhibition by verapamil in real-time, from which kinetic parameters for Rh123 transport could be estimated by a mathematical modeling. The method here described to evaluate the dynamics of P-gp-mediated transport in primary intestinal epithelial cells would be instrumental in investigating the physiological function of P-gp and its inhibitors/inducers in vitro.