RESUMO
Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.
Assuntos
Apoptose , Autofagia , Neoplasias Colorretais/patologia , Proteínas de Membrana/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína Beclina-1 , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/ultraestrutura , Meios de Cultura Livres de Soro , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estaurosporina/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
OBJECTIVE: Accumulating evidence illustrates that sirtuins (SIRTs) regulate autophagy and apoptosis in cancer cells; however, the role of SIRT5 in gastric cancer (GC) cells remains unknown. In this study, we examined the role of SIRT5 in GC cells. METHODS: We detected SIRT5 protein levels in freshly collected samples from patients with GC. Next, we studied the function of SIRT5 in autophagy. Furthermore, the signaling pathway through which SIRT5 enhanced autophagy in GC cells was detected. In addition, we established a GC cell apoptosis model to analyze the role of SIRT5 in apoptosis. RESULTS: SIRT5 expression was downregulated in GC tissues. We discovered that SIRT5 promoted autophagy in GC cells. We demonstrated that SIRT5 enhanced autophagy in GC cells via the AMP-activated protein kinase-mammalian target of rapamycin signaling pathway. In addition, SIRT5 was degraded during apoptosis in GC cells. Meanwhile, we observed that calpains and caspase-related proteins were associated with SIRT5-related GC cell apoptosis. CONCLUSIONS: SIRT5 is a crucial regulator of autophagy and apoptosis in GC cell lines that can maintain the balance of autophagy and apoptosis.
Assuntos
Sirtuínas , Neoplasias Gástricas , Apoptose , Autofagia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Transdução de Sinais , Sirtuínas/genética , Sirtuínas/metabolismo , Neoplasias Gástricas/genéticaRESUMO
Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells. However, whether Ambra1 plays an important role in the autophagy pathway in colorectal cancer cells is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in CRC cell lines. To test this hypothesis, we confirmed autophagic activity in serum-starved SW620 CRC cells by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes (transmission electron microscopy) and LC3 protein levels (Western blotting). Ambra1 expression was detected by Western blot in SW620 cells treated with staurosporine or etoposide. Calpain and caspase inhibitors were employed to verify whether calpains and caspases were responsible for Ambra1 cleavage. To examine the role of Ambra1 in apoptosis, Ambra1 knockdown cells were treated with staurosporine and etoposide. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in colorectal cancer cell lines. Ambra1 expression decreased during staurosporine- or etoposide-induced apoptosis. Calpains and caspases may be responsible for Ambra1 degradation. When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis. In addition, starvation-induced autophagy decreased. Finally, Co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated SW620 cells, suggesting that Ambra1 regulates autophagy in CRC cells by interacting with Beclin1. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis.