Effect And Mechanism Of BNIP3 Regulating Apoptosis In Pancreatic Cancer

Objective: Hypoxia plays an important role in tumorigenesis and tumor cells take advantage of responses to survival through hypoxia-inducible genes. Bcl-2 nineteen-kilodalton interacting protein 3(BNIP3) was found to be involved in various cellular processes and considered as a key regulator of hypoxia-induced cell apoptosis. However, the function of BNIP3 in pancreatic cancer has not yet been fully elucidated.Methods:(1) Immunohistochemistry was used to assess the expression of BNIP3 in 70 pairs of pancreatic cancer tissues and adjacent pancreatic tissues, and its correlation with clinicopathological features of pancreatic cancer was also analyzed,(2) investigated the expression of BNIP3 and HIF-1? in six pancreatic cancer cell lines under normoxic and hypoxic condition by western blot.(3)Then we observed the change of reactive oxygen species(ROS), mitochondrial membrane potential(??m) and apoptosis associated proteins after regulating the expression of BNIP3 in pancreatic cancer cells.(4)Finally, we investigated the relationship of methylation and the expression of BNIP3, as well as whether methylation could suppress the binding capacity of HIF-1? to BNIP3 promoter by ChIP assay.Results:(1) BNIP3 positive rate of pancreatic cancer was 35.7% and 72.9% in adjacent tissues(P <0.05), the positive rate of BNIP3 in tumor tissues was lower than in nontumorous tissues. The analysis of correlation between BNIP3 expression and clinicopathologic factors of pancreatic carcinoma shows that BNIP3 expression was strongly correlated with tumor size, clinical stage, lymph node metastasis(P <0.05), but did not show association with gender, age and tumor location, differentiation, vascular invasion at a statistically level(P >0.05).(2) In contrast to HIF-1? expression, we found that hypoxia treatment had no effect to enhance the expression of BNIP3 in pancreatic cancer cells. Our results indicated that reintroduction of BNIP3 by transfection into pancreatic cancer cells caused loss of ??m, increased ROS production and finally irreversible cell apoptosis. The opposite effect was shown in silencing of BNIP3 by RNAi.(3)Then we confirmed that absence of BNIP3 in pancreatic cancer cells was related to the methylation of the gene, and demethylation by Aza-dC restored BNIP3 expression and sensitized pancreatic cancer cells to BNIP3-induced cell apoptosis.(4)Finally, the Ch IP assay showed methylation suppressed HIF-1? binding to BNIP3 promoter.Conclusion: Our findings indicated that BNIP3 acted as a pro-apoptotic protein in pancreatic cancer cells that might induce cell apoptosis through a mitochondrial pathway. Loss of BNIP3 expression was associated with methylation of HRE site impacted HIF-1? binding to BNIP3 promoter. These observations implied that BNIP3 reactivation might be a potential target in therapeutic applications for pancreatic cancer.