Methylation of BNIP3 in pancreatic cancer inhibits the induction of mitochondrial-mediated tumor cell apoptosis

Abstract

Bcl-2 interacting protein 3 (BNIP3) is involved in various cellular processes and is considered a key regulator of hypoxia-induced apoptosis. In the present study, the expression of BNIP3 in pancreatic cancer tissues, the correlation with clinicopathological characteristics and prognosis and the regulation of this protein in pancreatic cancer cell lines with regard to the induction of apoptosis were investigated. BNIP3 expression was significantly lower in pancreatic cancer tissues compared with normal epithelia and was associated with tumor size, clinical stage, and lymph node metastasis. The expression of BNIP3 correlated positively to the proapoptotic protein Bax and negatively to the antiapoptotic protein Bcl-2, whereas the induction of apoptosis by BNIP3 was independent of caspase 3 and 9 activation. The restoration of BNIP3 expression in pancreatic cancer cells in vitro, caused loss of ΔΨm, increase in ROS production, and apoptosis induction. The opposite effect was observed in pancreatic cancer cells, following BNIP3 silencing by RNAi. The absence of BNIP3 expression in pancreatic cancer cells was related to gene methylation that suppressed binding of HIF-1α to the BNIP3 promoter, whereas 5-Aza-2′-deoxycytidine (Aza-dC) treatment restored BNIP3 expression and sensitized pancreatic cancer cells to BNIP3-induced apoptosis. The findings indicated that BNIP3 was significantly downregulated in pancreatic cancer resulting in reduced apoptosis induction. Silencing of BNIP3 expression was associated with methylation of the hypoxia-responsive element (HRE) site that in turn inhibited the binding of HIF-1α to the BNIP3 promoter. The data suggest that BNIP3 reactivation is a potential target for therapeutic intervention against pancreatic cancer.

Stabilization of Hypoxia-Inducible Factors and BNIP3 Promoter Methylation Contribute to Acquired Sorafenib Resistance in Human Hepatocarcinoma Cells

“…In accordance with our results, several studies have related BNIP3 silencing with methylation of its promoter as a protective mechanism of cell death in leukemia, pancreatic, and colorectal tumors [10,13,15,18,57,58,60,61], resulting from DNMT1 activity by the mitogen-activated protein kinase in pancreatic cancer [13] and from DNMT1/DNMT3B in colorectal cancer [10]. In most cases, 5-Aza was used to restore normal BNIP3 expression, sensitizing pancreatic cancer cells via hypoxia-mediated apoptosis promotion [13,15,18] and busulfan-resistant myeloid leukemia cells by upregulation of proapoptotic proteins, including BNIP3 [60]. This phenomenon has been largely studied in colorectal cancer, where 5-Aza recovered BNIP3 expression as a biosensitizer pretreatment of irinotecan [61] and to increase chemosensitivity to 5-FU in colorectal cancer [10].…”

“…BNIP3 downregulation has been linked to poorer patient survival and cell proliferation in pancreatic, colorectal, renal, and HCC tumors [10,[12][13][14][15][16][17][18][56][57][58][59]. Erkan et al [14] found lower BNIP3 mRNA levels in an in vitro model and in four out of eight pancreatic cancer cell lines.…”

“…BNIP3, which may be activated by HIF-1α due to its HREs-containing promoter, is expressed in hypoxic tumoral areas, contributing to hypoxia-induced cell death [12]. In fact, loss of BNIP3 expression correlates with poorer survival of patients and chemoresistance in different types of cancer and is broadly subjected to epigenetic alterations, highlighting histone deacetylation and DNA methylation [10,[12][13][14][15][16][17][18].…”

Analysis of BNIP3 and BNIP3L/Nix expression in cybrid cell lines harboring two LHON-associated mutations.

“…We can also hypothesize that the level of BNIP3 protein modifies the response to some kind of cellular stress and a decrease in the BNIP3 pro-apoptotic protein level in our LHON cell model, could negatively affect apoptosis induction, what in turn can lead to cell survival as in the case of BNIP3-negative cancer cells (He et al, 2017;Li et al, 2017). Our analyses of lymphoblasts with the 11778G>A mutation treated with different concentrations of testosterone could support this hypothesis.…”

“…Tumors which showed loss of expression of BNIP3 were found to be more prone to metastases (Koop et al, 2009). Decreased expression of BNIP3 in some cases of cancers was found to be caused by epigenetic modification, hypermethylation, of the BNIP3 promoter (Okami et al, 2004;Lazarini et al, 2012;Li et al, 2017). Treatment of BNIP3-negative pancreatic and colorectal cancer cell lines with a DNA methylation inhibitor restored BNIP3 expression and increased chemosensitivity and cell death in those cell lines (He et al, 2017;Li et al, 2017).…”