Bcl-2 interacting protein 3 (BNIP3) is involved in various cellular processes and is considered a key regulator of hypoxia-induced apoptosis. BNIP3 was independent of caspase 3 and 9 activation. The restoration of BNIP3 expression in pancreatic cancer cells via a mitochondrial pathway The induction of apoptosis was further investigated in Patu8999 cells that exhibited detectable levels of BNIP3 expression and in the CaPan-1 cell line that was used as a model for overexpression of BNIP3. CaPan-1 OE-BNIP transfected cells enhanced the percentage of apoptotic cells as demonstrated by Annexin V staining, whereas the opposite effect was noted for si-BNIP3 treated cells (Figure ?(Figure5A).5A). An approximate 8-fold increase in the percentage of apoptosis was noted for the former (15.73%0.40% vs. 1.37%0.06%, P<0.05), whereas an approximate 2-fold decrease was noted for the latter (6.97%0.21% vs. 15.40%0.26%, P<0.05) (Figure ?(Figure5A).5A). The function of the BNIP3 protein with regard to the induction of apoptosis was further investigated by measurement of mitochondrial potential (m) and ROS. The changes in m were analyzed based on the green/red fluorescence ratio of JC-1 (Figure ?(Figure5B).5B). m in the OE-BNIP3 group was markedly higher (approximately 3-fold) compared with that in the vector and control groups (P<0.05), whereas the same parameter was lower (approximately 1.5-fold) following si-BNIP3 treatment compared with the control and siRNA scramble groups (P<0.05) (Figure ?(Figure5B).5B). The ROS assay indicated similar results, and ROS production was higher in OE-BNIP3 group compared with the vector and control groups (P<0.05), whereas transfection of Patu8988 cells with 820957-38-8 si-BNIP3 decreased the production of ROS compared to control and siRNA scramble groups (Figure ?(Figure5C)5C) (P<0.05). The results demonstrated that BNIP3 induces the mitochondrial pathway of apoptosis via the generation of ROS and the loss of m. Figure 5 Induction of apoptosis in pancreatic cancer cells by BNIP3 The expression of the apoptosis-associated proteins that regulated the expression of BNIP3 was investigated by Western blotting. AIF and Bax were upregulated (P<0.05, respectively), whereas Bcl-2 was downregulated in OE-BNIP3 cells compared with vector and control cells (Figure ?(Figure6)6) (P<0.05). Knockdown of BNIP3 protein by si-BNIP3 demonstrated 820957-38-8 the opposite effect (Figure ?(Figure6).6). Therefore AIF and Bax were downregulated, Rabbit Polyclonal to MAK whereas Bcl-2 was upregulated in si-BNIP3 cells compared with control and si-scramble cells (Figure ?(Figure6)6) (P<0.05). The regulation of BNIP3 in CaPan-1 and Patu8988 cells exerted minimal effect on the expression of cleaved caspases 3 and 9 (P>0.05 for both). The findings demonstrated that BNIP3 induced apoptosis by the activation of AIF and Bax, and by the suppression of Bcl-2. In addition, BNIP3 induced the mitochondrial apoptotic pathway, which was independent of the expression of caspases 3 and 9 (Figure ?(Figure66). Figure 6 Correlation between BNIP3 and apoptosis-associated proteins in pancreatic cancer cells Silencing of BNIP3 gene expression in pancreatic cancer cells by methylation Previous studies have shown that methylation could silence the BNIP3 gene in tumors via epigenetic mechanisms [18]. In order to confirm the relationship between DNA methylation and BNIP3 silencing, we used MS-PCR to examine the methylation status of the six pancreatic cancer cell lines (Figure ?(Figure7A).7A). A total of five out of the six pancreatic cancer cell lines indicated increased methylation levels in the promoter region of the BNIP3 gene that were associated with silencing of BNIP3 expression, whereas Patu8988 cells that exhibited low BNIP3 expression displayed partial methylation (26%) (Figure ?(Figure7A).7A). The expression of BNIP3 following treatment with the methyltransferase inhibitor Aza-dC (Figure ?(Figure7B)7B) was further examined. Treatment of Capan-1 cells with 0.1, 0.5 and 1 M of Aza-dC resulted in a dose dependent increase (P<0.05 for 0.5 and 1 M) in the expression of BNIP3 compared to solvent control (0 M), whereas the same treatment in Patu8988 cells did not exhibit a significant change in BNIP3 expression levels (Figure ?(Figure7B).7B). TUNEL assay further demonstrated that the increase in BNIP3 expression following 0.5 and 1 M of Aza-dC treatment was associated with the induction of apoptosis (Figure ?(Figure7C).7C). A an approximate 2.5-fold increase in the apoptotic population of the cells was demonstrated at 1 M of Aza-dC treatment that in turn caused a 6-fold increase in the protein levels of BNIP3 compared with solvent control cells (Figure ?(Figure7B7B and 820957-38-8 ?and7C).7C). The findings indicated that BNIP3 silencing in pancreatic cells was related, at least in part, to gene methylation. Demethylation by Ada-dC could restore BNIP3 expression and sensitize pancreatic cancer cells to BNIP3-induced apoptosis. Figure 7 Analysis of BNIP3 methylation and demethylation Suppression of binding between HIF-1.