Expression Of Bcl-2/adenovirus E1B 19-kDa-interacting Protein 3 Research Articles

The Role of BNIP3 and Blocked Autophagy Flux in Arsenic-Induced Oxidative Stress-Induced Liver Injury in Rats.

Arsenic is a widely distributed environmental toxic substance in nature. Chronic arsenic exposure can cause permanent damage to the liver, resulting in the death of poisoned patients. However, the mechanism of liver damage caused by arsenic poisoning is yet unclear. Here, four different concentrations of sodium arsenite (NaAsO2) (0mg/L (control group), 25mg/L, 50mg/L, and 100mg/L group)were established to induce liver injury in rats. Taking this into account, the relationship and potential mechanisms of oxidative stress, Bcl-2/adenovirus E1B-19-kDa-interacting protein 3 (BNIP3), and inhibition of autophagy flux in liver injury caused by arsenic poisoning were studied. The results indicated that long-term exposure to NaAsO2 could induce oxidative stress, leading to high expression of BNIP3, thereby impaired autophagy flux, and ultimately resulting in liver damage. This research provides an important basis for future research on liver damage caused by chronic arsenic exposure and prevention and treatment with BNIP3 as the target.

Sevoflurane postconditioning alleviates hypoxia-reoxygenation injury of cardiomyocytes by promoting mitochondrial autophagy through the HIF-1/BNIP3 signaling pathway.

BackgroundSevoflurane postconditioning (SpostC) can alleviate hypoxia-reoxygenation injury of cardiomyocytes; however, the specific mechanism remains unclear. This study aimed to investigate whether SpostC promotes mitochondrial autophagy through the hypoxia-inducible factor-1 (HIF-1)/BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) signaling pathway to attenuate hypoxia-reoxygenation injury in cardiomyocytes.MethodsThe H9C2 cardiomyocyte hypoxia/reoxygenation model was established and treated with 2.4% sevoflurane at the beginning of reoxygenation. Cell damage was determined by measuring cell viability, lactate dehydrogenase activity, and apoptosis. Mitochondrial ultrastructural and autophagosomes were observed by transmission electron microscope. Western blotting was used to examine the expression of HIF-1, BNIP3, and Beclin-1 proteins. The effects of BNIP3 on promoting autophagy were determined using interfering RNA technology to silence BNIP3.ResultsHypoxia-reoxygenation injury led to accumulation of autophagosomes in cardiomyocytes, and cell viability was significantly reduced, which seriously damaged cells. Sevoflurane postconditioning could upregulate HIF-1α and BNIP3 protein expression, promote autophagosome clearance, and reduce cell damage. However, these protective effects were inhibited by 2-methoxyestradiol or sinBNIP3.ConclusionSevoflurane postconditioning can alleviate hypoxia-reoxygenation injury in cardiomyocytes, and this effect may be achieved by promoting mitochondrial autophagy through the HIF-1/BNIP3 signaling pathway.

Panax Notoginseng Saponins Attenuate Myocardial Ischemia-Reperfusion Injury Through the HIF-1α/BNIP3 Pathway of Autophagy.

Panax Notoginseng Saponins (PNS) is a formula of Chinese medicine commonly used for treating ischemia myocardial in China. However, its mechanism of action is yet unclear. This study investigated the effect and the mechanism of PNS on myocardial ischemia-reperfusion injury (MIRI) through the hypoxia-inducible factor 1α (HIF-1α)/bcl-2/adenovirus E1B19kDa-interacting protein3 (BNIP3) pathway of autophagy. We constructed a rat model of myocardial injury and compared among 4 groups (n = 10, each): the sham-operated group (Sham), the ischemia-reperfusion group (IR), the PNS low-dose group, and the PNS high-dose group were pretreated with PNS (30 and 60 mg/kg, respectively). Serum creatine kinase, malonaldehyde (MDA), lactate dehydrogenase, myocardial tissue superoxide dismutase, and reactive oxygen species were detected in rats with myocardial ischemia-reperfusion after the intervention of PNS. The rat myocardial tissue was examined using hematoxylin and eosin (H&E) staining, and the mitochondria of myocardial cells were observed using transmission electron microscopy. The expressions of microtubule-associated protein light chain 3 (LC3), HIF-1α, BNIP3, Beclin-1, and autophagy-related gene-5 (Atg5) in rat myocardial tissue were detected using Western blotting. The results showed that PNS was significantly protected against MIRI, as evidenced by the decreasing in the concentration of serum CK, MDA, lactate dehydrogenase, and myocardial tissue superoxide dismutase, reactive oxygen species, the attenuation of myocardial tissue histopathological changes and the mitochondrial damages of myocardial cells, and the increase of mitochondria autophagosome in myocardial cells. In addition, PNS significantly increased the expression of LC3 and the ratio of LC3II/LC3I in rat myocardial tissue. Moreover, PNS significantly increased the expression of HIF-1α, BNIP3, Atg5, and Beclin-1 in rat myocardial tissue. The protective effect of PNS on MIRI was mainly due to its ability to enhance the mitochondrial autophagy of myocardial tissue through the HIF-1α/BNIP3 pathway.

Expression Analysis of caspase-6, caspase-9 and BNIP3 in Prostate Cancer:

Altered regulation of cell death is a feature of human cancer. The aim of this study was to explore whether the expression of the proapoptotic proteins caspase-6, caspase-9, and Bcl-2/adenovirus E1B-19kDa-interacting protein3 (BNIP3) is altered in prostate cancers. We analyzed the expression of caspase-6, caspase-9, and BNIP3 in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. Normal glandular cells expressed caspase-6 and BNIP3 proteins in 10 (9.3%) and 9 (8.4%) prostate tissues, respectively. By contrast, the prostate cancers expressed caspase-6 and BNIP3 in 65 (60.7%) and 69 (64.5%) cases, respectively. Prostate intraepithelial neoplasia (PIN) showed caspase-6 and BNIP3 expression in 65% and 65% of cases, respectively. We observed caspase-9 expression in 40 (37.4%) normal, 8 (40%) PIN, and 45 (42.1%) cancer tissues. None of the expression of caspase-6, caspase-9 or BNIP3 was associated with pathological characteristics such as tumor size, patient age, Gleason score, or tumor stage. Our data showed that prostate cancer and PIN cells display higher expression of the proapoptotic proteins caspase-6 and BNIP3 than normal cells. Neo-expression of these proteins from the PIN stage suggests that apoptosis deregulation might occur in the early stage of prostate carcinogenesis, and that altered expression of proapoptotic proteins may be a feature of prostate cancer.

Effect of anesthesia factor on lung injury in patients undergoing thoracoscopic radical lung cancer surgery: combination of thoracic paravertebral block with dexmedetomidine mixed with ropivacaine and general anesthesia

Objective To investigate the effect of anesthesia factor on lung injury in patients undergoing thoracoscopic radical lung cancer surgery and to evaluate efficacy of combination of thoracic paravertebral block(TPVB) with dexmedetomidine mixed with ropivacaine and general anesthesia. Methods One hundred patients of both sexes, aged 18-64 yr, with body mass index of 18-25 kg/m2, of American Society of Anesthesiologists physical statusⅡor Ⅲ, scheduled for elective thoracoscopic radical lung cancer surgery, were divided into 5 groups(n=20 each) using a random number table: general anesthesia group(group G), TPVB with ropivacaine combined with general anesthesia group(group R), intravenously infused dexmedetomidine combined with general anesthesia group(group Div), intravenously infused dexmedetomidine plus TPVB with ropivacaine combined with general anesthesia group(group Div+ R), and TPVB with dexmedetomidine mixed with ropivacaine combined with general anesthesia group(group Dtp+ R). In group R, TPVB was performed under ultrasound guidance, two-point method was selected according to the position of intercostal space at surgical incision, and 0.5% ropivacaine 10 ml was injected into each puncture site.Dexmedetomidine 0.5 μg/kg was intravenously infused over 10 min in group Div.Dexmedetomidine was intravenously infused for TPVB in group Div+ R.TPVB solution contained dexmedetomidine 0.5 μg/kg and ropivacaine in group Dtp+ R.Anesthesia was then induced and maintained by IV infusion of propofol and remifentanil.The intraoperative consumption of propofol and remifentanil and development of adverse reactions such as hypoxemia, hypotension and bradycardia were recorded.Normal lung tissues around the tumor margin were obtained immediately after tumor resection for determination of the expression of hypoxia-inducible factor 1 alpha(HIF-1α), BCL2/adenovirus E1B 19kDa interacting protein 3(BNIP3) and microtubule-associated protein 1 light chain 3 Ⅱ(LC3 Ⅱ)(by Western blot), contents of tumor necrosis factor-alpha(TNF-α) and interleukin-6(IL-6) in lung tissues(by enzyme-linked immunosorbent assay) and cell apoptosis(by TUNEL) and for examination of the pathological changes(with a light microscope) which were scored.Apoptosis index was calculated. Results The amount of propofol consumed was significantly lower in Div+ R and Dtp+ R groups than in the other three groups, and the amount of remifentanil consumed was significantly higher in G and Div groups than in the other three groups(P<0.05). The incidence of hypertension and tachycardia was significantly lower in R and Div groups than in group G(P<0.05). The incidence of hypotension was significantly lower in R, Div and Dtp+ R groups than in group Div+ R(P<0.05). The incidence of bradycardia was significantly higher in Div and Div+ R groups than in group R(P<0.05). Compared with G and R groups, apoptosis index, contents of TNF-α and IL-6 and lung injury scores were significantly decreased, and the expression of HIF-1α, BNIP3 and LC3Ⅱ was up-regulated in Div, Div+ R and Dtp+ R groups(P<0.05). Compared with group Div, the TNF-α content and lung injury scores were significantly decreased, and the expression of HIF-1α and LC3Ⅱ was up-regulated in Div+ R and Dtp+ R groups, and the IL-6 content was significantly decreased in group Dtp+ R(P<0.05). Conclusion Combination of TPVB with dexmedetomidine mixed with ropivacaine and general anesthesia produces better efficacy in reducing lung injury in patients undergoing thoracoscopic radical lung cancer surgery. Key words: Dexmedetomidine; Amides; Nerve block; Anesthesia, general; Lung neoplasms

MicroRNA-210 Protects PC-12 Cells Against Hypoxia-Induced Injury by Targeting BNIP3.

MicroRNA (miR)-210 is the most consistently and predominantly up-regulated miR in response to hypoxia in multiple cancer cells. The roles of miR-210 in rat adrenal gland pheochromocytoma (PC-12) cells remain unknown. We aimed to explore the possible effect of miR-210 in neonatal brain injury. We explored the potential molecular mechanism by using PC-12 cells under hypoxia. Scramble miRs, miR-210 mimic, miR-210 inhibitor or its negative control were respectively transfected into PC-12 cells. Cell viability, migration, invasion and apoptosis were also assessed to evaluate hypoxia-induced cell injury. The expression level of miR-210 was identified by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Apoptosis-related protein expression as well as key kinases in the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signal pathway was studied by Western blot analysis. Hypoxia suppressed cell viability, migration and invasion, but promoted apoptosis through activation of mitochondrial- and caspase-dependent pathways. Hypoxia markedly induced up-regulation of miR-210 in PC-12 cells. Overexpression of miR-210 protected PC-12 cells against hypoxia-induced injury. Bcl-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) was proven to be a target gene of miR-210 in PC-12 cells. miR-210 overexpression ameliorated the hypoxia-induced injury in PC-12 cells by down-regulating BNIP3. Hypoxia-induced alterations of key kinases in the PI3K/AKT/mTOR signal pathway were affected by aberrant expression of BNIP3. These findings suggested that miR-210 protected PC-12 cells against hypoxia-induced injury by targeting BNIP3, involving the PI3K/AKT/mTOR signal pathway.

Nutrition deficiency promotes apoptosis of cartilage endplate stem cells in a caspase-independent manner partially through upregulating BNIP3.

Nutrition deficiency is reported to induce apoptosis of chondrocytes and degeneration of cartilage endplate (CEP) in rabbit. Cartilage endplate stem cells (CESCs) are important for the integrity of structure and function of CEP. Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) has been reported to regulate apoptosis, autophagy, and cytoprotection. In this study, we aimed to determine whether nutrition deficiency induces apoptosis of CESCs, and whether or not the BNIP3-related pathway is activated in CESCs during nutrition deficiency. CESCs isolated from degenerated human CEP were cultured under normal or nutrition-deficient condition. Then, apoptosis was analyzed by flow cytometry. The expression and intracellular localization of BNIP3 were detected by quantitative real-time polymerase chain reaction, western blot analysis, and immunofluorescence assay, respectively. Mitochondrial membrane potential (MMP) and caspase-3 activity were measured by JC-1 staining and caspase-3 activity assay. Our results showed that nutrition deficiency promotes apoptosis and BNIP3 expression in CESCs. Notably, knockdown of BNIP3 could partially decrease nutrition deficiency-induced apoptosis of CESCs. In addition, nutrition deficiency could also induce upregulation of BNIP3, resulting in mitochondrial translocation of BNIP3 and loss of MMP in CESCs in a time-dependent manner. However, nutrition deficiency showed no effects on caspase-3 activity in CESCs. In summary, nutrition deficiency may promote CESC apoptosis partially through upregulating BNIP3, which might lead to activation of the BNIP3-related pathway and apoptosis of CESCs in a caspase-independent manner.

BNIP3 Protein Suppresses PINK1 Kinase Proteolytic Cleavage to Promote Mitophagy

Mutations in PINK1 (PTEN-induced putative kinase 1) cause early onset familial Parkinson's disease (PD). PINK1 accumulates on the outer membrane of damaged mitochondria followed by recruiting parkin to promote mitophagy. Here, we demonstrate that BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a mitochondrial BH3-only protein, interacts with PINK1 to promote the accumulation of full-length PINK1 on the outer membrane of mitochondria, which facilitates parkin recruitment and PINK1/parkin-mediated mitophagy. Inactivation of BNIP3 in mammalian cells promotes PINK1 proteolytic processing and suppresses PINK1/parkin-mediated mitophagy. Hypoxia-induced BNIP3 expression results in increased expression of full-length PINK1 and mitophagy. Consistently, expression of BNIP3 in Drosophila suppresses muscle degeneration and the mitochondrial abnormality caused by PINK1 inactivation. Together, the results suggest that BNIP3 plays a vital role in regulating PINK1 mitochondrial outer membrane localization, the proteolytic process of PINK1 and PINK1/parkin-mediated mitophagy under physiological conditions. Functional up-regulation of BNIP3 may represent a novel therapeutic strategy to suppress the progression of PD.

Expression of BNIP3 and its correlations to hypoxia-induced autophagy and clinicopathological features in salivary adenoid cystic carcinoma.

The expression of Bcl-2/adenovirus E1B 19 kDa-interacting protein3 (BNIP3) has been explored in many human malignancies, but not in adenoid cystic carcinoma (ACC). This study investigated the clinical significance of expression of BNIP3 in ACC tissues and cells and elucidated its correlations to hypoxia-induced autophagy. Immunohistochemical and immunofluorescence staining were used to explore BNIP3, HIF-1α and LC3 expression. BNIP3 was positively expressed in 41 cases (63.1%), and was significantly correlated with histological grade (P= 0.001). HIF-1α was positively expressed in 52 cases (80.0%) and was significantly correlated with TNM stage (P= 0.023) and histological grade (P= 0.024). LC3 was positively expressed in 37 cases (56.9%) and was significantly correlated with TNM stage (P= 0.019). The expression of BNIP3 was correlated with HIF-1α expression (P= 0.011). The overall survival in the negative BNIP3 expression group tended to be better than in the positive BNIP3 expression (P= 0.011). In vitro experiment, BNIP3 immunofluorescence staining was detected in cells treated with CoCl2 (for hypoxic condition). The data indicated that BNIP3 plays a vital role in the tumorigenesis of adenoid cystic carcinoma and could be a new target for gene therapy of adenoid cystic carcinoma.

The interplay between epigenetic silencing, oncogenic KRas and HIF-1 regulatory pathways in control of BNIP3 expression in human colorectal cancer cells

Bcl-2/adenovirus E1B-19kDa-interacting protein 3 (BNIP3) is an important mediator of cell survival and a member of the Bcl-2 family of proteins that regulate programmed cell death and autophagy. We have previously established a link between the expression of oncogenic HRas and up-regulation of BNIP3 and the control of autophagy in cancer cells. However, in view of varied expression of BNIP3 in different tumor types and emerging uncertainties as to the role of epigenetic silencing, oncogenic regulation and the role of BNIP3 in cancer are still poorly understood.In the present study we describe profound effect of KRas on the expression of methylated BNIP3 in colorectal cancer cells and explore the interplay between HIF-1, hypoxia pathway and oncogenic KRas in this context. We observed that BNIP3 mRNA remains undetectable in aggressive DLD-1 cells harboring G13D mutant KRAS and HT-29 colorectal cancer cells unless the cells are exposed to demethylating agents such as 5-aza-2′-deoxycytidine. Following this treatment BNIP3 expression remains uniquely dependent on the Ras activity. We found that hypoxia or pharmacological activation of HIF-1 alone contributes to, but is not sufficient for efficient induction of BNIP3 mRNA transcription in cells lacking mutant KRas activity. The up-regulation of BNIP3 by KRas in this setting is mediated by the MAPK pathway, and is attenuated by the respective inhibitors (PD98059, U0126). Thus, we demonstrate the novel mechanism where activity of Ras is essential for 5-aza-2′-deoxycytidine-mediated BNIP3 expression. Moreover, we found that 5-aza-2′-deoxycytidine-mediated or enforced up-regulation of BNIP3 in DLD-1 cells results in KRas-dependent resistance to 5-Fluorouracil.

MiR-210 suppresses BNIP3 to protect against the apoptosis of neural progenitor cells

MiR-210 is a hypoxia-inducible factor (HIF)-1 target gene and is the most consistently and predominantly upregulated miRNA in response to hypoxia in various cancer cell lines. Our recent study shows that hypoxia increased miR-210 expression in neural progenitor cells (NPCs) in a time-dependent manner. However, the role of miR-210 in NPCs remains unknown. Following the identification of the miR-210 putative target genes, we demonstrated that the Bcl-2 adenovirus E1B 19kDa-interacting protein 3 (BNIP3), which is regulated by HIF-1 and activates cell death, is regulated by miR-210 in NPCs under hypoxia. Moreover, the over-expression of miR-210 decreased apoptosis in NPCs, and the inhibition of miR-210 expression remarkably increased the number of TUNEL-positive NPCs by 30% in response to hypoxia. Importantly, miR-210 mimics reduced both BNIP3 protein expression and the translocation of AIF into the nucleus, which reduced cell death, whereas miR-210 inhibitors reversed this process, leading to cell death during hypoxia. Taken together, we report a novel feedback loop of BNIP3 regulation in NPCs under hypoxia. HIF-1 is activated under hypoxia and then induces the expression of both BNIP3 and miR-210. The upregulation of miR-210 then directly suppresses BNIP3 expression to maintain the survival of NPCs under hypoxia. This negative feedback regulation might partially contribute to protection against hypoxia-induced cell death via the inhibition of AIF nuclear translocation.

Effect of Jinlong capsule on proliferation and apoptosis of human pancreatic cancer cells BxPC-3

ObjectiveTo study the possible roles of Jinlong capsule (JLC) on the proliferation and apoptosis of human pancreatic cancer cells BxPC-3. MethodsThe human pancreatic cancer cells BxPC-3 were treated with JLC at the concentration of 0.05-1.00 mg/mL for 24-120 h. The inhibition rate of JLC on human pancreatic cancer cells BxPC-3 was detected by 3-(4,5-dimethiylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Flow cytometry was employed to measure cell apoptosis using Annexin V-FITC/Propidium iodide (AV-FITC/PI) method. Cell cycles were determined by PI staining. The expression of S100 Calcium binding protein A4 (S100A4) in cell matrix was measured by enzyme-linked immunosorbent assay (ELISA). The expression levels of apoptosis-related protein such as BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), B-cell lymphoma/leukemia-2 (Bcl-2) and Cys-teinylaspartate specific proteinase 3 (Caspase-3) were detected by Western blotting. ResultsJLC significantly inhibited the proliferation of human pancreatic cancer cells BxPC-3 in a dose-dependent and time-dependent manner. JLC promoted cell apoptosis and maintained cell cycle in S and G2/M phase rather than G1/G0 phase. The expression of S100A4 in the cell matrix was reduced. The expression of cell apoptotic protein BNIP3 was increased while Bcl-2 was decreased. ConclusionJLC can inhibit the proliferation of human pancreatic cancer cells BxPC-3 by stimulating cell apoptosis, arresting the cell cycle at S and G2/M phase which blocks the circulation of normal cell cycle and reducing the expression of S100A4 protein. Higher pro-apoptosis protein BNIP3 and lower anti-apoptosis protein Bcl-2 levels were found, which may be related to the apoptotic effects of JLC.

Different molecular mechanisms involved in spontaneous and oxidative stress-induced mitochondrial fragmentation in tripeptidyl peptidase-1 (TPP-1)-deficient fibroblasts

NCLs (neuronal ceroid lipofuscinoses) form a group of eight inherited autosomal recessive diseases characterized by the intralysosomal accumulation of autofluorescent pigments, called ceroids. Recent data suggest that the pathogenesis of NCL is associated with the appearance of fragmented mitochondria with altered functions. However, even if an impairement in the autophagic pathway has often been evoked, the molecular mechanisms leading to mitochondrial fragmentation in response to a lysosomal dysfunction are still poorly understood. In this study, we show that fibroblasts that are deficient for the TPP-1 (tripeptidyl peptidase-1), a lysosomal hydrolase encoded by the gene mutated in the LINCL (late infantile NCL, CLN2 form) also exhibit a fragmented mitochondrial network. This morphological alteration is accompanied by an increase in the expression of the protein BNIP3 (Bcl2/adenovirus E1B 19 kDa interacting protein 3) as well as a decrease in the abundance of mitofusins 1 and 2, two proteins involved in mitochondrial fusion. Using RNAi (RNA interference) and quantitative analysis of the mitochondrial morphology, we show that the inhibition of BNIP3 expression does not result in an increase in the reticulation of the mitochondrial population in LINCL cells. However, this protein seems to play a key role in cell response to mitochondrial oxidative stress as it sensitizes mitochondria to antimycin A-induced fragmentation. To our knowledge, our results bring the first evidence of a mechanism that links TPP-1 deficiency and oxidative stress-induced changes in mitochondrial morphology.

Expression Analysis of caspase-6, caspase-9 and BNIP3 in Prostate Cancer

Altered regulation of cell death is a feature of human cancer. The aim of this study was to explore whether the expression of the proapoptotic proteins caspase-6, caspase-9, and Bcl-2/adenovirus E1B-19kDa-interacting protein3 (BNIP3) is altered in prostate cancers. We analyzed the expression of caspase-6, caspase-9, and BNIP3 in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. Normal glandular cells expressed caspase-6 and BNIP3 proteins in 10 (9.3%) and 9 (8.4%) prostate tissues, respectively. By contrast, the prostate cancers expressed caspase-6 and BNIP3 in 65 (60.7%) and 69 (64.5%) cases, respectively. Prostate intraepithelial neoplasia (PIN) showed caspase-6 and BNIP3 expression in 65% and 65% of cases, respectively. We observed caspase-9 expression in 40 (37.4%) normal, 8 (40%) PIN, and 45 (42.1%) cancer tissues. None of the expression of caspase-6, caspase-9 or BNIP3 was associated with pathological characteristics such as tumor size, patient age, Gleason score, or tumor stage. Our data showed that prostate cancer and PIN cells display higher expression of the proapoptotic proteins caspase-6 and BNIP3 than normal cells. Neo-expression of these proteins from the PIN stage suggests that apoptosis deregulation might occur in the early stage of prostate carcinogenesis, and that altered expression of proapoptotic proteins may be a feature of prostate cancer.

Activation of Ras Up-regulates Pro-apoptotic BNIP3 in Nitric Oxide-induced Cell Death

Nitric oxide (NO) produced by NO synthases causes nitration and nitrosylation of cellular factors. We have shown previously that endogenously produced or exogenously added NO induces expression of BNIP3 (Bcl-2/adenovirus E1B 19 kDa-interacting protein 3), leading to death of macrophages (Yook, Y.-H., Kang, K.-H., Maeng, O., Kim, T.-R., Lee, J.-O., Kang, K.-i., Kim, Y.-S., Paik, S.-G., and Lee, H. (2004) Biochem. Biophys. Res. Commun. 321, 298-305). We now provide evidence that Ras mediates NO-induced BNIP3 expression via the MEK/ERK/hypoxia-inducible factor (HIF)-1 pathway. (a) ras-Q61L, a constitutively active form of Ras, up-regulated BNIP3 protein expression by enhancing Bnip3 promoter activity, and ras-S17N, a dominant-negative form, and ras-C118S, an S-nitrosylation mutant, blocked NO-induced BNIP3 expression, suggesting that Ras acts downstream of NO and that NO activates Ras by nitrosylation. (b) U0126, a specific MEK inhibitor, completely abolished BNIP3 expression and the stimulation of promoter activity by NO and Ras, whereas 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, SB203580, and wortmannin, specific inhibitors of soluble guanylyl cyclase, p38 MAPK, and phosphatidylinositol 3-kinase, respectively, had no effect. Ras, MEK1/2, and ERK1/2 were sequentially activated by NO treatment of macrophages. (c) Mutation of the HIF-1-binding site (hypoxia-response element) in the Bnip3 promoter abolished BNIP3 induction, and HIF-1alpha was strongly induced by NO. (d) Transient expression of activated Ras promoted macrophage death, as did NO, and this Ras-mediated cell death was inhibited by silencing BNIP3 expression. These results suggest that NO-induced death of macrophages is mediated, at least in part, by BNIP3 induction.

Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3

Arsenic trioxide (As(2)O(3)) has shown considerable efficacy in treating hematological malignancies with induction of programmed cell death (PCD) type I, apoptosis. However, the mechanisms underlying the antitumor effect of As(2)O(3) on solid tumors are poorly defined. Previously, we reported that As(2)O(3) induced autophagic cell death (PCD type II) but not apoptosis in human malignant glioma cell lines. The purpose of this study was to elucidate the molecular pathway leading to autophagic cell death. In this study, we demonstrated that the cell death was accompanied by involvement of autophagy-specific marker, microtubule-associated protein light chain 3 (LC3), and damage of mitochondrial membrane integrity, but not by caspase activation. Analysis by cDNA microarray, RT-PCR, and Western blot showed that cell death members of Bcl-2 family, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and its homologue BNIP3-like (BNIP3L), were upregulated in As(2)O(3)-induced autophagic cell death. Exogenous expression of BNIP3, but not BNIP3L, induced autophagic cell death in malignant glioma cells without As(2)O(3) treatment. When upregulation of BNIP3 induced by As(2)O(3) was suppressed by a dominant-negative effect of the transmembrane-deleted BNIP3 (BNIP3 Delta TM), autophagic cell death was inhibited. In contrast, BNIP3 transfection augmented As(2)O(3)-induced autophagic cell death. These results suggest that BNIP3 plays a central role in As(2)O(3)-induced autophagic cell death in malignant glioma cells. This study adds a new concept to characterize the pathways by which As(2)O(3) acts to induce autophagic cell death in malignant glioma cells.