Inhibition of NADPH oxidase-related oxidative stress-triggered signaling by honokiol suppresses high glucose-induced human endothelial cell apoptosis

Angiopathy is a major complication of diabetes. Abnormally high blood glucose is a crucial risk factor for endothelial cell damage. Nuclear factor-kappaB (NF-kappaB) has been demonstrated as a mediated signaling in hyperglycemia or oxidative stress-triggered apoptosis of endothelial cells. Here we explored the efficacy of honokiol, a small molecular weight natural product, on NADPH oxidase-related oxidative stress-mediated NF-kappaB-regulated signaling and apoptosis in human umbilical vein endothelial cells (HUVECs) under hyperglycemic conditions. The methods of morphological Hoechst staining and annexin V/propidium iodide staining were used to detect apoptosis. Submicromolar concentrations of honokiol suppressed the increases of NADPH oxidase activity, Rac-1 phosphorylation, p22(phox) protein expression, and reactive oxygen species production in high glucose (HG)-stimulated HUVECs. The degradation of IkappaBalpha and increase of NF-kappaB activity were inhibited by honokiol in HG-treated HUVECs. Moreover, honokiol (0.125-1 microM) also suppressed HG-induced cyclooxygenase (COX)-2 upregulation and prostaglandin E(2) production in HUVECs. Honokiol could reduce increased caspase-3 activity and the subsequent apoptosis and cell death triggered by HG. These results imply that inhibition of NADPH oxidase-related oxidative stress by honokiol suppresses the HG-induced NF-kappaB-regulated COX-2 upregulation, apoptosis, and cell death in HUVECs, which has the potential to be developed as a therapeutic agent to prevent hyperglycemia-induced endothelial damage.     

lncRNA PVT1沉默对高糖诱导的血管内皮细胞增殖、凋亡及氧化应激的影响*

目的: 探讨抑制lncRNA PVT1对高糖诱导的血管内皮细胞的增殖,凋亡和氧化应激的影响。方法: 体外培养人脐静脉内皮细胞(HUVECs),分为四组:对照组(5.5 mmol/L葡萄糖),高糖组(30 mmol/L葡萄糖),高糖+siNC组(30 mmol/L葡萄糖+siNC,细胞转染阴性对照组),高糖+siPVT1组(30 mmol/L葡萄糖+siPVT1,抑制lncRNA PVT1组)。采用荧光定量PCR的方法检测转染后PVT1的表达水平。MTT检测siPVT1(短片段干扰RNA PVT1)对高糖诱导的HUVECs细胞增殖能力的影响。流式细胞术检测siPVT1对高糖诱导的HUVECs细胞ROS和凋亡水平。Western blot检测HUVECs细胞中凋亡相关蛋白如Bax,Bcl-2和cleaved-caspase-3的表达水平。结果: 与对照组比较,转染siPVT1后,PVT1的表达水平显著降低(P＜0.05)。MTT结果显示,与对照组比较,培养24 h和48 h后高糖组中HUVECs细胞增殖活力均显著降低,与高糖+siNC组(阴性对照组)比较,培养24 h和48 h后,高糖+siPVT1组中的HUVECs细胞增殖活力显著增加(P＜0.05)。流式细胞术检测结果表明,与对照组比较,高糖组HUVECs细胞中ROS和凋亡率均显著增加;和高糖+siNC组比较,高糖+siPVT1组中HUVECs细胞中ROS和凋亡率均有减少(P＜0.05)。Western blot结果表明,与对照组比较,高糖组中cleaved-caspase-3和Bax表达水平均显著上调,Bcl-2的表达水平显著下调(P＜0.05,P＜0.01)。与高糖+siNC组比较,高糖+siPVT1组cleaved-caspase-3和Bax表达水平显著下调,Bcl-2的表达显著上调(P＜0.05,P＜0.01)。结论: 抑制lncRNA PVT1可以显著增加高糖诱导的HUVECs细胞增殖活力,减轻氧化应激,抑制细胞凋亡。     

Hsa-miRNA-29a protects against high glucose-induced damage in human umbilical vein endothelial cells

Sustained exposure to high glucose (HG) results in dysfunction of vascular endothelial cells. Hence, diabetic patients often suffer from secondary vascular damages, such as vascular sclerosis and thrombogenesis, which may eventually cause cardiovascular problems. Thus, elucidating how HG results in vascular endothelial cell damage and finding an approach for prevention are important to prevent and treat vascular damages in diabetic patients. In the current study, we first showed that 72-hour exposure to HG-decreased hsa-miRNA-29a and increased the expression of Bcl-2 associated X protein (Bax), which subsequently inhibited Bcl-2 and promoted the expression of apoptotic protease activating factor-1 and activation of caspase-3, thus directly triggering the mitochondrial apoptotic pathway in human umbilical vein endothelial cells (HUVECs). Study of the underlying mechanism showed that hsa-miRNA-29a/Bax plays an essential role in the decreased proliferation and increased apoptosis of HUVECs induced by HG, and overexpression of hsa-miRNA-29a effectively inhibits HG-induced apoptosis and restores the proliferation and tube formation of HUVECs exposed to HG by inhibiting its target gene Bax. In short, our study demonstrates that hsa-miRNA-29a is a promising target for the prevention and treatment of vascular injury in diabetic patients.     

Apoptosis and cell recovery in response to oxidative stress in p53-deficient prostate carcinoma cells

We have studied the effects of different concentrations of H(2)O(2) on the proliferation of PC-3 prostate carcinoma cells. Since this cell line lacks functional p53, we sought to characterize whether apoptotic response to the oxidative insult was altered such that, unlike in cells containing functional p53 apoptosis may be reduced and replaced by other mechanisms of cellular arrest and death. We did not observe necrosis in PC-3 cells treated with H(2)O(2) concentrations of up to 500 microM. In the presence of 50 microM H(2)O(2), arrest was observed in the G2-phase of the cell cycle, along with p53-independent apoptosis. In the presence of 500 microM H(2)O(2), addition of l-buthionine sulfoximine increased the percentage of apoptotic cell death. Senescence-associated cell arrest was never observed. Moreover, some of the treated cells seemed to be resistant to oxidative damage. These cells re-entered the cell cycle and proliferated normally. Analysis of the expression of p21(waf1) and of p21 protein levels, as well as the activity of caspase-3 and caspase-8, allowed us to characterize some aspects of the arrest of PC-3 cells in G2 and the apoptotic response to oxidative stress in the absence of functional p53.     

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

Cell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells.     

The effect of NF-��B pathway on proliferation and apoptosis of human umbilical vein endothelial cells induced by intermittent high glucose

Our previous study found that blocking nuclear factor (NF)-κB signaling could protect human umbilical vein endothelial cells (HUVECs) from apoptosis and proliferation inhibition due to high glucose (HG). Intermittent HG makes glucose toxicity more significant. In this study, we aimed to investigate the effect of NF-κB pathway on HUVECs induced by intermittent HG (a daily alternating 5.5 or 30.5 mmol/l glucose). A recombinant adenovirus containing a RNAi cassette targeting the NF-κB/p65 gene was produced, and its silencing effect on p65 gene was detected by Western blot analysis in HUVECs cultured with intermittent HG. The subsequent effect on proliferation of HUVECs in the indicated conditions was measured by the AlamarBlue assay. The Bcl-2 expression was also detected by Western blot. The results showed that the expression of p65 protein could be inhibited efficiently by the RNAi adenovirus. Intermittent HG also induced the translocation of NF-κB in HUVECs. Inhibition of NF-κB with the RNAi adenovirus could prevent the effects. At the 6th day after HUVECs were exposed to intermittent HG, the proliferation of HUVECs with Ad-1566 was significantly higher than that of HUVECs with Ad-DEST (P < 0.01). Knockdown of NF-κB/p65 up-regulated the Bcl-2 expression of HUVECs under intermittent HG conditions (P < 0.01). These findings concluded that the NF-κB/p65-targeting RNAi adenovirus is an important tool, which can efficiently inhibit the expression of p65 gene in HUVECs. Intermittent HG reduces HUVECs proliferation with a concomitant increase in apoptosis. Knockdown of NF-κB/p65 partly protected HUVECs from proliferation inhibition and may reduce apoptosis.     

MiR-455-5p ameliorates HG-induced apoptosis,oxidative stress and inflammatory via targeting SOCS3 in retinal pigment epithelial cells

Diabetic retinopathy (DR) remains the leading cause of blindness in adults with diabetes mellitus. Numerous microRNAs (miRNAs) have been identified to modulate the pathogenesis of DR. The main purpose of this study was to evaluate the potential roles of miR-455-5p in high glucose (HG)-treated retinal pigment epithelial (RPE) cells and underlying mechanisms. Our present investigation discovered that the expression of miR-455-5p was apparently downregulated in ARPE-19 cells stimulated with HG. In addition, forced expression of miR-455-5p markedly enhanced cell viability and restrained HG-induced apoptosis accompanied by decreased BCL2-associated X protein (Bax)/B-cell leukemia/lymphoma 2 (Bcl-2) ratio and expression of apoptotic marker cleaved caspase-3 during HG challenged. Subsequently, augmentation of miR-455-5p remarkably alleviated HG-triggered oxidative stress injury as reflected by decreased the production of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) content as well as NADPH oxidase 4 expression, concomitant with enhanced the activities of superoxide dismutase, catalase, and GPX stimulated with HG. Furthermore, enforced expression of miR-455-5p effectively ameliorated HG-stimulated inflammatory response as exemplified by repressing the secretion of inflammatory cytokines interleukin 1β (IL-1β), IL-6, and tumour necrosis factor-α in ARPE-19 cells challenged by HG. Most importantly, we successfully identified suppressor of cytokine signaling 3 (SOCS3) as a direct target gene of miR-455-5p, and miR-455-5p negatively regulated the expression of SOCS3. Mechanistically, restoration of SOCS3 abrogated the beneficial effects of miR-455-5p on apoptosis, accumulation of ROS, and inflammatory factors production in response to HG. Taken together, these findings demonstrated that miR-455-5p relieved HG-induced damage through repressing apoptosis, oxidant stress, and inflammatory response by targeting SOCS3. The study gives evidence that miR-455-5p may serve as a new potential therapeutic agent for DR treatment.     

Hexosamine induction of oxidative stress, hypertrophy and laminin expression in renal mesangial cells: effect of the anti-oxidant alpha-lipoic acid

We have previously shown that one of the potential mediators of the deleterious effects of high glucose on extracellular matrix protein (ECM) expression in renal mesangial cells is its metabolic flux through the hexosamine biosynthesis pathway (HBP). Here, we investigate further whether the hexosamines induce oxidative stress, cell-cycle arrest and ECM expression using SV-40-transformed rat mesangial (MES) cells and whether the anti-oxidant alpha-lipoic acid will reverse some of these effects. Culturing renal MES cells with high glucose (HG, 25 mM) or glucosamine (GlcN, 1.5 mM) for 48 h stimulates laminin gamma1 subunit expression significantly approximately 1.5 +/- 0.2- and 1.9 +/- 0.3-fold, respectively, when compared to low glucose (LG, 5 mM). Similarly, HG and GlcN increase the level of G0/G1 cell-cycle progression factor cyclin D1 significantly approximately 1.7 +/- 0.2- and 1.4 +/- 0.04-fold, respectively, versus LG (p < 0.01 for both). Azaserine, an inhibitor of glutamine:fruc-6-PO(4) amidotransferase (GFAT) in the HBP, blocks the HG-induced expression of laminin gamma1 and cyclin D1, but not GlcN's effect because it exerts its metabolic function distal to GFAT. HG and GlcN also elevate reactive oxygen species (ROS) generation, pro-apoptotic caspase-3 activity, and lead to mesangial cell death as revealed by TUNEL and Live/Dead assays. FACS analysis of cell-cycle progression shows that the cells are arrested at G1 phase; however, they undergo cell growth and hypertrophy as the RNA/DNA ratio is significantly (p < 0.05) increased in HG or GlcN-treated cells relative to LG. The anti-oxidant alpha-lipoic acid (150 microM) reverses ROS generation and mesangial cell death induced by HG and GlcN. Alpha-lipoic acid also reduces HG and GlcN-induced laminin gamma1 and cyclin D1 expression in MES cells. In addition, induction of diabetes in rats by streptozotocin (STZ) increases both laminin gamma1 and cyclin D1 expression in the renal cortex and treatment of the diabetic rats with alpha-lipoic acid (400 mg kg(-1) body weight) reduces the level of both proteins significantly (p < 0.05) when compared to untreated diabetic rats. These results support the hypothesis that the hexosamine pathway mediates mesangial cell oxidative stress, ECM expression and apoptosis. Anti-oxidant alpha-lipoic acid reverses the effects of high glucose, hexosamine and diabetes on oxidative stress and ECM expression in mesangial cells and rat kidney.     

MicroRNA-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of X-linked inhibitor of apoptotic protein in endothelial cells

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that have emerged as one of the central players of gene expression regulation. Endothelial cell apoptosis plays a fundamental role in the development of atherosclerosis. This study was designed to determine the effect of miR-513a-5p on apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) and miR-513a-5p expression levels were determined. MiR-513a-5p target gene indentification, validation, and signalling pathways were investigated. Treatment of HUVECs with TNF-α and LPS up-regulated miR-513a-5p expressions more than 2-fold compared to control (P < 0.05). Inhibition of miR-513a-5p by antisense (AS) miR-513a-5p reversed TNF-α and LPS induced apoptosis (P < 0.01). Transfection of HUVECs with miR-513a-5p mimics also induced apoptosis (P < 0.01). Treatment of HUVECs with TNF-α and LPS attenuated X-linked inhibitor of apoptosis (XIAP) while increased caspase-3 expression, poly ADP-ribose polymerase (PARP) cleavage, and p53 expression. These effects were reversed by inhibition of miR-513a-5p. Of those miR-513a-5p candidate target genes, we identified and validated XIAP as a miR-513a-5p target gene. Targeting of the XIAP 3′-untranslated region by miR-513a-5p using luciferase reporter assay resulted in attenuated luciferase activity. Transfection of HUVECs with AS miR-513a-5p increased XIAP protein expression while miR-513a-5p mimics attenuated XIAP expression. These results together suggest that miR-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of XIAP in HUVECs.     

Cariporide inhibits high glucose-mediated adhesion of monocyte-endothelial cell and expression of intercellular adhesion molecule-1

The aim of this study was to examine whether cariporide, a new inhibitor of Na(+)/H(+) exchanger 1 (NHE-1), may inhibit high glucose-induced monocyte-endothelial cell adhesion and the expression of intercellular adhesion molecule-1 (ICAM-1). Cultured endothelial cells were incubated with normal glucose control (5.5 mM), cariporide control (5.5 mM glucose plus 10 microM cariporide), hyperosmolarity (5.5 mM glucose plus 16.5 mM mannitol), high glucose (HG, 22 mM), low-concentration cariporide (22 mM glucose plus 0.1 microM cariporide), medium-concentration cariporide (22 mM glucose plus 1 muM cariporide), and high-concentration cariporide (22 mM glucose plus 10 microM cariporide) for 24 h. Monocytes were isolated from peripheral human blood. Adhered monocytes were quantified by measuring their protein content. ICAM-1 expression and NHE-1 activity was determined with enzyme-linked immunosorbent assay (ELISA) and pH-sensitive fluorescent spectrophotometry. Exposure of endothelial cells to HG for 24 h caused an increase of adhesion of monocytes to endothelial cells and an increased expression of ICAM-1. However, these effects were reversed by treatment with cariporide (0.1, 1, 10 microM) in a concentration-dependent manner. Furthermore, cariporide (1 microM) was able to inhibit the activation of NHE-1 induced by HG in endothelial cells. These findings suggest that cariporide might inhibit HG-mediated monocyte-endothelial cell adhesion and expression of ICAM-1 by inhibiting the activation of NHE-1.     

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

Cell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells.     

Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used

INTRODUCTION: In vitro exposure of cells to a fluorochrome-labeled inhibitor of caspases (FLICA) labels cells after caspase activation and arrests further progress of apoptotic cell death. The labeled apoptotic cells can be quantified in relation to time of apoptosis induction with flow cytometry. Loss of membrane integrity (late apoptosis and cell death) was measured with exposure to propidium iodide (PI). From the labeling patterns with FLICA and PI the apoptotic cell death kinetics was calculated. METHODS: HL60 cells and human umbilical vein endothelial cells (HUVECs) were incubated in the presence of the fluorescent inhibitor of caspases, FAM-VAD-FMK (20 mM, FLICA) for up to 48 h. Apoptosis was induced by Camptothecin (CPT, 0.15 microM) or by a mixture of tumour necrosis factor alpha (TNF-alpha, 3 nM)-Cycloheximide (CHX, 50 microM). Samples were counterstained with PI. RESULTS: Incubation of HL60 cells with CPT induced apoptosis in 92% of cells within the first 18 h at a rate of 5% per hour while incubation with TNF-alpha/CHX resulted in apoptosis in 76% of the cells within the first 6 h at a rate of 12% per hour. Incubation of HUVECs with TNF-alpha/CHX induced apoptosis in 65% of the cells within the first 18 h at a rate of 3.7% per hour during the first 6 h of the incubation. During incubation with TNF-alpha/CHX the remaining viable HL60 cells and HUVECs entered apoptosis within 48 h at an approximate rate of 0.2 per hour. However, on the road of the cell death, HL60 cells showed a transit from the viable (FLICA-/PI-) to early (FLICA+/PI-) and further to late apoptotic phase (FLICA+/PI+), while HUVECs entered directly from the viable to the late apoptotic stage. CONCLUSION: Apoptotic turnover rate depends on the stimulus used to induce apoptosis, while the type of the cell determines the way of the transition within the apoptotic cascade.     

Myricetin ameliorates high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells

Endothelial dysfunction is recognized as the initial detectable stage of cardiovascular disease, a serious complication of diabetes. In this study, we evaluated effects of myricetin on high glucose (HG)-elicited oxidative damage in human umbilical vein endothelial cells (HUVECs). The cells were pre-incubated with myricetin and then treated with HG to induce apoptosis. The effect of myricetin on viability was investigated by MTT assay. The levels of lipid peroxidation (LPO) were determined by thiobarbituric acid (TBA) method. The protein expression of Bax, Bcl-2 and caspase-3 was measured by western blot analysis. Moreover, the effect of myricetin on total antioxidant capacity (TAC) and total thiol molecules was also determined. Our results showed that myricetin was able to markedly restore the viability of endothelial cells under oxidative stress. Myricetin reduced HG-caused increase in LPO levels. Also, TAC and total thiol molecules were notably elevated by myricetin. Incubation with myricetin decreased the protein expression levels of Bax, whereas it increased the expression levels of the Bcl-2, compared with HG treatment alone. Furthermore, myricetin significantly decreased cleaved caspase-3 protein expression. It is concluded that myricetin may protect HUVECs from oxidative stress induced by HG via increasing cell TAC and reducing Bax/Bcl-2 protein ratio, and caspase-3 expression.     

Low expression of miR-186-5p regulates cell apoptosis by targeting toll-like receptor 3 in high glucose–induced cardiomyocytes

To investigate the effect and mechanism of microRNA-186-5p (miR-186-5p) on the apoptosis in high glucose (HG)–treated cardiomyocytes. Diabetic cardiomyopathy model was established in cardiomyocytes by stimulating with HG. The expressions of miR-186-5p and toll-like receptor 3 (TLR3) were detected by quantitative polymerase chain reaction or Western blot analysis, respectively. Apoptosis was detected in HG-treated cardiomyocytes by flow cytometry and Western blot analysis. The interaction between miR-186-5p and TLR3 was explored by bioinformatics analysis and luciferase activity assay. Results showed that miR-186-5p expression was downregulated in HG-treated cardiomyocytes and its overexpression reversed HG-induced apoptosis and cleaved caspase-3 protein expression. Moreover, TLR3 was indicated as a target of miR-186-5p and regulated by miR-186-5p. Knockdown of TLR3 suppressed HG-induced apoptosis and cleaved caspase-3 protein expression. Besides, restoration of TLR3 ablated the effect of miR-186-5p on cell apoptosis. Collectively, miR-186-5p attenuated HG-induced apoptosis by regulating TLR3 in cardiomyocytes, providing novel biomarker for treatment of diabetic cardiomyopathy.     

Knockdown of miR-423-5p simultaneously upgrades the eNOS and VEGFa pathways in ADSCs and improves erectile function in diabetic rats

This study aimed to explore the possibility of miR-423-5p modified adipose-derived stem cell (ADSCs) therapy on streptozotocin (STZ)-induced diabetes mellitus erectile dysfunction (DMED) rats. MiR-423-5p was knocked down in ADSCs. ADSCs, NC-miR-ADSCs and miR-ADSCs were co-cultured with human umbilical vein endothelial cells (HUVECs). Normal and high glucose media were supplemented. The supernatant and HUVECs were collected for assessment of eNOS and VEGFa expression, cell proliferation, and apoptosis. HUVECs co-cultured with ADSCs or miR-ADSCs exhibited higher eNOS and VEGFa protein expression levels compared to DM groups. MiR-ADSCs enhanced HUVEC proliferation compared to the ADSCs and NC-miR-ADSCs. Lower apoptotic rates were observed when HUVECs were co-cultured with miR-ADSCs, compared to ADSCs and NC-miR-ADSCs. Fifteen male Sprague-Dawley (SD) rats aged 12 weeks were induced to develop diabetes mellitus by intraperitoneal injection with STZ, and five healthy SD rats were used as normal controls. Eight weeks after developing diabetes, the rats received ADSCs and miR-ADSCs via injection into the corpora cavernosa, whereas normal controls and DM controls were injected with saline. Erectile function and histological assessment of penile tissues were performed 8 weeks after injection. The ICP/MAP indicated that erectile function was impaired in the DM rats compared with the normal group. Injection of ADSCs and miR-ADSCs improved erectile function significantly and was associated with the overexpression of eNOS and VEGFa. MiR-423-5p knockdown in ADSCs ameliorated high glucose-mediated damage to HUVECs and improved erectile function in DM rats by inducing eNOS and VEGFa overexpression, indicating that miR-423-5p may be a potential target in the treatment of DMED.     

CDIP, a novel pro-apoptotic gene, regulates TNFalpha-mediated apoptosis in a p53-dependent manner

We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-alpha expression tightly correlates with CDIP expression, and that inhibition of TNF-alpha signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-alpha is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-alpha impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 --> CDIP --> TNF-alpha apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy.     

Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria

Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO-induced cell death is mediated by mitochondria. Addition of NO donors, 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP) or 1 mM diethylenetriamine-NO adduct (NOC-18), to PC12 cells resulted in a steady-state level of 1-3 microM: NO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24-h incubation of PC12 cells with NO donors (SNAP or NOC-18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80-100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO-induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl-Val-Ala-fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase-3- and caspase-3-processing-like proteases. Caspase-1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase-3- and caspase-3-processing-like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochrome c from mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC-18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochrome c, and subsequent caspase activation. NO-induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.     

Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures

Evidence has accumulated showing that pharmacological inhibition of proteasome activity can both induce and prevent neuronal apoptosis. We tested the hypothesis that these paradoxical effects of proteasome inhibitors depend on the degree of reduced proteasome activity and investigated underlying mechanisms. Murine cortical cell cultures exposed to 0.1 microM MG132 underwent widespread neuronal apoptosis and showed partial inhibition of proteasome activity down to 30-50%. Interestingly, administration of 1-10 microM MG132 almost completely blocked proteasome activity but resulted in reduced neuronal apoptosis. Similar results were produced in cortical cultures exposed to other proteasome inhibitors, proteasome inhibitor I and lactacystin. Administration of 0.1 microM MG132 led to activation of a mitochondria-dependent apoptotic signaling cascade involving cytochrome c, caspase-9, caspase-3 and degradation of tau protein; such activation was markedly reduced with 10 microM MG132. High doses of MG132 prevented the degradation of inhibitor of apoptosis proteins (IAPs) cIAP and X chromosome-linked IAP, suggesting that complete blockade of proteasome activity interferes with progression of apoptosis. In support of this, addition of high doses of proteasome inhibitors attenuated apoptosis of cortical neurons deprived of serum. Taken together, the present results indicate that inhibition of proteasome activity can induce or prevent neuronal cell apoptosis through regulation of mitochondria-mediated apoptotic pathways and IAPs.