Mitochondrial pathway mediated by reactive oxygen species involvement in α-hederin-induced apoptosis in hepatocellular carcinoma cells

AIM To investigate the antitumor activity of α-hederin in hepatocellular carcinoma(HCC) cells and its underlying mechanisms in vitro and in vivo.METHODS SMMC-7721, Hep G-2 and Huh-7 HCC cells were cultured in vitro and treated with α-hederin(0, 5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L, 25 μmol/L, 30 μmol/L, 35 μmol/L, 40 μmol/L, 45 μmol/L, 50 μmol/L, 55 μmol/L, or 60 μmol/L) for 12 h, 24 h, or 36 h, and cell viability was then detected by the Cell Counting Kit-8. SMMC-7721cells were treated with 0, 5 μmol/L, 10 μmol/L, or 20 μmol/L α-hederin for 24 h with or without DL-buthionineS,R-sulfoximine(2 mmol/L) or N-acetylcysteine(5 mmol/L) pretreatment for 2 h, and additional assays were subsequently performed. Apoptosis was observed after Hoechst staining. Glutathione(GSH) and adenosine triphosphate(ATP) levels were measured using GSH and ATP Assay Kits. Intracellular reactive oxygen species(ROS) levels were determined by measuring the oxidative conversion of 2',7'-dichlorofluorescin diacetate. Disruption of the mitochondrial membrane potential was evaluated using JC-1 staining. The protein levels of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C were detected by western blotting. The antitumor efficacy of α-hederin in vivo was evaluated in a xenograft tumor model.RESULTS The α-hederin treatment induced apoptosis of HCC cells. The apoptosis rates in the control, low-dose α-hederin(5 μmol/L), mid-dose α-hederin(10 μmol/L) and highdose α-hederin(20 μmol/L) groups were 0.90% ± 0.26%, 12% ± 2.0%, 21% ± 2.1% and 37% ± 3.8%, respectively(P 0.05). The α-hederin treatment reduced intracellular GSH and ATP levels, induced ROS, disrupted the mitochondrial membrane potential, increased the protein levels of Bax, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C, and decreased Bcl-2 expression. The α-hederin treatment also inhibited xenograft tumor growth in vivo. CONCLUSION The α-hederin saponin induces apoptosis of HCC cells via the mitochondrial pathway mediated by increased intracellular ROS and may be an effective treatment for human HCC.     

The inhibition in tumor necrosis factor-α-induced attenuation in endothelial thrombomodulin expression by carvedilol is mediated by nuclear factor-κB and reactive oxygen species

Carvedilol, a nonselective β-adrenoceptor antagonist, has been shown to possess antioxidant effects and reduce the risk of hospitalization and death in patients with severe congestive heart failure, which is featured by the activation of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), and leads to thrombotic complications. Thrombomodulin (TM) plays protective roles against thrombosis. Treatment of ECs with TNF-α resulted in a down-regulation in the TM expression in a time-dependent manner. Pre-treatment of ECs with carvedilol (1 and 10 μM) for 1 h significantly up-regulated the TM expression in ECs in response to TNF-α. When ECs were pre-treated with a nuclear factor-κB (NF-κB) inhibitor, i.e., parthenolide, their TNF-α-mediated down-regulation of TM expression was inhibited. Pre-treatment of ECs with carvedilol inhibited the NF-κB-DNA binding activity in ECs induced by TNF-α. Our findings provide insights into the mechanisms by which carvedilol exerts anti-thrombotic effects by inducing TM expression in ECs in response to pro-inflammatory stimulation.     

Protein farnesylation is requisite for mitochondrial fuel-induced insulin release: Further evidence to link reactive oxygen species generation to insulin secretion in pancreatic β-cells

Several lines of recent evidence implicate regulatory roles for reactive oxygen species (ROS) in islet function and insulin secretion. The phagocyte-like NADPH oxidase (Nox2) has recently been shown to be one of the sources of ROS in the signaling events leading to glucose stimulated insulin secretion (GSIS). We recently reported inhibition of glucose- or mitochondrial fuel-induced Nox2-derived ROS by a specific inhibitor of protein farnesyl transferse (FTase; FTI-277), suggesting that activation of FTase might represent one of the upstream signaling events to Nox2 activation. Furthermore, FTase inhibitors (FTI-277 and FTI-2628) have also been shown to attenuate GSIS in INS 832/13 cells and normal rodent islets. Herein, we provide further evidence to suggest that inhibition of FTase either by pharmacological (e.g., FTI-277) or gene silencing (siRNA-FTase) approaches markedly attenuates mitochondrial fuel-stimulated insulin secretion (MSIS) in INS 832/13 cells. Together, our findings further establish a link between nutrient-induced Nox2 activation, ROS generation and insulin secretion in the pancreatic β-cell.     

In vitro reversal of hyperglycemia normalizes insulin action in fat cells from type 2 diabetes patients: is cellular insulin resistance caused by glucotoxicity in vivo?

Chronic hyperglycemia promotes the development of insulin resistance. The aim of this study was to investigate whether cellular insulin resistance is secondary to the diabetic state in human type 2 diabetes. Subcutaneous fat biopsies were taken from 3 age-, sex-, and body mass index (BMI)-matched groups with 10 subjects in each group: type 2 diabetes patients with either good (hemoglobin A(1c) [HbA(1c)] < 7%, G) or poor (HbA(1c) > 7.5%, P) metabolic control and healthy control subjects (C). Insulin action in vitro was studied by measurements of glucose uptake both directly after cell isolation and following a 24-hour incubation at a physiological glucose level (6 mmol/L). The relationship with insulin action in vivo was addressed by employing the euglycemic clamp technique. Freshly isolated fat cells from type 2 diabetes patients with poor metabolic control had approximately 55% lower maximal insulin response (1,000 microU/mL) on glucose uptake (P <.05) compared to C. Cells from P were more insulin-resistant (P <.05) than cells from G at a low (5 microU/mL) but not at a high (1,000 microU/mL) insulin concentration, suggesting insulin insensitivity. However, following 24 hours of incubation at physiological glucose levels, insulin resistance was completely reversed in the diabetes cells and no differences in insulin-stimulated glucose uptake were found among the 3 groups. Insulin sensitivity in vivo assessed with hyperinsulinemic, euglycemic clamp (M-value) was significantly associated with insulin action on glucose uptake in fresh adipocytes in vitro (r = 0.50, P <.01). Fasting blood glucose at the time of biopsy and HbA(1c), but not serum insulin, were negatively correlated to insulin's effect to stimulate glucose uptake in vitro (r = -0.36, P =.064 and r = - 0.41, P <.05, respectively) in all groups taken together. In the in vivo situation, fasting blood glucose, HbA(1c), and serum insulin were all negatively correlated to insulin sensitivity (M-value; r = -0.62, P<.001, r= -0.61, P<.001, and r = -0.56, p <.01, respectively). Cell size, waist-to-hip ration (WHR), and BMI correlated negatively with insulin's effect to stimulate glucose uptake both in vitro (r = -0.55, P <.01, r = -0.54, P <.01, and r = -0.43, P <.05, respectively) and in vivo (r = -0.43, P <.05, r = -0.50, P <.01, and r = -0.36, P <.05, respectively). Multiple regression analyses revealed that adipocyte cell size and WHR independently predicted insulin resistance in vitro. Furthermore, insulin sensitivity in vivo could be predicted by fasting blood glucose and serum insulin levels. We conclude that insulin resistance in fat cells from type 2 diabetes patients is fully reversible following incubation at physiological glucose concentrations. Thus, cellular insulin resistance may be mainly secondary to the hyperglycemic state in vivo.     

Is insulin resistance caused by defects in insulin's target cells or by a stressed mind?

The importance of understanding insulin action is emphasized by the increasing prevalence of insulin resistance in various populations and by the fact that it plays an important pathophysiological role in many common disorders, for example, diabetes, obesity, hypertension and dyslipidemia. The primary factors responsible for the development of insulin resistance are so far unknown, although both genetic and environmental factors are involved. The genetic defects responsible for the common forms of insulin resistance, for example, in type 2 diabetes, are largely unidentified. Some studies from our group as well as by other investigators suggest that cellular insulin resistance is reversible and that it may be secondary to factors in the in vivo environment. These may include insulin-antagonistic action of hormones like catecholamines, glucocorticoids, sex steroids and adipokines as well as dysregulation of autonomic nervous activity and they could contribute to the early development of insulin resistance. Some of these factors can directly impair glucose uptake capacity and this might be due to alterations in key proteins involved in insulin's intracellular signaling pathways. This article briefly summarizes proposed mechanisms behind cellular and whole-body insulin resistance. In particular, we question the role of intrinsic defects in insulin's target cells as primary mechanisms in the development of insulin resistance in type 2 diabetes and we suggest that metabolic and neurohormonal factors instead are the main culprits.     

Phenylacetic acid stimulates reactive oxygen species generation and tumor necrosis factor-α secretion in vascular endothelial cells

Tumor necrosis factor (TNF)-α and oxidative stress are considered to play crucial roles in atherosclerosis and vascular calcification. "Uremic toxins" detected in patients with chronic kidney disease (CKD) could cause impaired signal transduction and dysfunction in many organs. Since phenylacetic acid (PAA), identified as one of the uremic toxins, has an inhibiting property of monocytes as well as osteoblastic cells, we examined the effects of PAA on TNF-α secretion and oxidative stress in vascular endothelial cells. In human aortic endothelial cells, TNF-α secretion was assessed after treatment with PAA using an ELISA kit and following the manufacturer's instructions. For determination of reactive oxygen species (ROS), 8-hydroxydeoxyguanosine (8-OHdG) in the culture medium was measured in the presence or absence of PAA. Treatment with PAA in aortic endothelial cells for 24 h significantly stimulated TNF-α secretion in a dose-dependent manner ranging between 0.5 and 5 mM. On the other hand, the 8-OHdG level in the culture medium was significantly increased in the cells incubated with 1 mM PAA for 12 h. To determine if PAA-induced TNF-α secretion is mediated by ROS production, the effect of free radical scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) was examined. It was found that PAA-induced TNF-α secretion was significantly inhibited by TEMPOL. Our findings indicate that PAA stimulates TNF-α secretion at least in part through ROS production in aortic endothelial cells. The plasma PAA level was reported to be approximately 3.5 mM in end-stage CKD patients, whereas it was <5 μM in healthy subjects; thus, PAA could be involved in the pathological changes of the vasculature in CKD.     

Deferasirox is a powerful NF-��B inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging

Background

Usefulness of iron chelation therapy in myelodysplastic patients is still under debate but many authors suggest its possible role in improving survival of low-risk myelodysplastic patients. Several reports have described an unexpected effect of iron chelators, such as an improvement in hemoglobin levels, in patients affected by myelodysplastic syndromes. Furthermore, the novel chelator deferasirox induces a similar improvement more rapidly. Nuclear factor-κB is a key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including myelodysplastic syndromes.

Design and Methods

We evaluated deferasirox activity on nuclear factor-κB in myelodysplastic syndromes as a possible mechanism involved in hemoglobin improvement during in vivo treatment. Forty peripheral blood samples collected from myelodysplastic syndrome patients were incubated with 50 μM deferasirox for 18h.

Results

Nuclear factor-κB activity dramatically decreased in samples showing high basal activity as well as in cell lines, whereas no similar behavior was observed with other iron chelators despite a similar reduction in reactive oxygen species levels. Additionally, ferric hydroxyquinoline incubation did not decrease deferasirox activity in K562 cells suggesting the mechanism of action of the drug is independent from cell iron deprivation by chelation. Finally, incubation with both etoposide and deferasirox induced an increase in K562 apoptotic rate.

Conclusions

Nuclear factor-κB inhibition by deferasirox is not seen from other chelators and is iron and reactive oxygen species scavenging independent. This could explain the hemoglobin improvement after in vivo treatment, such that our hypothesis needs to be validated in further prospective studies.     

NADPH oxidase-derived superoxide anion mediates angiotensin II-induced cardiac hypertrophy

Cardiac hypertrophy is an adaptive response to increases in blood pressure. Recent studies indicate that the hypertrophic process is associated with increases in intracellular oxidative stress in cardiomyocytes. We hypothesize that superoxide anion mediates the hypertrophic response and that antioxidant therapy may be effective in attenuating cardiac hypertrophy. Neonatal rat cardiac myocytes were stimulated with angiotensin II (AngII, 1 microM) with and without various antioxidants. N-acetylcysteine (NAC, 10 mM) and probucol (50 microM), and to a lesser extent, vitamin C (500 microM) and reduced glutathione (1 mM), inhibited AngII-induced [(3)H]-leucine uptake and atrial natriuretic factor (ANF) promoter activity. The hypertrophic response is mediated by superoxide anion (O(2)(-).) since cell-permeable polyethylene glycol (PEG)-conjugated superoxide dismutase (50 U/ml), but not PEG-catalase (500 U/ml), attenuated AngII-induced [(3)H]-leucine uptake and ANF promoter activity. Furthermore, NAC blocked AngII-induced increase in myocardial oxidative stress, decreased the expression of ANF and myosin light chain-2v, and inhibited the re-organization of cytoskeletal proteins, desmin and alpha-actinin. These effects of AngII were abolished by angiotensin type 1 receptor blocker, losartan, but not type 2 receptor blocker, PD123319. Indeed, co-administration of losartan (10 mg/kg/d, 14 d) or NAC (200 mg/kg/d, 14 d) inhibited AngII-induced O(2)(-). production and cardiac hypertrophy in rats without affecting blood pressure. These findings indicate that the generation of O(2)(-). contributes to oxidant-induced hypertrophic response and suggest that antioxidant therapy may have beneficial effects in cardiac hypertrophy.     

NADPH氧化酶在瘦素诱导肝星状细胞株HSC-T6产生活性氧中的作用及其机制

目的 研究NADPH氧化酶(NOX)是否参与瘦素诱导肝星状细胞(HSC)产生活性氧(ROS),并探讨其机制. 方法取对数生长期的HSC-T6细胞,随机分为9组:瘦素组、瘦素+ROS生成系统抑制剂[包括氯化二亚苯基碘嗡(DPI)、鱼藤酮、甲双吡丙酮、别嘌呤醇和吲哚美辛]组成的干预组、瘦素+JAK抑制剂AG490组成的干预组、正常对照组和阴性对照组.HSC-T6细胞经药物作用1、12、24 h后,荧光显微镜和(或)流式细胞术观察细胞内二氯荧光素强度来反应ROS水平;分光光度计检测NADPH的吸光度,以NADPH的消耗量表示NOX活性;逆转录聚合酶链反应检测Rac1和p22Phox的mRNA相对表达量.数据分析用单因素方差分析、SNK-q检验或秩和检验,P＜0.05为差异有统计学意义.结果 HSC-T6细胞加入瘦素(100 ng/ml)作用1 h后,荧光强度较正常对照组明显升高(92.91±4.19比27.56±6.27,P＜0.01);DPI(20 μmol/L)和AG490(50 μ mol/L)能抑制ROS的产生,荧光强度值分别为37.35±4.66和53.12±6.63,均低于瘦素组的92.91±4.19(q值分别为31.78和22.76,P值均＜0.01).瘦素作用HSC-T6细胞1 h后,NADPH的消耗量较正常对照组明显升高[(1.90±0.22)pmol·min-1·mg1比(0.76±0.06)pmol·min-1·mg-1,P＜0.05)],而作用12、24 h后的消耗量明显高于1 h时的消耗量(x2值均为7.54,P值均＜0.05);DPI及AG490干预能抑制瘦素诱导NOX活性的上调(q值分别为16.58和16.23,P值均＜0.05).瘦素作用12 h后,HSC-T6细胞内Racl和p22Phox的mRNA相对表达量明显高于正常对照组(分别为0.41±0.13比0.14±0.08和0.45±0.12比0.20±0.08,P值均＜0.05).结论 瘦素诱导HSC产生的ROS主要来源于NOX,其机制可能是瘦素通过JAK信号通路直接激活NOX,并通过上调NOX的亚基表达使NOX活性持续增高而产生大量ROS.     

Mitochondrial reactive oxygen species generation in obese non-diabetic and type 2 diabetic participants

Aims/hypothesis  The aim of this study was to measure mitochondrial reactive oxygen species (ROS) production directly from skeletal muscle biopsies obtained from obese insulin-resistant non-diabetic and type 2 diabetic participants. Methods  Ten lean healthy, ten obese non-diabetic and ten type 2 diabetic participants received a euglycaemic–hyperinsulinaemic clamp to measure whole body insulin sensitivity. Mitochondria were isolated from skeletal muscle biopsies, and mitochondrial ATP synthesis and hydrogen peroxide production were measured ex vivo under conditions that maximally stimulate ATP synthesis and ROS production using chemiluminescent and fluorescent techniques, respectively. Results  Compared with lean controls, both obese non-diabetic and type 2 diabetic participants were resistant to insulin, and had a reduced rate of mitochondrial ATP production. Obese insulin-resistant participants had a decreased rate of mitochondrial ROS production, while ROS production rate in participants with type 2 diabetes was similar to that in lean healthy participants. In non-diabetic participants, the rate of ROS production was strongly correlated with the rate of ATP synthesis and the glucose disposal rate measured with the euglycaemic–hyperinsulinaemic clamp. The ROS/ATP ratio in obese insulin-resistant participants was similar to that in lean insulin-sensitive participants, while the ratio was significantly elevated in type 2 diabetes participants. Conclusions/interpretation  Since, in absolute terms, the maximal capacity for mitochondrial ROS production was not increased in either obese insulin-resistant participants or in type 2 diabetic participants, these results do not favour a role for increased mitochondrial ROS production in the pathogenesis of insulin resistance in human skeletal muscle. However, care should be taken in extrapolating these ex vivo observations to the in vivo situation.     

NADPH氧化酶在氧化应激中的作用

NADPH氧化酶（Nox）是血管内生成活性氧簇（ROS）的主要酶体，在外来信号刺激下激活或失活，从而迅速升高或降低细胞内的ROS水平。ROS过多与炎性反应、甲状腺功能减退、糖尿病和心血管疾病的发生密切相关。Nox亚单位的表达与氧化应激、动脉硬化的发生相关；Nox在有丝分裂活跃的细胞和组织如肿瘤细胞中过度表达，使ROS水平升高。糖尿病患者细胞Nox的活性、p22^phox亚单位mRNA和蛋白质的表达均明显升高，p22^phoxmRNA的升高与糖化血红蛋白（Hb）A1C水平和糖尿病病程呈正相关。通过抑制Nox减少ROS的产生，为有效防治ROS相关的临床疾病提供新思路和新的药物靶点。     

胰岛素抵抗HepG2细胞模型的建立及鉴定

目的 建立呈胰岛素抵抗的ＨｅｐＧ２细胞模型。方法 将ＨｅｐＧ２细胞置于１０＾－７ｍｏｌ／Ｌ胰岛素培养液中２４小时,使ＨｅｐＧ２细胞胰岛素受体充分下调,检测燕比较下调和未下调的ＨｅｐＧ２细胞,糖,脂类代谢水平,发现：下调ＨｅｐＧ２细胞胰岛素受体减少了５６％,将ＤＲ－ＨｅｐＧ２细胞于不同浓度胰岛素培养液中,胰岛素受体数 明显减少,当培养液中胰岛素的浓度为１０＾－７ｍｏｌ／Ｌ时,ＤＲ－ＨｅｐＧ２细胞胰岛     

Resistin induces insulin resistance by both AMPK-dependent and AMPK-independent mechanisms in HepG2 cells

Resistin is a 12.5-KDa cysteine-rich peptide that has been implicated in the impairment of glucose homeostasis via the AMP-activated protein kinase (AMPK) pathway in a rodent model. However, the role resistin plays in humans is controversial. This study investigated the effect of resistin on glucose metabolism and insulin signaling using human recombinant resistin and small interfering RNA (siRNA) against AMPKα2 to treat the human liver HepG2 cells. The mRNA of key genes involved in glucose metabolism and the insulin-signaling pathway were detected by real-time RT-PCR. Phosphorylation levels of Akt and AMPK were measured by western blot. The incorporation of D-[U–¹⁴C] glucose into glycogen was quantitated by liquid scintillation counting. The results demonstrate that resistin stimulated expressions of glucose-6-phosphatase (G6Pase), phosphoenolypyruvate carboxykinase (PEPCK), and suppressor of cytokine signaling 3 (SOCS-3), repressed the expressions of insulin receptor substrate 2(IRS-2) and glucose transporter 2(GLUT2). In addition, resistin inhibited the insulin-induced phosphorylation of Akt independent of AMPK. In conclusion, our findings suggest that resistin induces insulin resistance in HepG2 cells at least partly via induction of SOCS-3 expression and reduction of Akt phosphorylation through an AMPK-independent mechanism. Resistin also increases glucose production via AMPK-mediated upregulated expression of the genes encoding hepatic gluconeogenic enzymes, G6Pase, and PEPCK.     

Increasing uncoupling protein-2 in pancreatic beta cells does not alter glucose-induced insulin secretion but decreases production of reactive oxygen species

Aims/hypothesis Levels of uncoupling protein-2 (UCP2) are regulated in the pancreatic beta cells and an increase in the protein level has been associated with mitochondrial uncoupling and alteration in glucose-stimulated insulin secretion. However, it is not clear whether an increase in uncoupling protein-2 per se induces mitochondrial uncoupling and affects ATP generation and insulin secretion. Materials and methods Transgenic mice with beta cell-specific overexpression of the human UCP2 gene and INS-1 cells with doxycycline-inducible overproduction of the protein were generated and the consequences of increased levels of UCP2 on glucose-induced insulin secretion and on parameters reflecting mitochondrial uncoupling were determined. Results In transgenic mice, an increase in beta cell UCP2 protein concentration did not significantly modify plasma glucose and insulin levels. Glucose-induced insulin secretion and elevation in the ATP/ADP ratio were unaltered by an increase in UCP2 level. In INS-1 cells, a similar increase in UCP2 level did not modify glucose-induced insulin secretion, cytosolic ATP and ATP/ADP ratio, or glucose oxidation. Increased levels of UCP2 did not modify the mitochondrial membrane potential and oxygen consumption. Increased UCP2 levels decreased cytokine-induced production of reactive oxygen species. Conclusion/interpretation The results obtained in transgenic mice and in the beta cell line do not support the hypothesis that an increase in UCP2 protein per se uncouples the mitochondria and decreases glucose-induced insulin secretion. In contrast, the observation that increased UCP2 levels decrease cytokine-induced production of reactive oxygen species indicates a potential protective effect of the protein on beta cells, as observed in other cell types. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorised users. N. Produit-Zengaffinen and N. Davis-Lameloise contributed equally to this work.     

NADPH oxidase-derived superoxide impairs calcium transients and contraction in aged murine ventricular myocytes

Since aging increases oxidative stress, we analyzed the contribution of reactive oxygen species (ROS) to the contractile dysfunction of aged ventricular myocytes and investigated whether short-term interference with ROS formation could normalize contractile performance.