Cyanidin 3-glucoside protects 3T3-L1 adipocytes against H2O2- or TNF-alpha-induced insulin resistance by inhibiting c-Jun NH2-terminal kinase activation

Anthocyanins are naturally occurring plant pigments and exhibit an array of pharmacological properties. Our previous study showed that black rice pigment extract rich in anthocyanin prevents and ameliorates high-fructose-induced insulin resistance in rats. In present study, cyanidin 3-glucoside (Cy-3-G), a typical anthocyanin most abundant in black rice was used to examine its protective effect on insulin sensitivity in 3T3-L1 adipocytes exposed to H(2)O(2) (generated by adding glucose oxidase to the medium) or tumor necrosis factor alpha (TNF-alpha). Twelve-hour exposure of 3T3-L1 adipocytes to H(2)O(2) or TNF-alpha resulted in the increase of c-Jun NH(2)-terminal kinase (JNK) activation and insulin receptor substrate 1 (IRS1) serine 307 phosphorylation, concomitantly with the decrease in insulin-stimulated IRS1 tyrosine phosphorylation and cellular glucose uptake. Blocking JNK expression using RNA interference efficiently prevented the H(2)O(2)- or TNF-alpha-induced defects in insulin action. Pretreatment of cells with Cy-3-G reduced the intracellular production of reactive oxygen species, the activation of JNK, and attenuated H(2)O(2)- or TNF-alpha-induced insulin resistance in a dose-dependent manner. In parallel, N-acetyl-cysteine, an antioxidant compound, did not exhibit an attenuation of TNF-alpha-induced insulin resistance. Taken together, these results indicated that Cy-3-G exerts a protective role against H(2)O(2)- or TNF-alpha-induced insulin resistance in 3T3-L1 adipocytes by inhibiting the JNK signal pathway.     

Modulation of agonist responses at the A(1) adenosine receptor by an irreversible antagonist,receptor-G protein uncoupling and by the G protein activation state

Potency and intrinsic activity of agonists depend on ligand structure, but are also regulated by receptor-G protein stoichiometry. A potential functional reserve in adenosine A(1) receptor-mediated G protein activation was investigated by stimulation of guanosine-5'-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding by the full agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) and the partial agonist 5'-deoxy-5'-methylthioadenosine (MeSA). Pretreatment of rat brain membranes with the irreversible antagonist 1-propyl-3-[3-[[4-(fluorosulfonyl)benzoyl]oxy]-propyl]-8-cyclopentylxanthine revealed no classical receptor reserve for either agonist. The functional significance of the G protein coupling state of the receptor and occupancy of G proteins by guanine nucleotides was assessed after partial uncoupling of receptor-G protein complexes with N-ethylmaleimide and in the presence of increasing GDP concentrations. Agonist EC(50) values in G protein activation were increased after NEM pretreatment and at higher GDP concentrations, and a decrease in the relative intrinsic activity of MeSA was observed. The shift of agonist concentration-response curves to the right, the decrease in maximal effects and the decrease in relative intrinsic activity of the partial agonist point to a functional reserve which has to be attributed to GDP-free receptor-G protein complexes. The mechanisms of action of FSCPX, NEM and GDP were fully consistent with the two-state model of receptor activation. The apparent reserve revealed by GDP reflects a shift from spontaneously active GDP-free receptor-G protein complexes (RG)(*), which can bind [35S]GTPgammaS, to (RG) occupied by GDP. The abundance of (RG)(*) is favored by agonists and by the absence of GDP.     

Effect of 15-lipoxygenase metabolites, 15-(S)-HPETE and 15-(S)-HETE on chronic myelogenous leukemia cell line K-562: reactive oxygen species (ROS) mediate caspase-dependent apoptosis

Growth inhibitory effects of 15-lipoxygenase-1 [13-(S)-HPODE and 13-(S)-HODE] and 15-lipoxygenase-2 [15-(S)-HPETE and 15-(S)-HETE] (15-LOX-1 and LOX-2) metabolites and the underlying mechanisms were studied on chronic myeloid leukemia cell line (K-562). The hydroperoxy metabolites, 15-(S)-HPETE and 13-(S)-HPODE rapidly inhibited the growth of K-562 cells by 3h with IC(50) values, 10 and 15microM, respectively. In contrast, the hydroxy metabolite of 15-LOX-2, 15-(S)-HETE, showed 50% inhibition only at 40microM by 6h and 13-(S)-HODE, hydroxy metabolite of 15-LOX-1, showed no significant effect up to 160microM. The cells exposed to 10microM of 15-(S)-HPETE and 40microM of 15-(S)-HETE showed typical apoptotic features like release of cytochrome c, caspase-3 activation and PARP-1 (poly(ADP) ribose polymerase-1) cleavage. A flow cytometry based DCFH-DA analysis and inhibitory studies with DPI, a pharmacological inhibitor of NADPH oxidase, NAC (N-acetyl cysteine) and GSH revealed that NADPH oxidase-mediated generation of ROS is responsible for caspase-3 activation and subsequent induction of apoptosis in the K-562 cell line.     

Distinct effects of N-ethylmaleimide on formyl peptide- and cyclopiazonic acid-induced Ca2+ signals through thiol modification in neutrophils

In this study, we demonstrate that N-ethylmaleimide (NEM), a cell permeable thiol-alkylating agent, enhanced the [Ca2+]i rise caused by stimulation with cyclopiazonic acid (CPA), a sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibitor, in rat neutrophils. In addition, NEM attenuated the formyl-Met-Leu-Phe (fMLP)-induced [Ca2+]i rise whether NEM was added to cells prior to or after fMLP stimulation. Moreover, application of NEM after fMLP activation in the absence of external Ca2+ inhibited the Ca2+ signal upon addition of Ca2+ to the medium. Similar patterns were also obtained by using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a cell impermeable dithiol-oxidizing agent, which replaced NEM in the CPA- and fMLP-induced [Ca2+]i rise experiments. Treatment with dithiothreitol (DTT), a cell permeable dithiol-reducing agent, N-acetyl-l-cysteine (NAC), a cell permeable monothiol-reducing agent, and tris-(2-carboxyethyl)phosphine (TCEP), a cell impermeable reductant without a thiol group, all rescued the fMLP-induced Ca2+ signal from NEM. Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) C6, inositol trisphosphate receptor (IP3R) 2 and IP3R3. NEM had no effect on the mitochondrial membrane potential. NEM could restore the polarization and F-actin accumulation of fMLP-treated cells to those of the control. In the absence of external Ca2+, NEM rendered the CPA-induced [Ca2+]i elevation persistently but inhibited the fMLP-induced Ca2+ spike, which was reversed by tris-(2-cyanoethyl)phosphine (TCP), a cell permeable reductant without a thiol group. DTNB did not affect the Ca2+ spike caused by fMLP. These results indicate that through protein thiol oxidation, NEM affects the receptor-activated and the store depletion-derived Ca2+ signals in an opposing manner.     

Isoliquiritigenin impairs insulin signaling and adipocyte differentiation through the inhibition of protein-tyrosine phosphatase 1B oxidation in 3T3-L1 preadipocytes

Isoliquritigenin (ISL) is an abundant dietary flavonoid with a chalcone structure, which is an important constituent in Glycyrrhizae Radix (GR). ISL exhibits anti-oxidant activity, and this activity has been shown to play a beneficial role in various health conditions. However, it is unclear whether the anti-oxidant activity of ISL affects insulin signaling pathway and lipid accumulation of adipocytes. We sought to investigate the effects and molecular mechanisms of ISL on insulin-stimulated adipogenesis in 3T3-L1 cells. We investigated whether ISL attenuates insulin-induced Reactive Oxygen Species (ROS) generation, and whether ISL inhibits the lipid accumulation and the expression of adipogenic-genes during the differentiation of 3T3-L1 cells. ISL blocked the ROS generation, suppressed the lipid accumulation and the expression of adipocyte-specific proteins, which are increased in response to insulin stimulation during adipocyte differentiation of 3T3-L1 cells. We also investigated whether the anti-oxidant capacity of ISL is involved in regulating the molecular events of insulin-signaling cascade in 3T3-L1 adipocytes. ISL restores PTP1B activity by inhibiting PTP1B oxidation and IR/PI3K/AKT phosphorylation during the early stages of insulin-induced adipogenesis. Our findings show that the anti-oxidant capacity of ISL attenuated insulin IR/PI3K/AKT signaling through inhibition of PTP1B oxidation, and ultimately attenuated insulin-induced adipocyte differentiation of 3T3-L1 cells.     

Blockade of IGF-1 receptor tyrosine kinase has antineoplastic effects in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. Due to very poor 5-year-survival new therapeutic approaches are mandatory. Most HCCs express insulin-like growth factors and their receptors (IGF-R). As IGF-1R-mediated signaling promotes survival, oncogenic transformation and tumor growth and spread, it represents a potential target for innovative treatment strategies of HCC. Here we studied the antineoplastic effects of inhibiting IGF-1R signaling in HCC cells by the novel IGF-1R tyrosine kinase inhibitor NVP-AEW541. METHODS AND RESULTS: NVP-AEW541 induced a time- and dose-dependent growth inhibition in the human hepatoblastoma and hepatocellular carcinoma cell lines SK-Hep-1, Hep-3B, Hep-G2 and Huh-7. Measurement of LDH-release showed that the antineoplastic effect of NVP-AEW541 was not due to cytotoxicity. Instead NVP-AEW541 induced apoptosis as evidenced by both caspase-3 and -8 activation as well as by apoptosis-specific morphological and mitochondrial changes. In addition, nuclear degradation was monitored by DNA-laddering. NVP-AEW541-treatment suppressed the expression of the antiapoptotic proteins Bcl-2 and survivin, while the expression of the proapoptotic protein BAX was stimulated in a dose-dependent manner. Moreover, NVP-AEW541 arrested the cell cycle at the G1/S checkpoint. When NVP-AEW541 was combined with cytotoxic chemotherapy or with a specific epidermal growth factor receptor antibody additive antiproliferative effects were observed. INTERPRETATION: Inhibition of IGF-1R tyrosine kinase (IGF-1R-TK) by NVP-AEW541 induces growth inhibition, apoptosis and cell cycle arrest in human HCC cell lines without accompanying cytotoxicity. Thus, IGF-1R-TK inhibition may be a promising novel treatment approach in HCC.     

Effects of the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cells.

Chronic toxic effects of arsenic resulting from drinking water are a human health problem, especially in South-America and Asia. Arsenic is capable of influencing various cellular processes, causing adverse effects, including cancer. Although the exact mechanism of the action is not known, a correlation between oxidative stress, tumour promotion and arsenic exposure has been observed. We examined the effects of silymarin and quercetin, in counteracting oxidative stress produced by acute or sub-chronic sodium arsenite exposure. The stress responses to arsenite included an increase in the heat shock protein 70 kDa expression, lipid peroxidation assayed by conjugated dienes measure, and gamma-glutamyl-transpeptidase activity. We found that all these stress responses were eliminated by silymarin and quercetin in acute experiments. Both flavonoids diminished the conjugated dienes formation during sub-chronic cultures. Our results suggest that these antioxidant flavonoids, which may be easily incorporated into the diet, may afford a protective effect against arsenite-induced cytotoxicity.     

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A3 receptor

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A₁ and A_2A adenosine receptors (ARs), but not of A_2B and A₃ARs. Activation of the heterologously expressed human A₃AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4 h exposure to H₂O₂ (750 μM) but not in untransfected cells. The A₃ agonist IB-MECA (EC₅₀ 3.8 μM) and the non-selective agonist NECA (EC₅₀ 3.9 μM) protected A₃ AR-transfected cells against H₂O₂ in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N⁶-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A₃AR to inhibit forskolin-stimulated cAMP formation (IC₅₀ 66 nM) and, similarly, protected A₃-transfected HL-1 cells from apoptosis-inducing H₂O₂ with greater potency (IC₅₀ 35 nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.     

Free radical production induced by methamphetamine in rat striatal synaptosomes

The pro-oxidative effect of methamphetamine (METH) in dopamine terminals was studied in rat striatal synaptosomes. Flow cytometry analysis showed increased production of reactive oxygen species (ROS) in METH-treated synaptosomes, without reduction in the density of dopamine transporters. In synaptosomes from dopamine (DA)-depleted animals, METH did not induce ROS production. Reserpine, in vitro, completely inhibited METH-induced ROS production. These results point to endogenous DA as the main source of ROS induced by METH. Antioxidants and inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC) prevented the METH-induced oxidative effect. EGTA and the specific antagonist methyllycaconitine (MLA, 50 microM) prevented METH-induced ROS production, thus implicating calcium and alpha7 nicotinic receptors in such effect. Higher concentrations of MLA (>100 microM) showed nonspecific antioxidant effect. Preincubation of synaptosomes with METH (1 microM) for 30 min reduced [(3)H]DA uptake by 0%. The METH effect was attenuated by MLA and EGTA and potentiated by nicotine, indicating that activation of alpha(7) nicotinic receptors and Ca(2+) entry are necessary and take place before DAT inhibition. From these findings, it can be postulated that, in our model, METH induces DA release from synaptic vesicles to the cytosol. Simultaneously, METH activates alpha(7) nicotinic receptors, probably inducing depolarization and an increase in intrasynaptosomal Ca(2+). This would lead to DAT inhibition and NOS and PKC activation, initiating oxidation of cytosolic DA.     

Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca(2+)-dependent endonuclease

Seven structurally related flavonoids including luteolin, nobiletin, wogonin, baicalein, apigenin, myricetin and fisetin were used to study their biological activities on the human leukemia cell line, HL-60. On MTT assay, wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them. The cytotoxic effects of wogonin and fisetin were accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including DNA fragmentation, apoptotic bodies and the sub-G1 ratio. Treatment with an apoptosis-inducing concentration of wogonin or fisetin causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity. Further, cleavage of poly(ADP-ribose) polymerase (PARP) and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated HL-60 cells. An increase in the pro-apoptotic protein, bax, and a decrease in the anti-apoptotic protein, Mcl-1, were detected in fisetin- and wogonin-treated HL-60 cells. However, Bcl-2, Bcl-XL, and Bad all remained unchanged in wogonin- and fisetin-treated HL-60 cells. In vitro chromatin digestion revealed that endonuclease activity was profoundly enhanced in wogonin- and fisetin-treated HL-60 cells, and the addition of ethylenediaminetetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA) into the reaction blocked endonuclease activation and at an optimum pH of 7.5. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated wogonin- and fisetin-induced DNA ladders, PARP cleavage, and endonuclease activation. Pretreatment of HL-60 cells with N-acetyl-cysteine or catalase efficiently inhibited H(2)O(2) (200 microM)-induced apoptosis, but showed no inhibitory effect on wogonin- and fisetin-induced DNA ladders, caspase 3 activation, or bax protein induction. Decrease in endogenous ROS production was detected in wogonin- and fisetin-treated HL-60 cells by DCHF-DA assay. In conclusion, our experiments indicate that a decrease in intracellular peroxide level was involved in wogonin- and fisetin-induced apoptosis; activation of caspase 3 and endonuclease, induction of bax protein and suppression of Mcl-1 protein were detected in the process.     

Leptomycin B-induced apoptosis is mediated through caspase activation and down-regulation of Mcl-1 and XIAP expression, but not through the generation of ROS in U937 leukemia cells

Leptomycin B (LMB), which is originally isolated from Streptomyces, possesses anti-tumor properties in vivo and in vitro. Though it was previously reported that LMB induces cell cycle arrest and p53-mediated apoptosis in certain cancer cells, however, the mechanism by which LMB induces apoptosis remains poorly understood. Here, we investigated the mechanisms of apoptosis induced by LMB in U937 cells. Treatment with LMB concentration-dependently induced cytotoxicity and apoptosis in U937 cells that correlated temporally with activation of caspases and down-regulation of Mcl-1 and XIAP. LMB did not change the expressions of Bcl-2 or Bax. A broad spectrum caspase inhibitor, z-VAD-fmk, blocked caspase-3 activation and elevated the survival in LMB-treated U937 cells, suggesting that caspase-3 activation is critical for LMB-induced apoptosis. Interestingly, Bcl-2 overexpression that blocked cytochrome c release by LMB effectively attenuated the apoptotic response to LMB, suggesting that LMB-induced apoptosis is mediated through the mitochondrial pathway. Antioxidants or antioxidant enzymes had no effects on LMB-induced apoptosis. Data of flow cytometry analysis using 2',7'-dichlorofluorescein-diacetate further revealed no reactive oxygen species (ROS) generation by LMB, indicating that apoptosis induced by LMB is ROS-independent. However, the apoptotic response to LMB was not shown in U937 cells pretreated with the sulfhydryl group-containing antioxidant N-acetylcysteine (NAC). Further analysis suggested that NAC directly binds LMB and abolishes the apoptotic effects of LMB. Collectively, these findings suggest that LMB potently induces apoptosis in U937 cells, and LMB-induced apoptosis in U937 cells is related with cytochrome c release, activation of caspases, and selective down-regulation of Mcl-1 and XIAP.     

Apoptosis induced by extracellular ATP in the mouse neuroblastoma cell line N1E-115: studies on involvement of P2 receptors and adenosine

Adenosine triphosphate (ATP) can be released in large amounts from (damaged) cells, leading to locally high concentrations. In this study, we investigated the effect of such high concentrations of ATP on neuroblastoma cells. ATP (>or=30 microM) induced apoptosis in the mouse neuroblastoma cell line N1E-115. Activation of the ATP receptor P2X(7) is one of the routes via which ATP has been shown to induce apoptosis. Although the P2X(7) receptor was present in N1E-115 cells, both at the protein and mRNA level, studies with the P2X(7) receptor agonist benzoyl-benzoyl ATP showed that this receptor was not involved in ATP-induced apoptosis. It has been shown previously that adenosine induces apoptosis in N1E-115 cells after transport inside the cell. In this study, both dipyridamole, a nucleoside transport protein blocker, and uridine, a substrate for this transporter, were able to block ATP-induced apoptosis. This indicated that ATP had to be broken down to adenosine to induce apoptosis. The ecto-nucleotidase inhibitors 6-N,N-diethyl-beta-dibromomethylene-D-adenosine-5'-triphosphate (ARL67156) and alpha,beta-methylene adenosine 5'-diphosphate (AOPCP) commonly used to slow breakdown of ATP did not inhibit ATP breakdown appreciably, while the ATP antagonist PPADS inhibited the breakdown of AMP to adenosine; PPADS was also the only compound capable of inhibiting ATP-induced apoptosis. We conclude that the main route of ATP-induced apoptosis in N1E-115 cells was via breakdown to adenosine.     

Inhibition of arterial contraction by dinitrosyl-iron complexes: critical role of the thiol ligand in determining rate of nitric oxide (NO) release and formation of releasable NO stores by S-nitrosation

The inhibition of arterial tone produced by two nitric oxide (NO) derivatives of biological relevance, dinitrosyl-iron complexes with cysteine (DNIC-CYS) or with glutathione (DNIC-GSH), was compared. Both compounds induced vasorelaxation within the same concentration range (3-300 nM) in endothelium-denuded rat aortic rings. Consistent with a faster rate of NO release from DNIC-CYS than from DNIC-GSH, the relaxant effect of DNIC-CYS was rapid in onset and tended to recover with time, whereas the one of DNIC-GSH developed slowly and was sustained. In addition, DNIC-GSH (0.3 and 1 microM) but not DNIC-CYS (1 microM) induced, even after washout of the drug, a persistent hyporesponsiveness to vasoconstrictors and a relaxant effect of low molecular weight thiols like N-acetylcysteine (NAC, which can displace NO from preformed NO stores). Both effects of DNIC-GSH were associated with elevation of cyclic GMP content and were attenuated by NO scavengers or a cyclic GMP-dependent protein kinases inhibitor. In rings previously exposed to DNIC-GSH, addition of mercuric chloride (which can cleave the cysteine-NO bond of S-nitrosothiols) elicited relaxation, completely blunted the one of NAC and also abolished the persistent elevation of NO content. In conclusion, this study shows that whereas both DNIC-CYS and DNIC-GSH elicited a NO release-associated relaxant effect in isolated arteries, only DNIC-GSH induced an inhibition of contraction which persisted after drug removal. The persistent effect of DNIC-GSH was attributed to the formation of releasable NO stores in arterial tissue, most probably as S-nitrosothiols. Thus, the nature of the thiol ligand plays a critical role in determining the mechanisms and duration of the effect of LMW-DNIC in arteries.     

Protection of rats against 3-butene-1,2-diol-induced hepatotoxicity and hypoglycemia by N-acetyl-l-cysteine

3-Butene-1,2-diol (BDD), an allylic alcohol and major metabolite of 1,3-butadiene, has previously been shown to cause hepatotoxicity and hypoglycemia in male Sprague-Dawley rats, but the mechanisms of toxicity were unclear. In this study, rats were administered BDD (250 mg/kg) or saline, ip, and serum insulin levels, hepatic lactate levels, and hepatic cellular and mitochondrial GSH, GSSG, ATP, and ADP levels were measured 1 or 4 h after treatment. The results show that serum insulin levels were not causing the hypoglycemia and that the hypoglycemia was not caused by an enhancement of the metabolism of pyruvate to lactate because hepatic lactate levels were either similar (1 h) or lower (4 h) than controls. However, both hepatic cellular and mitochondrial GSH and GSSG levels were severely depleted 1 and 4 h after treatment and the mitochondrial ATP/ADP ratio was also lowered 4 h after treatment relative to controls. Because these results suggested a role for hepatic cellular and mitochondrial GSH in BDD toxicity, additional rats were administered N-acetyl-l-cysteine (NAC; 200 mg/kg) 15 min after BDD administration. NAC treatment partially prevented depletion of hepatic cellular and mitochondrial GSH and preserved the mitochondrial ATP/ADP ratio. NAC also prevented the severe depletion of serum glucose concentration and the elevation of serum alanine aminotransferase activity after BDD treatment without affecting the plasma concentration of BDD. Thus, depletion of hepatic cellular and mitochondrial GSH followed by the decrease in the mitochondrial ATP/ADP ratio was likely contributing to the mechanisms of hepatotoxicity and hypoglycemia in the rat.     

Emodin induces apoptosis in human promyeloleukemic HL-60 cells accompanied by activation of caspase 3 cascade but independent of reactive oxygen species production

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active constituent of Rheum palmatum, and showed inhibitory activity on lipopolysaccharide-induced NO production in our previous study. However, the apoptosis-inducing activity of emodin has remained undefined. Among three structurally related anthraquinones, including emodin, physcion, and chrysophanol, emodin showed the most potent cytotoxic effects on HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells. Emodin at apoptosis-inducing concentrations causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity, according to cleavage of caspase 3 substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase 3 fragments being detected in emodin- but not physcion- or chrysophanol-treated HL-60 cells. A decrease in the anti-apoptotic protein, Mcl-1, was detected in emodin-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bad remained unchanged. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated emodin-induced DNA ladders, associated with the blockage of PARP and D4-GDI cleavage. Free radical scavenging agents including NAC, catalase, SOD, ALL, DPI, L-NAME and PDTC showed no preventive effect on emodin-induced apoptotic responses, whereas NAC, CAT and PDTC prevented HL-60 cells from ROS (H(2)O(2))-induced apoptosis through inhibition of caspase 3 cascades. Induction of catalase, but not SOD, activity was detected in emodin-treated HL-60 cells by in gel activity assays, and H(2)O(2)-induced intracellular peroxide level was significantly reduced by prior treatment of emodin in HL-60 cells. Our experiments provide evidence that emodin is an effective apoptosis inducer in HL-60 cells through activation of the caspase 3 cascade, but that it is independent of ROS production.     

Involvement of advanced lipooxidation end products (ALEs) and protein oxidation in the apoptotic actions of nitric oxide in insulin secreting RINm5F cells

We have explored the impact of nitric oxide (NO) exposure on oxidation damage of lipids, and proteins, and the contribution of this type of damage to the activation of the apoptotic program in insulin secreting RINm5F cells. Exposure of cells to NO donors and to interleukin-1 beta (IL-1beta) led to generation of lipooxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Addition of superoxide dismutase (SOD) and catalase (Cat) to cells decreased by 50% MDA and 4-HNE production induced by IL-1beta. Over-expression of Mn-SOD in cells conferred a remarkable decrease (75%) in IL-1beta-induced lipid peroxidation. These data suggest that peroxynitrite (ONOO(-)) mediates peroxidative damage to lipids in this cell system. Inhibitors of advanced lipooxidation end products (ALEs) formation such as aminoguanidine (AG) and pyridoxamine (PM) prevented partially apoptotic events triggered by NO such as DNA fragmentation, caspase-3 activation and cytochrome c release from mitochondria. These findings indicate that ALEs are involved in NO-induced apoptosis. In fact, NO-induced carbonylation of PARP protein preceded its apoptotic degradation and inhibitors of ALEs formation prevented both events. We thus propose that carbonylation of proteins is instrumental in linking NO-dependent lipid oxidation and apoptosis in this cell system.     

Induction of interleukin 8 release from the HMC-1 mast cell line: synergy between stem cell factor and activators of the adenosine A(2b) receptor

The HMC-1 mast cell line has both adenosine A(3) and A(2b) receptors on its surface, but only agonists of the A(2b) receptor are effective at releasing interleukin 8. Object of this study was to look for co-factors for adenosine A(2b) receptor activation. There was a powerful and statistically significant synergy for release of IL-8, both at the mRNA level (measured after 4 hr) and protein level (measured after 24 hr), between adenosine A(2b) receptor agonists and stem cell factor (SCF). Suitable concentrations for showing synergy were 100 ng/mL SCF and 3 microM 5'-N-ethylcarboxamidoadenosine (NECA). At these concentrations, the IL-8 released into the culture medium after SCF and NECA together was typically 3-5-fold greater in amount than the sum of the amounts of IL-8 released after exposure to the same concentrations of NECA and SCF separately. Since mast cells may be exposed to both adenosine and stem cell factor in the diseased lung, the synergy observed in this model system may have implications for asthma.