Targeting hepatoma using nitric oxide donor strategies.

AIMS: The study evaluated the role of increased intracellular nitric oxide (NO) concentration using NO donors or stably NO synthase-3 (NOS-3) overexpression during CD95-dependent cell death in hepatoma cells. The expression of cell death receptors and caspase activation, RhoA kinase activity, NOS-3 expression/activity, oxidative/nitrosative stress, and p53 expression were analyzed. The antitumoral activity of NO was also evaluated in the subcutaneous implantation of NOS-3-overexpressing hepatoma cells, as well NO donor injection into wild-type hepatoma-derived tumors implanted in xenograft mouse models. RESULTS: NO donor increased CD95 expression and activation of caspase-8 and 3 in HepG2, Huh7, and Hep3B cells. NOS-3 overexpression increased oxidative/nitrosative stress, p53 and CD95 expression, cellular Fas-associated death domain (FADD)-like IL-1beta converting enzyme (FLICE) inhibitory protein long (cFLIP(L)) and its short isoform (cFLIP(S)) shift, and cell death in HepG2 (4TO-NOS) cells. The inhibition of RhoA kinase and p53 knockdown using RNA interference reduced cell death in 4TO-NOS cells. The supplementation with hydrogen peroxide (H(2)O(2)) increased NOS-3 activity and cell death in 4TO-NOS cells. NOS-3 overexpression or NO donor injection into hepatoma-derived tumors reduced the size and increased p53 and cell death receptor expression in nude mice. INNOVATION AND CONCLUSIONS: The increase of intracellular NO concentration promoted oxidative and nitrosative stress, Rho kinase activity, p53 and CD95 expression, and cell death in cultured hepatoma cells. NOS-3-overexpressed HepG2 cells or intratumoral NO donor administration reduced tumor cell growth and increased the expression of p53 and cell death receptors in tumors developed in a xenograft mouse model.

Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes.

The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca2+ on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca2+ pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca2+ entrance was induced by A23187 in HepG2. Cell death, Ca2+ mobilization, NOS-1, -2 and -3 expression, AP-1 activation, and NO production were evaluated. GCDCA reduced intracellular Ca2+ concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and l-NAME enhanced, GCDCA-induced cell death. The reduction of Ca2+ entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca2+ entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca2+ concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca2+-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.

Mitochondrial-driven ubiquinone enhances extracellular calcium-dependent nitric oxide production and reduces glycochenodeoxycholic acid-induced cell death in hepatocytes.

Ca(2+) mobilization, nitric oxide (NO), and oxidative stress have been involved in cell death induced by hydrophobic bile acid in hepatocytes. The aim of the study was the elucidation of the effect of the antioxidant mitochondrial-driven ubiquinone (Mito Q) on the intracellular Ca(2+) concentration, NO production, and cell death in glycochenodeoxycholic acid (GCDCA)-treated HepG2 cells. The role of the regulation of the intracellular Ca(2+) concentration by Ca(2+) chelators (EGTA or BAPTA-AM), agonist of Ca(2+) entrance (A23187) or NO (L-NAME or NO donor), was assessed during Mito Q cytoprotection in GCDCA-treated HepG2 cells. Cell death, NO synthase (NOS)-1, -2, and -3 expression, Ca(2+) mobilization, and NO production were evaluated. GCDCA reduced the intracellular Ca(2+) concentration and NOS-3 expression and enhanced cell death in HepG2. NO donor prevented and L-NAME enhanced GCDCA-induced cell death. The reduction of Ca(2+) entry by EGTA, but not its release from intracellular stores by BAPTA-AM, reduced the expression of NOS-3 and enhanced cell death in control and GCDCA-treated cells. Mito Q prevented the reduction of intracellular Ca(2+) concentration, NOS-3 expression, NO production, and cell death in GCDCA-treated HepG2 cells. The conclusion is that the recovery of Ca(2+)-dependent NOS-3 expression by Mito Q may be considered an additional cytoprotective property of an antioxidant.

The reduction of cell death and proliferation by p27(Kip1) minimizes DNA damage in an experimental model of genotoxicity.

Hepatocellular carcinoma (HCC) is the fifth most commonly occurring cancer worldwide. The expression of p27 has been related to reduced severity of tumor grade and recurrence of HCC. The study assessed the role of p27 on the cell proliferation and death, and DNA mutagenesis in experimental genotoxicity induced by aflatoxin B1 (AFB(1)) in cultured hepatocytes obtained from control and p27(Kip1) deficient mice. The overexpression of p27 was assessed with wild type p27(Kip1) expression vector in HepG2 cells. The expression of p27, p21 and p53 was assessed in well and poorly-differentiated liver tumors. DNA damage and cell death induced by AFB(1) were related to a reduction of p27 and p21 expression in cultured hepatocytes. AFB(1)-induced nuclear phosphorylated (Ser 10) p27 degradation was related to a rise of nuclear KIST, Rsk-1 and Rsk-2 expression and cytoplasm phosphorylated (Thr 198) p27 expression. The overexpression of p27 reduced cell proliferation, cell death and DNA damage in AFB(1)-treated hepatocytes. The enhanced survival of patients with well differentiated compared to poorly-differentiated tumors was related to high expression of p27, p21 and p53 in liver sections. The study showed that the p27 reduced cell proliferation and death, as well as the accumulation of DNA damage in hepatocarcinogenesis.

Cellular density and cell type are the key factors in growth inhibition induced by 2,5bis [1-aziridinyl]-1,4 benzoquinone (DZQ).

BACKGROUND: Cell density regulates the expression of various antioxidant enzymes in cell culture. The aim of this study was to study the effect of 2,5 bis-[1-aziridinyl]-1,4 benzoquinone (DZQ), an antitumor quinone bioactivated by NQO1, on HeLa and HepG2 cells cultured at various cell densities. MATERIALS AND METHODS: Quinone toxicity was determined by a colorimetric growth inhibition assay. NQO1 and catalase activities were measured spectrophotometrically in soluble fractions, and NQO1 polypeptide was quantified by immunostaining with a commercial polyclonal antiserum. RESULTS: As reported previously, NQO1 activity was much higher in confluent HeLa cells than in sparse cells. However, HepG2 cultures showed an opposite pattern in the regulation of this antioxidant enzyme, sparse cell cultures showing higher NQO1 activity similar to that found in confluent HeLa cells. The expression pattern of catalase activity was similar to that of NQO1 in HeLa cells, but this activity was constant and cell density-independent in HepG2. The growth inhibition effect of DZQ, correlated with NQO1 activity within a given cell type, but HepG2 was always much more sensitive to DZQ than HeLa cells, even under conditions where NQO1 activity was high in HeLa but low in HepG2. CONCLUSION: These results suggest that NQO1 activity is a major factor for DZQ bioactivation, but this enzyme is not likely the sole factor involved in the growth inhibition mediated by DZQ. Since part of the cytotoxic effect of DZQ is mediated by H2O2, other antioxidant enzymes, mainly catalase, could modulate the different growth inhibition found between HeLa and HepG2 cells. In confluent HeLa cells, the higher activity of NQO1 coincides with an increment of catalase activity, thus, reducing the oxidative stress produced by the H2O2 formed.

Differential regulation of hepatic apoptotic pathways by dietary olive and sunflower oils in the aging rat.

In this work we have studied how dietary fat affects aging-related changes in a number of factors that regulate rat hepatic apoptosis. Animals were fed lifelong with two experimental diets containing either virgin olive oil or sunflower oil as dietary fat. Caspases of the intrinsic and extrinsic pathways of apoptosis, Bcl-2 and Bax polypeptide levels, and plasma membrane neutral sphingomyelinase activity were determined at 6, 12, and 24 months of age. Caspase-8/10 activity (a marker of the extrinsic pathway) was not affected by either aging or dietary fat, but activities of both caspase-9 (a marker of the intrinsic pathway) and caspase-3 (an executioner caspase) were significantly depressed in liver from animals fed on a sunflower oil-based diet. These decreases were not observed in animals fed with a diet based on virgin olive oil, which also resulted in significantly lower Bcl-2/Bax ratios. On the other hand, in comparison with sunflower, dietary olive oil decreased oxidative stress in liver from aged rats, resulting in lower levels of membrane hydroperoxides and higher coenzyme Q levels in plasma membrane. Plasma membrane Mg(2+)-dependent neutral sphingomyelinase was strongly activated in aged rats fed on the sunflower oil diet, but no aging-related increase was observed in animals fed on the olive oil diet. Our results support that dietary oil can alter significantly the susceptibility of hepatocytes to different apoptotic stimuli by altering both pro- and anti-apoptotic mediators, which reinforces the importance of the diet in aging studies. Because virgin olive oil may increase susceptibility of hepatocytes to apoptosis induced through the intrinsic pathway under conditions of decreased oxidative stress, our results may have important implications to understand the potential beneficial effects of that edible oil against liver carcinogenesis during aging.

Enhanced anti-oxidant protection of liver membranes in long-lived rats fed on a coenzyme Q10-supplemented diet.

Coenzyme Q10 supplementation increases life-span of rats fed on a diet enriched with polyunsaturated fatty acids (Quiles, J.L., Ochoa, J.J., Huertas, J.R., Mataix, J., 2004b. Coenzyme Q supplementation protects from age-related DNA double-strand breaks and increased lifespan in rats fed on a PUFA-rich diet. Exp. Gerontol. 39, 189-194). Our study was set as a first attempt to establish a mechanistic link between life span extension and CoQ10 supplementation. When rats were fed on a PUFAn-6 plus CoQ10 diet, levels of CoQ10 were increased in plasma membrane at every time point compared to control rats fed on a PUFAn-6-alone diet. Ratios of CoQ9 to CoQ10 were significantly lower at every time point in both liver plasma membranes and homogenates of CoQ10-supplemented animals. CoQ10 supplementation did not affect cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1), which increased significantly with aging, but plasma membrane-bound NQO1 decreased significantly in the CoQ10-supplemented group at 12 months, when maximal incorporation of exogenous CoQ10 was observed. Neither aging nor the diet affected NADH-cytochrome b5 reductase levels. Glutathione-dependent anti-oxidant activities such as cytosolic glutathione-S-transferase (GST) and microsomal Se-independent glutathione peroxidase decreased with aging and supplementation with CoQ10 attenuated this decay. 2,2' Azobis amidinopropane (AAPH)-induced oxidation of membranes was significantly higher in aged rats, and supplementation with CoQ10 also inhibited this increase. Consistent with higher CoQ10 levels and enhanced anti-oxidant protection, plasma membrane Mg2+-dependent neutral sphingomyelinase was inhibited by dietary CoQ10 in aged rats. Our results support the involvement of thiol-dependent mechanisms in the potentiation of the anti-oxidant capacity of membranes in CoQ10-supplemented rats, further supporting the potentially beneficial anti-oxidative role of dietary CoQ10 during aging. The possibility that a decreased CoQ9/CoQ10 ratio in animals fed on the PUFAn-6-rich plus CoQ10 diet could also influence longevity is also discussed.

Dicoumarol relieves serum withdrawal-induced G0/1 blockade in HL-60 cells through a superoxide-dependent mechanism.

This work was set to study how dicoumarol affects the cell cycle in human myeloid leukemia HL-60 cells. Cells were accumulated in G0/1 after serum deprivation. However, when cells were treated with 5 microM dicoumarol in serum-free medium, a significant increment in the number of cells in S-phase was observed. Inhibition of G0/1 blockade was confirmed by the increase of thymidine incorporation, the phosphorylation of retinoblastoma protein, and the promotion of cell growth in long-term treatments in the absence of serum. Dicoumarol treatment increased superoxide levels, but did not affect peroxide. Increase of cellular superoxide was essential for inhibition of G0/1 blockade, since scavenging this reactive species with a cell-permeable form of SOD and the SOD mimetics 2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine (ambroxol, 100 microM) and copper[II]diisopropyl salicylate (CuDIPS, 10 microM) completely abolished the effect of dicoumarol. However, N-acetyl-cysteine, overexpression of Bcl-2 or a cell-permeable form of catalase were not effective. 5-Methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione (ES936), a mechanism-based irreversible inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), did not promote S phase entry, and dicoumarol still inhibited G0/1 blockade in the presence of ES936. We demonstrate that dicoumarol inhibits the normal blockade in G0/1 in HL-60 cells through a mechanism involving superoxide, but this effect is not dependent solely on the inhibition of the NQO1 catalytic activity. Our results send a precautionary message about use of dicoumarol to elucidate cellular processes involving oxidoreductases.

Antioxidant response induced by serum withdrawal protects HL-60 cells against inhibition of NAD(P)H:quinone oxidoreductase 1.

We have previously shown that inhibition of NAD(P)H:quinone acceptor oxidoreductase 1 with dicoumarol decreases growth and viability of HL-60 cells in the absence of serum. Here we demonstrate that culturing HL-60 cells in serum-free medium in the presence of dicoumarol results in a significant potentiation of apoptosis. However, when cells were preincubated for 24 h without serum before they were treated with dicoumarol, the effect of the inhibitor on cell growth and death was much lower. We have investigated cellular changes induced in HL-60 cells by removal of serum that could account for protection against the effects of dicoumarol. Serum removal induced significant increases of NAD(P)H:quinone acceptor oxidoreductase 1, particularly at 32 h after serum withdrawal. Total amounts of ubiquinone in cells were unchanged but, its reduction state paralleled the observed increase in quinone reductase activity. Levels of the antiapoptotic protein Bcl-2 were also significantly increased after serum removal. Our results indicate that removal of serum evokes an antioxidant protective response that make HL-60 cells less sensitive to cell death induced by inhibition of NAD(P)H:quinone acceptor oxidoreductase 1 with dicoumarol.

Hydrogen peroxide- and cell-density-regulated expression of NADH-cytochrome b5 reductase in HeLa cells.

Environmental conditions regulate the expression of different antioxidant enzymes in cell culture. We have studied the effect of cell density and hydrogen peroxide on the expression of NADH-cytochrome b5 reductase in HeLa cells. Polypeptide levels of the NADH-cytochrome b5 reductase increased about three fold in confluent HeLa cells compared to sparse cells. Addition of H2O2 to HeLa cells altered expression levels of the NADH-cytochrome b5 reducatase in a concentration-dependent way, being sparse cells more sensitive to H2O2 addition than confluent cells. The presence of pyruvate, a H2O2 scavenger, produced a significant increment (200%) in the levels of NADH-cytochrome b5 reductase in sparse cells, but less increase (25%) in confluent cells, suggesting that generation of endogenous H2O2 could repress NADH-cytochrome b5 reductase expression, particularly in sparse cultures. Accordingly, confluent HeLa cells showed significantly lower levels of reactive oxygen species than cells in sparse cultures. Addition of tert-butylhydroquinone, a compound which generates reactive oxygen species through redox cycling, also reduced expression of the NADH-cytochrome b5 reductase. Increments in several antioxidant enzymes taking place during confluency could participate in the increase of NADH-cytochrome b5 reductase expression by reducing reactive oxygen species levels in cells. Overall, our results support that acute oxidative stress caused by H2O2 inhibits the expression levels of NADH-cytochrome b5 reductase, most likely due to inhibition of SP1 transcriptional activity. On the other hand, adaptation to H2O2 involved increased expression of the cytochrome b5 reductase, supporting the existence of additional regulatory mechanisms.

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1.

The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as "hydrophilic" and "hydrophobic" (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372-1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H(2)O(2)-Fe(2+). Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme.

NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance.

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.

A novel plasma membrane quinone reductase and NAD(P)H:quinone oxidoreductase 1 are upregulated by serum withdrawal in human promyelocytic HL-60 cells.

We have studied changes in plasma membrane NAD(P)H:quinone oxidoreductases of HL-60 cells under serum withdrawal conditions, as a model to analyze cell responses to oxidative stress. Highly enriched plasma membrane fractions were obtained from cell homogenates. A major part of NADH-quinone oxidoreductase in the plasma membrane was insensitive to micromolar concentrations of dicumarol, a specific inhibitor of the NAD(P)H:quinone oxidoreductase 1 (NQOI, DT-diaphorase), and only a minor portion was characterized as DT-diaphorase. An enzyme with properties of a cytochrome b5 reductase accounted for most dicumarol-resistant quinone reductase activity in HL-60 plasma membranes. The enzyme used mainly NADH as donor, it reduced coenzyme Q0 through a one-electron mechanism with generation of superoxide, and its inhibition profile by p-hydroxymercuribenzoate was similar to that of authentic cytochrome b5 reductase. Both NQO1 and a novel dicumarol-insensitive quinone reductase that was not accounted by a cytochrome b5 reductase were significantly increased in plasma membranes after serum deprivation, showing a peak at 32 h of treatment. The reductase was specific for NADH, did not generate superoxide during quinone reduction, and was significantly resistant to p-hydroxymercuribenzoate. The function of this novel quinone reductase remains to be elucidated whereas dicumarol inhibition of NQO1 strongly potentiated growth arrest and decreased viability of HL-60 cells in the absence of serum. Our results demonstrate that upregulation of two-electron quinone reductases at the plasma membrane is a mechanism evoked by cells for defense against oxidative stress caused by serum withdrawal.