Pyrrolidine dithiocarbamate induces bovine cerebral endothelial cell death by increasing the intracellular zinc level

The antioxidant and metal-chelating effects of pyrrolidine dithiocarbamate (PDTC) have been extensively studied. PDTC prevents cell death induced by various insults. However, PDTC itself may cause cell death in selected experimental paradigms. PDTC induced bovine cerebral endothelial cell death. However, in serum-depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. The metal chelators bathocuproine disulfonic acid, o-phenanthroline, bathophenanthroline disulfonic acid, and N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN) prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper or zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper or zinc in serum may mediate the cytotoxic effect of PDTC. The potency of zinc for PDTC-induced endothelial cell death was greater than that of copper. Zn-EDTA did not block PDTC-induced cell death, whereas Ca-EDTA and Cu-EDTA were able to prevent this PDTC effect. PDTC increased the intracellular fluorescence of the zinc probe dye N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide, which was quenched by TPEN or various EDTA preparations but not by Zn-EDTA. Results suggest that an increase in intracellular zinc concentration is required in PDTC-induced cerebral endothelial cell death.     

PDTC, metal chelating compound, induces G1 phase cell cycle arrest in vascular smooth muscle cells through inducing p21Cip1 expression: involvement of p38 mitogen activated protein kinase

Pyrrolidine dithiocarbamate (PDTC), a metal chelating compound, is known to induce cell death in vascular smooth muscle cells (VSMC). However, the molecular mechanism for PDTC-induced VSMC death is not well understood. Addition of PDTC reduced cell growth and DNA synthesis on VSMC in low density conditions. However, in serum depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. Several metal chelators prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper, iron, and zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper, iron, and zinc in serum may mediate the cytotoxic effect of PDTC. At low VSMC density in 10% FBS, treatment of PDTC, which induced a cell-cycle block in G1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 or p53 by PDTC was not observed. Finally, we determined PDTC-mediated signaling pathway involved in VSMC death. Among relevant pathways, PDTC induced marked activation of p38MAPK and JNK. Expression of dominant negative p38MAPK and SB203580, a p38MAPK specific inhibitor, blocked PDTC-dependent p38MAPK, growth inhibition, and p21 expression. These data demonstrate that the p38MAPK pathway participates in p21 induction, which consequently leads to decrease of cyclin D1/cdk4 and cyclin E/cdk2 complexes and PDTC-dependent VSMC growth inhibition. In conclusion, an understanding of the molecular mechanisms of PDTC in VSMC provides a theoretical basis for clinical approaches using antioxidant therapies in atherosclerosis.     

Pyrrolidine dithiocarbamate inhibits TNF-alpha-dependent activation of NF-kappaB by increasing intracellular copper level in human aortic smooth muscle cells

Pyrrolidine dithiocarbamate (PDTC) is a metal-chelating compound that acts as antioxidant or pro-oxidant and is widely used to study redox regulation of cell function. In the present study, we investigated effects of PDTC and another antioxidant, N-acetyl-l-cysteine (NAC), on TNF-alpha-dependent activation of NF-kappaB in human aortic smooth muscle cells (HASMC). Treatment of the cells with TNF-alpha induced the activation of p65/p50 heterodimer NF-kappaB and increased the mRNA levels of monocyte chemoattractant protein (MCP)-1. Pretreatment with PDTC markedly suppressed the NF-kappaB activation and expression of MCP-1 by inhibiting IkappaB-alpha degradation. In contrast, NAC had no effect. PDTC concomitantly increased the intracellular levels of copper, and bathocuproinedisulfonic acid, a non-cell-permeable chelator of Cu(1+), inhibited the PDTC-induced increase in intracellular copper level and reversed the PDTC effects on IkappaB-alpha, NF-kappaB, and MCP-1. These results indicate that TNF-alpha-dependent expression of MCP-1 in HASMC is tightly regulated by NF-kappaB and that intracellular copper level is crucial for the TNF-alpha-dependent activation of NF-kappaB in HASMC.     

Activation of heat shock factor 1 by pyrrolidine dithiocarbamate is mediated by its activities as pro-oxidant and thiol modulator

Pyrrolidine dithiocarbamate (PDTC) is known to inhibit NF-kappa B, which plays a critical role(s) as an immediate early mediator of immune and inflammatory responses. Here we show that PDTC induces heat shock factor 1 (HSF1) activation and heat shock protein expression, while other antioxidants such as butylated hydroxytoluene (BHT), n-propylgallate (PG), ascorbic acid (AA), and N-acetyl-L-cysteine (NAC) do not. Since PDTC exerts other functions than antioxidant, e.g., a pro-oxidant, metal chelator, and thiol group modulator, we examined which of these activities is responsible for the PDTC-induced HSF1 activation. PDTC-induced HSF1 activation was not prevented by metal chelators, EDTAs, indicating that the metal chelating effect of PDTC is not linked to the HSF1 activation. PDTC increased intracellular GSSG level. In addition, PDTC-induced activation of HSF1 was significantly inhibited by NAC and a thiol-reducing agent dithiothreitol (DTT), while it was partially prevented by other antioxidants, AA, BHT, and PG. These results suggest that the activation of HSF1 by PDTC may be due to its activities as pro-oxidant and thiol group modulator rather than anti-oxidant.     

Intracellular zinc increase inhibits p53(-/-) pancreatic adenocarcinoma cell growth by ROS/AIF-mediated apoptosis

We show that treatment with non-toxic doses of zinc in association to the ionophore compound pyrrolidine dithiocarbamate (PDTC) inhibits p53(-/-) pancreatic cancer cell growth much more efficiently than gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Both the metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine and the radical scavenger N-acetyl-l-cysteine are able to recover cell growth inhibition by Zn/PDTC, demonstrating that this effect depends on the increased levels of intracellular zinc and of reactive oxygen species (ROS). Zn/PDTC treatment induces a strong apoptotic cell death that is associated to ROS-dependent nuclear translocation of the mitochondrial factor AIF, but not to the regulation of apoptotic genes and caspase activation. Primary fibroblasts are more resistant than pancreatic cancer cells to Zn/PDTC treatment and exhibit a lower basal and Zn/PDTC-induced enhancement of intracellular zinc. We show that Zn/PDTC induces p53 proteasomal degradation and that the proteasome inhibitor MG132 further increases fibroblast growth inhibition by Zn/PDTC, suggesting that p53 degradation plays an important role in fibroblast resistance to Zn/PDTC.     

Pyrrolidine dithiocarbamate induces cyclooxygenase-2 expression in NIH 3T3 fibroblast cells

Pyrrolidine dithiocarbamate (PDTC) is a metal chelating compound that can exert either pro-oxidant or antioxidant effects in different situations. Several studies demonstrate that it can inhibit cyclooxygenase-2 (COX-2) expression, which may be due to its antioxidant activity. Here, we found that PDTC rather increased COX-2 expression in NIH 3T3. The increase of COX-2 expression was inhibited by adding bathocuproline disulfonic acid, a non-permeable specific copper chelator, in the incubation medium. This result suggests that PDTC exerts its effect by transporting redox-active copper ions into the cells. In support of this observation, PDTC did not induce COX-2 expression in a serum-free environment. When PDTC was added with copper in the serum-free medium, it acted as the inducer of COX-2 expression. In addition, pretreatment of N-acetyl-L-cystein or dithiothreitol, other antioxidants, inhibited the PDTC-induced COX-2 expression. Our data indicate that PDTC induces COX-2 expression in NIH 3T3 cells, which may be due to its activities as a copper chelator and a pro-oxidant.     

Enhanced proliferation and altered intracellular zinc levels in early- and late-passage mouse aorta smooth muscle cells

Cell growth and DNA synthesis were studied from a cultured early- and late- passage mouse aorta smooth muscle cell (MASMC) because the proliferation of vascular smooth muscle cell (VSMC) is a key factor in development of atherosclerosis. In this study, the cells were cultured in fetal bovine serum (FBS) and stimulated by growth factors such as thrombin and platelet-derived growth factor-BB (PDGF-BB). Compared to the number of early-passage MASMC (passage 3 to 9) the number of late-passage MASMC (passage 30 to 40) in a normal serum state was increased 2 fold at Day 1,3 and 6 in culture, respectively. Incorporation of [³H] thymidine into DNA induced by serum, PDGF and thrombin in late-passage MASMC was greater than those in early-passage MASMC. We also examined whether intracellular zinc levels would be an aging factor or not. The intracellular zinc level in, early- and late-passage MASMC was monitored by using the zinc probe dyeN-(6-methoxy-8-quinolyl)-p-toluenesulfonamide. It is interested that late-passage MASMC increased the intracellular fluorescence level of zinc, more than the early passage MASMC did. The alterations of intracellular zinc level occur concurrently with changes in MASMC proliferation rate during aging. This data suggest that the age-associated changes in zinc concentrations may provide a newin vitro model for the study of smooth muscle cell differentiation.     

Redox regulation of cell proliferation by pyrrolidine dithiocarbamate in murine thymoma cells transplanted in vivo.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic compound largely used in cell biological studies and known to exert either antioxidant or pro-oxidant effects. Recently, its antitumoral activity has been proposed on the basis of its antioxidant and proapoptotic effects. In the present study, we evaluated the effect of increasing i.p. doses of PDTC on the growth of a strain of highly malignant thymoma cells inoculated in the peritoneum of inbred Balb/c mice. PDTC treatment increased the number of thymoma cells in a dose-dependent manner, enhancing the percentage of proliferating tumor cells. PDTC exerted regulatory effects on cell cycle distribution, decreasing the expression of cell cycle inhibitors. Alterations in the production of intracellular reactive oxygen species, levels of oxidized glutathione, and intracellular levels of the redox-active metals iron and copper were also observed. The above results represent the first evidence that PDTC may induce in vivo cell proliferation in a murine thymoma cell model. In addition, we suggest that the ability of PDTC to bind and transport metals inside the cell and its pro-oxidant property may be factors underlying its effects on thymoma cell proliferation and cell cycle distribution.     

Prooxidant and cytotoxic action of N-acetylcysteine and glutathione in combinations with vitamin B12b

Prooxidant and cytotoxic effects of thiols N-acetylcysteine (NAC) and glutathione (GSH) were studied in combinations with vitamin B_12b. Both GSH and NAC at physiological doses when combined with B_12b were shown to cause initiation of apoptosis. It was established that the prooxidant action of NAC (or GSH) combined with B_12b, i.e., generation and accumulation of hydrogen peroxide in culture medium, led to intractellular oxidative stress and cell redox imbalance. These effects are completely prevented by nonthiol antioxidants catalase and pyruvate. The chelators of iron phenanthroline and deferoxamine do not suppress the H₂O₂ accumulation in culture medium, but inhibit cell death induced by NAC combined with B_12b or by GSH combined with B_12b. Therefore, the thiols GSH or NAC in combination with vitamin B_12b reveal prooxidant properties and induce, with participation of intracellular iron, apoptotic HEp-2 cell death.     

<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> induces aponecrosis in human aortic smooth muscle cells

Background  

The intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis.     

Pyrrolidine dithiocarbamate is a potent antioxidant against hypochlorous acid-induced protein damage

The antioxidant potential of the dithiol compound pyrrolidine dithiocarbamate (PDTC) against protein damage induced by hypochlorous acid (HOCl) was investigated. The effects of PDTC were compared to those of reduced glutathione (GSH) and N-acetylcysteine (NAC). PDTC markedly and in a concentration-dependent manner inhibited HOCl-induced inactivation of alpha(1)-antiproteinase, protein carbonyl formation on serum albumin and oxidation of human low-density lipoprotein. The direct scavenging of HOCl by PDTC was demonstrated by two quantitative methods, oxidation of ferrocyanide and chlorination of monochlorodimedon. In all assay systems, PDTC was two to three times more potent than GSH and NAC, while diethyldithiocarbamate was about as effective as PDTC. These data demonstrate that PDTC is a potent antioxidant against HOCl-induced protein oxidative damage, suggesting that PDTC might be useful in the prevention and treatment of inflammatory conditions.     

Effects of Selenium on Liver and Muscle Contents and Urinary Excretion of Zinc,Copper, Iron and Manganese

Selenium is a main component of glutathione peroxidase (GPX), a key antioxidant enzyme. Other elements, such as zinc, copper, manganese and iron, are also involved in the pathogenesis of oxidative damage as well as in other important metabolic pathways. The effects of selenium supplementation on the metabolism of these elements have yield controversial results .The aim of this study is to analyse the effects of selenium supplementation on liver, muscle and urinary excretion of zinc, copper, iron and manganese in a situation of oxidative stress, such as protein deficiency. The experimental design included four groups of adult male Sprague–Dawley rats, which received the Lieber–DeCarli control diet, an isocaloric 2 % protein-containing diet and another similar two groups to which selenomethionine (6 mg/l liquid diet) was added. After sacrifice (5 weeks later), muscle, liver and serum selenium were determined, as well as muscle, liver and urinary zinc, copper, manganese and iron and liver GPX activity and liver malondialdehyde. Selenium addition led to decreased liver copper, increased muscle copper, increased copper excretion and increased liver iron, whereas zinc and manganese parameters were essentially unaltered. Muscle, liver and serum selenium were all significantly correlated with liver GPX activity.     

Intracellular redox state as determinant for melatonin antiproliferative vs cytotoxic effects in cancer cells

Abstract

Melatonin is an endogenous indolamine, classically known as a light/dark regulator. Besides classical functions, melatonin has also showed to have a wide range of antitumoral effects in numerous cancer experimental models. However, no definite mechanism has been described to explain the whole range of antineoplasic effects. Here we describe a dual effect of melatonin on intracellular redox state in relation to its antiproliferative vs cytotoxic actions in cancer cells. Thus, inhibition of proliferation correlates with a decrease on intracellular reactive oxygen species (ROS) and increase of antioxidant defences (antioxidant enzymes and intracellular gluthation,GSH levels), while induction of cell death correlates with an increase on intracellular ROS and decrease of antioxidant defences. Moreover, cell death can be prevented by other well-known antioxidants or can be increased by hydrogen peroxide. Thus, tumour cell fate will depend on the ability of melatonin to induce either an antioxidant environment—related to the antiproliferative effect or a prooxidant environment related to the cytotoxic effect.     

Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism

To account for the observed anticancer properties of plant polyphenols, we have earlier proposed a mechanism which involves the mobilization of endogenous copper ions by polyphenols leading to the generation of reactive oxygen species (ROS) that serve as proximal DNA cleaving agents and lead to cell death. Over the last decade we have proceeded to validate our hypothesis with considerable success. As a further confirmation of our hypothesis, in this paper we first show that oral administration of copper to rats leads to elevated copper levels in lymphocytes. When such lymphocytes with a copper overload were isolated and treated with polyphenols EGCG, genistein and resveratrol, an increased level of DNA breakage was observed. Further, preincubation of lymphocytes having elevated copper levels with the membrane permeable copper chelator neocuproine, resulted in inhibition of polyphenol induced DNA degradation. However, membrane impermeable chelator of copper bathocuproine, as well as iron and zinc chelators were ineffective in causing such inhibition in DNA breakage, confirming the involvement of endogenous copper in polyphenol induced cellular DNA degradation. It is well established that serum and tissue concentrations of copper are greatly increased in various malignancies. In view of this fact, the present results further confirm our earlier findings and strengthen our hypothesis that an important anticancer mechanism of plant polyphenols could be the mobilization of intracellular copper leading to ROS-mediated cellular DNA breakage. In this context, it may be noted that cancer cells are under considerable oxidative stress and increasing such stress to cytotoxic levels could be a successful anticancer approach.     

Selenium, iron, copper, and zinc levels and copper-to-zinc ratios in serum of patients at different stages of viral hepatic diseases

Viral hepatic diseases, especially those induced by the hepatitis B virus, can progress into more serious pathological outcomes and eventually to hepatocellular carcinoma. A growing body of evidence indicates that many trace elements play important roles in a number of carcinogenic processes that proceed through various mechanisms. To examine the status of trace elements during the development of hepatic carcinoma, we determined the selenium, iron, copper, and zinc levels and copper-to-zinc ratios in the serum of patients at different stages of viral hepatic disease. We observed significant changes in the selenium, iron copper, and zinc levels in the serum of patients having hepatocellular carcinoma, relative to those of healthy controls (p<0.05). The mean serum copper level in patients with hepatocellular carcinoma was significantly higher than that of the control group. In contrast, the mean selenium, iron, and zinc levels in patients having hepatocellular carcinoma were significantly lower than those of the control group. In addition, the mean zinc level in the serum of patients with hepatic cirrhosis was significantly lower than that of the control group (p<0.05). Moreover, we found markedly elevated Cu: Zn ratios (p<0.05) in patients having hepatic cirrhosis or hepatocellular carcinoma. Our findings imply that the levels of some trace elements, such as selenium, iron, copper, and zinc, and Cu:Zn ratios, might serve as biomarkers for the increased severity of viral hepatic damage.     

Prooxidant and cytotoxic action of N-acetylcysteine and glutathione combined with vitamin Bl2b

We studied the prooxidant and cytotoxic action of thiols N-acetylcystein (NAC) and glutathione (GSH) combined with vitamin Bl2b. The synergism of action of the thiols and Bl2b resulted in human carcinoma cell damage was found. It was shown that GSH and NAC in physiological doses combined with Bl2b caused the initiation of apoptosis. It was established that prooxidant action of the thiols combined with vitamin Bl2b, i. e. generation and accumulation of hydrogen peroxide in culture medium, led to intracellular oxidative stress and injury of cell redox system. These effects were completely abolished by nonthiol antioxidants catalase and pyruvate. The chelators of iron phenanthroline and deferoxamine did not suppress the H2O2 accumulation in culture medium but significantly inhibited the cell death induced by the thiols combined with Bl2b. Therefore, the thiols GSH and NAC widely used as antioxidants, in combination with vitamin Bl2b show prooxidant characteristics and induce, with the participation of intracellular iron, apoptotic HEp-2 cell death.     

N-acetyl-L-cysteine in the presence of Cu<Superscript>2+</Superscript> induces oxidative stress and death of granule neurons in dissociated cultures of rat cerebellum

Addition into the culture medium of the antioxidant N-acetylcysteine (NAC, 1 mM) in the presence of Cu²⁺ (0.0005-0.001 mM) induced intensive death of cultured rat cerebellar granule neurons, which was significantly decreased by the zinc ion chelator TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine). However, the combined action of NAC and Zn²⁺ did not induce destruction of the neurons. Measurement of the relative intracellular concentration of Zn²⁺ with the fluorescent probe FluoZin-3 AM or of free radical production using a CellROX Green showed that incubation of the culture for 4 h with Cu²⁺ and NAC induced an intensive increase in the fluorescence of CellROX Green but not of FluoZin-3. Probably, the protective effect of TPEN in this case could be mediated by its ability to chelate Cu²⁺. Incubation of cultures in a balanced salt solution in the presence of 0.01 mM Cu²⁺ caused neuronal death already after 1 h if the NAC concentration in the solution was within 0.005–0.05 mM. NAC at higher concentrations (0.1–1 mM) together with 0.01mM Cu²⁺ did not cause the death of neurons. These data imply that the antioxidant NAC can be potentially harmful to neurons even in the presence of nanomolar concentrations of variable valence metals.     

Synchronization of Cultured Vascular Smooth Muscle Cells Following Reversal of Quiescence Induced by Treatment with the AntioxidantN-Acetylcysteine

Smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. Recently we demonstrated the thiol antioxidantN-acetylcysteine (NAC) inhibits constitutive NF-κB/Rel activity and growth of vascular SMCs. Here we show that treatment of human and bovine aortic SMC with the thiol antioxidant NAC causes cells to exit the cell cycle and remain quiescent as determined by a greatly reduced incorporation of [³H]thymidine and G₀/G₁DNA content. Removal of NAC from the culture medium stimulates SMCs to synchronously reenter the cell cycle as judged by induction of cyclin D1 and B-mybgene expression during mid and late G₁phase, respectively, and induction of histone gene expression and [³H]thymidine incorporation during S phase. The time course of cyclin D1, B-myb,and histone gene expression after NAC removal was similar to that of serum-deprived cells induced to resume cell cycle progression by the addition of fetal bovine serum to the culture medium. Taken together, these results indicate that NAC treatment causes SMCs to enter a reversible G₀quiescent, growth-arrested state. Thus, NAC provides an important new method for synchronizing SMCs in culture.