Bucillamine Inhibits UVB-Induced MAPK Activation and Apoptosis in Human HaCaT Keratinocytes and SKH-1 Hairless Mouse Skin.

Ultraviolet B (UVB) radiation is known as a culprit in skin carcinogenesis. We have previously reported that bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine), a cysteine derivative with antioxidant and anti-inflammatory capacity, protects against UVB-induced p53 activation and inflammatory responses in mouse skin. Since MAPK signaling pathways regulate p53 expression and activation, here we determined bucillamine effect on UVB-mediated MAPK activation in vitro using human skin keratinocyte cell line HaCaT and in vivo using SKH-1 hairless mouse skin. A single low dose of UVB (30 mJ cm-2 ) resulted in increased JNK/MAPK phosphorylation and caspase-3 cleavage in HaCaT cells. However, JNK activation and casaspe-3 cleavage were inhibited by pretreatment of HaCaT cells with physiological doses of bucillamine (25 and 100 µm). Consistent with these results, bucillamine pretreatment in mice (20 mg kg-1 ) inhibited JNK/MAPK and ERK/MAPK activation in skin epidermal cells at 6-12 and 24 h, respectively, after UVB exposure. Moreover, bucillamine attenuated UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in mouse skin. These findings demonstrate that bucillamine inhibits UVB-induced MAPK signaling, cell proliferation and apoptosis. Together with our previous report, we provide evidence that bucillamine has a photoprotective effect against UV exposure.

Photoprotective effects of bucillamine against UV-induced damage in an SKH-1 hairless mouse model.

UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non-DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N-(2-mercapto-2-methylpropionyl)-l-cysteine] is a cysteine-derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB-induced photodamage using the SKH-1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230 mJ cm(-2)) on the dorsal skin of SKH-1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6 h after the last UVB exposure. Bucillamine pretreatment (20 mg kg(-1) of body weight, administered subcutaneously) markedly attenuated UVB-mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser-15 and Ser-20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.

A lipid-soluble iron chelator alters cell cycle regulatory protein binding in breast cancer cells compared to normal breast cells.

The lipid-soluble iron chelator desferri-exochelin (D-Exo) causes reversible cell cycle arrest in normal human mammary epithelial cells (NHMEC) but triggers apoptotic cell death in human breast cancer cells. We studied the effects of iron chelation with D-Exo on cell cycle regulatory proteins in cultures of NHMEC and MCF-7 breast cancer cells. In co-immunoprecipitation studies, D-Exo inhibited binding of cyclins A and E to cyclin dependent kinase 2 (CDK2) in NHMEC, but in MCF-7 cells binding of these cyclins to CDK2 was enhanced. D-Exo treatment markedly increased expression of p53 and increased binding of p21 to CDK2 in the MCF-7 cells but not in NHMEC. Therefore differences in effects of iron chelation on cell cycle protein binding in cancer cells compared to normal cells may trigger apoptosis in cancer cells while normal breast cells are spared.

A mycobacterial iron chelator, desferri-exochelin, induces hypoxia-inducible factors 1 and 2, NIP3, and vascular endothelial growth factor in cancer cell lines.

Hypoxia is a key phenomenon in tumor behavior, selecting for resistance to apoptosis, conferring resistance to radiotherapy and chemotherapy, and also inducing angiogenic factors such as vascular endothelial growth factor (VEGF). Exochelins are naturally evolved iron chelators produced by Mycobacterium tuberculosis. Because iron chelation has been reported to activate the hypoxia-inducible factor (HIF), we investigated the effects of an exochelin [desferri-exochelin (DFE) 772SM] on this hypoxia-inducible pathway and downstream target genes. DFE induced HIF-1alpha and HIF-2alpha transcription factors regulating the hypoxic response in the breast tumor cell line MDA468. DFE was 10 times more potent and more rapid in onset of effect than the clinically used iron chelator deferoxamine. The expression of downstream hypoxia-responsive target genes VEGF and the proapoptotic protein NIP3 was activated by transcription. MDA468 proliferation was inhibited via HIF-independent pathways, related to other effects of iron chelation. DFE inhibited effects of VEGF on endothelial cell proliferation. DFE potentially could be useful in cancer therapy by inducing apoptosis via NIP3 in conjunction with other non-HIF-related growth inhibitory pathways and blocking endothelial proliferation despite the presence of VEGF.

The exochelins of pathogenic mycobacteria: unique, highly potent, lipid- and water-soluble hexadentate iron chelators with multiple potential therapeutic uses.

SIGNIFICANCE: Exochelins are lipid- and water-soluble siderophores of Mycobacterium tuberculosis with unique properties that endow them with exceptional pharmacologic utility. Exochelins can be utilized as probes to decipher the role of iron in normal and pathological states, and, since they rapidly cross cell membranes and chelate intracellular iron with little or no toxicity, exochelins are potentially useful for the treatment of a number of iron-dependent pathological phenomena. RECENT ADVANCES: In animal models, exochelins have been demonstrated to have promise for the treatment of transfusion-related iron overload, restenosis after coronary artery angioplasty, cancer, and oxidative injury associated with acute myocardial infarction and transplantation. CRITICAL ISSUES: To be clinically effective, iron chelators should be able to rapidly enter cells and chelate iron at key intracellular sites. Desferri-exochelins, and other lipid-soluble chelators, can readily cross cell membranes and remove intracellular free iron; whereas deferoxamine, which is lipid insoluble, cannot do so. Clinical utility also requires that the chelators be nontoxic, which, we hypothesize, includes the capability to prevent iron from catalyzing free radical reactions which produce •OH or other reactive oxygen species. Lipid-soluble iron chelators currently available for clinical application are bidentate (deferiprone) or tridentate (desferasirox) molecules that do not block all six sites on the iron molecule capable of catalyzing free radical reactions. In contrast, desferri-exochelins are hexadentate molecules, and by forming a one-to-one binding relationship with iron, they prevent free radical reactions. FUTURE DIRECTIONS: Clinical studies are needed to assess the utility of desferri-exochelins in the treatment of iron-dependent pathological disorders.

Prolonged administration of a dithiol antioxidant protects against ventricular remodeling due to ischemia-reperfusion in mice.

The prolonged production of reactive oxygen species due to ischemia-reperfusion (I/R) is a potential cause of the pathological remodeling that frequently precedes heart failure. We tested the ability of a potent dithiol antioxidant, bucillamine, to protect against the long-term consequences of I/R injury in a murine model of myocardial infarction. After transiently occluding the left anterior descending coronary artery for 30 min, saline or bucillamine (10 microg/g body wt) was injected intravenously as a bolus within the first 5 min of reperfusion. The antioxidant treatment continued with daily subcutaneous injections for 4 wk. There were no differences in infarct sizes between bucillamine- and saline-treated animals. After 4 wk of reperfusion, cardiac hypertrophy was decreased by bucillamine treatment (ventricular weight-to-body weight ratios: I/R + saline, 4.5 +/- 0.2 mg/g vs. I/R + bucillamine, 4.2 +/- 0.1 mg/g; means +/- SE; P < 0.05). Additionally, the hearts of bucillamine-treated mice had improved contractile function (echocardiographic measurement of fractional shortening) relative to saline controls: I/R + saline, 32 +/- 3%, versus I/R + bucillamine, 41 +/- 4% (P < 0.05). Finally, I/R-induced injury in the saline-treated mice was accompanied by a fetal pattern of gene expression determined by ribonuclease protection assay that was consistent with pathological cardiac hypertrophy and remodeling [increased atrial natriuretic peptide, beta-myosin heavy chain (MHC), skeletal alpha-actin; decreased sarco(endo)plasmic reticulum Ca2+ ATPase 2a, and alpha-MHC-to-beta-MHC ratio]. These changes in gene expression were significantly attenuated by bucillamine. Therefore, treatment with a dithiol antioxidant for 4 wk after I/R preserved ventricular function and prevented the abnormal pattern of gene expression associated with pathological cardiac remodeling.

Desferri-Exochelin, a lipid-soluble, hexadentate iron chelator, effectively removes tissue iron.

Chronic iron-overload is damaging to the heart, liver, and other organs. Better iron chelators are needed to treat this serious medical condition. The uptake and distribution of the lipid-soluble, hexadentate iron chelator desferri-Exochelin 772SM (D-Exo) is studied and its efficacy in removing iron from tissue in rodent models is evaluated. After an intravenous bolus of tritiated D-Exo to rats, counts rapidly disappeared from the blood and rapidly appeared in 15 organs studied, usually peaking within 15 min. There was considerable uptake in the heart and liver, 2 organs especially susceptible to damage from clinical iron overload. To assess actual decreases in cardiac and hepatic iron in response to D-Exo, mice loaded with 42 mg of iron dextran (2100 mg/kg) were studied. Untreated, iron-loaded mice sacrificed 9 weeks later had a 4-fold increase in cardiac iron and a 20-fold increase in hepatic iron compared with controls that were not iron-loaded. In iron-loaded mice treated with 7 mg of D-Exo intraperitoneally (i.p.) 4 days/week for 8 weeks (total 224 mg), tissue iron, measured by atomic absorption, was reduced by 20% in the liver and 25% in the heart (P < 0.01 for each organ). During the first 8 h after a D-Exo dose, iron was excreted in the urine. Mice treated with D-Exo gained weight normally and showed no evidence of toxicity. In conclusion, in this iron-overload mouse model, D-Exo administered intravenously or i.p. rapidly diffuses into multiple organs, including the heart and liver, and effectively removes iron without apparent toxicity.

Cell permeable iron chelators as potential cancer chemotherapeutic agents.

Iron is an essential micronutrient for the growth and function of all cells. It is, therefore, an attractive target for chemotherapeutic compounds. Numerous studies in vitro and in vivo provide evidence that iron chelators may be effective antitumor agents. Lipophilic iron chelators that are readily cell permeable and can bind intracellular iron stores may selectively kill cancer cells without damaging normal cells. In this review we discuss the role of iron in cellular processes and how these processes differ between normal and neoplastic cells. We also review the effects on normal and cancer cell growth of several lipophilic iron chelators.

Paradoxical effects of iron chelation on growth of vascular endothelial cells.

Endothelial cell (EC) and vascular smooth muscle cell (VSMC) interactions play critical roles in restenosis following vascular injury. We examined the effects of intracellular iron chelation on endothelial cell cycle progression and VSMC modulation of endothelial cell growth. A diffusible, lipid-soluble iron chelator that rapidly enters cells, desferri-exochelin 772SM (D-Exo), was studied in human endothelial cells and VSMCs. In both cell types D-Exo reversibly halted cell cycle progression from G0/G1 phase to S phase and from S phase to G2/M phase and increased expression of hypoxia-inducible factor 1alpha (HIF-1alpha). D-Exo increased secretion of vascular endothelial growth factor (VEGF), a downstream target of HIF-1alpha, in VSMCs, but there was no VEGF production in endothelial cells. D-Exo was 25-fold more potent than the lipid-insoluble iron chelator deferoxamine, which does not readily enter cells. Intracellular iron chelation with D-Exo directly inhibits endothelial cell growth but indirectly stimulates endothelial cell growth by increasing VEGF release by VSMCs.

Regulation of oxygen delivery during induced polycythemia in exercising dogs.

Previous studies have concluded that polycythemia decreases oxygen delivery primarily because of a large fall in cardiac output associated with a rise in systemic vascular resistance that has been attributed to increased blood viscosity. However, because other studies have shown that polycythemia may not reduce oxygen delivery, an alternative hypothesis is that cardiac output falls in response to a rising oxygen content, thereby maintaining oxygen delivery constant. To determine whether oxygen content participates in the regulation of cardiac output during polycythemia, we studied eight chronically instrumented dogs trained to exercise on a treadmill. The dogs underwent exchange transfusion with packed red blood cells containing methemoglobin, which caused an increase in hematocrit from 35 +/- 1 to 50 +/- 1% and in viscosity, with little change in oxygen content. The expected fall in exercise cardiac output failed to occur after exchange transfusion with red blood cells containing methemoglobin (7.5 +/- 4 vs. 6.8 +/- 0.5 l/min; P = not significant), and there was no rise in systemic vascular resistance. Methylene blue was then administered intravenously to facilitate conversion of methemoglobin to oxyhemoglobin, which increased oxygen content (12.8 +/- 0.9 vs. 18.4 +/- 0.9 vol%; P < 0.01) with no change in hematocrit or viscosity. Resting cardiac output did not change significantly, but there was a significant decrease in exercise output (6.8 +/- 0.5 vs. 5.8 +/- 0.4 l/min; P < 0.05). Thus we conclude that the fall in cardiac output seen in acute polycythemia results in part from the regulation of oxygen delivery and is not due solely to increased blood viscosity.

Bucillamine: a potent thiol donor with multiple clinical applications.

Bucillamine has potential to attenuate or prevent damage during myocardial infarction, cardiac surgery and organ transplantation. Bucillamine, a cysteine derivative that contains two donatable thiol groups, is capable of replenishing the thiol group in glutathione, thereby reactivating this endogenous defense against oxidant injury. Bucillamine rapidly enters cells by the same mechanism that normally transports the amino acid cysteine. Bucillamine is a more potent thiol donor than other cysteine derivatives: approximately 16-fold more potent than N-acetylcysteine (Mucomyst(R)) in vivo. In addition bucillamine appears to have additional anti-inflammatory effects unrelated to its antioxidant effect. Oral bucillamine is used clinically in Asia for treatment of rheumatoid arthritis. There is a strong preclinical evidence that parenteral infusion of this agent is efficacious in acute settings characterized by inflammation and oxidative stress. In an investigator-blinded, rigorous intact dog model, consisting of 90 min of coronary artery occlusion and 48 h of reperfusion, bucillamine, given i.v. during the first 3 h of reperfusion, substantially reduced myocardial infarct size. Livers exposed to 24 h of cold ischemia were markedly protected by bucillamine in several transplantation models. In Phase I human studies in normal volunteers, bucillamine at doses up to 25 mg/kg/h i.v. for 3 h elicited no serious toxicity. On the basis of pharmacokinetic analyses of blood levels during these studies it was concluded that bucillamine, infused at i.v. doses > or =10 mg/kg/h for 3 h to humans could be expected to be therapeutically effective in myocardial infarction, organ transplantation and other acute inflammatory syndromes.

Effect on ribonucleotide reductase of novel lipophilic iron chelators: the desferri-exochelins.

Desferri-exochelins are siderophores secreted by Mycobacterium tuberculosis that are both lipid- and water-soluble and have a high binding affinity for iron. Desferri-exochelin 772SM inhibits DNA replication and ribonucleotide reductase activity at 10-fold less concentration than the lipid-insoluble iron chelator deferoxamine, which is currently in clinical use. Neither chelator can extract iron directly from ribonucleotide reductase. However, because of its lipid-solubility and high binding affinity, desferri-exochelin is able to enter cells rapidly and access intracellular iron, while deferoxamine has limited capacity to cross the cell membrane.

Bucillamine, a thiol antioxidant, prevents transplantation-associated reperfusion injury.

Ischemia/reperfusion (I/R) injury is a serious potential threat to outcomes in organ transplantation and other clinical arenas in which there is temporary interruption of blood flow. I/R is a frequent cause of primary failure in organ transplantation. We hypothesized that the antioxidant bucillamine, a potent sulfhydryl donor, would protect against I/R injury in high-risk organ transplants. Because livers subjected to prolonged ischemia and very fatty livers are highly susceptible to severe I/R injury, we studied the effect of bucillamine in three animal models of liver transplantation: two ex vivo models of isolated perfused livers, either normal or fatty rat livers, and an in vivo model of syngenic orthotopic liver transplants in rats. In all models, livers were deprived of oxygen for 24 h before either ex vivo reperfusion or transplantation. In the ex vivo models, bucillamine treatment significantly improved portal vein blood flow and bile production, preserved normal liver architecture, and significantly reduced liver enzyme release and indices of oxidative stress. Moreover, bucillamine treatment significantly increased levels of reduced glutathione in the liver and lowered levels of oxidized glutathione in both liver and blood. In rats subjected to liver transplants, bucillamine significantly enhanced survival and protected against hepatic injury. Possible mechanisms of this protection include prevention of excessive accumulation of toxic oxygen species, interruption of redox signaling in hepatocytes, and inhibition of macrophage activation. This study demonstrates the potential utility of bucillamine or other cysteine-derived thiol donors for improving outcomes in organ transplantation and other clinical settings involving I/R injury.

Thiol antioxidants inhibit the adjuvant effects of aerosolized diesel exhaust particles in a murine model for ovalbumin sensitization.

Although several epidemiological studies indicate a correlation between exposure to ambient particulate matter and adverse health effects in humans, there is still a fundamental lack of understanding of the mechanisms involved. We set out to test the hypothesis that reactive oxygen species are involved in the adjuvant effects of diesel exhaust particles (DEP) in a murine OVA sensitization model. First, we tested six different antioxidants, N-acetylcysteine (NAC), bucillamine (BUC), silibinin, luteolin, trolox (vitamin E), and ascorbic acid, for their ability to interfere in DEP-mediated oxidative stress in vitro. Of the six agents tested, only the thiol antioxidants, BUC and NAC, were effective at preventing a decrease in intracellular reduced glutathione:glutathione disulfide ratios, protecting cells from protein and lipid oxidation, and preventing heme oxygenase 1 expression. Therefore, we selected the thiol antioxidants for testing in the murine OVA inhalation sensitization model. Our data demonstrate that NAC and BUC effectively inhibited the adjuvant effects of DEP in the induction of OVA-specific IgE and IgG1 production. Furthermore, NAC and BUC prevented the generation of lipid peroxidation and protein oxidation in the lungs of OVA- plus DEP-exposed animals. These findings indicate that NAC and BUC are capable of preventing the adjuvant effects of inhaled DEP and suggest that oxidative stress is a key mechanistic component in the adjuvant effect of DEP. Antioxidant treatment strategies may therefore serve to alleviate allergic inflammation and may provide a rational basis for treating the contribution of particulate matter to asthmatic disease.