RESUMO
Significance: Cancer is a complex and heterotypic structure with a spatial organization that contributes to challenges in therapeutics. Enzymes associated with producing the gasotransmitter hydrogen sulfide (H2S) are differentially expressed in tumors. Indeed, critical and paradoxical roles have been attributed to H2S in cancer-promoting characteristics by targeting both cancer cells and their milieu. This review focuses on the evidence and knowledge gaps of H2S on the tumor redox microenvironment and the pharmacological effects of H2S donors on cancer biology. Recent Advances: Endogenous and pharmacological concentrations of H2S evoke different effects on the same cell type: physiological H2S concentrations have been associated with tumor development and progression. In contrast, pharmacological concentrations have been associated with anticancer effects. Critical Issues: The exact threshold between the promotion and inhibition of tumorigenesis by H2S is largely unknown. The main issues covered in this review include H2S-modulated signaling pathways that are critical for cancer cells, the potential effects of H2S on cellular components of the tumor microenvironment, temporal modulation of H2S in promoting or inhibiting tumor progression (similar to observed for inflammation), and pharmacological agents that modulate H2S and which could play a role in antineoplastic therapy. Future Directions: Given the complexity and heterogeneity of tumor composition, mechanistic studies on context-dependent pharmacological effects of H2S donors for cancer therapy are necessary. These studies must determine the critical signaling pathways and the cellular components involved to allow advances in the rational use of H2S donors as antineoplastic agents. Antioxid. Redox Signal. 40, 250-271.
Assuntos
Gasotransmissores , Sulfeto de Hidrogênio , Neoplasias , Humanos , Sulfeto de Hidrogênio/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Gasotransmissores/metabolismo , Transdução de Sinais , Carcinogênese , Microambiente TumoralRESUMO
Objective- PDI (protein disulfide isomerase A1) was reported to support Nox1 (NADPH oxidase) activation mediated by growth factors in vascular smooth muscle cells. Our aim was to investigate the molecular mechanism by which PDI activates Nox1 and the functional implications of PDI in Nox1 activation in vascular disease. Approach and Results- Using recombinant proteins, we identified a redox interaction between PDI and the cytosolic subunit p47phox in vitro. Mass spectrometry of crosslinked peptides confirmed redox-dependent disulfide bonds between cysteines of p47phox and PDI and an intramolecular bond between Cys 196 and 378 in p47phox. PDI catalytic Cys 400 and p47phox Cys 196 were essential for the activation of Nox1 by PDI in vascular smooth muscle cells. Transfection of PDI resulted in the rapid oxidation of a redox-sensitive protein linked to p47phox, whereas PDI mutant did not promote this effect. Mutation of p47phox Cys 196, or the redox active cysteines of PDI, prevented Nox1 complex assembly and vascular smooth muscle cell migration. Proximity ligation assay confirmed the interaction of PDI and p47phox in murine carotid arteries after wire injury. Moreover, in human atheroma plaques, a positive correlation between the expression of PDI and p47phox occurred only in PDI family members with the a' redox active site. Conclusions- PDI redox cysteines facilitate Nox1 complex assembly, thus identifying a new mechanism through which PDI regulates Nox activity in vascular disease.
Assuntos
Dissulfetos/química , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , NADPH Oxidase 1/metabolismo , NADPH Oxidases/química , Isomerases de Dissulfetos de Proteínas/química , Animais , Movimento Celular , Células Cultivadas , Ativação Enzimática , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Oxirredução , Superóxidos/metabolismoRESUMO
NADPH oxidases derived reactive oxygen species (ROS) play an important role in vascular function and remodeling in hypertension through redox signaling processes. Previous studies demonstrated that protein disulfide isomerase (PDI) regulates Nox1 expression and ROS generation in cultured vascular smooth muscle cells. However, the role of PDI in conductance and resistance arteries during hypertension development remains unknown. The aim of the present study was to investigate PDI expression and NADPH oxidase dependent ROS generation during hypertension development. Mesenteric resistance arteries (MRA) and thoracic aorta were isolated from 6, 8, and 12 week-old spontaneously hypertensive (SHR) and Wistar rats. ROS production (dihydroethidium fluorescence), PDI (WB, imunofluorescence), Nox1 and NOX4 (RT-PCR) expression were evaluated. Results show a progressive increase in ROS generation in MRA and aorta from 8 to 12 week-old SHR. This effect was associated with a concomitant increase in PDI and Nox1 expression only in MRA. Therefore, suggesting a positive correlation between PDI and Nox1 expression during the development of hypertension in MRA. In order to investigate if this effect was due to an increase in arterial blood pressure, pre hypertensive SHR were treated with losartan (20 mg/kg/day for 30 days), an AT1 receptor antagonist. Losartan decreased blood pressure and ROS generation in both vascular beds. However, only in SHR MRA losartan treatment lowered PDI and Nox1 expression to control levels. In MRA PDI inhibition (bacitracin, 0.5 mM) decreased Ang II redox signaling (p-ERK 1/2). Altogether, our results suggest that PDI plays a role in triggering oxidative stress and vascular dysfunction in resistance but not in conductance arteries, increasing Nox1 expression and activity. Therefore, PDI could be a new player in oxidative stress and functional alterations in resistance arteries during the establishment of hypertension.
RESUMO
Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI-mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell-free system, PDI inhibitors scrRNase (100 µg/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by ~40%), whereas PDIred diminished (by ~60%) superoxide generation. No change occurred after incubation with PDI serine-mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by ~70%. Thus, oxidized PDI supports, whereas reduced PDI down-regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol-mediated interaction with p47(phox) in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47(phox). PDI-p47(phox) association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47(phox) in cytosol. Incubation of purified PDI (>80% reduced) and p47(phox) in vitro promoted their arachidonate-dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re)cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox-dependent enzyme complex organizer.
Assuntos
Membrana Celular/enzimologia , Citosol/enzimologia , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Superóxidos/metabolismo , Substituição de Aminoácidos , Antibacterianos/farmacologia , Bacitracina/farmacologia , Membrana Celular/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Mutação de Sentido Incorreto , NADPH Oxidases/genética , Oxirredução/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
Chronic stimulation of beta-adrenoceptors with isoproterenol induces alteration of vascular reactivity and increases local pro-inflammatory cytokines. We investigated whether fenofibrate and pioglitazone, PPAR-alpha and -gamma agonists, respectively, improve the changes in vascular reactivity induced by isoproterenol. Wistar rats received isoproterenol (0.3 mg x kg x day, SC) or vehicle (CT) plus fenofibrate (alpha, 100 mg x kg x day, PO), pioglitazone (gamma, 2.5 mg.kg.day, PO), or water for 7 days. In aortas, isoproterenol treatment enhanced the maximal response (Rmax) to phenylephrine (10 to 10 M) compared to CT as previously demonstrated. The effects of endothelium removal (E-) or L-NAME incubation (100 microM) on the phenylephrine response were smaller in isoproterenol-treated animals compared to CT while superoxide dismutase (SOD, 150 U/mL) significantly reduced the Rmax to phenylephrine to CT levels. Neither fenofibrate nor pioglitazone changed the effects induced by isoproterenol in aorta. E-, L-NAME, or SOD effects were similar between CTalpha and CT. However, pioglitazone per se increased Rmax to phenylephrine (CT: 59 +/- 4 versus CTgamma: 72 +/- 5 % of contraction to KCl). E- or L-NAME effects were reduced in CTgamma compared to CT, and SOD normalized the altered reactivity to phenylephrine in the CTgamma group. In conclusion, neither fenofibrate nor pioglitazone ameliorates the altered vascular reactivity present in aorta from isoproterenol-treated rats. Moreover, pioglitazone per se induced endothelial dysfunction and increased phenylephrine-induced contraction in aorta.