Cisplatin is a widely used antineoplastic agent in the treatment of head and neck cancer. However, it is highly nephrotoxic. Oxidative stress is the main mechanism responsible for cisplatin-induced ...nephrotoxicity. The aim of this study was to characterize cisplatin-induced nephrotoxicity, oxidative stress in peripheral blood mononuclear cells, and the relationship between them. Twenty-four patients were included in the study. Patients had their blood collected prior to cisplatin administration, and 5 and 20 days after initiating therapy, to assess renal function and to determine oxidative stress with MitoSOX™Red, H
2
DCF-DA, and Amplex
®
Red tests. Renal function was assessed by measuring serum creatinine, creatinine clearance, and blood urea nitrogen (BUN). Serum creatinine and creatinine clearance were used to grade nephrotoxicity using Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Compared to baseline values, the mean BUN and serum creatinine increased 135 and 100%, respectively, 5 days after cisplatin infusion. Mean creatinine clearance showed a 43% decrease compared to baseline value. Non-statistically significant changes in superoxide anion (O
2
•−
), hydrogen peroxide (H
2
O
2
), and general reactive oxygen species production occurred. A higher production of H
2
O
2
was correlated with variation in serum creatinine, and was associated with higher grades for serum creatinine increases and creatinine clearance reductions. Linear regression analyses showed an association between H
2
O
2
production and serum creatinine, creatinine clearance, and BUN levels. These results were observed for 5 days following cisplatin administration. In conclusion, H
2
O
2
production was significantly related to changes in all renal parameters that were evaluated, following the cisplatin infusion.
BlL, a galactose-binding C-type lectin purified from Bothrops leucurus snake venom, exhibits anticancer activity. The current study was designed to elucidate the cellular mechanisms by which BlL ...induces melanoma cell death. The viabilities of B16-F10 melanoma cells and HaCaT keratinocytes treated with BlL were evaluated. Necrotic and apoptotic cell death, cytosolic Ca2+ levels, mitochondrial Ca2+ transport and superoxide levels were assessed in B16-F10 melanoma cells exposed to BlL. We found that treatment with BlL caused dose-dependent necrotic cell death in B16-F10 melanoma cells. Conversely, the viability of non-tumorigenic HaCaT cells was not affected by similar doses of BlL. BlL-induced B16-F10 necrosis was preceded by a significant (2-fold) increase in cytosolic calcium concentrations and a significant (3-fold) increase in mitochondrial superoxide generation. It is likely that BlL treatment triggers B16-F10 cell death via mitochondrial permeability transition (MPT) pore opening because the pharmacological MPT inhibitors bongkrekic acid and Debio 025 greatly attenuated BlL-induced cell death. Experiments evaluating mitochondrial Ca2+ transport in permeabilized B16-F10 cells strongly supported the hypothesis that BlL rapidly stimulates cyclosporine A-sensitive Ca2+-induced MPT pore opening. We therefore conclude that BlL causes selective B16-F10 melanoma cell death via dysregulation of cellular Ca2+ homeostasis and Ca2+-induced opening of MPT pore.
•BlL, a galactose-binding C-type lectin, causes death in cultured B16-F10 melanoma cells.•BlL-induced melanoma cell death involves dysregulation of cellular Ca2+ homeostasis.•BlL-induced melanoma cell death involves opening of mitochondrial permeability transition pore.
Different regimens of food restriction have been associated with protection against obesity, diabetes and CVD. In the present study, we hypothesised that food restriction would bring benefits to ...atherosclerosis- and diabetes-prone hypercholesterolaemic LDL-receptor knockout mice. For this purpose, 2-month-old mice were submitted to an intermittent fasting (IF) regimen (fasting every other day) over a 3-month period, which resulted in an overall 20 % reduction in food intake. Contrary to our expectation, epididymal and carcass fat depots and adipocyte size were significantly enlarged by 15, 72 and 68 %, respectively, in the IF mice compared with the ad libitum-fed mice. Accordingly, plasma levels of leptin were 50 % higher in the IF mice than in the ad libitum-fed mice. In addition, the IF mice showed increased plasma levels of total cholesterol (37 %), VLDL-cholesterol (195 %) and LDL-cholesterol (50 %). As expected, in wild-type mice, the IF regimen decreased plasma cholesterol levels and epididymal fat mass. Glucose homeostasis was also disturbed by the IF regimen in LDL-receptor knockout mice. Elevated levels of glycaemia (40 %), insulinaemia (50 %), glucose intolerance and insulin resistance were observed in the IF mice. Systemic inflammatory markers, TNF-α and C-reactive protein, were significantly increased and spontaneous atherosclerosis development were markedly increased (3-fold) in the IF mice. In conclusion, the IF regimen induced obesity and diabetes and worsened the development of spontaneous atherosclerosis in LDL-receptor knockout mice. Although being efficient in a wild-type background, this type of food restriction is not beneficial in the context of genetic hypercholesterolaemia.
TNFα is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the ...hypothalamus, TNFα can activate thermogenesis and modulate food intake. Here we show that high concentration TNFα in the hypothalamus leads to increased O2 consumption/CO2 production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by β3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as β3-adrenergic receptor, peroxisomal proliferator-activated receptor-γ coactivator-1α, and uncoupling protein-1, are increased. In the hypothalamus, TNFα produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNFα-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of β3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNFα action to be important mediator of the wastage syndrome in cachexia.
The mechanisms involved in the thermogenic actions of TNF-1α, which impacts on the accelerated wastage syndrome in conditions such as cancer and sepsis, are discussed.
Ca2+ and P(i) accumulation by mitochondria triggers a number of alterations leading to nonspecific increase in inner membrane permeability Kowaltowski, A. J., et al. (1996) J. Biol. Chem. 271, ...2929-2934. The molecular nature of the membrane perturbation that precedes oxidative damage is still unknown. EPR spectra of spin probes incorporated in submitochondrial particles (SMP) and in model membranes suggest that Ca(2+)-cardiolipin (CL) complexation plays an important role. Ca(2+)-induced lipid domain formation was detected in SMP but not in mitoplasts, in SMP extracted lipids, or in CL-containing liposomes. The results were interpreted in terms of Ca2+ sequestration of CL tightly bound to membrane proteins, in particular the ADP-ATP carrier, and formation of CL-enriched strongly immobilized clusters in lipid shells next to boundary lipid. The in-plane lipid and protein rearrangement is suggested to cause increased reactive oxygen species production in succinate-supplemented, antimycin A-poisoned SMP, favoring the formation of carbon-centered radicals, detected by EPR spin trapping. Removal of tightly bound CL is also proposed to cause protein aggregation, facilitating intermolecular thiol oxidation. Lipid peroxidation was also monitored by the disappearance of the nitroxide EPR spectrum. The decay was faster for nitroxides in a more hydrophobic environment, and was inhibited by butylated hydroxytoluene, by EGTA, or by substituting Mg2+ for Ca2+. In addition, Ca2+ caused an increase in permeability, evidenced by the release of carboxyfluorescein from respiring SMP. The results strongly support Ca2+ binding to CL as one of the early steps in the molecular mechanism of Ca(2+)-induced nonspecific inner mitochondrial membrane permeabilization.
Statins are efficient cholesterol-lowering medicines utilized worldwide. However, 10% of patients suffer from adverse effects specially related to skeletal muscle function. Pro- or anti-oxidant ...effects of statins have been reported. Here we hypothesized that statins induce muscle mitochondrial oxidative stress leading to mitochondrial permeability transition (MPT) which may explain statin muscle toxicity. Thus, our aims were to investigate the effects of statin chronic treatment on muscle mitochondrial respiration rates, MPT and redox state indicators in the context of hypercholesterolemia. For this purpose, we studied muscle biopsies of the hypercholesterolemic LDL receptor knockout mice (
LDLr
-/-
)
treated with pravastatin during 3 months. Plantaris, but not soleus muscle of treated mice showed significant inhibition of respiration rates induced by ADP (–14%), oligomycin (–20%) or FCCP (–40%). Inhibitions of respiratory rates were sensitive to EGTA (Ca
2+
chelator), cyclosporin A (MPT inhibitor), ruthenium red (inhibitor of mitochondria Ca
2+
uptake) and coenzyme Q
10
(antioxidant), indicating that pravastatin treatment favors Ca
2+
induced MPT. Diet supplementation with creatine (antioxidant) also protected treated mice against pravastatin sensitization to Ca
2+
induced MPT. Among several antioxidant enzymes analyzed, only catalase activity was increased by 30% in plantaris muscle of pravastatin treated mice. Oxidized lipids, but not proteins biomarkers were identified in treated
LDLr
-/-
plantaris muscle. Taken together, the present results suggest that chronic pravastatin administration to a model of familial hypercholesterolemia promotes mitochondrial dysfunctions in plantaris muscle that can be counteracted by antioxidants administered either
in vitro
(CoQ
10
) or
in vivo
(creatine). Therefore, we propose that inhibition of muscle mitochondrial respiration by pravastatin leads to an oxidative stress that, in the presence of calcium, opens the permeability transition pore. This mitochondrial oxidative stress caused by statin treatment also signals for cellular antioxidant system responses such as catalase upregulation. These results suggest that the detrimental effects of statins on muscle mitochondria could be prevented by co-administration of a safe antioxidant such as creatine or CoQ10.
Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed ...in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells.
Mitochondrial redox imbalance and high Ca2+ uptake induce the opening of the permeability transition pore (PTP) that leads to disruption of energy‐linked mitochondrial functions and triggers cell ...death in many disease states. In this review, we discuss the major results from our studies investigating the consequences of NAD(P)‐transhydrogenase (NNT) deficiency, and of statins treatment for mitochondrial functions and susceptibility to Ca2+‐induced PTP. We highlight the aggravation of high fat diet‐induced fatty liver disease in the context of NNT deficiency and the role of antioxidants in the prevention of statins toxicity to mitochondria.
Mitochondrial redox imbalance and high Ca
uptake induce the opening of the permeability transition pore (PTP) that leads to disruption of energy-linked mitochondrial functions and triggers cell death ...in many disease states. In this review, we discuss the major results from our studies investigating the consequences of NAD(P)-transhydrogenase (NNT) deficiency, and of statins treatment for mitochondrial functions and susceptibility to Ca
-induced PTP. We highlight the aggravation of high fat diet-induced fatty liver disease in the context of NNT deficiency and the role of antioxidants in the prevention of statins toxicity to mitochondria.
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