Dysregulation of endoplasmic reticulum stress response in skin wounds in a streptozotocin-induced diabetes mouse model.

Dysfunction in key cellular organelles has been linked to diabetic complications. This study intended to investigate the alterations in the unfolded protein response (UPR), autophagy, and mitochondrial function, which are part of the endoplasmic reticulum (ER) stress response, in wound healing (WH) under diabetes conditions. WH mouse models were used to evaluate the UPR, autophagy, mitochondrial fusion, fission, and biogenesis as well as mitophagy in the skin of control and diabetic mice at baseline and 10 days after wounding. The autophagic flux in response to high-glucose conditions was also evaluated in keratinocyte and fibroblast cell cultures. WH was impaired in the diabetic mouse model, and we found that the UPR and autophagy pathways were activated in skin wounds of control mice and in the non-wounded skin of diabetic mice. Moreover, high-glucose conditions induced autophagy in the keratinocyte and fibroblast cell cultures. However, mitophagy did not change in the skin of diabetic mice or the wounded skin. In addition, mitochondrial fusion was activated in control but not in the skin wounds of diabetic mice, while mitochondrial biogenesis is downregulated in the skin of diabetic mice. In conclusion, the activation of the UPR, autophagy, and mitochondrial remodeling are crucial for a proper WH. These results suggest that the increase in ER stress and autophagy in the skin of diabetic mice at baseline significantly escalated to pathological levels after wounding, contributing to impaired WH in diabetes.

Mitochondrial respiration in thoracic perivascular adipose tissue of diabetic mice.

Introduction: Thoracic perivascular adipose tissue (tPVAT) has a phenotype resembling brown AT. Dysfunctional tPVAT appears to be linked to vascular dysfunction. Methods: We evaluated uncoupling protein 1 (UCP1) expression by Western blot, oxidative stress by measuring lipid peroxidation, the antioxidant capacity by HPLC and spectrophotometry, and mitochondrial respiration by high-resolution respirometry (HRR) in tPVAT, compared to inguinal white AT (iWAT), obtained from non-diabetic (NDM) and streptozocin-induced diabetic (STZ-DM) mice. Mitochondrial respiration was assessed by HRR using protocol 1: complex I and II oxidative phosphorylation (OXPHOS) and protocol 2: fatty acid oxidation (FAO) OXPHOS. OXPHOS capacity in tPVAT was also evaluated after UCP1 inhibition by guanosine 5'-diphosphate (GDP). Results: UCP1 expression was higher in tPVAT when compared with iWAT in both NDM and STZ-DM mice. The malondialdehyde concentration was elevated in tPVAT from STZ-DM compared to NDM mice. Glutathione peroxidase and reductase activities, as well as reduced glutathione levels, were not different between tPVAT from NDM and STZ-DM mice but were lower compared to iWAT of STZ-DM mice. OXPHOS capacity of tPVAT was significantly decreased after UCP1 inhibition by GDP in protocol 1. While there were no differences in the OXPHOS capacity between NDM and STZ-DM mice in protocol 1, it was increased in STZ-DM compared to NDM mice in protocol 2. Moreover, complex II- and FAO-linked respiration were elevated in STZ-DM mice under UCP1 inhibition. Conclusions: Pharmacological therapies could be targeted to modulate UCP1 activity with a significant impact in the uncoupling of mitochondrial bioenergetics in tPVAT.

Altered mitochondrial epigenetics associated with subchronic doxorubicin cardiotoxicity.

Doxorubicin (DOX), a potent and broad-spectrum antineoplastic agent, causes an irreversible, cumulative and dose-dependent cardiomyopathy that ultimately leads to congestive heart failure. The mechanisms responsible for DOX cardiotoxicity remain poorly understood, but seem to involve mitochondrial dysfunction on several levels. Epigenetics may explain a portion of this effect. Since mitochondrial dysfunction may affect the epigenetic landscape, we hypothesize that this cardiac toxicity may result from epigenetic changes related to disruption of mitochondrial function. To test this hypothesis, eight-week-old male Wistar rats (n=6/group) were administered 7 weekly injections with DOX (2mgkg-1) or saline, and sacrificed two weeks after the last injection. We assessed gene expression patterns by qPCR, global DNA methylation by ELISA, and proteome lysine acetylation status by Western blot in cardiac tissue from saline and DOX-treated rats. We show for the first time that DOX treatment decreases global DNA methylation in heart but not in liver. These differences were accompanied by alterations in mRNA expression of multiple functional gene groups. DOX disrupted cardiac mitochondrial biogenesis, as demonstrated by decreased mtDNA levels and altered transcript levels for multiple mitochondrial genes encoded by both nuclear and mitochondrial genomes. Transcription of genes involved in lipid metabolism and epigenetic modulation were also affected. Western blotting analyses indicated a differential protein acetylation pattern in cardiac mitochondrial fractions of DOX-treated rats compared to controls. Additionally, DOX treatment increased the activity of histone deacetylases. These results suggest an interplay between mitochondrial dysfunction and epigenetic alterations, which may be a primary determinant of DOX-induced cardiotoxicity.

Glucose and Lipid Dysmetabolism in a Rat Model of Prediabetes Induced by a High-Sucrose Diet.

Glucotoxicity and lipotoxicity are key features of type 2 diabetes mellitus, but their molecular nature during the early stages of the disease remains to be elucidated. We aimed to characterize glucose and lipid metabolism in insulin-target organs (liver, skeletal muscle, and white adipose tissue) in a rat model treated with a high-sucrose (HSu) diet. Two groups of 16-week-old male Wistar rats underwent a 9-week protocol: HSu diet (n = 10)-received 35% of sucrose in drinking water; Control (n = 12)-received vehicle (water). Body weight, food, and beverage consumption were monitored and glucose, insulin, and lipid profiles were measured. Serum and liver triglyceride concentrations, as well as the expression of genes and proteins involved in lipid biosynthesis were assessed. The insulin-stimulated glucose uptake and isoproterenol-stimulated lipolysis were also measured in freshly isolated adipocytes. Even in the absence of obesity, this rat model already presented the main features of prediabetes, with fasting normoglycemia but reduced glucose tolerance, postprandial hyperglycemia, compensatory hyperinsulinemia, as well as decreased insulin sensitivity (resistance) and hypertriglyceridemia. In addition, impaired hepatic function, including altered gluconeogenic and lipogenic pathways, as well as increased expression of acetyl-coenzyme A carboxylase 1 and fatty acid synthase in the liver, were observed, suggesting that liver glucose and lipid dysmetabolism may play a major role at this stage of the disease.

Glucose uptake and lipid metabolism are impaired in epicardial adipose tissue from heart failure patients with or without diabetes.

Type 2 diabetes mellitus is a complex metabolic disease, and cardiovascular disease is a leading complication of diabetes. Epicardial adipose tissue surrounding the heart displays biochemical, thermogenic, and cardioprotective properties. However, the metabolic cross-talk between epicardial fat and the myocardium is largely unknown. This study sought to understand epicardial adipose tissue metabolism from heart failure patients with or without diabetes. We aimed to unravel possible differences in glucose and lipid metabolism between human epicardial and subcutaneous adipocytes and elucidate the potential underlying mechanisms involved in heart failure. Insulin-stimulated [(14)C]glucose uptake and isoproterenol-stimulated lipolysis were measured in isolated epicardial and subcutaneous adipocytes. The expression of genes involved in glucose and lipid metabolism was analyzed by reverse transcription-polymerase chain reaction in adipocytes. In addition, epicardial and subcutaneous fatty acid composition was analyzed by high-resolution proton nuclear magnetic resonance spectroscopy. The difference between basal and insulin conditions in glucose uptake was significantly decreased (P= 0.006) in epicardial compared with subcutaneous adipocytes. Moreover, a significant (P< 0.001) decrease in the isoproterenol-stimulated lipolysis was also observed when the two fat depots were compared, and it was strongly correlated with lipolysis, lipid storage, and inflammation-related gene expression. Moreover, the fatty acid composition of these tissues was significantly altered by diabetes. These results emphasize potential metabolic differences between both fat depots in the presence of heart failure and highlight epicardial fat as a possible therapeutic target in situ in the cardiac microenvironment.

Doxorubicin-induced cardiotoxicity: from bioenergetic failure and cell death to cardiomyopathy.

Doxorubicin (DOX) is an anticancer anthracycline that presents a dose-dependent and cumulative cardiotoxicity as one of the most serious side effects. Several hypotheses have been advanced to explain DOX cardiac side effects, which culminate in the development of life-threatening cardiomyopathy. One of the most studied mechanisms involves the activation of DOX molecule into a more reactive semiquinone by mitochondrial Complex I, resulting in increased oxidative stress. The present review describes and critically discusses what is known about some of the potential mechanisms of DOX-induced cardiotoxicity including mitochondrial oxidative damage and loss of cardiomyocytes. We also discuss alterations of mitochondrial metabolism and the unique characteristics of DOX delayed toxicity, which can also interfere on how the cardiac muscle handles a "second-hit stress." We also present pharmaceutical and nonpharmaceutical approaches that may decrease DOX cardiac alterations in animal models and humans and discuss the limitations of each strategy.

Ipilimumab and vemurafenib: two different routes for targeting melanoma.

Melanoma, a malignant tumor of melanocytes, causes the majority (75%) of all skin cancer-related deaths. The overall efficacy of different anti-cancer therapies on metastatic melanoma is quite limited, due to its high resistance to all forms of conventional treatments, including chemotherapy, radiotherapy and immunotherapy, leading to low patient survival rates. The present review identifies possible strategies for the treatment of advanced melanoma and describes two novel agents, Ipilimumab and Vemurafenib, which may now be useful for clinical practice. Ipilimumab, a humanized, IgG1 monoclonal antibody, acts through immune-modulation since it blocks cytotoxic T-lymphocyte- associated antigen-4 (CTLA-4), producing favourable antitumor immune system responses and reducing tolerance to tumor-associated antigens. Vemurafenib is a novel oral small-molecule kinase inhibitor with high selectivity and efficacy toward a specific mutated oncogenic BRAF-signalling mediator. The mechanism of action of Vemurafenib involves selective inhibition of the mutated BRAF(V600E) kinase that leads to reduced signalling through the aberrant MAPK pathway. However, as patients commonly develop Vemurafenib resistance, clinical trials of Vemurafenib in combination with Ipilimumab or other targeted or cytotoxic chemotherapeutic agents may provide more effective regimens with longterm clinical benefits, emphasizing the importance of simultaneously targeting several pathways. As both drugs had only modest effects on median survival, new therapeutic combinations are needed, such as BRAF inhibitors with MEK inhibitors or combinations of immunomodulators and pathway inhibitors. Such strategies should have the potential of maximizing antitumor effect while minimizing and improving clinical benefit. Nevertheless, these two new agents open a promising view into an effective management of melanoma.

Rapid human melanoma cell death induced by sanguinarine through oxidative stress.

Sanguinarine is a natural isoquinoline alkaloid derived from the root of Sanguinaria canadensis and from other poppy fumaria species, and is known to have a broad spectrum of pharmacological properties. Here we have found that sanguinarine, at low micromolar concentrations, showed a remarkably rapid killing activity against human melanoma cells. Time-lapse videomicroscopy showed rapid morphological changes compatible with an apoptotic cell death, which was further supported by biochemical markers, including caspase activation, poly(ADP-ribose) polymerase (PARP) cleavage and DNA breakdown. Pan-caspase inhibition blocked sanguinarine-induced cell death. Sanguinarine treatment also induced an increase in intracellular calcium concentration, which was inhibited by dantrolene, and promoted cleavage of BAP-31, thus suggesting a putative role for Ca(2+) release from endoplasmic reticulum and a cross-talk between endoplasmic reticulum and mitochondria in the anti-melanoma action of sanguinarine. Sanguinarine disrupted the mitochondrial transmembrane potential (ΔΨm), released cytochrome c and Smac/DIABLO from mitochondria to cytosol, and induced oxidative stress. Overexpression of Bcl-XL by gene transfer did not prevent sanguinarine-mediated cell death, oxidative stress or release of mitochondrial apoptogenic proteins. However, preincubation with N-acetyl-l-cysteine (NAC) prevented sanguinarine-induced oxidative stress, PARP cleavage, release of apoptogenic mitochondrial proteins, and cell death. Pretreatment with glutathione (GSH) also inhibited the anti-melanoma activity of sanguinarine. Thus, pretreatment with the thiol antioxidants NAC and GSH abrogated the killing activity of sanguinarine. Taking together, our data indicate that sanguinarine is a very rapid inducer of human melanoma caspase-dependent cell death that is mediated by oxidative stress.

Edelfosine and perifosine disrupt hepatic mitochondrial oxidative phosphorylation and induce the permeability transition.

Edelfosine and perifosine are alkylphospholipids that have been intensively studied as potential antitumor agents. Apoptotic cell death caused by these two compounds is mediated, at least in part, through mitochondria. Additionally, previous works demonstrated that edelfosine induces changes in mitochondrial membrane permeability that are somehow reduced by using cyclosporin A. Therefore, the objective of the present study was not only to confirm mitochondrial permeability transition but also identify direct effects of both ether lipids on mitochondrial hepatic fractions, namely on mitochondrial oxidative phosphorylation and generation of hydrogen peroxide (H(2)O(2)) through the respiratory chain. Results show that edelfosine and perifosine inhibit mitochondrial respiration and decrease transmembrane electric potential. However, despite these effects, edelfosine and perifosine were still able to induce mitochondrial permeability transition in non-energized mitochondria. Interestingly, edelfosine decreased H(2)O(2) production through the respiratory chain. In conclusion, the present work demonstrates previously unknown alterations of mitochondrial physiology directly induced by edelfosine and perifosine. The study is relevant in the understanding of mitochondrial-target effects of both compounds, as well as to acknowledge possible toxic responses in non-tumor organs.

Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells.

The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine.

Berberine as a promising safe anti-cancer agent - is there a role for mitochondria?

Metabolic regulation is largely dependent on mitochondria, which play an important role in energy homeostasis. Imbalance between energy intake and expenditure leads to mitochondrial dysfunction, characterized by a reduced ratio of energy production (ATP production) to respiration. Due to the role of mitochondrial factors/events in several apoptotic pathways, the possibility of targeting that organelle in the tumor cell, leading to its elimination is very attractive, although the safety issue is problematic. Berberine, a benzyl-tetra isoquinoline alkaloid extracted from plants of the Berberidaceae family, has been extensively used for many centuries, especially in the traditional Chinese and Native American medicine. Several evidences suggest that berberine possesses several therapeutic uses, including anti-tumoral activity. The present review supplies evidence that berberine is a safe anti-cancer agent, exerting several effects on mitochondria, including inhibition of mitochondrial Complex I and interaction with the adenine nucleotide translocator which can explain several of the described effects on tumor cells.

Mitochondrial toxicity of the phyotochemicals daphnetoxin and daphnoretin--relevance for possible anti-cancer application.

Daphnetoxin is a daphnane type orthoester diterpene found exclusively in plants of the family Thymelaeaceae while daphnoretin, a bis-coumarin derivative that is the major constituent of the bark of some plants of this family, can also be found in Leguminosae and Rutaceae. These two compounds are recognized to have different biological effects, including a possible anti-cancer activity. The subject of the present research was to compare their mitochondrial toxicity and also investigate a possible selectivity towards tumor cell lines. Wistar rat liver mitochondria and three distinct cell lines were used to investigate compound-induced toxicity. The results indicate that both test compounds are toxic to isolated mitochondrial fractions, especially when used at concentrations higher than 100 microM. However, daphnetoxin presented the highest toxicity including increased proton leak in the inner mitochondrial membrane, increased induction of the mitochondrial permeability transition pore, inhibition of ATP synthase and inhibition of the mitochondrial respiratory chain. Both compounds also inhibited cell proliferation, regardless of the cell line used. Up to the maximal concentration tested in cells, no mitochondrial effects were detected by vital epifluorescence imaging, indicating that inhibition of cell proliferation may also originate from mitochondrial-independent mechanisms. The results warrant careful assessment of toxicity vs. pharmacology benefits of both molecules.