Survivin, a key player in cancer progression, increases in obesity and protects adipose tissue stem cells from apoptosis.

Adipose tissue (AT) has a central role in obesity-related metabolic imbalance through the dysregulated production of cytokines and adipokines. In addition to its known risk for cardiovascular disease and diabetes, obesity is also a major risk for cancer. We investigated the impact of obesity for the expression of survivin, an antiapoptotic protein upregulated by adipokines and a diagnostic biomarker of tumor onset and recurrence. In a cross-sectional study of 111 subjects classified by body mass index, circulating levels of survivin and gene expression in subcutaneous AT were significantly higher in obese patients and positively correlated with leptin. Within AT, survivin was primarily detected in human adipocyte-derived stem cells (hASCs), the adipocyte precursors that determine AT expansion. Remarkably, survivin expression was significantly higher in hASCs isolated from obese patients that from lean controls and was increased by proinflammatory M1 macrophage soluble factors including IL-1ß. Analysis of survivin expression in hASCs revealed a complex regulation including epigenetic modifications and protein stability. Surprisingly, obese hASCs showed survivin promoter hypermethylation that correlated with a significant decrease in its mRNA levels. Nonetheless, a lower level of mir-203, which inhibits survivin protein translation, and higher protein stability, was found in obese hASCs compared with their lean counterparts. We discovered that survivin levels determine the susceptibility of hASCs to apoptotic stimuli (including leptin and hypoxia). Accordingly, hASCs from an obese setting were protected from apoptosis. Collectively, these data shed new light on the molecular mechanisms governing AT expansion in obesity through promotion of hASCs that are resistant to apoptosis, and point to survivin as a potential new molecular player in the communication between AT and tumor cells. Thus, inhibition of apoptosis targeting survivin might represent an effective strategy for both obesity and cancer therapy.

Unveiling the Metabolic Changes on Muscle Cell Metabolism Underlying p-Phenylenediamine Toxicity.

Rhabdomyolysis is a disorder characterized by acute damage of the sarcolemma of the skeletal muscle leading to release of potentially toxic muscle cell components into the circulation, most notably creatine phosphokinase (CK) and myoglobulin, and is frequently accompanied by myoglobinuria. In the present work, we evaluated the toxicity of p-phenylenediamine (PPD), a main component of hair dyes which is reported to induce rhabdomyolysis. We studied the metabolic effect of this compound in vivo with Wistar rats and in vitro with C2C12 muscle cells. To this aim we have combined multi-omic experimental measurements with computational approaches using model-driven methods. The integrative study presented here has unveiled the metabolic disorders associated to PPD exposure that may underlay the aberrant metabolism observed in rhabdomyolys disease. Animals treated with lower doses of PPD (10 and 20 mg/kg) showed depressed activity and myoglobinuria after 10 h of treatment. We measured the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) in rats after 24, 48, and 72 h of PPD exposure. At all times, treatment with PPD at higher doses (40 and 60 mg/kg) showed an increase of AST and ALT, and also an increase of lactate dehydrogenase (LDH) and CK after 24 h. Blood packed cell volume and hemoglobin levels, as well as organs weight at 48 and 72 h, were also measured. No significant differences were observed in these parameters under any condition. PPD induce cell cycle arrest in S phase and apoptosis (40% or early apoptotic cells) on mus musculus mouse C2C12 cells after 24 h of treatment. Incubation of mus musculus mouse C2C12 cells with [1,2-13C2]-glucose during 24 h, subsequent quantification of 13C isotopologues distribution in key metabolites of glucose metabolic network and a computational fluxomic analysis using in-house developed software (Isodyn) showed that PPD is inhibiting glycolysis, non-oxidative pentose phosphate pathway, glycogen turnover, and ATPAse reaction leading to a reduction in ATP synthesis. These findings unveil the glucose metabolism collapse, which is consistent with a decrease in cell viability observed in PPD-treated C2C12 cells and with the myoglubinuria and other effects observed in Wistar Rats treated with PPD. These findings shed new light on muscle dysfunction associated to PPD exposure, opening new avenues for cost-effective therapies in Rhabdomyolysis disease.

Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase.

OBJECTIVE: Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes. METHODS: ZAG action on glucose uptake and insulin action was analyzed. ß1 and ß2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR. RESULTS: ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific ß1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via ß1-AR, whereas inhibition of insulin action is dependent on ß2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG. CONCLUSIONS: ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a ß2-AR- and PP2A-dependent manner.

Disruption of GIP/GIPR axis in human adipose tissue is linked to obesity and insulin resistance.

CONTEXT: Glucose-dependent insulinotropic peptide (GIP) has a central role in glucose homeostasis through its amplification of insulin secretion; however, its physiological role in adipose tissue is unclear. OBJECTIVE: Our objective was to define the function of GIP in human adipose tissue in relation to obesity and insulin resistance. DESIGN: GIP receptor (GIPR) expression was analyzed in human sc adipose tissue (SAT) and visceral adipose (VAT) from lean and obese subjects in 3 independent cohorts. GIPR expression was associated with anthropometric and biochemical variables. GIP responsiveness on insulin sensitivity was analyzed in human adipocyte cell lines in normoxic and hypoxic environments as well as in adipose-derived stem cells obtained from lean and obese patients. RESULTS: GIPR expression was downregulated in SAT from obese patients and correlated negatively with body mass index, waist circumference, systolic blood pressure, and glucose and triglyceride levels. Furthermore, homeostasis model assessment of insulin resistance, glucose, and G protein-coupled receptor kinase 2 (GRK2) emerged as variables strongly associated with GIPR expression in SAT. Glucose uptake studies and insulin signaling in human adipocytes revealed GIP as an insulin-sensitizer incretin. Immunoprecipitation experiments suggested that GIP promotes the interaction of GRK2 with GIPR and decreases the association of GRK2 to insulin receptor substrate 1. These effects of GIP observed under normoxia were lost in human fat cells cultured in hypoxia. In support of this, GIP increased insulin sensitivity in human adipose-derived stem cells from lean patients. GIP also induced GIPR expression, which was concomitant with a downregulation of the incretin-degrading enzyme dipeptidyl peptidase 4. None of the physiological effects of GIP were detected in human fat cells obtained from an obese environment with reduced levels of GIPR. CONCLUSIONS: GIP/GIPR signaling is disrupted in insulin-resistant states, such as obesity, and normalizing this function might represent a potential therapy in the treatment of obesity-associated metabolic disorders.

Novel Phenazine 5,10-Dioxides Release OH in Simulated Hypoxia and Induce Reduction of Tumour Volume In Vivo.

Phenazine 5,10-dioxides (PDOs) are a new class of bioreductive cytotoxins, which could act towards tumours containing hypoxic regions. The PDOs selective-hypoxic bioreduction was probed in vitro; however, the mechanism of action has not been completely explained. Besides, PDOs in vivo antitumour activities have not been demonstrated hitherto. We study the mechanism of hypoxic/normoxic cytotoxicity of PDO representative members. Electron spin resonance is used to confirm (•)OH production, alkaline comet assay to determine genotoxicity, and gel electrophoresis and flow cytometry to analyze DNA fragmentation and cell cycle distribution. Chemically induced rat breast tumours are employed to evaluate in vivo activities. For the most selective cytotoxin, 7(8)-bromo-2-hydroxyphenazine 5,10-dioxide (PDO1), exclusive hypoxic (•)OH production is evidenced, while for the unselective ones, (•)OH is produced in both conditions (normoxia and simulated hypoxia). In normoxia (Caco-2 cells), PDO1 induces cell-cycle arrest and DNA fragmentation but does not significantly induce apoptosis neither at IC(50) nor IC(80). No difference in the comet-assay scores are observed in normoxia and simulated hypoxia being the unselective 2-amino-7(8)-bromophenazine 5,10-dioxide (PDO2) the most genotoxic. The in vivo efficacy with the absence of systemic toxicity of PDO1 and PDO2 is checked out. Results from this study highlight the potential of PDOs as new therapeutics for cancer.

Study of the potential association of adipose tissue GLP-1 receptor with obesity and insulin resistance.

The increase in glucagon-like peptide-1 (GLP-1) activity has emerged as a useful therapeutic tool for the treatment of type 2 diabetes mellitus. The actions of GLP-1 on ß-cells and the nervous and digestive systems are well known. The action of this peptide in adipose tissue (AT), however, is still poorly defined. Furthermore, no relationship has been established between GLP-1 receptor (GLP-1R) in AT and obesity and insulin resistance (IR). We provide evidence for the presence of this receptor in AT and show that its mRNA and protein expressions are increased in visceral adipose depots from morbidly obese patients with a high degree of IR. Experiments with the 3T3-L1 cell line showed the lipolytic and lipogenic dose-dependent effect of GLP-1. Moreover, GLP-1 stimulated lipolysis in 3T3-L1 adipocytes in a receptor-dependent manner involving downstream adenylate cyclase/cAMP signaling. Our data also demonstrate that the expression of the GLP-1R in AT correlated positively with the homeostasis model assessment index in obese IR subjects. Furthermore, prospective studies carried out with patients that underwent biliopancreatic diversion surgery showed that subjects with high levels of GLP-1R expression in AT, which indicates a deficit of GLP-1 in this tissue, were those whose insulin sensitivity improved after surgery, suggesting the potential relationship between AT GLP-1R and insulin sensitivity amelioration in obese subjects. Altogether these results indicate that the GLP-1/GLP-1R system in AT represents another potential candidate for improving insulin sensitivity in obese patients.

Phagocytic activity is impaired in type 2 diabetes mellitus and increases after metabolic improvement.

OBJECTIVE: 1) To evaluate whether peripheral blood mononuclear cells (PBMCs) from type 2 diabetic patients present an impairment of phagocytic activity; 2) To determine whether the eventual impairment in phagocytic activity is related to glycemic control and can be reversed by improving blood glucose levels. METHODS: 21 type 2 diabetic patients and 21 healthy volunteers were prospectively recruited for a case-control study. In addition, those patients in whom HbA1c was higher than 8% (n = 12) were hospitalized in order to complete a 5-day intensification treatment of blood glucose. Phagocytic activity was assessed by using a modified flow cytometry procedure developed in our laboratory based on DNA/RNA viable staining to discriminate erythrocytes and debris. This method is simple, highly sensitive and reproducible and it takes advantage of classic methods that are widely used in flow cytometry. RESULTS: Type 2 diabetic patients showed a lower percentage of activated macrophages in comparison with non-diabetic subjects (54.00±18.93 vs 68.53±12.77%; p = 0.006) Significant negative correlations between phagocytic activity and fasting glucose (r = -0.619, p = 0.004) and HbA1c (r = -0.506, p = 0.019) were detected. In addition, multiple linear regression analyses showed that either fasting plasma glucose or HbA1c were independently associated with phagocytic activity. Furthermore, in the subset of patients who underwent metabolic optimization a significant increase in phagocytic activity was observed (p = 0.029). CONCLUSIONS: Glycemic control is related to phagocytic activity in type 2 diabetes. Our results suggest that improvement in phagocytic activity can be added to the beneficial effects of metabolic optimization.

ABCG2 is required to control the sonic hedgehog pathway in side population cells with stem-like properties.

The Sonic Hedgehog (Hh) pathway has been implicated in the maintenance of stem or progenitor cells in many adult tissues. Importantly, abnormal Hh pathway activation is also associated with initiation of neoplasia, but its role in tumor growth is still unclear. Here, we demonstrate that cyclopamine, a plant-derived alkaloid product used to inhibit the Hh signaling pathway, reduces the Side Population (SP) obtained by Hoechst 33342 (Ho342) dye measurements. In addition, cyclopamine is able to modulate, along with oxysterols and other products, the ABCG2 transporter by increasing Ho342 and mitoxantrone uptake. Therefore, if the SP is solely measured as a Ho342 dye extruding fraction, this may be significantly modulated by the inhibition of ABCG2 transport fraction, independently from the action of cyclopamine on the Hh pathway. Our results indicate that ABCG2 may act in the upstream regulation of the Hh signaling pathway to protect the stemness of the SP compartment, giving support to the cancer stem cell hypothesis and suggesting that ABCG2 is not only critical for increased resistance to anticancer agents.

New betulinic acid derivatives induce potent and selective antiproliferative activity through cell cycle arrest at the S phase and caspase dependent apoptosis in human cancer cells.

New semisynthetic derivatives of betulinic acid (BA) RS01, RS02 and RS03 with 18-45 times improved cytotoxic activity against HepG2 cells, were tested for their ability to induce apoptosis and cell cycle arrest in HepG2, HeLa and Jurkat cells. All the compounds induced significant increase in the population at the S phase more effectively than BA. RS01, RS02 and RS03 were also found to be potent inducers of apoptosis with RS01 being markedly more potent than BA, suggesting that the introduction of the imidazolyl moiety is crucial for enhancing the induction of apoptosis and the cell cycle arrest. The mechanism of apoptosis induction has been studied in HepG2 cells and found to be mediated by activation of the postmitochondrial caspases-9 and -3 cascade and possibly by mitochondrial amplification loop involving caspase-8. These facts were corroborated by detection of mitochondrial cytochrome c release and DNA fragmentation. Because RS01, RS02 and RS03 exhibited significant improved antitumor activity with respect to BA, they may be promising new agents for the treatment of cancer. In particular, RS01 is the most promising compound with an IC(50) value 45 times lower than BA on HepG2 cells and 61 times lower than the one found for the non-tumoral Chang liver cells.

Study of benzo[a]phenazine 7,12-dioxide as selective hypoxic cytotoxin-scaffold. Identification of aerobic-antitumoral activity through DNA fragmentation.

Phenazine 5,10-dioxides are prodrugs for antitumor therapy that undergo hypoxic-selective bioreduction to form cytotoxic species. Here we investigate the expanded system benzo[a]phenazine 7,12-dioxides as selective hypoxic cytotoxin-scaffold. The clonogenic survival of V79 cells on aerobic and anaerobic conditions, conduct us to study antiproliferative activity on Caco-2 tumoral cells in normoxia. Electrochemical, DNA-interaction and DNA-damage studies were performed to establish the mode of action. The results demonstrated the potential biological properties of the studied scaffold being derivatives 6-10 structural hits for further chemical-modifications to become into therapeutics for solid tumors. Compounds 6 and 8 with cytotoxicity against V79 cells in both conditions (aerobia and anaerobia) were also cytotoxic against Caco-2 tumoral cells in aerobiosis.

A quinoxaline 1,4-di-N-oxide derivative induces DNA oxidative damage not attenuated by vitamin C and E treatment.

Some anticancer compounds are pro-drugs which give rise to toxic species through enzymatic reduction. The quinoxaline-di-N-oxide derivative Q-85 HCl (7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride) is a bioreductive compound selectively toxic in hypoxia. Due to the possibility of secondary tumors the study of the genotoxic capability of antitumoral drugs is very important. The aim of this study was to assess the ability of Q-85 HCl to produce reactive oxygen species (ROS) and oxidative DNA damage in Caco-2 cells, both in hypoxia and in well-oxygenated conditions. Secondly, we attempted to evaluate the effect of vitamins C and E under hypoxic and normoxic conditions, in order to determine if these antioxidant substances modify Q-85 HCl effect in hypoxic cells or possibly exert a protective action in normal cells. Caco-2 cells were treated with Q-85 HCl for 2h, at high concentrations in normoxia (0.1-5 microM) and at low concentrations in hypoxia (0.002-0.1 microM). In normoxia, a dose-related significant increase in intracellular ROS level was evident; in hypoxia all the concentrations produced very high level of ROS. Just after the treatment and 24h later, oxidative DNA damage was evaluated by the modified comet assay after post-digestion of the cells with formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (Endo III). Q-85 HCl treatment evoked a significant dose-dependent increase in the total comet score of the cells both in hypoxia and normoxia, indicating that this compound or some metabolite is able to oxidize purine and pyrimidine bases. After 24h DNA damage caused by the compound was completely repaired with only one exception: cells treated with the highest concentration of Q-85 HCl in hypoxia and post-digested with FPG. Vitamin C (5-100 microM) and vitamin E (500-400 microM) did not have a pro-oxidant effect in Caco-2 cells. Treatment of cells with vitamin C (10 microM) or vitamin E (100 microM) did not significantly reduce oxidative DNA damage in hypoxia and normoxia. In conclusion, the use of these vitamins would not hinder toxicity against hypoxic cells, but a protective effect in normoxic cells was not evident.

DNA damage induced by a quinoxaline 1,4-di-N-oxide derivative (hypoxic selective agent) in Caco-2 cells evaluated by the comet assay.

The DNA damage induced by 7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride (Q-85 HCl) in Caco-2 cells under hypoxic and well-oxygenated conditions has been studied by using the comet assay. This compound has shown a good in vitro profile of high selective toxicity in hypoxia, but its mechanism of action is unknown. The DNA damage has been evaluated by performing the comet assay after a 2-h treatment with Q-85 HCl (0.1, 0.2, 0.4 microM in hypoxia; 20, 40 microM in well-oxygenated conditions). The number of cells in apoptosis has also been assessed by flow cytometry analysis of Annexin V-FITC staining. The capability of the cells to repair the DNA damage and the proliferation rate was evaluated at different times after the treatment (24-168 h). Under hypoxic conditions, a clear dose-dependent increase in the number of nuclei with a comet was observed (comet score: 132 +/- 13, 343 +/- 30 and 399 +/- 1; control comet score: 42 +/- 14). Under well-oxygenated conditions, the number of nuclei with comet increased significantly with respect to the control (comet score: 273 +/- 14 and 312 +/- 9; control comet score: 27 +/- 4). Cells in apoptosis were not detected by the comet assay nor by flow cytometry. The recovery from DNA damage was time- and concentration-dependent in hypoxia (cells treated with the highest concentration still showed DNA damage after 72 h) and rather time-dependent in well-oxygenated conditions (DNA was completely repaired after 24 h). In conclusion, Q-85 HCl acts by DNA damage and not only the reduced intermediate is genotoxic but also some other derivatives and Q-85 HCl itself may be acting.

Selective toxicity of a quinoxaline 1,4-di-N-oxide derivative in human tumour cell lines.

The presence of hypoxic cells in human solid tumours is one of the causes of tumour resistance to conventional therapy, and is also associated with processes that promote the tumour progression. Different chemical agents have been designed in order to take advantage of the particular metabolic characteristics of hypoxic regions. These drugs, called bioreductive agents, are activated inside the hypoxic cells to give active species that, in the presence of oxygen, are oxidised back to the non-toxic parent compound. Several quinoxaline 1,4-di-N-oxides have been described as potential bioreductive agents, and among them, 7-cloro-3-[[(N,N-dimethylamino)propy]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrocloride (Q-85 HCl) appeared to be the most promising one. In the present work, the selective cytotoxicity of Q-85 HCl was studied in several human tumour cell lines of different origin (Caco2, MCF-7, HT-29 and Tk-10). Cell viability was calculated after 2 h treatment under hypoxic and well-oxygenated conditions. The potency (the concentration that gives 1% of cell survival) in hypoxia and hypoxia cytotoxicity ratio (HCR = potency in oxygenated conditions/potency in hypoxia) were calculated after a 14-day clonogenic assay. Q-85 HCl was more toxic in hypoxia than in well-oxygenated cells in all the tumour cell lines. The best profile of potency in hypoxia (0.4 micromol/L) and selectivity (HCR=155) was found in CaCo-2 cells. Altogether, these results suggest an in vitro biological profile for Q-85 HCl that makes it an interesting candidate for the development as a bioreductive agent.