Evangelia Tsiani

Stimulation of muscle cell glucose uptake by resveratrol through sirtuins and AMPK

Although recent studies in vitro and in vivo indicate that the polyphenol resveratrol (RSV) has anti-diabetic properties, the exact mechanisms involved are not known. In the present study, we examined the effects of RSV and the mechanism of regulation of glucose uptake in skeletal muscle cells. In L6 myotubes RSV (100 microM) induced maximum stimulation of glucose (2DG) uptake (201+/-8.90% of control, p<0.001), an effect that was similar to insulin action. RSV-stimulated glucose uptake was abolished by AMPK inhibition. In the presence of the sirtuin inhibitor nicotinamide, RSV-stimulated 2DG uptake and AMPK phosphorylation were abolished. RSV did not stimulate significant translocation of GLUT4 or GLUT1 transporters. However, treatment with indinavir, a GLUT4 specific inhibitor, blocked RSV-stimulated glucose uptake. We propose that RSV elevates glucose uptake in muscle cells through a mechanism that involves sirtuins and AMPK and possibly stimulation of GLUT4 transporter intrinsic activity.

Metformin inhibits growth and enhances radiation response of non-small cell lung cancer (NSCLC) through ATM and AMPK.

Background:We examined the potential of metformin (MET) to enhance non-small cell lung cancer (NSCLC) responses to ionising radiation (IR).Methods:Human NSCLC cells, mouse embryonic fibroblasts from wild-type and AMP-activated kinase (AMPK) α1/2-subunit−/− embryos (AMPKα1/2−/−-MEFs) and NSCLC tumours grafted into Balb/c-nude mice were treated with IR and MET and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays and immunohistochemistry (IHC).Results:Metformin (2.5 μM–5 mM) inhibited proliferation and radio-sensitised NSCLC cells. Metformin (i) activated the ataxia telengiectasia-mutated (ATM)–AMPK–p53/p21cip1 and inhibited the Akt–mammalian target of rapamycin (mTOR)–eIF4E-binding protein 1 (4EBP1) pathways, (ii) induced G1 cycle arrest and (iii) enhanced apoptosis. ATM inhibition blocked MET and IR activation of AMPK. Non-small cell lung cancer cells with inhibited AMPK and AMPKα1/2−/−-MEFs were resistant to the antiproliferative effects of MET and IR. Metformin or IR inhibited xenograft growth and combined treatment enhanced it further than each treatment alone. Ionising radiation and MET induced (i) sustained activation of ATM–AMPK–p53/p21cip1 and inhibition of Akt–mTOR–4EBP1 pathways in tumours, (ii) reduced expression of angiogenesis and (iii) enhanced expression of apoptosis markers.Conclusion:Clinically achievable MET doses inhibit NSCLC cell and tumour growth and sensitise them to IR. Metformin and IR mediate their action through an ATM–AMPK-dependent pathway. Our results suggest that MET can be a clinically useful adjunct to radiotherapy in NSCLC.

Naringenin, a citrus flavonoid, increases muscle cell glucose uptake via AMPK.

Naringenin, a flavonoid found in high concentrations in grapefruit, has been reported to have antioxidant, antiatherogenic, and anticancer effects. Effects on lipid and glucose metabolism have also been reported. Naringenin is structurally similar to the polyphenol resveratrol, that has been reported to activate the SIRT1 protein deacetylase and to have antidiabetic properties. In the present study we examined the direct effects of naringenin on skeletal muscle glucose uptake and investigated the mechanism involved. Naringenin stimulated glucose uptake in L6 myotubes in a dose- and time-dependent manner. Maximum stimulation was seen with 75 microM naringenin for 2 h (192.8+/-24%, p<0.01), a response comparable to maximum insulin response (190.1+/-13%, p<0.001). Similar to insulin, naringenin did not increase glucose uptake in myoblasts indicating that GLUT4 glucose transporters may be involved in the naringenin-stimulated glucose uptake. In addition, naringenin did not have a significant effect on basal or insulin-stimulated Akt phosphorylation while significantly increased AMPK phosphorylation/activation. Furthermore, silencing of AMPK, using siRNA approach, abolished the naringenin-stimulated glucose uptake. The SIRT1 inhibitors nicotinamide and EX527 did not have an effect on naringenin-stimulated AMPK phosphorylation and glucose uptake. Our data show that naringenin increases glucose uptake by skeletal muscle cells in an AMPK-dependent manner.

Facilitative glucose transporters: Implications for cancer detection, prognosis and treatment.

It is long recognized that cancer cells display increased glucose uptake and metabolism. In a rate-limiting step for glucose metabolism, the glucose transporter (GLUT) proteins facilitate glucose uptake across the plasma membrane. Fourteen members of the GLUT protein family have been identified in humans. This review describes the major characteristics of each member of the GLUT family and highlights evidence of abnormal expression in tumors and cancer cells. The regulation of GLUTs by key proliferation and pro-survival pathways including the phosphatidylinositol 3-kinase (PI3K)-Akt, hypoxia-inducible factor-1 (HIF-1), Ras, c-Myc and p53 pathways is discussed. The clinical utility of GLUT expression in cancer has been recognized and evidence regarding the use of GLUTs as prognostic or predictive biomarkers is presented. GLUTs represent attractive targets for cancer therapy and this review summarizes recent studies in which GLUT1, GLUT3, GLUT5 and others are inhibited to decrease cancer growth.

Resveratrol enhances prostate cancer cell response to ionizing radiation. Modulation of the AMPK, Akt and mTOR pathways

BackgroundProstate cancer (PrCa) displays resistance to radiotherapy (RT) and requires radiotherapy dose escalation which is associated with greater toxicity. This highlights a need to develop radiation sensitizers to improve the efficacy of RT in PrCa. Ionizing radiation (IR) stimulates pathways of IR-resistance and survival mediated by the protein kinase Akt but it also activates the metabolic energy sensor and tumor suppressor AMP-Activated Protein Kinase (AMPK). Here, we examined the effects of the polyphenol resveratrol (RSV) on the IR-induced inhibition of cell survival, modulation of cell cycle and molecular responses in PrCa cells.MethodsAndrogen-insensitive (PC3), sensitive (22RV1) PrCa and PNT1A normal prostate epithelial cells were treated with RSV alone (2.5-10 μM) or in combination with IR (2-8 Gy). Clonogenic assays, cell cycle analysis, microscopy and immunoblotting were performed to assess survival, cell cycle progression and molecular responses.ResultsRSV (2.5-5 μM) inhibited clonogenic survival of PC3 and 22RV1 cells but not of normal prostate PNT1A cells. RSV specifically sensitized PrCa cells to IR, induced cell cycle arrest at G1-S phase and enhanced IR-induced nuclear aberrations and apoptosis. RSV enhanced IR-induced expression of DNA damage (γH2Ax) and apoptosis (cleaved-caspase 3) markers as well as of the cell cycle regulators p53, p21cip1 and p27kip1. RSV enhanced IR-activation of ATM and AMPK but inhibited basal and IR-induced phosphorylation of Akt.ConclusionsOur results suggest that RSV arrests cell cycle, promotes apoptosis and sensitizes PrCa cells to IR likely through a desirable dual action to activate the ATM-AMPK-p53-p21cip1/p27kip1 and inhibit the Akt signalling pathways.

Lovastatin Sensitizes Lung Cancer Cells to Ionizing Radiation: Modulation of Molecular Pathways of Radioresistance and Tumor Suppression

Introduction: In this study, we investigated the effect of the 3-hydroxy-3-methylgutaryl-CoA reductase inhibitor lovastatin, as a sensitizer of lung cancer cells to ionizing radiation (IR). Methods: A549 lung adenocarcinoma cells were treated with 0 to 50 &mgr;M lovastatin alone or in combination with 0 to 8 Gy IR and subjected to clonogenic survival and proliferation assays. To assess the mechanism of drug action, we examined the effects of lovastatin and IR on the epidermal growth factor (EGF) receptor and AMP-activated kinase (AMPK) pathways and on apoptotic markers and the cell cycle. Results: Lovastatin inhibited basal clonogenic survival and proliferation of A549 cells and sensitized them to IR. This was reversed by mevalonate, the product of 3-hydroxy-3-methylgutaryl-CoA reductase. Lovastatin attenuated selectively EGF-induced phosphorylation of EGF receptor and Akt, and IR-induced Akt phosphorylation, in a mevalonate-sensitive fashion, without inhibition on extracellular signal-regulated kinase 1/2 phosphorylation by either stimulus. IR phosphorylated and activated the metabolic sensor and tumor suppressor AMPK, but lovastatin enhanced basal and IR-induced AMPK phosphorylation. The drug inhibited IR-induced expression of p53 and the cyclin-dependent kinase inhibitors p21cip1 and p27kip1, but caused a redistribution of cells from G1-S phase (control and radiated cells) and G2-M phase (radiated cells) of cell cycle into apoptosis. The latter was also evident by induction of nuclear fragmentation and cleavage of caspase 3 by lovastatin in both control and radiated cells. Conclusions: We suggest that lovastatin inhibits survival and induces radiosensitization of lung cancer cells through induction of apoptosis, which may be mediated by a simultaneous inhibition of the Akt and activation of the AMPK signaling pathways.

Resveratrol inhibits neointimal formation after arterial injury through an endothelial nitric oxide synthase-dependent mechanism

Revascularization procedures used for treatment of atherosclerosis often result in restenosis. Resveratrol (RSV), an antioxidant with cardiovascular benefits, decreases neointimal formation after arterial injury by a mechanism that is still not fully clarified. Our main objective was to address the role of nitric oxide synthases (NOSes) and more specifically the endothelial-NOS (eNOS) isoform as a mediator of this effect. RSV (4 mg/kg/day, s.c.) alone or in combination with the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) (2 mg/kg/day, s.c.) was given to Sprague-Dawley rats beginning at 3 days before arterial (carotid or aortic) injury. RSV reduced neointimal formation by 50% (P<0.01), decreased intimal cell proliferation by 37% (P<0.01) and reduced inflammatory markers such as PECAM and MMP-9 mRNA. These effects of RSV were all abolished by coadministration of l-NAME. Oral RSV (beginning at 5 days before arterial injury) reduced neointimal thickness after femoral wire injury in mice, however this effect was not observed in eNOS knockout mice. This is the first report of RSV decreasing neointimal cell proliferation and neointimal growth through an eNOS-dependent mechanism.

Anticancer Effects of Rosemary (Rosmarinus officinalis L.) Extract and Rosemary Extract Polyphenols

Cancer cells display enhanced growth rates and a resistance to apoptosis. The ability of cancer cells to evade homeostasis and proliferate uncontrollably while avoiding programmed cell death/apoptosis is acquired through mutations to key signaling molecules, which regulate pathways involved in cell proliferation and survival. Compounds of plant origin, including food components, have attracted scientific attention for use as agents for cancer prevention and treatment. The exploration into natural products offers great opportunity to evaluate new anticancer agents as well as understand novel and potentially relevant mechanisms of action. Rosemary extract has been reported to have antioxidant, anti-inflammatory, antidiabetic and anticancer properties. Rosemary extract contains many polyphenols with carnosic acid and rosmarinic acid found in highest concentrations. The present review summarizes the existing in vitro and in vivo studies focusing on the anticancer effects of rosemary extract and the rosemary extract polyphenols carnosic acid and rosmarinic acid, and their effects on key signaling molecules.

Inhibition of human lung cancer cell proliferation and survival by wine

BackgroundCompounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events.MethodsHuman NSCLC adenocarcinoma A549 and H1299 cells were used. Cell proliferation was assessed by thymidine incorporation. Clonogenic assays were used to assess cell survival. Immunoblotting was used to examine total and phosphorylated levels of Akt, Erk and p53.ResultsIn A549 cells red wine inhibited cell proliferation and reduced clonogenic survival at doses as low as 0.02%. Red wine significantly reduced basal and EGF-stimulated Akt and Erk phosphorylation while it increased the levels of total and phosphorylated p53 (Ser15). Control experiments indicated that the anti-proliferative effects of wine were not mediated by the associated contents of ethanol or the polyphenol resveratrol and were independent of glucose transport into cancer cells. White wine also inhibited clonogenic survival, albeit at a higher doses (0.5-2%), and reduced Akt phosphorylation. The effects of both red and white wine on Akt phosphorylation were also verified in H1299 cells.ConclusionsRed wine inhibits proliferation of lung cancer cells and blocks clonogenic survival at low concentrations. This is associated with inhibition of basal and EGF-stimulated Akt and Erk signals and enhancement of total and phosphorylated levels of p53. White wine mediates similar effects albeit at higher concentrations. Our data suggest that wine may have considerable anti-tumour and chemoprevention properties in lung cancer and deserves further systematic investigation in animal models of lung cancer.

Response of Bone Turnover Markers and Cytokines to High-Intensity Low-Impact Exercise.

INTRODUCTION Low-impact, high-intensity interval exercise (HIE) was used to investigate the postexercise response in bone turnover markers and cytokines. METHODS Twenty-three recreationally active males (21.8 ± 2.4 yr) performed one HIE bout on a cycle ergometer at 90% maximum workload. The total duration of the exercise was 12 min and included six 1-min high-intensity exercise intervals separated by 1-min rest intervals. Blood samples were collected before exercise, 5 min, 1 h, and 24 h after exercise and were analyzed for serum levels of anti- and pro-inflammatory cytokines (interleukin 10 [IL-10], interleukin 6 [IL-6], interleukin 1-alpha [IL-1α], interleukin 1-beta [IL-1β], and tumor necrosis factor alpha [TNF-α]) and markers of bone formation (bone alkaline phosphatase [BAP], osteoprotegerin [OPG]) and resorption (amino-terminal cross-linking propeptide [NTX], receptor activator of NF-κB ligand [RANKL]). RESULTS There was a significant (P < 0.05) time effect for all bone turnover markers. Post hoc analysis showed that BAP, OPG, and RANKL significantly increased from baseline to 5 min after exercise (10.9%, 13.5%, and 34.2%, P < 0.05). At 1 h after exercise, only BAP was significantly higher than baseline (9.5%, P = 0.010) and remained higher than baseline at 24 h (10.9%, P = 0.001). NTX was significantly lower than baseline 24 h after exercise (-14.6%, P = 0.046). Significant (P < 0.05) time effects were also observed for IL-1α, IL-1β, IL-6, and TNF-α, with all four significantly increasing 5 min after exercise and returning to baseline levels 1 h after exercise. The postexercise changes in bone formation markers correlated positively with the anti-inflammatory cytokine (IL-10) and negatively with the pro-inflammatory cytokines while NTX correlated positively with a pro-inflammatory cytokine, TNF-α (P < 0.05). CONCLUSIONS These results suggest that HIE stimulates a response in bone turnover markers and cytokines and that a correlation exists between immune and skeletal responses to this form of exercise. This type of exercise may benefit individuals for whom high-impact exercise might be contraindicated.