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Experimental & Molecular Medicine (2016) 48, e210; doi:10.1038/emm.2015.116 & 2016 KSBMB. All rights reserved 2092-6413/16 www.nature.com/emm

ORIGINAL ARTICLE

Resveratrol augments ER stress and the cytotoxic effects of glycolytic inhibition in neuroblastoma by downregulating Akt in a mechanism independent of SIRT1 Regina M Graham1, Fiorela Hernandez2,4, Nataly Puerta3,5, Guillermo De Angulo2, Keith A Webster3 and Steven Vanni1 Cancer cells typically display increased rates of aerobic glycolysis that are correlated with tumor aggressiveness and a poor prognosis. Targeting the glycolytic pathway has emerged as an attractive therapeutic route mainly because it should spare normal cells. Here, we evaluate the effects of combining the inhibition of glycolysis with application of the polyphenolic compound resveratrol (RSV) in neuroblastoma (NB) cancer cell lines. Inhibiting glycolysis with 2-deoxy-D-glucose (2-DG) significantly reduced NB cell viability and was associated with increased endoplasmic reticulum (ER) stress and Akt activity. Administration of 2-DG increased the expression of the ER molecular chaperones GRP78 and GRP94, the prodeath protein C/EBP homology protein (CHOP) and the phosphorylation of Akt at S473, T450 and T308. Combined treatment with both RSV and 2-DG reduced GRP78, GRP94 and Akt phosphorylation but increased CHOP and NB cell death when compared with the administration of 2-DG alone. The selective inhibition of Akt activity also decreased 2-DG-induced GRP78 and GRP94 expression and increased CHOP expression, suggesting that Akt can modulate ER stress. Protein phosphatase 1α (PP1α) was activated by RSV, as indicated by a reduction in PP1α phosphorylation at T320. Pretreatment of cells with tautomycin, a selective PP1α inhibitor, prevented the RSV-mediated decrease in Akt phosphorylation, suggesting that RSV enhances 2-DG-induced cell death by activating PP1 and downregulating Akt. The RSV-mediated inhibition of Akt in the presence of 2-DG was not prevented by the selective inhibition of SIRT1, a known target of RSV, indicating that the effects of RSV on this pathway are independent of SIRT1. We propose that RSV inhibits Akt activity by increasing PP1α activity, thereby potentiating 2-DG-induced ER stress and NB cell death. Experimental & Molecular Medicine (2016) 48, e210; doi:10.1038/emm.2015.116; published online 19 February 2016

INTRODUCTION Neuroblastoma (NB), which is presumed to arise from neuronal precursor cells that originate from the neural crest during embryonic development, is the most common pediatric extracranial tumor and the fourth most common malignancy during childhood. NB affects very young children, with approximately one-third of affected children diagnosed in infancy and two-thirds diagnosed by the age of 5 years. More than half of affected children over the age of 1 year have metastatic disease at the time of diagnosis.1 In children without metastatic disease or infants under the age of 18 months, the 1

prognosis is very good. However, the prognosis for high-risk patients is extremely poor, and these include children with MYCN-amplified tumors, unfavorable tumor biology and/or the presence of distant metastasis. Despite aggressive multimodal treatment including surgery, high-dose chemotherapy, radiotherapy and stem cell transplant followed by retinoid maintenance therapy, these children often relapse and succumb to the disease. Newer therapies, including treatment with an anti-GD2 antibody in combination with retinoic acid, and multiple ongoing clinical trials offer some hope; however, the effects of aggressive treatment regimens induce significant

Department of Neurological Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA; 2Miami Children’s Hospital, Miami, FL, USA and Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, FL, USA 4Current address: MD Anderson, Houston, TX, USA. 5Current address: European Research Institute for the Biology of Aging (ERIBA), University of Groningen, Groningen, The Netherlands. Correspondence: Dr RM Graham, Department of Neurological Surgery, School of Medicine, 1095 NW 14 Terrace, Room 5-27, Miami, FL 33136, USA. Email: [email protected] Received 9 February 2015; revised 7 September 2015; accepted 22 September 2015 3

Resveratrol blocks Akt and UPR in neuroblastoma RM Graham et al 2

morbidity and, as a result, children may not qualify for additional clinical trials.2 This underscores the need for more effective and less toxic treatment options. The fact that cancer cells display increased aerobic glycolysis compared with normal cells is known as the Warburg effect. This effect has been exploited for imaging tumors using fluorodeoxyglucose positron emission tomography for many years. Fluorodeoxyglucose is an analog of glucose that is rapidly taken up by highly glycolytic cancer cells. The use of fluorodeoxyglucose positron emission tomography is increasing in NB, confirming the glycolytic nature of these tumors.3 The activation of oncogenes, the loss of tumor suppressors