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.

Introduction: Low survival rates and severe side effects of current neuroblastoma treatments underline the need for more effective less toxic therapies. Compared to normal cells, cancer cells demonstrate high glucose consumption even under aerobic conditions, therefore; targeting the glycolytic pathway in cancer cells as a potential therapy is currently an area of intense investigation. The polyphenolic compound resveratrol which can be found in grapes, peanuts, red wine and purple grape juice, has demonstrated antitumor activities both in vitro and in vivo. Here we investigate the potential of inhibiting glycolysis in combination with resveratrol treatment. Methods: To inhibit glycolysis we used the glucose analog, 2-Deoxy-D-glucose (2-DG). N-MYC amplified neuroblastoma cells (NB1691) were exposed to low dose 2-DG (0.5 and 2mM) and resveratrol 10uM. Cell viability was determined at 72hr using MTS assay. Western blotting was used to determine cell signaling pathways and apoptotic protein levels. The role of the NAD+ dependent HDAC sirtuin 1 (sirt1) was determined using 50uM of the inhibitor sirtinol. Results: While 2-DG did reduce cell viability (2mM:82+4.4% of control), the combination of 2-DG and resveratrol significantly reduced cell viability (20+2.2%) compared to resveratrol alone (69+2.8%). Western blot analysis indicated a robust activation of AKT with 2-DG with treatment, however this activation was blocked with 10uM resveratrol. Furthermore, western blot analysis revealed that this was caspase dependent cell death. PI3K/Akt inhibitors wortmannin and LY-294002 (10uM each) also reduced cell viability in combination with 2-DG to approximately 40% compared to non-treated, confirming the protective role of AKT signaling in response to 2-DG treatment. To determine if resveratrol, a well-known activator of sirt1, inhibited AKT via sirt1, cells were pretreated with sirtinol. Inhibiting sirt1 did not prevent resveratrol's inhibition of AKT. Conclusions: The oncoprotein AKT mediates signaling processes involved in evading apoptosis and promoting cell proliferation and recently has been linked to glycolysis. Resveratrol significantly increased 2-DG-induced cell death by inhibiting the 2-DG induced increase in AKT activity independent of sirt1. Here we show that combining resveratrol with 2-DG, two relatively non-toxic agents can significantly reduce neuroblastoma cell viability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3214. doi:1538-7445.AM2012-3214