Control of Glycolytic Flux by AMP-Activated Protein Kinase in Tumor Cells Adapted to Low pH.

Tumor cells grow in nutrient- and oxygen-deprived microenvironments and adapt to the suboptimal growth conditions by altering their metabolic pathways. This adaptation process commonly results in a tumor phenotype that displays a high rate of aerobic glycolysis and aggressive tumor characteristics. The glucose regulatory molecule, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), is a bifunctional enzyme that is central to glycolytic flux and is downstream of the metabolic stress sensor AMP-activated protein kinase (AMPK), which has been suggested to modulate glycolysis and possibly activate isoforms of PFKFB, specifically PFKFB3 expressed in tumor cells. Our results demonstrated that long-term low pH exposure induced AMPK activation, which resulted in the up-regulation of PFKFB3 and an increase in its serine residue phosphorylation. Pharmacologic activation of AMPK resulted in an increase in PFKFB3 as well as an increase in glucose consumption, whereas in contrast, inhibition of AMPK resulted in the down-regulation of PFKFB3 and decreased glycolysis. PFKFB3 overexpression in DB-1 tumor cells induced a high rate of glycolysis and inhibited oxygen consumption, confirming its role in controlling glycolytic flux. These results show that low pH is a physiological stress that can promote a glycolytic phenotype commonly associated with tumorigenesis. The implications are that the tumor microenviroment contributes to tumor growth and treatment resistance.

Exploiting tyrosinase expression and activity in melanocytic tumors: quercetin and the central role of p53.

Melanoma is an aggressive tumor that expresses the pigmentation enzyme tyrosinase. Tyrosinase expression increases during tumorigenesis, which could allow for selective treatment of this tumor type by strategies that use tyrosinase activity. Approaches targeting tyrosinase would involve gene transcription or signal transduction pathways mediated by p53 in a direct or indirect manner. Two pathways are proposed for exploiting tyrosinase expression: (a) a p53-dependent pathway leading to apoptosis or arrest and (b) a reactive oxygen species-mediated induction of endoplasmic reticulum stress in p53 mutant tumors. Both strategies could use tyrosinase-mediated activation of quercetin, a dietary polyphenol that induces the expression of p53 and modulates reactive oxygen species. In addition to antitumor signaling properties, activation of quercetin could complement conventional cancer therapy by the induction of phase II detoxification enzymes resulting in p53 stabilization and transduction of its downstream targets. In conclusion, recent advances in tyrosinase enzymology, prodrug chemistry, and modern chemotherapeutics present an intriguing and selective multitherapy targeting system where dietary bioflavonoids could be used to complement conventional cancer treatments.

Quercetin abrogates chemoresistance in melanoma cells by modulating deltaNp73.

BACKGROUND: The alkylating agent dacarbazine (DTIC) has been used in the treatment of melanoma for decades, but when used as a monotherapy for cancer only moderate response rates are achieved. Recently, the clinical use of temozolomide (TMZ) has become the more commonly used analog of DTIC-related oral agents because of its greater bioavailability and ability to cross the blood brain barrier. The response rates achieved by TMZ are also unsatisfactory, so there is great interest in identifying compounds that could be used in combination therapy. We have previously demonstrated that the bioflavonoid quercetin (Qct) promoted a p53-mediated response and sensitized melanoma to DTIC. Here we demonstrate that Qct also sensitizes cells to TMZ and propose a mechanism that involves the modulation of a truncated p53 family member, deltaNp73. METHODS: DB-1 melanoma (p53 wildtype), and SK Mel 28 (p53 mutant) cell lines were treated with TMZ (400 microM) for 48 hrs followed by Qct (75 microM) for 24 hrs. Cell death was determined by Annexin V-FITC staining and immunocytochemical analysis was carried out to determine protein translocation. RESULTS: After treatment with TMZ, DB-1 cells demonstrated increased phosphorylation of ataxia telangiectasia mutated (ATM) and p53. However, the cells were resistant to TMZ-induced apoptosis and the resistance was associated with an increase in nuclear localization of deltaNp73. Qct treatment in combination with TMZ abolished drug insensitivity and caused a more than additive induction of apoptosis compared to either treatment alone. Treatment with Qct, caused redistribution of deltaNp73 into the cytoplasm and nucleus, which has been associated with increased p53 transcriptional activity. Knockdown of deltaNp73 restored PARP cleavage in the TMZ treated cells, confirming its anti-apoptotic role. The response to treatment was predominantly p53 mediated as the p53 mutant SK Mel 28 cells showed no significant enhancement of apoptosis. CONCLUSION: This study demonstrates that Qct can sensitize cells to TMZ and that the mechanisms of sensitization involve modulation of p53 family members.