Original Contribution Radiosensitization by 2-deoxy-D-glucose and 6-aminonicotinamide involves activation of redox sensitive ASK1-JNK/p38MAPK signaling in head and neck cancer cells Pradeep Kumar Sharma a,b , Bilikere Srinivasa Dwarakanath a , Rajeev Varshney a,c,n a Institute of Nuclear Medicine and Allied Sciences, DRDO, Brig. S K Mazumdar Road, Delhi-110 054, India b University School of Biotechnology, Guru Gobind Singh Indraprastha University, sector-16 C, Dwarka, New Delhi-110075, India c 533, O/o SA to RM, Secretary Defence R&D & DG DRDO, DRDO Bhawan, Ministry of Defence, Government of India, Rajaji Marg, New Delhi-110 105, India article info Available online 21 July 2012 Keywords: 2-deoxy-D-glucose 6-aminonicotinamide Oxidative stress ASK1 Redox alterations abstract Our previous studies on simultaneous inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) and pentose phosphate activity by 6-aminonicotinamide (6-AN) have been shown to induce oxidative stress mediated selective radiosensitization in wide range of human malignant cells. However, the mechanism of radiosensitization induced by this combination (2-DG þ6-AN) is not completely under- stood. Since activation of apoptotic signal regulating kinase (ASK1) and subsequent apoptosis are implicated in oxidative stress response, the role of ASK1 activation in radiosensitization by this combination was investigated in the present study. Our results demonstrated that redox alterations induced by this combination activated ASK1 and subsequent apoptosis during radiosensitization of head and neck carcinoma cells (KB). In addition, mRNA and protein expression of thioredoxin and thioredoxin reductase decreased significantly under similar treatment conditions. Further, the down- stream targets such as JNK and p38MAPK were also activated by this combination, and their pharmacological inhibition by SP600125 and SB201291 respectively resulted in suppression of 2-DG þ6-AN mediated apoptosis in irradiated KB cells. Interestingly, the activation of ASK1 was mediated by hydrogen peroxide rather than superoxide anions as PEG-catalase but not PEG-SOD suppressed its activation. Our observations clearly suggest that redox alterations by inhibition of glucose metabolism serves as a molecular switch that activate ASK1-JNK/p38MAPK signaling in malignant cells during radiosensitization by 2-DG þ6-AN. The present study emphasizes the impor- tance of redox alterations in determining radiosensitivity of tumor cells that may greatly influence the outcome of radiation therapy. & 2012 Elsevier Inc. All rights reserved. Introduction Evasion from apoptosis is one of the hallmarks of cancer phenotype that has been suggested as one of the factors responsible for their resistance towards variety of treatments including radio- therapy and pharmacological drugs [1]. Cancer cells often exhibit an elevated level of reactive oxygen species (ROS) than their normal counterparts and therefore, maintenance of redox homeostasis has been suggested as an important mechanism of their survival [2]. Moreover, enhanced aerobic glycolysis (the Warburg effect) and pentose phosphate pathway (PPP) activity have been shown to protect cancer cells from oxidative damage by maintaining their redox homeostasis via producing more pyruvate and NADPH respectively [3–5]. Further, many lines of evidences have clearly shown that either glucose deprivation or inhibition of glucose metabolism resulted in oxidative stress-mediated activation of apoptotic signaling and subsequent cytotoxicity in malignant cells [6–11]. Therefore, it has been hypothesized that inhibition of glucose metabolism may shift the cellular redox homeostasis in favor of increasing intracellular ROS leading to oxidative stress- mediated activation of apoptotic signaling and render cancer cells more susceptible to radiation-induced cytotoxicity. Although, it is known that 2-deoxy-D-glucose (2-DG) inhibits both glycolysis and PPP, however, a great degree of heterogeneity in 2-DG-induced radiosensitization has been observed in variety Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine 0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2012.07.001 Abbreviations: 2-DG, 2-deoxy-D-Glucose; 6-AN, 6-aminonicotinamide; ROS, reactive oxygen species; PPP, pentose phosphate pathway; Trx, thioredoxin; ASK1, Apoptosis signal-regulating kinase 1; JNK, c-Jun N-terminal kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; MAPKK, mitogen- activated protein kinase kinase; DCFDA, 2’7’dichlorofluorescein diacetate; DHE, Dihydroethidium; PBS, phosphate buffered saline; NAC, N-acetyl cysteine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Redox sensor red CC-1, 2, 3, 4, 5, 6-pentafluorotetramethyldihydrorosamine) n Corresponding author at: 533, O/o SA to RM, Secretary Defence R&D & DG DRDO, DRDO Bhawan, Ministry of Defence, Government of India, Rajaji Marg, New Delhi-110 105, India. Fax: þ91 01123792628. E-mail addresses: rvarshney@rediffmail.com, rvarshney@hqr.drdo.in (R. Varshney). Free Radical Biology and Medicine 53 (2012) 1500–1513