Media was then aspirated from the remaining plates and replaced with fresh media containing fenretinide (0.54.0 M). of GSH appeared to be most dependent onde novobiosynthesis rather than recycling. RNAi-mediated knockdown of important GSH regulatory enzymes -glutamylcysteine synthetase or glutathione disulfide reductase partially reversed the hypoxia-induced resistance to fenretinide, and increasing GSH levels usingN-acetylcysteine augmented the hypoxia-induced resistance in a cell line-specific manner. These observations are consistent with the conclusion that this role of the GSH antioxidant system in modulating the sensitivity of ESFT cells to fenretinide is usually heterogeneous depending on environment and cell type. This is likely to limit the value of targeting GSH as a therapeutic strategy to overcome hypoxia-induced drug resistance in ESFT. Whether targeting the GSH antioxidant system in conjunction with other therapeutics may benefit some patients with ESFT remains to be seen. == Introduction == Glutathione (-glutamylcysteinylglycine, GSH) is the most abundant non-protein thiol in mammalian cells that functions both as a homeostatic redox buffer and in cellular defense against free radicals and reactive electrophiles[1],[2]. In cancer cells, GSH has a dual role both in preventing the initiation of cancer and promoting the progression of disease through GSH-dependent drug resistance mechanisms[2],[3],[4]. Consequently, anticancer strategies targeting the GSH redox system such asredox chemotherapyhave been developed to modulate the intracellular redox environment with the expectation this would sensitize cancer cells to therapy[5]leading to improved therapeutic response and end result. Although redox chemotherapy is an emerging anticancer strategy, it has not been as successful in the clinic as preclinical studies indicated it might[6],[7]. We propose that this may reflect the presence of hypoxic regions in some tumours that confer resistance to apoptosis and increase the metastatic potential of malignant cells as previously reported[8],[9]. Hypoxia-induced drug resistance mechanisms have been widely explained[10],[11],[12], but the molecular mechanisms are not fully resolved. In this study we have hypothesised that hypoxia may cause up-regulation of the GSH antioxidant system in ESFT thereby attenuating the efficacy of some therapeutics. Three important enzymes are involved in the regulation of cellular GSH at the level of biosynthesis (-glutamylcysteine synthetase, GCS), regeneration/recycling (glutathione disulfide reductase, GRD) and breakdown (-glutamyltranspeptidase, GGT). GCS is the initiating and rate-limiting enzyme in the biosynthesis of GSH within cells, whereas GRD and GGT are involved in the regeneration and breakdown respectively of oxidized GSH (glutathione disulfide, GSSG). GCS is the target enzyme for inhibition by the GSH-depleting agentL-buthionine S, R-sulfoximine Rabbit polyclonal to HIRIP3 (BSO)[13]which has been exploited to sensitize some cancer Cyclosporin D cells to cytotoxic drugs such as melphalan and cisplatin[14],[15]. However GGT might Cyclosporin D also be a useful therapeutic target because it is usually up-regulated in many aggressive cancers where it is important in the maintenance of cellular GSH redox homeostasis[16]. Ewing’s sarcoma is the second most common bone malignancy found in children and young adults[17], and belongs to the Ewing’s sarcoma family of tumours (ESFT). ESFT are characterised by non-random gene rearrangements between theEWSgene on chromosome 22q12 and anETSgene family member; thet(11;22)(q24;q12) chromosome rearrangement that produces the chimeric EWS/Fli1 fusion gene is the most frequently described. These pathognomonic gene fusions are major drivers of the development and maintenance of the ESFT malignant phenotype[17],[18]. Current treatment for patients with metastatic ESFT includes a combination of surgery, radiotherapy and chemotherapy, incorporating doxorubicin, vincristine, cyclophosphamide, and etoposide[19]. Despite this intensive treatment regime, the 5 12 months survival rate for patients with metastatic disease at diagnosis is usually less than 25%[20], prompting the need for Cyclosporin D new treatment methods for this cancer. Fenretinide (N-(4-hydroxyphenyl)-retinamide) is a synthetic retinoic acid derivative that has documented cytotoxicity in a wide variety of malignant cell types[21],[22],[23], including ESFT[10],[24],[25]. Fenretinide induces cell death in malignant cells through overproduction of ROS that is thought to activate the intrinsic cell death cascade[22],[24]. This mechanism of action and minimal toxicity in both adults[26],[27]and children[28],[29]makes fenretinide a stylish candidate for the potential treatment of ESFT. Previous studies from our laboratory have exhibited that fenretinide kills ESFT cellsin vitroat concentrations achievablein vivo[24],[25]. Furthermore we have Cyclosporin D identified the cellular antioxidant GSH as a determinant of the sensitivity of ESFT cells to fenretinide in normoxia[25]. Whether GSH plays a similar role in hypoxia-induced drug resistance was the major question for this study. We therefore investigated the effect of hypoxia on GSH and its regulatory enzymes and the response of ESFT cells to selected therapeutics including fenretinide in normoxia and hypoxia. The results presented here demonstrate that this involvement of GSH in hypoxia-induced drug resistance in ESFT is usually cell line-specific, and might therefore be tumour-specific. These observations suggest that optimal patient benefit from redox-based therapies targeting the GSH.
