br Grant support This work was supported by

Grant support This work was supported by INSERM, the “Ligue Contre le Cancer” (Equipe Labellisée LIGUE 2012; grant EL2012NCC/DH), the “Fédération Nationale Enfants et Santé” and the “Société Française de lutte contre les Cancers et les leucémies de l\'Enfant et de l\'Adolescent(“appel à projet 2013”).
Acknowledgments
Introduction Osteosarcoma is the most frequent primary bone malignancy, mainly attacks adolescents [1]. The combined chemotherapy with intensive dose has greatly improved the overall survival for osteosarcoma patients to over 70% [2]. However, the prognosis remains poor for those with metastasis, or for those who relapse, and survival rates only reach 20–30% [3,4]. Doxorubicin, methotrexate and Cisplatin are commonly used as anticancer drugs in osteosarcoma [5] for the last 20 years, and there has been no improvement in the survival of those osteosarcoma patients, who acquisite the drug-resistant phenotype. Thus, it is urgent to recognize the drug-resistance mechanism of osteosarcoma and to provide novel therapeutic options for this disease. Hypoxia-induced drug resistance has been confirmed for a variety of anti-tumor agents in various types of tumors [8,6,7], and even in osteosarcoma [9]. Such hypoxia markers as hypoxia-inducible factor-1 (HIF-1), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CA IX) are detectable in osteosarcomas [10], and correlate with poor progress of osteosarcoma patients, suggesting the important role of hypoxia in the survival of osteosarcoma INCB 018424 [11,12], and implying the potential importance of hypoxia as a target to antagonize drug resistance in osteosarcoma. However, it is indicated that the drug resistance in osteosarcoma is independent on the upregulated HIF-1α, suggesting other hypoxia-related signaling may be more relevant in the drug resistance to osteosarcoma. Multiple other signaling pathways are deregulated in hypoxia and may exert regulatory roles in the hypoxia-induced drug resistance. Wild-type p53 is confirmed to be inactivated in some tumor cells by hypoxia [14,13]. c-jun, activator protein-1 (AP-1), Phosphoinositol-3-kinase (PI3K) pathway and nuclear factor kappa-B (NF-κB) have also been indicated to involve in the hypoxia-induced drug resistance, mainly by inhibiting the drug-induced apoptosis [16,15,17]. And the target inhibiting of these signaling pathways sensitizes cells to cytotoxic agents under the condition of hypoxia, implying these markers as possible targets to counteract the hypoxia-induced drug resistance. AMP-activated protein kinase (AMPK) is the most important sensor of cellular energy [19,18], and is also activated by hypoxia as a compensatory response to the reduced mitochondrial respiration [20]. The heterotrimeric AMPK composes of two regulatory subunits and one catalytic subunit, which is activated the increased AMP/ATP ratio [21]. Then the activity was promoted of upstream kinases and phosphatases that control AMPK phosphorylation and dephosphorylation at the Thr-172 [22,23], particularly by the tumor suppressor LKB1, which phosphorylates the catalytic subunit of AMPK in an AMP-dependent manner [24,25]. AMPK is also activated by the Ca2 /calmodulin-dependent protein kinase kinase (CaMKK) [27,26], independently of cellular AMP levels. In addition, the activation of AMPK can also be induced in response to oxidant stress [28,29] with or without hypoxia. However, the role of the AMPK activation in hypoxia in osteosarcoma is still unclarified.
Materials and methods
Results
Discussion The role of hypoxia in the drug sensitivity has not been universally concluded, varying according to the type of tumor and the drug used [35,36]. It was observed to be resistant in rhabdomyosarcoma, Ewing’s sarcoma and neuroblastoma [36,37]. Evidence exists of the importance of hypoxia in osteosarcoma [39,38]. In particular, the patients with a moderate or strong expression of HIF-1α showed significantly shorter overall survival (OS) and disease-free survival (DFS), compared with HIF-1α negative/weak expression [40]. And recently, a significant hypoxia-induced drug resistance in osteosarcoma cells has been confirmed in intro[9], highlighting the potential importance of hypoxia in the drug resistance in osteosarcoma. However, though the HIF-1α was also found to be upregulated by hypoxia, it was irrelevant to such drug resistance.