In this paper we focused on Ewing

In this paper, we focused on Ewing sarcoma (ES), a rapidly growing, highly malignant bone tumor developing metastases in the vast majority of patients unless multiagent chemotherapy is applied [16]. ES is the second most frequent bone tumor in childhood and adolescence, and is characterized by the presence of EWS–ETS gene rearrangements [17]. Recently, some authors have reported that, in ES, hypoxia enhances the malignant phenotype through an upregulation of the EWS–FLI1 fusion gene by hypoxia-inducible factor (HIF)-1α [18]. HIF-1α and HIF-1β are the main mediators of the hypoxic response that favors cell proliferation through the control of the expression of numerous genes that regulate glucose uptake, metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis, and lactic carbachol production [19], [20]. Interestingly, glycogen granules accumulate in normal or cancer cells in a HIF-1-dependent manner [21], and the positive glycogen reaction to the cytochemical periodic acid-Schiff (PAS) stain is an additional feature that is currently used for the diagnosis of ES in parallel to the molecular detection of the EWS-FLI1 fusion gene [22].
Material and methods
Results
Discussion The role of mitochondria in cancer development and progression is a fundamental and complex issue [32], [33]. Since Warburg's theory [3], it is now widely accepted that tumor cells drive a metabolic shift characterized by the suppression of OXPHOS combined with the activation of glycolysis as the main pathway for ATP synthesis [34]. In this study, we verified this metabolic alteration in ES, an extremely aggressive, poorly differentiated cancer of putative mesenchymal origin. According to the bioenergetic signature (BEC index) [26], [27], and to the staining with the Mytotraker dye, we found that ES cells have a significantly higher mitochondrial activity with respect to normal fibroblasts. ES cells also showed a higher GAPDH expression, with particular regard to the SK-N-MC cells, reflecting a higher glycolytic activity. In keeping with the in vitro high glucose consumption of ES cells, the sensitivity, specificity, and accuracy of FDG-PET in this tumor have relevant impacts on treatment [35]. Using this approach, we therefore concluded that ES cells develop a compensatory response to adverse local environment that facilitates tumor progression through the simultaneous activation of mitochondrial respiration and upregulation of glycolysis. This is in contrast with the classical Warburg theory, in which the complete loss of function of mitochondria in cancer is postulated. The role of mitochondria in cancer is still controversial, however, and in many instances tumor cells have been demonstrated to carry fully functional mitochondria [36], possibly due to the ability of cancer cells to recycle these organelles [37]. In an effort to consider V-ATPase as a candidate therapeutic target for ES, we focused on the inhibitory effect of this proton pump on tumor growth. First, to directly correlate the cell number inhibition with impairment of V-ATPase expression we used a more specific targeting technique than BF-1 treatment. siRNA silencing strongly affected V0c mRNA expression and resulted in a significant reduction of the number of ES cells. We chose the V0c subunit because its inhibition was very effective to impair growth and chemoresistance also in other tumors [13], [29]. Moreover, the V0c subunit is the target of the V-ATPase inhibitor BF-1 [58] that already showed to be effective in ES cells, as described above. Secondly, we used OME which is known to block V-ATPase through the binding to the V1A subunit [59]. Among the different proton transporter/ion inhibitors, OME and other related drugs have sparked great interest in cancer [48], [60]. Two important attractive features of these agents are that they require acidic conditions to be converted into the active form, thus providing the possibility of tumor-specific selection, and that their use in long-term therapy produces only limited side effects [61]. Proton pump inhibitors have been previously used in sarcoma and bone tumor patients at low dosage, and only to prevent gastric lesions as a side effect of the treatment with conventional drugs. Therefore, proton pump inhibitors have been never described and published in sarcomas as an anti-cancer therapy. We obtained a significant reduction of the number of cells that survived under low pHe with either siRNA, or OME. Moreover, we observed a dose-dependent effect of OME treatment.