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  • Interestingly several ES cell lines


    Interestingly, several ES cell lines are null for p53, possibly because the EWS-FLI1 chimeric protein can directly and indirectly interfere with p53 activity [38], [39]. The inhibition, loss and/or mutation of p53, one of the most common events in tumorigenesis, results in a decreased oxygen consumption and increased glycolysis [40], [41], also by enhancing HIF-1α levels [42]. As a confirmation, Knowles et al. demonstrated that ES cell lines express HIF-1α [43]. In turn, HIF-1α may induce EWS-FLI1 expression in a time-dependent manner [18], [19]. Notably, A-673 and SK-N-MC show higher levels of HIF1α activation when compared to RD-ES and SK-ES-1 cell lines [43]. An excessive glycolytic activity in tumors can also be a consequence of microenvironmental alterations. In fact, cancer cells are induced to produce large amounts of metabolic TAPI-1 generated by glycolysis, glucose utilization, and lactate production. To prevent acidity-induced apoptosis, tumor cells are forced to enhance proton efflux, leading to a significant decrease of pH in the extracellular environment [6], [7], [8], [9]. On the other side, both human and animal tumor cells have a slightly alkaline pHi (pH7.1-7.2) [44], [45], suggesting that, despite lower than normal levels of pHe, cancer cells are capable to effectively reduce the excess of protons in the cytoplasm by active transport across the plasma membrane and storage within the lysosomal compartment. Lysosomes and lysosome-related organelles constitute a system of acidic compartments that interconnect the inside of the cell with the extracellular space via secretory and endocytic pathways. We verified TAPI-1 the extension of the lysosomal compartment and activity in ES cells, and compared ES cells with hFG. In this paper, we used a primary culture of human fibroblasts as a control, also with the aim to demonstrate the selectiveness and effectiveness of the suggested targeting in the ES cells with respect to normal cells. Lysosomes and lysosome-related organelles were rather scanty in all ES cell lines, in sharp contrast to hFG. The observed presence of autolysosomes in ES cells further supports the hypothesis of a survival mechanism under acidic stress, as already demonstrated in other cancers [46]. In fact, even if ES cells have only a few lysosomes, the cytotoxic effect of autophagy inhibitor bafilomycin A1 is very high, especially under acidic conditions. Of note, BF-1 treatment was completely ineffective in normal human cells, also at pH6.5. Although the level of specificity is still controversial, the main target of BF-1 is the proton pump V-ATPase. Thus, we wondered if V-ATPase could represent an effective target for the treatment of ES. V-ATPase is the major electrogenic pump of vacuolar membranes, and is crucial to establish and maintain intracellular pH gradients across specialized organelle membranes, including the trans-Golgi network, secretory granules, endosomes, and lysosomes [47]. V-ATPase has recently gained great attention in cancer, especially with regard to its role in microenvironment acidification and in metastasis [10], [47]. Interestingly, among the many mechanisms that regulate the tumor microenvironment, V-ATPases are especially valuable because they can be blocked by proton pump inhibitors [48], [49]. First, we looked at the V-ATPase expression and localization. V-ATPase was constantly expressed in ES cells and at the highest level both for V0c and V1B2 subunits in SK-N-MC, the ES cell line with the highest GAPDH expression and with the highest susceptibility to the BF-1 treatment. Notably, the signal for V-ATPase in ES cells was localized inside the cell and on the cell membrane. The membrane expression of V-ATPase is possibly due to improper sorting of normal cellular components and was proven to be functional also in other tumors [10]. Immunohistochemical analysis in breast cancer cells has revealed a higher plasmalemmal V-ATPase in highly metastatic cells compared to low metastatic cells [50], [51]. Accordingly, our data suggest that V-ATPase on the membrane of ES cells is an additional factor that possibly contributes to the formation of metastases [52]. V-ATPase is usually recycled from the plasma membrane for the formation of endosomes that fuse with vesicles with hydrolytic enzymes derived from the Golgi apparatus to generate new lysosomes. Accordingly, in non permeabilized cells, we also observed the presence of V-ATPase localization in clusters closed to the plasma membrane and possibly associated with the invagination of the membrane to form endosomes. In agreement with results obtained with the level of expression of V-ATPase, we found the highest acidification rate in SK-N-MC cells. Based on our findings, glycolytic ES cells have a strong proton secretion activity that contribute to form an acidic microenvironment and that is directly related to V-ATPase expression. The low pHe has been repeatedly implicated as playing a key role both in cell transformation and in the active progression and maintenance of the neoplastic process [48]. We then verified the consequence of the acidification activity exerted by the V-ATPase activity in ES cells. Adachi and Tannock induced intratumoral acidification in an in vivo cancer model by using the vasodilating drug captopril, and simultaneously obtained a significant delay in tumor growth [53]. Consistent with these in vivo data, we found that under acidic (6.5) in vitro pH conditions, the number of ES cells is significantly decreased. However, the number of apoptotic cell is not affected whereas the percentage of cells in G0–G1 phase is increased. Thus, the reduction in cell number is a consequence of a delay of the cell cycle rather than of a cytotoxic effect of the acidic pHe. On the other hand, the ability to degrade gelatin and bone collagen and the clonogenic efficiency is significantly increased. An increased invasion ability under acidic conditions is a well-known feature of tumor cells that is due to an increase of matrix metalloproteinase [48], [54]. In ES, the observed collagen and gelatin degradation is possibly a consequence of the secretion of MMP-9 [55], [56], [57]. In this tumor, the increased invasive capacity and the enhanced cloning efficiency has been previously demonstrated also under hypoxia [18], which, however, directly concurs to the acidification of tumor microenvironment. Consequently, in ES cells the ability to survive and to invade the surrounding tissues under acidic conditions is increased, possibly through the activation of V-ATPase.