Intracellular proton pumps as targets in chemotherapy: V-ATPases and cancer

1 Intracellular proton pumps as targets in chemotherapy: V- ATPases and cancer Agustín Hernández*, Gloria Serrano-Bueno, José R. Pérez-Castiñeira, and Aurelio Serrano Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Avda. Américo Vespucio 49, 41092 Seville, Spain. * to whom correspondence should be addressed at IBVF-CSIC, Avda. Americo Vespucio 49, 41092 Seville, Spain. Phone: +34 954 489500. Fax +34 954 460065. E-mail: [email protected] Keywords: V-ATPase, acidification, proton gradients, Bafilomycin, Concanamycin, Salicylihalamide, apoptosis, traffic. Running title: V-ATPases as targets in cancer chemotherapy. Curr Pharm Des. 2012;18(10):1383-94 DOI : 10.2174/138161212799504821 2 Abstract Cancer cells show a metabolic shift that makes them overproduce protons; this has the potential to disturb the cellular acid-base homeostasis. However, these cells show cytoplasmic alkalinisation, increased acid extrusion and endosome-dependent drug resistance. Vacuolar type ATPases (V-ATPases), toghether with other transporters, are responsible to a great extent for these symptoms. These multisubunit proton pumps are involved in the control of cytosolic pH and the generation of proton gradients (positive inside) across endocellular membrane systems like Golgi, endosomes or lysosomes. In addition, in tumours, they have been determined to play an important role in the acidification of the intercellular medium. This importance makes them an attractive target for control of tumour cells. In the present review we portray the major characteristics of this kind of proton pumps, we provide some recent insights on their in vivo regulation, an overview of the consequences that V-ATPase inhibition carries for the tumour cell, such as cell cycle arrest or cell death, and a brief summary of the studies related to cancer made recently with commercially available inhibitors for this kind of proton pump. Some new approaches to affect V-ATPase function are also suggested in the light of recent knowledge on the regulation of this proton pump. 3 In tumor cells, NADH is recycled to its oxidised form by reducing pyruvate to lactic acid, instead of being transported to and being processed at the mitochondria. As a consequence, two H+ per glucose molecule consumed are released into the cytosol and need to be disposed of in order to maintain cytosolic pH homeostasis. Strikingly, tumour cells not only maintain a near-neutral pH, but they often display a slightly alkaline cytosol in comparison to normal cells, probably as an acquired advantage against apoptosis induction [1]. The most obvious way to dispose of H+ is to extrude them to the intercellular space. This has some added advantages for the tumour cell: lactate and H+ are immunosuppresors that impair cytotoxic T lymphocyte metabolism [2]. Moreover, an acid external medium promotes invasion and cell proliferation by providing the optimal conditions for proteases like cathepsins or matrix metalloproteases that can degrade the intercellular matrix and promote angiogenesis [3] and even help the scape of highly proliferating or metastatic cells from the tumour into adjacent tissues or the blood stream [4]. All this, together with the fact that solid tumours typically display deficient perfusion, especially at the inner core, complicates cytosolic homeostasis because steep H+ gradients need to be maintained across the plasma membrane of neoplastic cells in order to keep H+ from flooding back into the cytoplasm. V-ATPases represent the sole primary H+ transporters in endosomes, Golgi cisternae and lysosomes in mammalian cells. In other organisms, such as parasitic trypanosomatids –responsible for diseases like malaria, Kala azar, sleeping sickness or chagas disease- H+-pumping pyrophosphatases comprise a second set of pumps and may be an important pharmacological target to fight these ailments [5, 6]. V-ATPases are usually absent from the cell surface, but it is naturally located at the plasma membrane of some cell types such as those at the vas deferens and in osteocytes [7, 8]. In tumour cells, its presence at the plasma membrane has been reported [9, 10]. How this location is 4 achieved by a usually intracellular protein is still obscure, but examples of other proteins of similar origin being found in the extracellular environment are known in neoplasic cells, e.g. the cathepsins [11]. It is thought that plasmalemmal V-ATPases play a significant role in proton extrusion and regulation of cytosolic pH in mammalian cells [12]. At any rate, plasma membrane V-ATPase abundance have been shown to correlate with metastatic and proliferative potential [9, 12]. ACIDIFICATION OF INTERNAL CELL COMPARTMENTS An overproduction of acid equivalents also has implications in internal organelles since many of them maintain pH values in their lumina that need to differ substantially from that of the cytoplasm. Under physiological conditions, maintenance of these proton gradients has been found to be necessary for vacuole fusion [13], glycosylation of proteins in the Golgi apparatus [14, 15], ligand-receptor dissociation and recycling in endosomes [16], endosomal proteolysis of ligands [17], and protein transport from endosomes to lysosomes [18]. In addition, drug sequestration is also dependent on the maintenance of lysosomal acidification [19]. The endo and exocytic pathways transport cargo and vesicles between different organelles (Fig. 1). As a gross simplification, it could be considered that the endoplasmic reticulum (ER) lies at one end of these pathways, while the lysosome and the plasma membrane would represent the other end. The ER maintains no pH difference compared with that of the cytosol. However, the closer we move towards the lysosome and the plasma membrane, H+ gradients (acid inside) maintained in the lumina of the different organelles become steeper (Fig. 1). 5 It must be noted that, although V-ATPases are the primary pumps driving the accumulation of protons inside single-membrane organelles, the final proton gradient formed is the result of the interaction of several other factors playing in favour or against the build up of such a gradient. For example, biological membranes are, to some extent, permeable to protons; also, secondary transporters consume accumulated protons to drive the translocation of substrates such as Na+ or amino acids. Conversely, the action of channels that dissipate the electrical gradient associated to the accumulation of positive charges (e.g. Clchannels) allow a greater accumulation of H+. In addition, resident proteins and solutes with (de)protonable residues provide buffering capacity to organelle lumina. For a detailed view on the determinants acting on these organelles, the reader may refer to more specialised reviews [20, 21]. Although an intervention on any of these factors should produce an alteration of luminal pH, many of these are difficult to target and the extent of the individual importance of some others is still unknown. Hence V-ATPase reveals itself as the best candidate for pharmacological intervention to affect luminal acidification. STRUCTURE OF V-ATPASES These H+-pumps are complex multisubunit enzymes capable of coupling the hydrolysis of ATP or, with a much lower affinity, GTP to the translocation of H+ across a membrane [22]. Its name derives from being first identified at the plant vacuolar membrane as a H+- pump different to those found in mitochondria and plasma membrane (F0F1 and P-type ATPases, respectively). All in all, the fully assembled mammalian holoenzyme displays a molecular mass nearing 900 kDa. 6 The V-ATPase was early observed to share some structural homology with the F0F1 ATPase of mitochondria and bacteria [23] and thus, much of its domain nomenclature is influenced by knowledge on the mitochondrial pump. Indeed it is considered likely that these two types of ATPases share a common ancestor that behaved as an ATP synthase, probably akin to that found in present archaea [24]. Similarly to bacterial and mitochondrial transporters, V-ATPases are composed of two different multisubunit domains: one composed of lipophylic polypeptides and the other by hydrophylic proteins. The hydrophylic domain, named V1 by similarity to the F1 domain in F0F1 ATPases, is composed by eight different polypeptides (termed always using capital letters, A to H) (Fig. 2). The stoichiometry of these polypeptides is three for A, B, E and G and one for C, D, H, and F. Polypeptides A and B form an hexamer complex alternating A’s with B’s; single components attach to this substructure and to each other mostly on the V0-proximal side. On their turn, polypeptides E and G extend their physical interaction further up to provide a bridge between the V1 domain and RAVE complexes involved in regulation of V0/V1 assembly [25]. On its turn, the V0 domain consists of a ring composed of five c subunits and a single c’’ one. In close contact with this, there are single copies of a, d and e subunits and, in the case of mammals, an accessory subunit termed Ac45. This last subunit may be specific of plasma membrane localised pumps [26]. Subunits A in the V1 domain have ATPase hydrolytic activity and, since subunits B immobilise the V1 domain through their interaction with the actin cytoskeleton [27, 28], the free energy released in these reactions is transmitted, with the help of subunitis Dfd, to the cc'' subunit ring providing torque for the rotation of the latter [29]. Noteworthy, subunits C, E, G, H, in the V1 domain, and subunit a in the V0 domain remain static 7 through its physical interaction with the A3B3 hexamer. This is important because subunit a contains two hydrophylic hemichannels. The first one feeds H+ from the external side of the membrane to the c and c’’ subunits. Protons are bound by glutamic acid residues in the core of these polypeptides and kept for a full turn with the help of the hydrophobic environment of the membrane until the loaded subunit c or c’’ reaches back subunit a and comes in contact with the second hemichannel. This second hemichannel permits the exit of the H+ from the luminal side of the membrane. Typically, V-ATPase rotors turn clockwise, as seen from the normal to the cytosolic plane of the membrane, and, in a single 360o rotation, up to two H+ are translocated per ATP consumed [29]. However, actual H+ stoichiometry depends on the isoform of subunit a that integrates the pump. Thus, in budding yeast, Stv1p isoform generates pumps that translocate a single H+ per ATP consumed, while Vph1p harbouring V-ATPases conform to the theoretical 2 H+/ATP [30]. The implications of this in V-ATPase regulation will be dealt with farther on. REGULATION OF V-ATPASE ACTIVITY Knowledge on regulation of V-ATPase activity has received a boost recently. The best characterised mechanism of its H+-pumping activity regulation is by assembly/dissasembly of the holoenzyme. Under physiological conditions, domains V0 and V1 are devoid of H+ transport or ATP hydrolytic activity, respectively, when not as part of the holoenzyme [31, 32]. This makes possible to use assembly of the pump as a comparatively simple mechanism to regulate dynamically the total H+ transport capacity on the envelope of an organelle. Domain V0 is synthesized and assembled in the ER and transported to the Golgi system via COP II coated vesicles where it will receive a V1 domain already assembled in 8 the cytosol [33]. However, this holoenzyme can disassemble and reassemble V1 and V0 domains in response to cell energy status. In yeast, it was early shown that vacuolar V- ATPases rapidly disassembled upon glucose deprivation but that it could reassemble when the sugar was added back to the growth medium [34]. This same behaviour has been observed in renal epithelial cells [35, 36] and, probably, it plays a role in other tissues, as it is suggested by the results reported on ovine rumen [37]. The first indication of a regulatory link between carbohydrate metabolism and reversible assembly of this H+-pump came from osteoclasts and kidney cells, where aldolase was identified as a an interacting partner with subunit E influencing the assembly of the holoenzyme [38]. Later it was determined that the enzymatic activity of aldolase was not required for its regulatory function [39]. Anyhow, this behaviour has been best studied in budding yeast and much of the following information comes from this model organism. Noteworthy, the mechanisms for assembly and disassembly are different: while disassembly requires an intact tubulin microtubular network, reassembly of the holoenzyme depends on RAVE (regulator of the H+-ATPase of vacuolar and endosomal membranes), a protein complex that interact with subunits E, G and C and maintains them in an assembly-competent state [40]. In yeast, reversible (re)assembly of the holoenzyme has been proposed to derive from ras/cAMP/PKA pathway activation [41]. According to this model, intracellular glucose would activate the ras pathway leading to PKA activation through an increase in cAMP [41]. How PKA could be exerting its influence on assembly is still not clear; work by Voss et al. has shown a direct phosphorylation of subunit C by PKA that could influence V1/V0 assembly [42, 43]. In line with this, PKA has already been reported to phosphorylate the catalytic subunit A and regulate V-ATPase activity in human kidney cells [44], although no data on dissociation of the complex was reported in this case. Related to this, AMPK 9 (AMP-activated protein kinase) could be exerting an inhibitory role, opposed to activation by PKA, in kidney epidydimal cells [45, 46]; however, in this report AMPK would be affecting the H+-pump distribution rather than assembly. Alternatively, at least in yeast, glucose metabolism, or to be more exact, a high glycolytic flux, would translate into an increased cytosolic pH that could be sensed by V-ATPases through yet obscure mechanisms and this would drive their reassembly; activation of PKA would result as a downstream effect of the reactivation of H+ transport [47]. This is in agreement with subunit a being proposed as a pH sensor for the holoenzyme, both in yeast and in kidney epithelial cells [48-50]. Further work is needed to clarify the involvement of PKA in reassembly. It would be interesting to ascertain if this kinase shows any effect on RAVE. Nevertheless, extracellular pH has been shown to affect the ability of yeast V-ATPases to reversibly dissociate their V1 and V0 domains [51], although in this case cytosolic pH was shown not to be altered. In addition to this, other signalling pathways may also be contributing to the regulation of V-ATPases. In proximal tubule and kidney cells, PI3- kinase has been reported to activate V-ATPase activity and assembly [35, 36, 52], and in the former type of cells, angiotensin II could be exerting an activation of the enzyme through both PI3K and p38MAPK [53]. Other protein kinases, such as analogs of the yeast stress and cell-cycle related Dbf2p [54], may prove important for V-ATPase activity and assembly in mammalian cells in the future. Regulation of gene expression is a commonly encountered mechanism for enzyme activity regulation. However, as it is easily understandable for a multisubunit complex that is composed of at least 13 different polypeptides, the concerted gene regulation of all of them is cumbersome. In agreement with this, early studies supported the notion that V-ATPases 16 including the use of bafilomycin A1 (e.g. [101]). However, in this last case a point of caution must be exerted since bafilomycin A1 and other plecomacrolides have been seen to exert conflicting effects on autophagy and apoptosis induction in neurons depending on the concentration used [102]. No data are available on on other cell types or other V-ATPase inhibitors, but still the utilisation of plecomacrolides as potentiators of stress or druginduced apoptosis through inhibition of autophagy reveals itself as one of the most attractive potential uses for V-ATPase inhibiting compounds. Many of the chemotherapeutic drugs in present use are cationic molecules. As a consequence, they tend to get excluded from alkaline cytosols and accumulate into acidic compartments like the lysosome [103]. Not surprisingly then, cells that are able to maintain greater pH gradients in these compartments show a greater drug accumulation than normal cells [104] and cells showing a greater luminal acidification prowess are more chemoresistant than those that do not [105, 106]. Conversely, inhibition of V-ATPase activity leads to chemosensitivity [107, 108]. Other important effects are those related to iron uptake and Wnt/β-catenin signalling. Using global gene expression data Straud et al. found that the increased sensitivity to V- ATPase inhibitors observed in cancer cells was correlated to their greater dependence on iron uptake, presumably to compensate their ROS production [109] . Wnt/ β-catenin pathway is an important tumourigenic signal in many cancer cell types, e.g. colorectal [110], and ovarian cancers [111]. It has recently been assessed that the Wnt receptor complex component LRP6 binds Prorenin receptor as an adaptor to bind V-ATPase. Upon activation, LRP6 gets endocytosed and phosphorylated in an acidic environment to become active. Furthermore, inhibiton of V-ATPase activity using bafilomycin A1 or apicularen prevented Wnt signalling [112]. 17 Cell cycle arrest is a common outcome of V-ATPase inhibition and often preceds cell death. S-phase arrest has been reported as a consequence of iejimalide A and B action [113], but in general, G1 arrest has been observed. Increased expression of the G1/M transition inhibitor p21 has been found in these cases [114-116]. However, the cellular mechanisms by which this occurs are not known in detail. In HT-29 colon cancer cells, p53 was stabilised following intracellular compartment alkalinization but p21 induction was partially p53 independent [114]. On the other hand, G1 arrest was observed to depend on inhibition of the degradation of hypoxia-inducible factor 1 (HIF-1) [116]. However, the actual mechanism integrating HIF-1 degradation and V-ATPase inhibition is still unknown. A hint for this may come from the fact that HIF-1 is a transcription factor that induces p21 expression under conditions of hypoxia but that it is kept at low levels under normal aeration conditions [117]. In Hela cells, antisense experiments targeting c proteolipids induced necrosis [118]. However, cell death mechanisms related to V-ATPase inhibition in other reports is exclusively apoptosis so far [113, 119-123]. Most of these studies have been performed using plecomacrolides and, therefore, since there are no data yet for archazolids and indoles, and very few in the case of benzolactone enamides, differences may be found in future studies. In any case, apoptosis seems to follow the intrinsic pathway of through mitochondrial depolarization and liberation of cytochrome c to the cytosol in most cases[107, 119, 121] albeit, in EGFR overexpressing cancer cell lines, the extrinsic pathway of apoptosis has also been reported to play a decisive role through Fas/FasL in concanamycin B-induced cell death [124]. Anyhow, V-ATPase inhibition induced apoptosis is probably caspase-dependent in all cases [107, 119, 121]. 18 PHARMACOLOGICAL INHIBITORS OF V-ATPASES There is a vivid interest in the pharmacological intervention of V-ATPases, since this may prove helpful to understand a wide range of diseases, among which is cancer, but that also includes osteopetrosis and alzheimer's disease [125, 126]. As a consequence, the list of inhibitory compounds identified along the last 30 years is long and heterogeneous. We will only review here the most important types, with preference for those commercially available, and some strategies that may lead to new approaches towards V-ATPase activity regulation. Due to their particular nature, benzimidazole proton pump inhibitors deserve a detailed review and thus they will be dealt with in another review article in this issue [Ref de Milito]. A more comprehensive list and further details on these and other compounds are available in some excellent reviews [127-131]. Plecomacrolides and Derivatives The first specific inhibitor described for V-ATPases was bafilomycin A1 [132], but concanamycin A was first reported that same year [133]. Both compounds are collectively known as plecomacrolides and consist of a large macrocyclic lactone ring comprising 16- 18 carbons. Their general structures are shown in Fig. 5. Originally isolated from several species of Streptomyces bacteria [129], the total synthesis of bafilomycin A1 was achieved in 1997 [134] and that of concanamycin (concanamycin F) in 2001 [135]. Although it is now well established that the binding site of both compounds lies on subunit c and that causes the inhibition of proton transport through this proteolipid [136], there were some 19 indications that bafilomycin A1 may also bind to subunit a [137] and these have been confirmed recently [138]. All of them are very potent inhibitors of V-ATPases with IC50 in the low nanomolar range. This makes them useful in research to distinguish between different types of ATPases in a cell, since P-type and F0F1-type ATPases are not inhibited at concentrations achieving full inhibition of V-ATPases. However, being proteolipid c one of the most conserved subunits across species and there being no isoforms in humans, the downside is that both bafilomycins and concanamycins lack specificity towards different forms of V-ATPases. Recently, an unexpected activity of bafilomycin A1 was uncovered: using mitochondria isolated from rat liver, bafilomycin A1, in a concentration range between 50 and 250 nM, was shown to transport potassium across the inner mitochondrial membrane in a manner resembling that of valinomycin, causing swelling and depolarisation independently of any inhibition of the F0F1 ATPase [139]. These concentrations are above those needed for inhibition of V-ATPases in vitro, but may be meaningful to understand the effects observed in in vivo assays. For example, this might help to understand some unresolved effects of plecomacrolides, such as the induction of the expression of hypoxia-inducible factor 1 (HSF-1) and p21 [115, 116]. Similarly, both concanamycin A and bafilomycin A1 have been shown to induce nitric oxide synthase, c-Jun N-terminal kinase and NF-B concomitant to mitochondrial swelling and depolarisation in RAW 264.7 leukemia cells [140]. Using the information obtained in structural studies of bafilomycins, a series of simpler compounds that were still able to inhibit V-ATPases were described in the late 1990's [141, 142]. These compounds consist of an indole core, hence its name “indoles” (Fig. 5). Similarly to plecomacrolides, these compounds also bind subunit c [143, 144]. However, 20 as in the case of the plecomacrolides, it was thought for some time that they may bind to subunit a, a situation that could help explaining their preferential inhibition of osteoclast V-ATPases, as opposed to other mammalian V-ATPases [141, 142, 145], but this hypothesis is now abandoned [146]. Benzolactone Enamides Compounds sharing a benzolactone enamide core and a cytotoxic profile similar to that of plecomacrolides in NCI's 60-Cell screens [147] were identified by two independent groups in the late 1990's [148-150]. The first of this, salicylihalamide, was originally isolated from Haliclona sp sponges, lobatimides were from Aplidium lobatum tunicates, while apicularens were extracted from Chondromyces sp mixobacteria. This wide range of sources is probably misleading and chances are that they are all produced by symbiotic micro-organisms, most likely mixobacteria [147]. Chemical structures representative of this class of compounds are shown on Fig. 6. Total synthesis of these compounds were successfully reported a few years later [151-153] and several compounds showing similarities have been described, such as cruentaren, the oximidines or the closely related palmerolides. An excellent review on these latter compounds was published recently [129]. Also similar to plecomacrolides, benzolactone enamides are potent inhibitors of ATP- dependent H+-translocation showing IC50s in the nanomolar range [129, 154], and bind to the V0 domain [136, 154]. However, the binding sites of salicylihalamide and lobatimide on the V-ATPase complex are probably different to that of plecomacrolides since the former cannot compete binding of the latter type of compounds [136, 154, 155]. Interestingly, benzolactone enamides have shown selectivity towards animal V-ATPases, 21 as opposed to their homologous fungal proton pumps [147, 155]. Nevertheless, no information is yet available on the exact subunit benzolactone enamides bind. Alternative Strategies The major drawback of traditional V-ATPase inhibitors is their lack of cell-type or tissue specificity. This comes from a common mechanism of action based at binding V0 domain subunits and, in most instances, specifically on the highly conserved c subunit [136, 146, 154]. Thus, their potential use in cancer chemotherapy is hindered. However, there are alternative approaches and molecules that may give some hope. The development of systemic delivery methods for gene silencing in vivo may pave the way to effective cancer treatment, and this strategy is already being tested for other targets in several types of cancer [156]. Gene silencing constructs against tumour-characteristic V-ATPase subunits, either delivered systemically or locally, could be effective at circumventing specificity issues. At any rate, silencing of single subunits in cell culture models have demonstrated its use to reduce tumour cell invasiveness in breast cancer [61]. Moreover, silencing of subunit c gene ATP6V0C inhibited dramatically the proliferation and metastatic potential of HCCLM3 xenografts in mice liver, albeit gene silencing only reduced expression to 60% of untreated controls [157]. Lipid modulation of V-ATPase activity may provide another alternative way for chemotherapeutic intervention. Cancer cells display dramatically increased rates of fatty acid synthesis [158], cholesterol uptake and synthesis [159] and dysregulated sphingolipid metabolism [160]. This makes them susceptible to pharmacological intervention and its the basis of many efforts and proposals (e.g. [161, 162]). Inhibition of any lipid synthesis is 22 inherently pleiotropic; however, from what we have seen above, both sphingolipids and sterols seem to be paramount for V-ATPase function [76-79] and own unpublished results]. Moreover, abnormal sterol-mediated inhibition of lumenal acidification may well lie at the basis of azole fungicide efficacy [77]. Therefore, administration of sphingolipid or cholesterol analogues may lead to effective inhibition of V-ATPases. Sphingolipid analogues have been tested in cancer models with cytotoxic results in brain, prostate and breast cancer. However, these studies did not evaluate the possibility of an induced V- ATPase dysfunction [160]. LDL receptor-mediated cholesterol uptake mechanisms are used to deliver sterol-conjugated compounds into the cell successfully [163]. In addition, tumour cells display a dramatic increase in LDL receptors and cholesterol uptake that can reach even 100-fold of that found in normal cells [164]. This could be used to increase the specificity of V-ATPase inhibition in cancer cells. Indeed several sterol analogues have been reported to inhibit V-ATPases in vitro, for example the well known antibiotic fusidic acid [165] and the marine sponge metabolites adociasulfates-1, -7 and -8 [166]. Nevertheless, no in vivo data are available yet. CONCLUDING REMARKS The control of cell proliferation through targeting lumenal, cytosolic and/or extracellular pH homeostasis is a strategy that is receiving increasing attention by the scientific community and may well prove helpful in the control of cancer progression if sufficient effort is put in developing appropriate small molecules. Compounds active against V- ATPases raise a great interest, as judged by the number of preclinical tests published and the fair amount of patents issued [167]. However, V-ATPase inhibitors still face a 23 tremendous challenge to be succesful: their high toxicity. In this sense, inhibition of V- ATPases in pancreas has been proposed to be responsible of increased risk of suffering induced glucose intolerance [168]. Therefore, studies on new derivatives are paramount, specially from those compounds that already show some kind of specificity. Alternatively, the search for new ways to inhibit V-ATPases, making use of different approaches (e.g. by targeting subunit a or by gene silencing) or properties of these proteins that have received little attention, like their sensitivity to the lipid environment, may be instrumental in regulating V-ATPases, and hence cell proliferation, in the future. ACKNOWLEDGEMENTS The authors wish to thank Ms I. Jiménez for her invaluable technical assistance. This work was funded by the Andalusian Regional Government and the Spanish Ministry of Science and Innovation through their support to PAIDI group BIO-261, and grants P07-CVI-3082, BFU2007-61887 and BFU2010-15622, all of them partially funded by the EU FEDER program. PAIDI group BIO-261 belongs to the CeiA3 and Andalucia TECH Campuses of International Excelence. 24 Figure Legends Fig. (1). Intracellular compartments and their internal characteristic pH values. Vesicle flux between single membrane organelles is shown by arrows. Fig. (2). Subunit structure of V-ATPases. Subunits in capital letters constitute the hydrophilic V1 domain; subunits in small letters comprise the V0 membrane-embedded domain. Asterisks denote the two hemi-channels involved in proton transport. Fig. (3). Cellular consequences of V-ATPase activity inhibition. Arrow heads indicate increase in response while blunt ends indicate diminution in the response. Fig. (4). Schematic representation of the (macro)autophagic process and autophagosome maturation. Fig. (5). Chemical structure of plecomacrolides and derivatives. A, bafilomycin A; B, concanamycin A; C, SB242784 (an indole). Fig. (6). Chemical structure of benzolactone enamides. A, salicylihalimide A; B, lobatamide A; C, apicularen A. 25 References [1] Lagadic-Gossmann, D, Huc, L, Lecureur, V. Alterations of intracellular pH homeostasis in apoptosis: origins and roles. Cell Death Differ 2004; 11(9): 953- 961. [2] Ferreira, LMR. Cancer metabolism: the Warburg effect today. 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