Search

A fine balance in reactive oxygen species (ROS) production and removal is of utmost importance for homeostasis of all cells and especially in highly proliferating cells that encounter increased ROS production due to enhanced metabolism. Consequently, increased production of these highly reactive molecules requires coupling with increased antioxidant defense production within cells. This coupling is observed in cancer cells that allocate significant energy reserves to maintain their intracellular redox balance. Glutathione (GSH), as a first line of defense, represents the most important, non-enzymatic antioxidant component together with the NADPH/NADP+ couple, which ensures the maintenance of the pool of reduced GSH. In this review, the central role of amino acids (AAs) in the maintenance of redox homeostasis in cancer, through GSH synthesis (cysteine, glutamate, and glycine), and nicotinamide adenine dinucleotide (phosphate) production (serine, and glutamine/glutamate) are illustrated. Special emphasis is placed on the importance of AA transporters known to be upregulated in cancers (such as system xc-light chain and alanine-serine-cysteine transporter 2) in the maintenance of AA homeostasis, and thus indirectly, the redox homeostasis of cancer cells. The role of the ROS varies (often described as a “two-edged sword") during the processes of carcinogenesis, metastasis, and cancer treatment. Therefore, the context-dependent role of specific AAs in the initiation, progression, and dissemination of cancer, as well as in the redox dependent sensitivity/resistance of the neoplastic cells to chemotherapy are highlighted. [ABSTRACT FROM AUTHOR]

Cancer cells are optimised for growth and survival via an ability to outcompete normal cells in their microenvironment. Many of these advantageous cellular adaptations are promoted by the pathophysiological hypoxia that arises in solid tumours due to incomplete vascularisation. Tumour cells are thus faced with the challenge of an increased need for nutrients to support the drive for proliferation in the face of a diminished extracellular supply. Among the many modifications occurring in tumour cells, hypoxia inducible factors (HIFs) act as essential drivers of key pro-survival pathways via the promotion of numerous membrane and cytosolic proteins. Here we focus our attention on two areas: the role of amino acid uptake and the handling of metabolic acid (CO2/H+) production. We provide evidence for a number of hypoxia-induced proteins that promote cellular anabolism and regulation of metabolic acid-base levels in tumour cells including amino-acid transporters (LAT1), monocarboxylate transporters, and acid-base regulating carbonic anhydrases (CAs) and bicarbonate transporters (NBCs). Emphasis is placed on current work manipulating multiple CA isoforms and NBCs, which is at an interesting crossroads of gas physiology as they are regulated by hypoxia to contribute to the cellular handling of CO2 and pHi regulation. Our research combined with others indicates that targeting of HIF-regulated membrane proteins in tumour cells will provide promising future anti-cancer therapeutic approaches and we suggest strategies that could be potentially used to enhance these tactics. [ABSTRACT FROM AUTHOR]

The hypoxic and acidic tumor environment necessitates intracellular pH (pHi) regulation for tumor progression. Carbonic anhydrase IX (CA IX; hypoxia-induced) is known to facilitate CO2 export and generate HCO3− in the extracellular tumor space. It has been proposed that HCO3− is re-captured by the cell to maintain an alkaline pHi. A diverse range of HCO3− transporters, coupled with a lack of a clear over-expression in cancers have limited molecular identification of this cellular process. Here, we report that hypoxia induces the Na+/HCO3− co-transporter (NBCe1) SLC4A4 mRNA expression exclusively in the LS174 colon adenocarcinoma cell line in a HIF1α dependent manner. HCO3− dependent pHi recovery observations revealed the predominant use of an NBC mechanism suggesting that reversal of a Cl−/HCO3− exchanger is not utilized for tumor cell pHi regulation. Knockdown of SLC4A4 via shRNA reduced cell proliferation and increased mortality during external acidosis and spheroid growth. pHi recovery from acidosis was partially reduced with knockdown of SLC4A4. In MDA-MB-231 breast cancer cells expressing high levels of SLC4A4 compared to LS174 cells, SLC4A4 knockdown had a strong impact on cell proliferation, migration, and invasion. SLC4A4 knockdown also altered expression of other proteins including CA IX. Furthermore the Na+/HCO3− dependent pHi recovery from acidosis was reduced with SLC4A4 knockdown in MDA-MB-231 cells. Combined our results indicate that SLC4A4 contributes to the HCO3− transport and tumor cell phenotype. This study complements the on-going molecular characterization of the HCO3− re-uptake mechanism in other tumor cells for future strategies targeting these potentially important drug targets. J. Cell. Physiol. 230: 1954-1963, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The efficacy of targeting pH disruption to induce cell death in the acidic and hypoxic tumor microenvironment continues to be assessed. Here we analyzed the impact of varying levels of hypoxia in acidic conditions on fibroblast and tumor cell survival. Across all cell lines tested, hypoxia (1% O2) provided protection against acidosis induced cell death compared to normoxia. Meanwhile severe hypoxia (0.1% O2) removed this protection and in some cases exacerbated acidosis-induced cell death. Differential survival between cell types during external acidosis correlated with their respective intracellular pH regulating capabilities. Cellular ATP measurements were conducted to determine their contribution to cell survival under these combined stresses. In general, hypoxia (1% O2) maintained elevated ATP levels in acidic conditions while severe hypoxia did not. To further explore this interaction we combined acidosis with ATP depletion using 2-deoxyglucose and observed an enhanced rate of cell mortality. Striking results were also observed with hypoxia providing protection against cell death in spite of a severe metabolic stress induced by a combination of acidosis and oligomycin. Finally, we demonstrated that both HIF1α and HIF2α expression were drastically reduced in hypoxic and acidic conditions indicating a sensitivity of this protein to cellular pH conditions. This knockdown of HIF expression by acidosis has implications for the development of therapies targeting the disruption of cellular pH regulation. Our results reinforce the proof of concept that acidosis and metabolic disruption affecting ATP levels could be exploited as a tumor cell killing strategy. J. Cell. Physiol. 228: 1854-1862, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Intense interest in the 'Warburg effect' has been revived by the discovery that hypoxia-inducible factor 1 (HIF1) reprogrammes pyruvate oxidation to lactic acid conversion; lactic acid is the end product of fermentative glycolysis. The most aggressive and invasive cancers, which are often hypoxic, rely on exacerbated glycolysis to meet the increased demand for ATP and biosynthetic precursors and also rely on robust pH-regulating systems to combat the excessive generation of lactic and carbonic acids. In this Review, we present the key pH-regulating systems and synthesize recent advances in strategies that combine the disruption of pH control with bioenergetic mechanisms. We discuss the possibility of exploiting, in rapidly growing tumours, acute cell death by 'metabolic catastrophe'. [ABSTRACT FROM AUTHOR]

The relationship between acidosis with in the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (pHi)-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX) and LS174Tr cells (inducible knock-down for ca9/ca12) were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pHo manipulations and hypoxia (1% O2) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pHi-regulating capacity of fibroblasts through inhibition of Na+/H+ exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pHiregulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pHi regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pHi-regulating CAs as an anti-cancer strategy. [ABSTRACT FROM AUTHOR]

A distinguishing phenotype of solid tumors is the presence of an alkaline cellular feature despite the surrounding acidic microenvironment. This phenotypic characteristic of tumors, originally described by Otto Warburg, arises due to alterations in metabolism of solid tumors. Hypoxic regions of solid tumors develop due to poor vascularization and in turn regulate the expression of numerous genes via the transcription factor HIF-1. Ultimately, the tumor microenvironment directs the development of tumor cells adapted to survive in an acidic surrounding where normal cells perish. The provision of unique pH characteristics in tumor cells provides a defining trait that has led to the pursuit of treatments that target metabolism, hypoxia, and pH-related mechanisms to selectively kill cancer cells. Numerous studies over the past decade involving the cancer-specific carbonic anhydrase IX have re-kindled an interest in pH disruption-based therapies. Although an acidification of the intracellular compartment is established as a means to induce normal cell death, the defining role of acid-base disturbances in tumor physiology and survival remains unclear. The aim of this review is to summarize recent data relating to the specific role of pH regulation in tumor cell survival. We focus on membrane transport and enzyme studies in an attempt to elucidate their respective functions regarding tumor cell pH regulation. These data are discussed in the context of future directions for the field of tumor cell acid-base-related research. J. Cell. Physiol. 226: 299-308, 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The anterior midgut of the larval yellow fever mosquito Aedes aegypti generates a luminal pH in excess of 10 in vivo and similar values are attained by isolated and perfused anterior midgut segments after stimulation with submicromolar serotonin. In the present study we investigated the mechanisms of strong luminal alkalinization using the intracellular fluorescent indicator BCECF-AM. Following stimulation with serotonin, we observed that intracellular pH (pHi) of the anterior midgut increased from a mean of 6.89 to a mean of 7.62, whereas pHi of the posterior midgut did not change in response to serotonin. Moreover, a further increase of pHi to 8.58 occurred when the pH of the luminal perfusate was raised to an in vivo-like value of 10.0. Luminal Zn2+ (10μmol l-1), an inhibitor of conductive proton pathways, did not inhibit the increase in pHi, the transepithelial voltage, or the capacity of the isolated tissue to alkalinize the lumen. Finally, the transapical voltage did not significantly respond to luminal pH changes induced either by perfusion with pH 10 or by stopping the luminal perfusion with unbuffered solution which results in spontaneous luminal alkalinization. Together, these results seem to rule out the involvement of conductive pathways for proton absorption across the apical membrane and suggest that a serotonin-induced alkaline pHi plays an important role in the generation of an alkaline lumen. [ABSTRACT FROM AUTHOR]

Posterior isolated gills of Neohelice (Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3-] and pH. Elevating [HCO3-] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference (Vte), a measure of ion transport. The elevation in [HCO3-] also induced a switch from acid secretion (-43.7 ± 22.5 µequivµkg-1µh-1) in controls to base secretion (84.7 ± 14.4 µequivµkg-1µh-1). The HCO3--induced Vte increase was inhibited by basolateral acetazolamide (200 µmol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vte response to HCO3- did not take place in Cl--free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in Vte, which was matched by a net increase in acid secretion of 67.8 ± 18.4 µequiv kg-1 h-1. This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and Na+-free conditions, but it still took place in Cl--free saline. Therefore, the cellular response to low pH is different from the HCO3--stimulated response. We also report V-H+-ATPase- and Na+-K+-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral Na+/H+ exchangers and Na+-K+-ATPase and apical anion exchangers participate in the HCO3--stimulated response, while CA, apical V-H+-ATPase and basolateral HCO3--dependent cotransporters mediate the response to low pH. [ABSTRACT FROM AUTHOR]

We investigated the involvement of carbonic anhydrase (CA) in mediating V-H+-ATPase translocation into the basolateral membrane in gills of alkalotic Squalus acanthias. Immunolabeling revealed that CA is localized in the same cells as V-H+-ATPase. Blood plasma from dogfish injected with acetazolamide [30 mg/kg at time (t) = 0 and 6 h] and infused with NaHCO3 for 12 h (1,000 +µeq·kg-1·h-1) had significantly higher plasma HCO+ concentration than fish that were infused with NaHCO3 alone (28.72 ± 0.41 vs. 6.57 ± 2.47 mmol/l, n = 3), whereas blood pH was similar in both treatments (8.03 ± 0.11 vs. 8.04 ± 0.11 pH units at r = 12 h). CA inhibition impaired V-H+-ATPase translocation into the basolateral membrane, as estimated from immunolabeled gill sections and Western blotting on gill cell membranes (0.24 ± 0.08 vs. 1.00 ± 0.28 arbitrary units, n = 3; P < 0.05). We investigated V-H+-ATPase translocation during a postfeeding alkalosis ("alkaline tide"). Gill samples were taken 24-26 h after dogfish were fed to satiety in a natural-like feeding regime. Immunolabeled gill sections revealed that V-H+-ATPase translocated to the basolateral membrane in the postfed fish. Confirming this result, V-H+-ATPase abundance was twofold higher in gill cell membranes of the postfed fish than in fasted fish (n = 4-5; P < 0.05). These results indicate that 1) intracellular H+ or HCO+ produced by CA (and not blood pH or HCO3-) is likely the stimulus that triggers the V-H+-ATPase translocation into the basolateral membrane in alkalotic fish and 2) V-H+-ATPase translocation is important for enhanced HCO3- secretion during a naturally occurring postfeeding alkalosis. [ABSTRACT FROM AUTHOR]

lsolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pHi) imaging with the pH-sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover pHi from an NH4CI-induced acidification in the absence of Na+ An apparent link between resting pHi and Na+-independent pHi recovery was made. We observed a unique pHi acidification event that was induced by extracellular Na+ addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the Na+-induced pH acidification, whereas the minority (23%) demonstrated an alkalinization of pHi under the same circumstances. The focus of this study was placed on the Na+-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that Na+ uptake was responsible for the intracellular acidification. Further experiments revealed that pHi acidification could be abolished when Na+ was allowed entry into the cell, but the activity of an electrogenic Na+-HCO-3 cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, Na+-induced membrane potential depolarization as observed via imaging of the voltage-sensitive dye bis-oxonol. We also demonstrated NBC immunoreactivity via Western blotting and immunohistochemistry in gill tissue. We propose a model for transepithelial Na+ uptake occurring via an apical Na+ channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells. [ABSTRACT FROM AUTHOR]

We have previously shown that continuous intravenous infusion of NaHCO3 for 24 h (∼1000 μmol kg-1 h-1) results in the relocation of V-H+-ATPase from the cytoplasm to the basolateral membrane in the gills of the Pacific dogfish. To further investigate this putative base-secretive process we performed similar experiments with the addition of colchicine, an inhibitor of cytoskeleton-dependent cellular trafficking processes. Blood pH and plasma total CO2 were significantly higher in the colchicines-treated, HCO3--infused fish compared with fish infused with HCO3- alone. The effect of colchicine was highest after 24 h of infusion (8.33±0.06 vs 8.02±0.03 pH units, 15.72±3.29 vs 6.74±1.34 mmol CO2 l-1, N=5). Immunohistochemistry and western blotting confirmed that colchicine blocked the transit of V-H+-ATPase to the basolateral membrane. Furthermore, western blotting analyses from whole gill and cell membrane samples suggest that the short-term (6 h) response to alkaline stress consists of relocation of V-H+-ATPases already present in the cell to the basolateral membrane, while in the longer term (24 h) there is both relocation of pre-existent enzyme and upregulation in the synthesis of new units. Our results strongly suggest that cellular relocation of V-H+-ATPase is necessary for enhanced HCO3- secretion across the gills of the Pacific dogfish. [ABSTRACT FROM AUTHOR]

The mechanistic target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. Dysregulation in the mTORC1 network underlies a wide array of pathological states, including metabolic diseases, neurological disorders, and cancer. Tumor cells are characterized by uncontrolled growth and proliferation due to a reduced dependency on exogenous growth factors. The genetic events underlying this property, such as mutations in the PI3K-Akt and Ras-Erk signaling networks, lead to constitutive activation of mTORC1 in nearly all human cancer lineages. Aberrant activation of mTORC1 has been shown to play a key role for both anabolic tumor growth and resistance to targeted therapeutics. While displaying a growth factor-independent mTORC1 activity and proliferation, tumors cells remain dependent on exogenous nutrients such as amino acids (AAs). AAs are an essential class of nutrients that are obligatory for the survival of any cell. Known as the building blocks of proteins, AAs also act as essential metabolites for numerous biosynthetic processes such as fatty acids, membrane lipids and nucleotides synthesis, as well as for maintaining redox homeostasis. In most tumor types, mTORC1 activity is particularly sensitive to intracellular AA levels. This dependency, therefore, creates a targetable vulnerability point as cancer cells become dependent on AA transporters to sustain their homeostasis. The following review will discuss the role of AA transporters for mTORC1 signaling in cancer cells and their potential as therapeutic drug targets. [ABSTRACT FROM AUTHOR]

The evolution of life from extreme hypoxic environments to an oxygen-rich atmosphere has progressively selected for successful metabolic, enzymatic and bioenergetic networks through which a myriad of organisms survive the most extreme environmental conditions. From the two lethal environments anoxia/high O 2 , cells have developed survival strategies through expression of the transcriptional factors ATF4, HIF1 and NRF2. Cancer cells largely exploit these factors to thrive and resist therapies. In this review, we report and discuss the potential therapeutic benefit of disrupting the major Myc/Hypoxia-induced metabolic pathway, also known as fermentative glycolysis or “Warburg effect”, in aggressive cancer cell lines. With three examples of genetic disruption of this pathway: glucose-6-phosphate isomerase (GPI), lactate dehydrogenases (LDHA and B) and lactic acid transporters (MCT1, MCT4), we illuminate how cancer cells exploit metabolic plasticity to survive the metabolic and energetic blockade or arrest their growth. In this context of NRF2 contribution to OXPHOS re-activation we will show and discuss how, by disruption of the cystine transporter xCT (SLC7A11), we can exploit the acute lethal phospholipid peroxidation pathway to induce cancer cell death by ‘ferroptosis’. [ABSTRACT FROM AUTHOR]