Intracellular Acidification Research Articles

Abstract 4987: ASS1 downregulation facilitates cancer cell survival in acidic environment

Abstract ”pH gradient reversal”, namely the alkalinization of intracellular pH (pHi) and acidification of extracellular pH (pHe), is a unique phenotype of cancer cells which enable cancer survival, migration and invasion. Hence, not surprisingly, pH gradient reversal is emerging as a new hallmark of cancer. Argininosuccinate synthase, ASS1, is a urea cycle enzyme found to be downregulated by methylation in multiple different types of cancers in correlation with increased metastasis and poor prognosis. We recently showed that ASS1 downregulation increases proliferation by channeling aspartate towards pyrimidine synthesis by CAD. We now show that under hypoxia and acidic environments, ASS1 is downregulated together with CAD by HIF1α as part of an essential network necessary for cancer cells’ survival, invasion and migration. Similar to germline deficiency of ASS1 which increases the levels of glutamine and ammonia, ASS1 downregulation by HIF1αenables the cancer cells to increase glutamine levels allowing its utilization for glutathione and importantly, for preservation of alkali pHi by elevating ammonia levels and buffering the acidity caused by cancers’ enhanced glycolysis and the consequent increase in lactic acid. Moreover, cancer cells which express ASS1 when grown in culture, downregulate its expression in hypoxic regions when grown in vivo, suggesting that these cancers might also be sensitive to drugs previously utilized only in ASS1 deficient cancers. Finally, we show that treating cancers with ASS1 deficiency with ammonia scavengers as given to ASS1 deficient patients, increases apoptosis and restricts migration, and is hence beneficial therapeutically. In summary, ASS1 downregulation is regulated differently at altered carcinogenic states, promoting proliferation as well as cancer survival and metastasis. Citation Format: Ayelet Erez, Alon Silberman, Adi Jacob, Shiran Limanovich, Odeya Boukobza1, Erez Persi, Rom Keshet, Alona Sarver, Joo Sang Lee, Alexander Brandis, Keren Bahar Halpern, Raya Eilam, Ben Pode, Yair Anikster, Igor Ulitsky, Sandesh Nagamani, Eytan Ruppin. ASS1 downregulation facilitates cancer cell survival in acidic environment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4987. doi:10.1158/1538-7445.AM2017-4987

Thermodynamics in Gliomas: Interactions between the Canonical WNT/Beta-Catenin Pathway and PPAR Gamma.

Gliomas cells are the site of numerous metabolic and thermodynamics abnormalities with an increasing entropy rate which is characteristic of irreversible processes driven by changes in Gibbs energy, heat production, intracellular acidity, membrane potential gradient, and ionic conductance. We focus our review on the opposing interactions observed in glioma between the canonical WNT/beta-catenin pathway and PPAR gamma and their metabolic and thermodynamic implications. In gliomas, WNT/beta-catenin pathway is upregulated while PPAR gamma is downregulated. Upregulation of WNT/beta-catenin signaling induces changes in key metabolic enzyme that modify their thermodynamics behavior. This leads to activation pyruvate dehydrogenase kinase 1(PDK-1) and monocarboxylate lactate transporter 1 (MCT-1). Consequently, phosphorylation of PDK-1 inhibits pyruvate dehydrogenase complex (PDH). Thus, a large part of pyruvate cannot be converted into acetyl-CoA in mitochondria and in TCA (tricarboxylic acid) cycle. This leads to aerobic glycolysis despite the availability of oxygen, named Warburg effect. Cytoplasmic pyruvate is, in major part, converted into lactate. The WNT/beta-catenin pathway induces also the transcription of genes involved in cell proliferation, cell invasiveness, nucleotide synthesis, tumor growth, and angiogenesis, such as c-Myc, cyclin D1, PDK. In addition, in gliomas cells, PPAR gamma is downregulated, leading to a decrease in insulin sensitivity and an increase in neuroinflammation. Moreover, PPAR gamma contributes to regulate some key circadian genes. Abnormalities in the regulation of circadian rhythms and dysregulation in circadian clock genes are observed in gliomas. Circadian rhythms are dissipative structures, which play a key role in far-from-equilibrium thermodynamics through their interactions with WNT/beta-catenin pathway and PPAR gamma. In gliomas, metabolism, thermodynamics, and circadian rhythms are tightly interrelated.

Pre-clinical pharmacology of AZD3965, a selective inhibitor of MCT1: DLBCL, NHL and Burkitt's lymphoma anti-tumor activity.

Tumors frequently display a glycolytic phenotype with increased flux through glycolysis and concomitant synthesis of lactate. To maintain glycolytic flux and prevent intracellular acidification, tumors efflux lactate via lactate transporters (MCT1-4). Inhibitors of lactate transport have the potential to inhibit glycolysis and tumor growth. We developed a small molecule inhibitor of MCT1 (AZD3965) and assessed its activity across a panel of cell lines. We explored its antitumor activity as monotherapy and in combination with doxorubicin or rituximab. AZD3965 is a potent inhibitor of MCT1 with activity against MCT2 but selectivity over MCT3 and MCT4. In vitro, AZD3965 inhibited the growth of a range of cell lines especially haematological cells. Inhibition of MCT1 by AZD3965 inhibited lactate efflux and resulted in accumulation of glycolytic intermediates. In vivo, AZD3965 caused lactate accumulation in the Raji Burkitt’s lymphoma model and significant tumor growth inhibition. Moreover, AZD3965 can be combined with doxorubicin or rituximab, components of the R-CHOP standard-of-care in DLBCL and Burkitt’s lymphoma. Finally, combining lactate transport inhibition by AZD3965 with GLS1 inhibition in vitro, enhanced cell growth inhibition and cell death compared to monotherapy treatment. The ability to combine AZD3965 with novel, and standard-of-care inhibitors offers novel combination opportunities in haematological cancers.

Protein mediated regulation of the NHE1 isoform of the Na+/H+ exchanger in renal cells. A regulatory role of Hsp90 and AKT kinase

Na+/H+ exchanger isoform one (NHE1) is a pH regulatory protein that is present in renal tissues and serves to remove protons from within cells and protect against intracellular acidification. NHE1 has a large 315 amino acid cytosolic regulatory domain that regulates the catalytic membrane domain. We examined protein-mediated regulation of NHE1 through the cytosolic domain. Affinity chromatography with the C-terminus of NHE1 yielded a number of NHE1 binding proteins including 14-3-3 protein, heat shock proteins (Hsp90 and Hsp70) and Na+/K+ ATPase. We confirmed that 14-3-3 and heat shock proteins bind to or regulate NHE1 but could not confirm that Na+/K+ ATPase binds to the intact protein. The Hsp90 inhibitor 17-AAG decreased NHE1 activity and NHE1 phosphorylation in MDCK cells but did not decrease protein levels. Additionally, 17-AAG decreased phospho-AKT levels. Direct inhibition of AKT with MK2206 decreased NHE1 activity, though this effect was not additive with the effect of 17-AAG. The results demonstrate that in renal cells, NHE1 is associated with several regulatory proteins including Hsp90, and that Hsp90 affects its function possibly through altered phosphorylation of the protein via the AKT kinase.

Extracellular acidosis and very low [Na+ ] inhibit NBCn1- and NHE1-mediated net acid extrusion from mouse vascular smooth muscle cells.

The electroneutral Na+ , HCO3- cotransporter NBCn1 and Na+ /H+ exchanger NHE1 regulate acid-base balance in vascular smooth muscle cells (VSMCs) and modify artery function and structure. Pathological conditions - notably ischaemia - can dramatically perturb intracellular (i) and extracellular (o) pH and [Na+ ]. We examined effects of low [Na+ ]o and pHo on NBCn1 and NHE1 activity in VSMCs of small arteries. We measured pHi by 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-based fluorescence microscopy of mouse mesenteric arteries and induced intracellular acidification by NH4+ prepulse technique. NBCn1 activity - defined as Na+ -dependent, amiloride-insensitive net base uptake with CO2 /HCO3- present - was inhibited equally when pHo decreased from 7.4 (22mm HCO3-/5% CO2 ) by metabolic (pHo 7.1/11mm HCO3-: 22±8%; pHo 6.8/5.5mm HCO3-: 61±7%) or respiratory (pHo 7.1/10% CO2 : 35±11%; pHo 6.8/20% CO2 : 56±7%) acidosis. Extracellular acidosis more prominently inhibited NHE1 activity - defined as Na+ -dependent net acid extrusion without CO2 /HCO3- present - at both pHo 7.1 (45±9%) and 6.8 (85±5%). Independently of pHo , lowering [Na+ ]o from 140 to 70mm reduced NBCn1 and NHE1 activity <20% whereas transport activities declined markedly (25-50%) when [Na+ ]o wasreduced to 35mm. Steady-state pHi decreased more during respiratory(ΔpHi /ΔpHo =71±4%) than metabolic (ΔpHi /ΔpHo =30±7%) acidosis. Extracellular acidification inhibits NBCn1 and NHE1 activity in VSMCs. NBCn1 is equivalently inhibited when pCO2 is raised or [HCO3-]o decreased. Lowering [Na+ ]o inhibits NBCn1 and NHE1 markedly only below the typical physiological and pathophysiological range. We propose that inhibition of Na+ -dependent net acid extrusion at low pHo protects against cellular Na+ overload at the cost of intracellular acidification.

A novel method for assessment of local pH in periplasmic space and of cell surface potential in yeast.

Yeast cells exhibit a negative surface potential due to negative charges at the cell membrane surface. Consequently, local concentrations of cations at the periplasmic membrane surface may be significantly increased compared to their bulk environment. However, in cell suspensions only bulk concentrations of cations can be measured directly. Here we present a novel method enabling the assessment of local pH at the periplasmic membrane surface which can be directly related to the underlying cell surface potential. In this proof of concept study using Saccharomyces cerevisiae cells with episomally expressed pH reporter, pHluorin, intracellular acidification induced by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was measured using synchronously scanned fluorescence spectroscopy (SSF). The analysis of titration curves revealed that the pH at the periplasmic surface of S. cerevisiae cells was about two units lower than the pH of bulk medium. This pH difference was significantly decreased by increasing the ionic strength of the bulk medium. The cell surface potential was estimated to amount to -130mV. Comparable results were obtained also with another protonophore, pentachlorophenol (PCP).

Inwardly Rectifying K+ Currents in Cultured Oligodendrocytes from Rat Optic Nerve are Insensitive to pH.

Inwardly rectifying K+ (Kir) channel expression signals at an advanced stage of maturation during oligodendroglial differentiation. Knocking down their expression halts the generation of myelin and produces severe abnormalities in the central nervous system. Kir4.1 is the main subunit involved in the tetrameric structure of Kir channels in glial cells; however, the precise composition of Kir channels expressed in oligodendrocytes (OLs) remains partially unknown, as participation of other subunits has been proposed. Kir channels are sensitive to H+; thus, intracellular acidification produces Kir current inhibition. Since Kir subunits have differential sensitivity to H+, we studied the effect of intracellular acidification on Kir currents expressed in cultured OLs derived from optic nerves of 12-day-old rats. Unexpectedly, Kir currents in OLs (2-4 DIV) did not change within the pH range of 8.0-5.0, as observed when using standard whole-cell voltage-clamp recording or when preserving cytoplasmic components with the perforated patch-clamp technique. In contrast, low pH inhibited astrocyte Kir currents, which was consistent with the involvement of the Kir4.1 subunit. The H+-insensitivity expressed in OL Kir channels was not intrinsic because Kir cloning showed no difference in the sequence reported for the Kir4.1, Kir2.1, or Kir5.1 subunits. Moreover, when Kir channels were heterologously expressed in Xenopus oocytes they behaved as expected in their general properties and sensitivity to H+. It is therefore concluded that Kir channel H+-sensitivity in OLs is modulated through an extrinsic mechanism, probably by association with a modulatory component or by posttranslational modifications.

Carbonic anhydrase inhibitors modify intracellular pH transients and contractions of rat middle cerebral arteries during CO2/HCO3– fluctuations

The CO2/HCO3– buffer minimizes pH changes in response to acid–base loads, HCO3– provides substrate for Na+,HCO3–-cotransporters and Cl–/HCO3–-exchangers, and H+ and HCO3– modify vasomotor responses during acid–base disturbances. We show here that rat middle cerebral arteries express cytosolic, mitochondrial, extracellular, and secreted carbonic anhydrase isoforms that catalyze equilibration of the CO2/HCO3– buffer. Switching from CO2/HCO3–-free to CO2/HCO3–-containing extracellular solution results in initial intracellular acidification due to hydration of CO2 followed by gradual alkalinization due to cellular HCO3– uptake. Carbonic anhydrase inhibition decelerates the initial acidification and attenuates the associated transient vasoconstriction without affecting intracellular pH or artery tone at steady-state. Na+,HCO3–-cotransport and Na+/H+-exchange activity after NH4+-prepulse-induced intracellular acidification are unaffected by carbonic anhydrase inhibition. Extracellular surface pH transients induced by transmembrane NH3 flux are evident under CO2/HCO3–-free conditions but absent when the buffer capacity and apparent H+ mobility increase in the presence of CO2/HCO3– even after the inhibition of carbonic anhydrases. We conclude that (a) intracellular carbonic anhydrase activity accentuates pH transients and vasoconstriction in response to acute elevations of pCO2, (b) CO2/HCO3– minimizes extracellular surface pH transients without requiring carbonic anhydrase activity, and (c) carbonic anhydrases are not rate limiting for acid–base transport across cell membranes during recovery from intracellular acidification.

Abstract PR12: Targeting eIF2α phosphorylation for cancer therapy

Abstract Solid tumor tumors contain hypoxic regions. Tumor hypoxia is co-present with reduced nutrients and usually intracellular acidification. Lack of O2 and nutrients, and intracellular acidification causes endoplasmic reticulum stress (ER). In addition to canonical hypoxia response, tumor cells activate integrated ER-stress response (IERSR) in a spatially and temporally regulated manner to enter into dormancy under hypoxia and de-activate IERSR to return to a proliferative state once blood supply is restored. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) branch of IERSR is a critical determinant of tumor survival under hypoxia. Temporally and spatially regulated eIF2α phosphorylation attenuates overall translation while favoring translation of a subset of mRNAs coding for transcription factors and accessory proteins. This results in re-programming of gene expression that favors tumor-cell survival. Once the hypoxic conditions are ameliorated eIF2α phosphorylation is reversed and tumor cells exit dormancy. Sustained eIF2α phosphorylation on the other hand not only reduces overall protein synthesis to inhibit proliferation but also re-programs gene expression to induce expression of pro-apoptotic proteins such as CHOP to induce cell death. We therefore hypothesized that pharmacological activation of eIF2α kinases to induce sustained eIF2a phosphorylation will cause selective demise of tumors. To test this hypothesis and develop mechanism specific anti-cancer agents we generated high throughput reporter assays and screened large chemical libraries for inducers of eIF2α phosphorylation. Our Screening effort combined with functional genetic studies, and synthesis and evaluation of focused libraries resulted in identification of highly potent and specific activators of three eIF2α kinases, heme-regulated inhibitor (HRI), Protein kinase R, and PKR-like ER-kinase (PERK). These agents downregulate expression of pro-proliferative proteins such as cyclin D1 and cyclin E while upregulating the expression of pro-apoptotic proteins such as CHOP and tumor suppressor proteins such as BRCA1; all dependent on eIF2α phosphorylation. We will present unpublished data on the structure activity relationship studies of the second generation activators of eIF2α kinases. We will also present in vivo data demonstrating that inducers of eIF2α phosphorylation fully abrogate proliferation of human breast and melanoma xenografts growth with no apparent toxicity. At the molecular level, they induce eIF2α phosphorylation, expression of pro-apoptotic protein CHOP and cell cycle inhibitor p27Kip1 while inhibiting Cyclin D1 expression in xenograft tumors. Tumor cells acquire resistance to targeted therapy by bypassing the signaling node inhibited by treatment; activating alternate oncogenic pathway or downstream signaling node. Because translation initiation is in the apex of many oncogenic and proliferative pathways we hypothesized that translation initiation inhibitors will abrogate proliferation of targeted-therapy resistant cancer cell lines independent of the primary therapy or the mechanism of resistance. Our studies in melanoma cancer cells resistant to B-Raf or B-Raf and MEK inhibitors demonstrate that activators of eIF2α kinases or inhibitors of eIF4F complex inhibit the proliferation of parental and drug resistant cancer-cells with the same efficiency. We will also present these data and on our progress in resolving the crystal structure of full-length HRI which should allow us to accelerate drug development by improving the specificity and potency of our current lead compounds. We conclude that our second generation activators of eIF2α kinases are potent and mechanism specific agents that can be developed for treatment of naïve and drug resistant tumors. This abstract is also being presented as Poster B20. Citation Format: Revital Yefidoff-Freedman, Ronghui Du, Fan Jing, Quingwen Zhang, Bertal H. Aktas. Targeting eIF2α phosphorylation for cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr PR12.

Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites.

Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 µM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3 % of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.

Polyamines preserve connexin 43-mediated gap junctional communication during intracellular hypercalcemia and acidosis.

Changes in the regulation, formation, and gating of connexin-based gap junction channels occur in various disorders. It has been shown that H and Ca are involved in the regulation of gap junctional communication. Ischemia-induced intracellular acidification and Ca overload lead to closure of gap junctions and inhibit an exchange by ions and small molecules throughout the network of cells in the heart, brain, and other tissues. In this study, we examined the role of the polyamines in the regulation of connexin 43 (Cx43)-based gap junction channels under elevated intracellular concentrations of hydrogen ([H]i) and calcium ([Ca]i) ions. Experiments, conducted in Novikoff and A172 human glioblastoma cells, which endogenously express Cx43, showed that polyamines prevent downregulation of Cx43-mediated gap junctional communication caused by elevated [Ca]i and [H]i, accompanying ischemic and other pathological conditions. siRNA knockdown of Cx43 significantly reduces gap junctional communication, indicating that Cx43 gap junctions are the targets for spermine regulation.

Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps.

The inflammatory microenvironment is commonly characterized by extracellular acidosis (pH < 7.35). Sensitivity to pH, CO2 or bicarbonate concentrations allows neutrophils to react to changes in their environment and to detect inflamed areas in the tissue. One important antimicrobial effector mechanism is the production of neutrophil extracellular traps (NETs), which are released during a programmed reactive oxygen species (ROS)-dependent cell death, the so-called NETosis. Although several functions of neutrophils have been analyzed under acidic conditions, the effect of extracellular acidosis on NETosis remains mainly unexplored and the available experimental results are contradictory. We performed a comprehensive study with the aim to elucidate the effect of extracellular acidosis on ROS-dependent NETosis of primary human neutrophils and to identify the underlying mechanisms. The study was performed in parallel in a CO2–bicabonate-buffered culture medium, which mimics in vivo conditions, and under HEPES-buffered conditions to verify the effect of pH independent of CO2 or bicarbonate. We could clearly show that extracellular acidosis (pH 6.5, 6.0, and 5.5) and intracellular acidification inhibit the release of ROS-dependent NETs upon stimulation of neutrophils with phorbol myristate acetate and immobilized immune complexes. Moreover, our findings suggest that the diminished NET release is a consequence of reduced ROS production and diminished glycolysis of neutrophils under acidic conditions. It was suggested previously that neutrophils can sense the border of inflamed tissue by the pH gradient and that a drop in pH serves as an indicator for the progress of inflammation. Following this hypothesis, our data indicate that an acidic inflammatory environment results in inhibition of extracellular operating effector mechanisms of neutrophils such as release of ROS and NETs. This way the release of toxic components and tissue damage can be avoided. However, we observed that major antimicrobial effector mechanisms such as phagocytosis and the killing of pathogens by neutrophils remain functional under acidic conditions.

Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH.

Many strains of lactic acid bacteria (LAB) and bifidobacteria have exhibited strain-specific capacity to produce γ-aminobutyric acid (GABA) via their glutamic acid decarboxylase (GAD) system, which is one of amino acid-dependent acid resistance (AR) systems in bacteria. However, the linkage between bacterial AR and GABA production capacity has not been well established. Meanwhile, limited evidence has been provided to the global diversity of GABA-producing LAB and bifidobacteria, and their mechanisms of efficient GABA synthesis. In this study, genomic survey identified common distribution of gad operon-encoded GAD system in Lactobacillus brevis for its GABA production among varying species of LAB and bifidobacteria. Importantly, among four commonly distributed amino acid-dependent AR systems in Lb. brevis, its GAD system was a major contributor to maintain cytosolic pH homeostasis by consuming protons via GABA synthesis. This highlights that Lb. brevis applies GAD system as the main strategy against extracellular and intracellular acidification demonstrating its high capacity of GABA production. In addition, the abundant GadA retained its activity toward near-neutral pH (pH 5.5–6.5) of cytosolic acidity thus contributing to efficient GABA synthesis in Lb. brevis. This is the first global report illustrating species-specific characteristic and mechanism of efficient GABA synthesis in Lb. brevis.

Focal photodynamic intracellular acidification as a cancer therapeutic

Cancer cells utilize an array of proton transporters to regulate intra- and extracellular pH to thrive in hypoxic conditions, and to increase tumor growth and metastasis. Efforts to target many of the transporters involved in cancer cell pH regulation have yielded promising results, however, many productive attempts to disrupt pH regulation appear to be non-specific to cancer cells, and more effective in some cancer cells than others. Following a review of the status of photodynamic cancer therapy, a novel light-activated process is presented which creates very focal, rapid, and significant decreases in only intracellular pH (pHi), leading to cell death. The light-activation of the H+ carrier, nitrobenzaldehyde, has been effective at initiating pH-induced apoptosis in non-cancerous and numerous cancerous cell lines in vitro, to include breast, prostate, and pancreatic cancers. Also, this intracellular acidification technique caused significant reductions in tumor growth rate and enhanced survival in mice bearing triple negative breast cancer tumors. The efficacy of an NBA-upconverting nanoparticle to kill breast cancer cells in vitro is described, as well as a discussion of the potential intracellular mechanisms underlying the pH-induced apoptosis.

Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression

Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles incancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but notSREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification.

Voltage Dependent Phosphatase Activity is Enhanced by Intracellular Acidification

Voltage sensitive phosphatases (VSPs) are PI(4,5)P2/PI(3,4,5)P3-5- and PI(3,4)P2/PI(3,4,5)P3-3-phosphatases. For non-mammalian VSPs, this activity is regulated by membrane voltage via a voltage sensor domain (VSD). For mammalian VSPs the VSD seems insensitive to voltage changes yet still essential for control of the phosphatase activity. Under physiological conditions, the non-mammalian VSPs strongly deplete PI(4,5)P2 in a voltage dependent manner. The physiological regulator of mammalian VSP activity remains elusive. VSPs are suggested to play a role in fertilization and development, where changes in intracellular pH are known to occur. They are found to be expressed in tissues like kidney, stomach, sperm, and ovary which are known to undergo such pH changes. Therefore, we speculated that intracellular pH might modulate VSP activity.To test this hypothesis we performed whole-cell patch-clamp experiments in CHO cells expressing diverse VSPs and fluorescent PI(4,5)P2 reported domains, the latter allowing for monitoring VSP activity by means of total internal reflection microscopy (TIRF-M). The whole-cell patch-clamp configuration allowed for control not only over membrane voltage but also intracellular pH by dialysing the cell with solutions with the desired pH.We find that acidification of the cytoplasm results in increased PI(4,5)P2 depletion, accompanied by a shift of the apparent voltage dependence towards more negative potentials. An increase in intracellular pH has the opposite effect. The voltage dependence of sensing currents was unaffected by the pH changes, suggesting that alterations of the VSD are not causal for the observed changes in voltage dependent activity. Similar effects were observed in all tested VSPs We conclude that the overall activity of the phosphatase is enhanced under acidic and diminished under alkaline conditions. Kinetic modeling predicts a shift in apparent voltage dependence under these circumstances that is in agreement with the observed shift.In conclusion, we suggest that intracellular pH can play a role in the regulation of the activity of VSPs.This work was supported by a research grant of the University Medical Center Giessen and Marburg (UKGM32/2011MR) to C.R.H and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to D.O.

Linseed polyol-assisted, microwave-induced synthesis of nano CuO embedded in polyol-polyester matrix: antifungal behavior and coating properties

Nano-sized CuO particles were prepared insitu via “one-pot, solvent-less” microwave-induced synthesis, by environmentally benign polyol process using polyol obtained from Linseed oil by (non-aqueous) condensation reaction, for the first time. Linseed polyol acts as solvent, base matrix providing functional groups, and stabilizer; the reaction is devoid of any additive, catalyst or solvent. In the prepared nanocomposites [LPE-CuO], CuO nanoparticles shaped somewhat like rods, assembled themselves in a “T-fashion”. The antifungal effects of synthesized particles were investigated against four clinical isolates of different Candida species. Results showed that the synthesized compound LPE-0.06 was found to be most effective, followed by LPE-0.05 and LPE-0.04. The rapid actions of the synthesized compounds on fungal cells suggested membrane-located targets. We investigated the effect of MIC90 values of these compounds on H+-ATPase mediated proton pumping by different Candida species. In the absence of glucose, the mean inhibition rate of H+ extrusion caused by MIC of LP was 28.09%, LPE: 33.04%, CuAc: 36.86%, LPE-0.04: 58.54%, LPE-0.05: 69.82% and LPE-0.06: 78.07%, respectively. Antifungal mechanism of action suggested that the synthesized compounds exert their antifungal activity by targeting H+-ATPase mediated proton pumping. Inhibition of H+-ATPase activity leads to intracellular acidification and consequently cell death. Results obtained in this study indicated strong antifungal activity of synthesized compounds against all the Candida species and suggest that the inhibition of fungal H+ ATPase is responsible for the antifungal activity of the test compounds. LPE-CuO can be employed as antifungal polyester and polyurethane coatings. The present work is consistent with the Principles (Principle 1,2,3,4,5,6,7,8, 12) of Green Chemistry.

Intracellular acidification increases adenosine transport in human umbilical vein endothelial cells

IntroductionAdenosine is taken up via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) at a physiological extracellular pH (pHo ∼7.4) in human umbilical vein endothelial cells (HUVECs). Acidic pHo increases the uptake of adenosine and 5-hydroxytryptamine (5HT) via hENT4 in this cell type. However, modulation of hENT1 and hENT2 transport activity by the pHi is unknown. We investigated whether hENT1 and hENT2-adenosine transport was regulated by acidic pHi. MethodsHUVECs loaded with a pH sensitive probe were subjected to 0.1–20 mmol/L NH4Cl pulse assay to generate 6.9–6.2 pHi. Before pHi started to recover, adenosine transport kinetics (0–500 μmol/L, 37 °C) in the absence or presence 1 or 10 μmol/L S-(4-nitrobenzyl)-6-thio-inosine (NBTI), 2 mmol/L hypoxanthine, 2 mmol/L adenine, 100 μmol/L 5HT, or 500 μmol/L adenosine, was measured. ResultsOverall adenosine transport (i.e., hENT1+hENT2) was semisaturable and partially inhibited by 1 μmol/L, but abolished by 10 μmol/L NBTI in cells non-treated or treated with NH4Cl. The initial velocity and non-saturable, lineal component for overall transport were increased after NH4Cl pulse. hENT1 and hENT2-mediated adenosine transport maximal capacity was increased by acidic pHi. hENT1 activity was more sensitive than hENT2 activity to acidic pHi. DiscussionhENT1 and hENT2-adenosine transport is differentially regulated by acidic pHi in HUVECs. These findings are important in pathologies associated with pHi alterations such as gestational diabetes mellitus.

Proton pump inhibition and cancer therapeutics: A specific tumor targeting or it is a phenomenon secondary to a systemic buffering?

One of the unsolved mysteries in oncology includes the strategies that cancer cells adopt to cope with an adverse microenvironment. However, we knew, from the Warburg's discovery that through their metabolism based on sugar fermentation, cancer cells acidify their microenvironment and this progressive acidification induces a selective pressure, leading to the development of very malignant cells entirely armed to survive in the hostile microenvironment generated by their own metabolism. In the last decades a primordial role for proton exchangers has been supported as a key tumor advantage in facing off the acidic milieu. Proton exchangers do not allow intracellular acidification through a continuous elimination of H+ either outside the cells or within the internal vacuoles. This article wants to comment a translational process through that led to the preclinical demonstration that a class of proton pump inhibitors (PPI) exploited worldwide for peptic ulcer treatment and gastroprotection are indeed powerful chemosensitizers as well. In this process we achieved the clinical proof of concept that PPI may well be included in new anti-cancer strategies with a solid background and rationale.

A pH-responsive sequential-disassembly nanohybrid for mitochondrial targeting.

Cationic materials have been reported as promising tools for targeting to mitochondria which are the "power houses" and "metabolic garbage keepers" of cells. However, their positive nature also restricts their in vivo application due to the quick clearance. Herein, we fabricated a nanohybrid consisting of the pH-responsive N-(2-hydroxypropyl)methacrylamide (HPMA) co-polymer (R-P) shells and positive mesoporous silica nanoparticle cores via electrostatic interaction. The anticancer drug, docetaxel (DTX), was encapsulated in the positive MSN cores (MSN-DTX). Once concealed by the anionic R-P shield, the assembled nanohybrid R-P@MSN-DTX will achieve prolonged blood circulation thereby leading to an enhanced EPR effect. At mildly acidic tumor environmental pH, first-stage charge reversion took place due to the hydrolysis of the amide bond on HPMA co-polymers. The de-attachment of the HPMA co-polymer occurred because of the positive charge repulsion and partial exposure of the positively charged MSN core promoted the cell internalization. The second-stage pH-responsiveness in the endo/lysosomes with a more acidic environment accelerates the disassembly of the nanohybrid and the leakage of the core facilitated the endo/lysosome escape and mitochondrial targeting with the help of intracellular compartmental acidity. Gathering up the characteristics of neutralized charge and stepwise pH-responsiveness, the R-P@MSN-DTX acquired a good tumor inhibition rate of 72.6% on nude mice. Our report provided a reference for systemic mitochondrial targeting achieved by the union of "assembly-disassembly".