Reversed pH Gradient Research Articles

Whole-Brain Intracellular pH Mapping of Gliomas Using High-Resolution 31P MR Spectroscopic Imaging at 7.0 T.

Malignant tumors commonly exhibit a reversed pH gradient compared with normal tissue, with a more acidic extracellular pH and an alkaline intracellular pH (pHi). In this prospective study, pHi values in gliomas were quantified using high-resolution phosphorous 31 (31P) spectroscopic MRI at 7.0 T and were used to correlate pHi alterations with histopathologic findings. A total of 12 participants (mean age, 58 years ± 18 [SD]; seven male, five female) with histopathologically proven, newly diagnosed glioma were included between September 2018 and November 2019. The 31P spectroscopic MRI scans were acquired using a double-resonant 31P/1H phased-array head coil together with a three-dimensional (3D) 31P chemical shift imaging sequence (5.7-mL voxel volume) performed with a 7.0-T whole-body system. The 3D volumetric segmentations were performed for the whole-tumor volumes (WTVs); tumor subcompartments of necrosis, gadolinium enhancement, and nonenhancing T2 (NCE T2) hyperintensity; and normal-appearing white matter (NAWM), and pHi values were compared. Spearman correlation was used to assess association between pHi and the proliferation index Ki-67. For all study participants, mean pHi values were higher in the WTV (7.057 ± 0.024) compared with NAWM (7.006 ± 0.012; P < .001). In eight participants with high-grade gliomas, pHi was increased in all tumor subcompartments (necrosis, 7.075 ± 0.033; gadolinium enhancement, 7.075 ± 0.024; NCE T2 hyperintensity, 7.043 ± 0.015) compared with NAWM (7.004 ± 0.014; all P < .01). The pHi values of WTV positively correlated with Ki-67 (R2 = 0.74, r = 0.78, P = .001). In conclusion, 31P spectroscopic MRI at 7.0 T enabled high-resolution quantification of pHi in gliomas, with pHi alteration associated with the Ki-67 proliferation index, and may aid in diagnosis and treatment monitoring. Keywords: 31P MRSI, pH, Glioma, Glioblastoma, Ultra-High-Field MRI, Imaging Biomarker, 7 Tesla Supplemental material is available for this article. © RSNA, 2023.

Abstract 6034: Detection of potassium channel (KCN) gene expression, often multiple and pH sensitive, in cancer genomic landscapes, as low to mid level genes of interest, suggests potassium transmembrane flow may enable proton efflux in a Goldilocks manner

Abstract A reversed pH gradient across cell membranes underlies multiple malignant features, including invasiveness. Extracellular low pH activates metalloproteases and intracellular high pH activates ATP citrate lyase, inactivates caspases, etc. Hydrogen ions attracted to inner membrane fixed anions are putatively displaced by potassium ion flow through membrane channels. Altered expression of potassium channel (KCN) genes sensitive to pH in invasive brain tumors (Beckner, Proc AACR, A3039, 2022) and 2 mutated KCN genes (1 pH sensitive) in U87 glioblastoma cells (Clark MJ et al, PLOS Genet 2010, e1000832) led to this investigation. Published studies with altered expression or mutations in KCN genes qualifying them as genes of interest (GOI) were compared. Both study types included breast, lung, gastrointestinal, brain, bone marrow, liver, melanocytic malignancies, and 8 others were in either. At least 1 figure (heatmap, Venn diagram, phylogenetic tree, etc.) or table (not Supplemental) had 1 or more KCN genes in a group of GOI. Average numbers of GOI with 95% CI for 23 expression and 14 mutation studies were 38.5 ± 0.4 and 28.4 ± 0.4. No KCN GOI was further examined in the studies. Expression was according to tumor types, inhibitors, siRNAs, regulators of pathways, transcription factors, location, etc. Mutations were single nucleotide polymorphisms, insertions, deletions, fusions, etc. Tissue from surgery, organoids, xenografts, cell lines, stem cells, etc. were analyzed. Of 27 KCN GOI in expression studies, 2/3 were detected with other KCN GOI. For 12 mutated KCN genes there were no other KCN genes mutated in the same study. In expression studies, 8 (34.8%) had multiple KCN GOI, differing from mutation studies, p=0.015, Fishers Exact. In expression studies, 8 found pH sensitive KCN GOI including KCNK1 (2X), K3, K5, K6, K12, K15 (2X), and KCNJ16. In mutation studies, KCNK9 was the only one found. The most frequently mutated KCN subfamily was KCNJ, KCNJ5 (2X) and KCNJ12 (2X). All expression studies included at least 1 repeat GOI (KCN or other). APOE, RET, and RTN1 were each GOI 3 times. Additionally, 40 expressed GOI (including 6 KCN GOI) were found 2X. A small cell lung cancer study had 6 repeats (30%) in 20 GOI (ASCL1, BCL2, KCNA1, RET, SCN3A, and SOX2). KCN gene multiplicity, often pH sensitive members, among low to mid level GOI consistent with a Goldilocks' level of expression, and repeated association with cancer genes supports a pH related measured/regulated role for potassium channels in malignancy. Mainly inward (and some outward for recirculation) potassium transmembrane flows in tumor cells potentially compete with hydrogen ions attracted to inner membrane anions to promote proton efflux by displacement with subsequent reversed pH gradients that aid invasion, cell survival, etc. Citation Format: Marie E. Beckner. Detection of potassium channel (KCN) gene expression, often multiple and pH sensitive, in cancer genomic landscapes, as low to mid level genes of interest, suggests potassium transmembrane flow may enable proton efflux in a Goldilocks manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6034.

Melatonin: Regulation of Prion Protein Phase Separation in Cancer Multidrug Resistance.

The unique ability to adapt and thrive in inhospitable, stressful tumor microenvironments (TME) also renders cancer cells resistant to traditional chemotherapeutic treatments and/or novel pharmaceuticals. Cancer cells exhibit extensive metabolic alterations involving hypoxia, accelerated glycolysis, oxidative stress, and increased extracellular ATP that may activate ancient, conserved prion adaptive response strategies that exacerbate multidrug resistance (MDR) by exploiting cellular stress to increase cancer metastatic potential and stemness, balance proliferation and differentiation, and amplify resistance to apoptosis. The regulation of prions in MDR is further complicated by important, putative physiological functions of ligand-binding and signal transduction. Melatonin is capable of both enhancing physiological functions and inhibiting oncogenic properties of prion proteins. Through regulation of phase separation of the prion N-terminal domain which targets and interacts with lipid rafts, melatonin may prevent conformational changes that can result in aggregation and/or conversion to pathological, infectious isoforms. As a cancer therapy adjuvant, melatonin could modulate TME oxidative stress levels and hypoxia, reverse pH gradient changes, reduce lipid peroxidation, and protect lipid raft compositions to suppress prion-mediated, non-Mendelian, heritable, but often reversible epigenetic adaptations that facilitate cancer heterogeneity, stemness, metastasis, and drug resistance. This review examines some of the mechanisms that may balance physiological and pathological effects of prions and prion-like proteins achieved through the synergistic use of melatonin to ameliorate MDR, which remains a challenge in cancer treatment.

Effect of Exogenous pH on Cell Growth of Breast Cancer Cells.

Breast cancer is the most common type of cancer in women and the most life-threatening cancer in females worldwide. One key feature of cancer cells, including breast cancer cells, is a reversed pH gradient which causes the extracellular pH of cancer cells to be more acidic than that of normal cells. Growing literature suggests that alkaline therapy could reverse the pH gradient back to normal and treat the cancer; however, evidence remains inconclusive. In this study, we investigated how different exogenous pH levels affected the growth, survival, intracellular reactive oxygen species (ROS) levels and cell cycle of triple-negative breast cancer cells from MDA-MB-231 cancer cell lines. Our results demonstrated that extreme acidic conditions (pH 6.0) and moderate to extreme basic conditions (pH 8.4 and pH 9.2) retarded cellular growth, induced cell death via necrosis and apoptosis, increased ROS levels, and shifted the cell cycle away from the G0/G1 phase. However, slightly acidic conditions (pH 6.7) increased cellular growth, decreased ROS levels, did not cause significant cell death and shifted the cell cycle from the G0/G1 phase to the G2/M phase, thereby explaining why cancer cells favored acidic conditions over neutral ones. Interestingly, our results also showed that cellular pH history did not significantly affect the subsequent growth of cells when the pH of the medium was changed. Based on these results, we suggest that controlling or maintaining an unfavorable pH (such as a slightly alkaline pH) for cancer cells in vivo could retard the growth of cancer cells or potentially treat the cancer.

Chemical and Energy Recovery Alternatives in SWRO Desalination through Electro-Membrane Technologies

Electro-membrane technologies are versatile processes that could contribute towards more sustainable seawater reverse osmosis (SWRO) desalination in both freshwater production and brine management, facilitating the recovery of materials and energy and driving the introduction of the circular economy paradigm in the desalination industry. Besides the potential possibilities, the implementation of electro-membrane technologies remains a challenge. The aim of this work is to present and evaluate different alternatives for harvesting renewable energy and the recovery of chemicals on an SWRO facility by means of electro-membrane technology. Acid and base self-supply by means of electrodialysis with bipolar membranes is considered, together with salinity gradient energy harvesting by means of reverse electrodialysis and pH gradient energy by means of reverse electrodialysis with bipolar membranes. The potential benefits of the proposed alternatives rely on environmental impact reduction is three-fold: (a) water bodies protection, as direct brine discharge is avoided, (b) improvements in the climate change indicator, as the recovery of renewable energy reduces the indirect emissions related to energy production, and (c) reduction of raw material consumption, as the main chemicals used in the facility are produced in-situ. Moreover, further development towards an increase in their technology readiness level (TRL) and cost reduction are the main challenges to face.

Tracing of intracellular pH in cancer cells in response to Taxol treatment

ABSTRACT Genetically encoded pH-sensors are the promising instrument for intracellular pH (pHi) registration. In tumor tissue the reversed pH gradient is known to be the important hallmark of cancer and regulator of tumor response on chemotherapy. However the effect of chemotherapeutic drugs on the pHi of tumor cells is largely unknown.Here we using genetically encoded ratiometric pH-sensor SypHer2 were able to monitor pHi in vitro in cell monolayer and tumor spheroids and in vivo in tumor xenografts. In tumor cell monolayer different pHi dynamic was revealed in the dying cell and division-arrested surviving cells. The treatment effect of taxol varied in monolayer and tumor spheroids and pHi changes were able to reflect these difference. The tend to pHi decrease in respect to taxol in vivo matched with results obtained for the cell monolayer. Also in both cases the cell cycle-arrest was the main treatment effect in contrast to tumor spheroid, where the cell death was the primary result.These findings elucidate the significance of pHi in the mechanisms of taxol action on cervical cancer cells and will be valuable for development of new approaches for cancer treatment.

Disruption of pH Dynamics Suppresses Proliferation and Potentiates Doxorubicin Cytotoxicity in Breast Cancer Cells.

The reverse pH gradient is a major feature associated with cancer cell reprogrammed metabolism. This phenotype is supported by increased activity of pH regulators like ATPases, carbonic anhydrases (CAs), monocarboxylate transporters (MCTs) and sodium–proton exchangers (NHEs) that induce an acidic tumor microenvironment, responsible for the cancer acid-resistant phenotype. In this work, we analyzed the expression of these pH regulators and explored their inhibition in breast cancer cells as a strategy to enhance the sensitivity to chemotherapy. Expression of the different pH regulators was evaluated by immunofluorescence and Western blot in two breast cancer cell lines (MDA-MB-231 and MCF-7) and by immunohistochemistry in human breast cancer tissues. Cell viability, migration and invasion were evaluated upon exposure to the pH regulator inhibitors (PRIs) concanamycin-A, cariporide, acetazolamide and cyano-4-hydroxycinnamate. Additionally, PRIs were combined with doxorubicin to analyze the effect of cell pH dynamic disruption on doxorubicin sensitivity. Both cancer cell lines expressed all pH regulators, except for MCT1 and CAXII, only expressed in MCF-7 cells. There was higher plasma membrane expression of the pH regulators in human breast cancer tissues than in normal breast epithelium. Additionally, pH regulator expression was significantly associated with different molecular subtypes of breast cancer. pH regulator inhibition decreased cancer cell aggressiveness, with a higher effect in MDA-MB-231. A synergistic inhibitory effect was observed when PRIs were combined with doxorubicin in the breast cancer cell line viability. Our results support proton dynamic disruption as a breast cancer antitumor strategy and the use of PRIs to boost the activity of conventional therapy.

PH gradient reversal fuels cancer progression.

pH gradient reversal refers to intracellular alkalization and extracellular acidification commonly seen in malignant tumors. To meet their high anabolic demand, cancer cells rewire their glucose metabolism from oxidative phosphorylation to lactate fermentation, which results in the excessive generation of protons. To avoid lethal cytosolic acidification, lactate-fermenting cancer cells activate multiple acid removal pathways leading to the acidification of the extracellular space. This acidification is often further intensified by the defective capacity of the disorganized tumor vasculature to dilute protons away from the cancer tissue. The cancer-specific proton equilibrium with highly alkaline cytosol and acidic extracellular space is emerging as a fundamental driving force for cancer growth. Here, we discuss how cancer cells establish and maintain reversed pH gradient, how pH gradient reversal fuels cancer progression, and how these mechanisms can be targeted in cancer therapy.

The Golgi: Keeping It Unapologetically Basic.

Tumor cells maintain a reverse pH gradient relative to normal cells, conferring cell-intrinsic and cell-extrinsic benefits that sustain tumor growth. In this issue of Cancer Discovery, Galenkamp and colleagues reveal that NHE7 mediates acidification of the trans-Golgi network in pancreatic ductal adenocarcinoma, which is critical for the maintenance of cytosolic pH and consequently tumor growth.See related article by Galenkamp et al., p. 822.

Targeting the Multidrug Transporter Ptch1 Potentiates Chemotherapy Efficiency.

One of the crucial challenges in the clinical management of cancer is resistance to chemotherapeutics. Multidrug resistance (MDR) has been intensively studied, and one of the most prominent mechanisms underlying MDR is overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) transporters. Despite research efforts to develop compounds that inhibit the efflux activity of ABC transporters and thereby increase classical chemotherapy efficacy, to date, the Food and Drug Administration (FDA) has not approved the use of any ABC transporter inhibitors due to toxicity issues. Hedgehog signaling is aberrantly activated in many cancers, and has been shown to be involved in chemotherapy resistance. Recent studies showed that the Hedgehog receptor Ptch1, which is over-expressed in many recurrent and metastatic cancers, is a multidrug transporter and it contributes to the efflux of chemotherapeutic agents such as doxorubicin, and to chemotherapy resistance. Remarkably, Ptch1 uses the proton motive force to efflux drugs, in contrast to ABC transporters, which use ATP hydrolysis. Indeed, the “reversed pH gradient” that characterizes cancer cells, allows Ptch1 to function as an efflux pump specifically in cancer cells. This makes Ptch1 a particularly attractive therapeutic target for cancers expressing Ptch1, such as lung, breast, prostate, ovary, colon, brain, adrenocortical carcinoma, and melanoma. Screening of chemical libraries have identified several molecules that are able to enhance the cytotoxic effect of different chemotherapeutic agents by inhibiting Ptch1 drug efflux activity in different cancer cell lines that endogenously over-express Ptch1. In vivo proof of concept has been performed in mice where combining one of these compounds with doxorubicin prevented the development of xenografted adrenocortical carcinoma tumors more efficiently than doxorubicin alone, and without obvious undesirable side effects. Therefore, the use of a Ptch1 drug efflux inhibitor in combination with classical or targeted therapy could be a promising therapeutic option for Ptch1-expressing cancers.

Systems analysis of intracellular pH vulnerabilities for cancer therapy

A reverse pH gradient is a hallmark of cancer metabolism, manifested by extracellular acidosis and intracellular alkalization. While consequences of extracellular acidosis are known, the roles of intracellular alkalization are incompletely understood. By reconstructing and integrating enzymatic pH-dependent activity profiles into cell-specific genome-scale metabolic models, we develop a computational methodology that explores how intracellular pH (pHi) can modulate metabolism. We show that in silico, alkaline pHi maximizes cancer cell proliferation coupled to increased glycolysis and adaptation to hypoxia (i.e., the Warburg effect), whereas acidic pHi disables these adaptations and compromises tumor cell growth. We then systematically identify metabolic targets (GAPDH and GPI) with predicted amplified anti-cancer effects at acidic pHi, forming a novel therapeutic strategy. Experimental testing of this strategy in breast cancer cells reveals that it is particularly effective against aggressive phenotypes. Hence, this study suggests essential roles of pHi in cancer metabolism and provides a conceptual and computational framework for exploring pHi roles in other biomedical domains.

Role of sodium hydrogen exchanger isoform 1 in tumor microenvironment

The reversed pH gradient across the cell membrane on account of intracellular alkalinization and extracellular acidosis is an importart characteristic of tumor microenvironment. Sodium hydrogen exchanger isoform 1 (NHE1) is ubiquitously expressed at the plasma membrane of many types of cells, which plays a cri-tical role in intracellular pH and cell volume homeostasis. NHE1 plays an important role in the regulation of tumor microenvironment and involves in tumor migration and invasion, which may be a potential new target in anti-cancer therapy. Key words: Acid-base equilibrium; Neoplasm invasiveness; Cell movement; Tumor microenvironment; Sodium hydrogen exchanger isoform 1

Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer

Current pharmacological treatments for lung cancer show very poor clinical outcomes, therefore, the development of novel anticancer agents with innovative mechanisms of action is urgently needed. Cancer cells have a reversed pH gradient compared to normal cells, which favours cancer progression by promoting proliferation, metabolic adaptation and evasion of apoptosis. In this regard, the use of ionophores to modulate intracellular pH appears as a promising new therapeutic strategy. Indeed, there is a growing body of evidence supporting ionophores as novel antitumour drugs. Despite this, little is known about the implications of pH deregulation and homeostasis imbalance triggered by ionophores at the cellular level. In this work, we deeply analyse for the first time the anticancer effects of tambjamine analogues, a group of highly effective anion selective ionophores, at the cellular and molecular levels. First, their effects on cell viability were determined in several lung cancer cell lines and patient-derived cancer stem cells, demonstrating their potent cytotoxic effects. Then, we have characterized the induced lysosomal deacidification, as well as, the massive cytoplasmic vacuolization observed after treatment with these compounds, which is consistent with mitochondrial swelling. Finally, the activation of several proteins involved in stress response, autophagy and apoptosis was also detected, although they were not significantly responsible for the cell death induced.Altogether, these evidences suggest that tambjamine analogues provoke an imbalance in cellular ion homeostasis that triggers mitochondrial dysfunction and lysosomal deacidification leading to a potent cytotoxic effect through necrosis in lung cancer cell lines and cancer stem cells.

Mild Alkalization Acutely Triggers the Warburg Effect by Enhancing Hexokinase Activity via Voltage-Dependent Anion Channel Binding.

To fully understand the glycolytic behavior of cancer cells, it is important to recognize how it is linked to pH dynamics. Here, we evaluated the acute effects of mild acidification and alkalization on cancer cell glucose uptake and glycolytic flux and investigated the role of hexokinase (HK). Cancer cells exposed to buffers with graded pH were measured for 18F-fluorodeoxyglucose (FDG) uptake, lactate production and HK activity. Subcellular localization of HK protein was assessed by western blots and confocal microscopy. The interior of T47D breast cancer cells was mildly alkalized to pH 7.5 by a buffer pH of 7.8, and this was accompanied by rapid increases of FDG uptake and lactate extrusion. This shift toward glycolytic flux led to the prompt recovery of a reversed pH gradient. In contrast, mild acidification rapidly reduced cellular FDG uptake and lactate production. Mild acidification decreased and mild alkalization increased mitochondrial HK translocation and enzyme activity. Cells transfected with specific siRNA against HK-1, HK-2 and voltage-dependent anion channel (VDAC)1 displayed significant attenuation of pH-induced changes in FDG uptake. Confocal microscopy showed increased co-localization of HK-1 and HK-2 with VDAC1 by alkaline treatment. In isolated mitochondria, acidic pH increased and alkaline pH decreased release of free HK-1 and HK-2 from the mitochondrial pellet into the supernatant. Furthermore, experiments using purified proteins showed that alkaline pH promoted co-immunoprecipitation of HK with VDAC protein. These findings demonstrate that mild alkalization is sufficient to acutely trigger cancer cell glycolytic flux through enhanced activity of HK by promoting its mitochondrial translocation and VDAC binding. This process might serve as a mechanism through which cancer cells trigger the Warburg effect to maintain a dysregulated pH.

Abstract P5-04-05: Targeting the pH regulatory mechanisms of breast cancer cells

Abstract Background: The abnormal regulation of H+ ions, leading to a reversed pH gradient in tumor cells in comparison to normal cells, is considered to be one of the hallmarks of cancer. This feature, however, has yet to be exploited as a therapeutic target. The aim of this study was to assess whether targeting proteins (CAIX, NHE1 and V-ATPase) that permit hypoxic cancer cell adaptation to acidosis in the tumor microenvironment can produce an effective therapeutic response in breast cancer, using 2D and 3D models. Method: Western blotting and gene expression analysis were performed on MCF-7, MDA-MB-231 and HBL-100 cancer cells to assess target protein expression in differing O2 conditions in 2D, while IHC was used to measure protein levels in 3D using multicellular tumor spheroids. Sulforhodamine B assays were executed to analyze the effects of inhibitors targeting CAIX, NHE1 and V-ATPase on breast cancer cell proliferation in 2D. 3D invasion assays were performed with MDA-MB-231 spheroids and explant tissue derived from human patients to see if CAIX inhibition had any effect on cancer cell invasion. An MDA-MB-231 xenograft model was used to investigate the effects of CAIX inhibition in vivo. Clonogenic assays were performed with MDA-MB-231 spheroids to evaluate whether any of the drugs combined effectively with irradiation. Results: 2D and 3D expression analysis showed that CAIX levels were extremely responsive to changes in O2 conditions in each of the cell lines, with HBL100 cells exhibiting the largest changes in both mRNA (42-fold increase) and protein (78-fold increase) levels at low (0.5%) O2 concentrations. NHE1 and V-ATPase mRNA/protein levels were, however, much more consistently expressed across the cell lines in different O2 conditions. Drugs targeting CAIX, NHE1 and V-ATPase had anti-proliferative effects on the breast cancer cells in 2D. Normoxic cancer cells were the most sensitive to drug treatment, acute hypoxic cancer cells showed increased resistance to the anti-proliferative effects of these drugs, while chronic hypoxic cells had IC50 values more similar to the normoxic cells. The results for the CAIX inhibitor were unexpected, as we had predicted that the increased levels of CAIX in the acute hypoxic cells would make them more sensitive to treatment. CAIX inhibition did, however, significantly reduce the invasion of cancer cells from both MDA-MB-231 spheroids (p≤0.01) and explant tissue (p≤0.001). Targeting pH regulation was also shown to have an effect in vivo on MDA-MB-231 xenografts, with CAIX inhibition significantly reducing the growth (p≤0.05) and proliferation (p≤0.05) of tumors within mice. Finally, clonogenic assays showed that drugs targeting both CAIX and NHE1 led to a significant reduction in colony number when combined with radiation (p≤0.05), compared to either drug individually or radiation treatment alone. Conclusions: This study shows that drugs targeting pH regulation molecules have potential in the treatment of breast cancer. This is highlighted by their ability to affect the proliferation and invasion of breast cancer cells, along with their ability to be combined with radiation. Of the 3 pH regulatory molecules, CAIX represents the target with the most promise. Citation Format: Meehan J, Ward C, Jarman E, Xintaropoulou C, Martinez-Perez C, Turnbull A, Supuran C, Dixon M, Kunkler I, Langdon SP. Targeting the pH regulatory mechanisms of breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-05.

Role of pHi, and proton transporters in oncogene-driven neoplastic transformation

The change of a normal, healthy cell to a transformed cell is the first step in the evolutionary arc of a cancer. While the role of oncogenes in this 'passage' is well known, the role of ion transporters in this critical step is less known and is fundamental to our understanding the early physiological processes of carcinogenesis. Cancer cells and tissues have an aberrant regulation of hydrogen ion dynamics leading to a reversal of the normal tissue intracellular to extracellular pH gradient (ΔpHi to ΔpHe). When this perturbation in pH dynamics occurs during carcinogenesis is less clear. Very early studies using the introduction of different oncogene proteins into cells observed a concordance between neoplastic transformation and a cytoplasmic alkalinization occurring concomitantly with a shift towards glycolysis in the presence of oxygen, i.e. 'Warburg metabolism'. These processes may instigate a vicious cycle that drives later progression towards fully developed cancer where the reversed pH gradient becomes ever more pronounced. This review presents our understanding of the role of pH and the NHE1 in driving transformation, in determining the first appearance of the cancer 'hallmark' characteristics and how the use of pharmacological approaches targeting pH/NHE1 may open up new avenues for efficient treatments even during the first steps of cancer development.

PH dependent transfer of nano-pores into membrane of cancer cells to induce apoptosis

Proper balance of ions in intracellular and extracellular space is the key for normal cell functioning. Changes in the conductance of membranes for ions will lead to cell death. One of the main differences between normal and cancerous cells is the low extracellular pHe and the reverse pH gradient: intracellular pHi is higher than extracellular pHe. We report here pH-selective transfer of nano-pores to cancer cells for the dis-regulation of balance of monovalent cations to induce cell death at mildly acidic pHe as it is in most solid tumors. Our approach is based on the pH-sensitive fusion of cellular membrane with the liposomes containing gramicidin A forming cation-conductive β-helix in the membrane. Fusion is promoted only at low extracellular pH by the pH (Low) Insertion Peptide (pHLIP®) attached to the liposomes. Gramicidin channels inserted into the cancer cells open flux of protons into the cytoplasm and disrupt balance of other monovalent cations, which induces cell apoptosis.

A cellular automaton model examining the effects of oxygen, hydrogen ions and lactate on early tumour growth

Some tumours are known to exhibit an extracellular pH that is more acidic than the intracellular, creating a 'reversed pH gradient' across the cell membrane and this has been shown to affect their invasive and metastatic potential. Tumour hypoxia also plays an important role in tumour development and has been directly linked to both tumour morphology and aggressiveness. In this paper, we present a hybrid mathematical model of intracellular pH regulation that examines the effect of oxygen and pH on tumour growth and morphology. In particular, we investigate the impact of pH regulatory mechanisms on the cellular pH gradient and tumour morphology. Analysis of the model shows that: low activity of the Na+/H+ exchanger or a high rate of anaerobic glycolysis can give rise to a "fingering" tumour morphology; and a high activity of the lactate/H+ symporter can result in a reversed transmembrane pH gradient across a large portion of the tumour mass. Also, the reversed pH gradient is spatially heterogeneous within the tumour, with a normal pH gradient observed within an intermediate growth layer within the spheroid. We also include a fractal dimension analysis of the simulated tumour contours, in which we compare the fractal dimensions of the simulated tumour surfaces with those found experimentally via photomicrographs.

Cancer cell bioenergetics and pH regulation influence breast cancer cell resistance to paclitaxel and doxorubicin

The multidrug resistance (MDR) phenotype, frequently observed during cancer treatment, is often associated with drug efflux pump activity. However, many other factors are also known to be involved. Cancer cells often rely on aerobic glycolysis for energy production; this is known as the "Warburg effect" and is used as a survival mechanism. Associated to this event, a reverse pH gradient across the cell membrane occurs, leading to cytosol alkalinization and extracellular acidification. In the present study, we investigated the role of different mechanisms involved in MDR, such as altered tumor microenvironment and energetic metabolism. The breast cancer cell line MCF-7, used as model, was exposed to two widely used antitumor drugs, paclitaxel (antimitotic agent) and doxorubicin (alkylating agent). Cancer pH regulation was shown to be crucial for malignant characteristics such as cell migration and drug resistance. Our results showed that a lower extracellular pH induced a higher migratory capacity and higher resistance to the studied chemotherapeutical compounds in MCF-7 cells. Besides the influence of the extracellular pH, the role of the tumor metabolism in the MDR phenotype was also investigated. Pre-treatment with different bioenergetic modulators led to cell ATP depletion and altered lactic acid production and glucose consumption, resulting in increased sensitivity to paclitaxel and doxorubicin. Overall, this study supports the potential use of compounds targeting cell metabolism and tumor microenvironment factors such as pH, as co-adjuvants in conventional chemotherapy.

Regulation of tumour intracellular pH: A mathematical model examining the interplay between H+ and lactate

Non-invasive measurements of pH have shown that both tumour and normal cells have intracellular pH (pHi) that lies on the alkaline side of neutrality (7.1–7.2). However, extracellular pH (pHe) is reported to be more acidic in some tumours compared to normal tissues. Many cellular processes and therapeutic agents are known to be tightly pH dependent which makes the study of intracellular pH regulation of paramount importance. We develop a mathematical model that examines the role of various membrane-based ion transporters in tumour pH regulation, in particular, with a focus on the interplay between lactate and H+ ions and whether the lactate/H+ symporter activity is sufficient to give rise to the observed reversed pH gradient that is seen is some tumours. Using linear stability analysis and numerical methods, we are able to gain a clear understanding of the relationship between lactate and H+ ions. We extend this analysis using perturbation techniques to specifically examine a rapid change in H+-ion concentrations relative to variations in lactate. We then perform a parameter sensitivity analysis to explore solution robustness to parameter variations. An important result from our study is that a reversed pH gradient is possible in our system but for unrealistic parameter estimates—pointing to the possible involvement of other mechanisms in cellular pH gradient reversal, for example acidic vesicles, lysosomes, golgi and endosomes.