Crosstalk with renal proximal tubule cells drives acidosis-induced inflammatory response and dedifferentiation of fibroblasts via p38-singaling.

BACKGROUND: Tubulointerstitial kidney disease associated microenvironmental dysregulation, like acidification, inflammation and fibrosis, affects tubule cells and fibroblasts. Micromilieu homeostasis influences intracellular signaling and intercellular crosstalk. Cell-cell communication in turn modulates the interstitial microenvironment. We assessed the impact of acidosis on inflammatory and fibrotic responses in proximal tubule cells and fibroblasts as a function of cellular crosstalk. Furthermore, cellular signaling pathways involved were identified. METHODS: HK-2 (human proximal tubule) and CCD-1092Sk (human fibroblasts), in mono and coculture, were exposed to acidic or control media for 3 or 48 h. Protein expression of inflammation markers (TNF, TGF-ß and COX-2), dedifferentiation markers (N-cadherin, vinculin, ß-catenin and vimentin), fibrosis markers (collagen III and fibronectin) and phospho- as well as total MAPK levels were determined by western blot. Secreted collagen III and fibronectin were measured by ELISA. The impact of MAPK activation was assessed by pharmacological intervention. In addition, necrosis, apoptosis and epithelial permeability were determined. RESULTS: Independent of culture conditions, acidosis caused a decrease of COX-2, vimentin and fibronectin expression in proximal tubule cells. Only in monoculture, ß-Catenin expression decreased and collagen III expression increased in tubule cells during acidosis. By contrast, in coculture collagen III protein expression of tubule cells was reduced. In fibroblasts acidosis led to an increase of TNF, COX-2, vimentin, vinculin, N-cadherin protein expression and a decrease of TGF-ß expression exclusively in coculture. In monoculture, expression of COX-2 and fibronectin was reduced. Collagen III expression of fibroblasts was reduced by acidosis independent of culture conditions. In coculture, acidosis enhanced phosphorylation of ERK1/2, JNK1/2 and p38 transiently in proximal tubule cells. In fibroblasts, acidosis enhanced phosphorylation of p38 in a sustained and very strong manner. ERK1/2 and JNK1/2 were not affected in fibroblasts. Inhibition of JNK1/2 and p38 under coculture conditions reduced acidosis-induced changes in fibroblasts significantly. CONCLUSIONS: Our data show that the crosstalk between proximal tubule cells and fibroblasts is crucial for acidosis-induced dedifferentiation of fibroblasts into an inflammatory phenotype. This dedifferentiation is at least in part mediated by p38 and JNK1/2. Thus, cell-cell communication is essential for the pathophysiological impact of tubulointerstitial acidosis.

Physicochemical aspects of the tumour microenvironment as drivers of vasculogenic mimicry.

Tumour vascularisation is vital for cancer sustainment representing not only the main source of nutrients and oxygen supply but also an escape route for single or clustered cancer cells that, once detached from the primary mass, enter the blood circulation and disseminate to distant organs. Among the mechanisms identified to contribute to tumour vascularisation, vasculogenic mimicry (VM) is gaining increasing interest in the scientific community representing an intriguing target for cancer treatment. VM indeed associates with highly aggressive tumour phenotypes and strongly impairs patient outcomes. Differently from vessels of healthy tissues, tumour vasculature is extremely heterogeneous and tortuous, impeding efficient chemotherapy delivery, and at the meantime hyperpermeable and thus extremely accessible to metastasising cancer cells. Moreover, tumour vessel disorganisation creates a self-reinforcing vicious circle fuelling cancer malignancy and progression. Because of the inefficient oxygen delivery and metabolic waste removal from tumour vessels, many cells within the tumour mass indeed experience hypoxia and acidosis, now considered hallmarks of cancer. Being strong inducers of vascularisation, therapy resistance, inflammation and metastasis, hypoxia and acidosis create a permissive microenvironment for cancer progression and dissemination. Along with these considerations, we decided to focus our attention on the relationship between hypoxia/acidosis and VM. Indeed, besides tumour angiogenesis, VM is strongly influenced by both hypoxia and acidosis, which could potentiate each other and fuel this vicious circle. Thus, targeting hypoxia and acidosis may represent a potential target to treat VM to impair tumour perfusion and cancer cell sustainment.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells.

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

A microenvironment of high lactate and low pH created by the blastocyst promotes endometrial receptivity and implantation.

RESEARCH QUESTION: Is the blastocyst's idiosyncratic metabolic production of lactate, and creation of a specialized microenvironment at the implatation site, an important mediator of maternal-fetal signalling to promote endometrial receptivity and implantation? DESIGN: Hormonally primed ECC-1 and Ishikawa cells were used to assess functional changes to the endometrial epithelium after exposure to lactic acid (LA), LA with neutralized pH (nLA) or acidic pH (pHL). Tight junction integrity (transepithelial resistance [TER]), cellular proliferation or changes to gene expression by RT-PCR were analysed. The effect of LA on Endometrial stromal cells decidualization and migratory capacity, and HUVEC endothelial tube formation and angiogenesis, were also assessed. RESULTS: Treatment of ECC-1 cells with 2.5 mM (P = 0.0037), 5 mM (P = 0.0044), 7.5 mM and 10 mM (P = 0.003) (P = 0.0021) LA significantly decreased the rate of cellular proliferation while TER was decreased with exposure to 2.5 mM LA (P = 0.024), 5 mM LA (P = 0.021) and 7.5 mM LA (P = 0.033). Exposure to nLA or pHL had no effect on proliferation or TER. Upregulation of GLUT4 (P = 0.002), GPR81 (P = 0.048), VEGF, SNAI1 (both P < 0.001) and RELA (P = 0.023) mRNA expression was observed after exposure of Ishikawa cells to combined LA plus pHL. Lactic acid increased the migratory capacity of decidualized stromal cells (P = 0.047) without changing the extent of decidualization. HUVEC tube formation was significantly increased by 5 mM LA exposure (P = 0.009). CONCLUSIONS: The identification of LA as an important mediator in the maternal-fetal dialogue underpinning implantation is supported. Further examination of the role of LA within the infertile or compromised endometrium could improve natural and assisted pregnancy success and needs further investigation.

Glycolysis-derived acidic microenvironment as a driver of endothelial dysfunction in systemic sclerosis.

OBJECTIVES: SSc is an autoimmune disease characterized by peripheral vasculopathy and skin and internal organ fibrosis. Accumulating evidence underlines a close association between a metabolic reprogramming of activated fibroblasts and fibrosis. This prompted us to determine the metabolism of SSc dermal fibroblasts and the effect on the vasculopathy characterizing the disease. METHODS: A Seahorse XF96 Extracellular Flux Analyzer was used to evaluate SSc fibroblast metabolism. In vitro invasion and capillary morphogenesis assays were used to determine the angiogenic ability of endothelial cells (ECs). Immunofluorescence, flow cytometry and real-time PCR techniques provided evidence of the molecular mechanism behind the impaired vascularization that characterizes SSc patients. RESULTS: SSc fibroblasts, compared with controls, showed a boosted glycolytic metabolism with increased lactic acid release and subsequent extracellular acidification that in turn was found to impair EC invasion and organization in capillary-like networks without altering cell viability. A molecular link between extracellular acidosis and endothelial dysfunction was identified as acidic ECs upregulated MMP-12, which cleaves and inactivates urokinase-type plasminogen activator receptor, impairing angiogenesis in SSc. Moreover, the acidic environment was found to induce the loss of endothelial markers and the acquisition of mesenchymal-like features in ECs, thus promoting the endothelial-to-mesenchymal transition process that contributes to both capillary rarefaction and tissue fibrosis in SSc. CONCLUSION: This study showed the relationship of the metabolic reprogramming of SSc dermal fibroblasts, extracellular acidosis and endothelial dysfunction that may contribute to the impairment and loss of peripheral capillary networks in SSc disease.

SOX2 as a novel contributor of oxidative metabolism in melanoma cells.

BACKGROUND: Deregulated metabolism is a hallmark of cancer and recent evidence underlines that targeting tumor energetics may improve therapy response and patient outcome. Despite the general attitude of cancer cells to exploit the glycolytic pathway even in the presence of oxygen (aerobic glycolysis or "Warburg effect"), tumor metabolism is extremely plastic, and such ability to switch from glycolysis to oxidative phosphorylation (OxPhos) allows cancer cells to survive under hostile microenvironments. Recently, OxPhos has been related with malignant progression, chemo-resistance and metastasis. OxPhos is induced under extracellular acidosis, a well-known characteristic of most solid tumors, included melanoma. METHODS: To evaluate whether SOX2 modulation is correlated with metabolic changes under standard or acidic conditions, SOX2 was silenced and overexpressed in several melanoma cell lines. To demonstrate that SOX2 directly represses HIF1A expression we used chromatin immunoprecipitation (ChIP) and luciferase assay. RESULTS: In A375-M6 melanoma cells, extracellular acidosis increases SOX2 expression, that sustains the oxidative cancer metabolism exploited under acidic conditions. By studying non-acidic SSM2c and 501-Mel melanoma cells (high- and very low-SOX2 expressing cells, respectively), we confirmed the metabolic role of SOX2, attributing SOX2-driven OxPhos reprogramming to HIF1α pathway disruption. CONCLUSIONS: SOX2 contributes to the acquisition of an aggressive oxidative tumor phenotype, endowed with enhanced drug resistance and metastatic ability.

Reciprocal regulation of two G protein-coupled receptors sensing extracellular concentrations of Ca2+ and H.

G protein-coupled receptors (GPCRs) are cell surface receptors that detect a wide range of extracellular messengers and convey this information to the inside of cells. Extracellular calcium-sensing receptor (CaSR) and ovarian cancer gene receptor 1 (OGR1) are two GPCRs that sense extracellular Ca(2+) and H(+), respectively. These two ions are key components of the interstitial fluid, and their concentrations change in an activity-dependent manner. Importantly, the interstitial fluid forms part of the microenvironment that influences cell function in health and disease; however, the exact mechanisms through which changes in the microenvironment influence cell function remain largely unknown. We show that CaSR and OGR1 reciprocally inhibit signaling through each other in central neurons, and that this is lost in their transformed counterparts. Furthermore, strong intracellular acidification impairs CaSR function, but potentiates OGR1 function. Thus, CaSR and OGR1 activities can be regulated in a seesaw manner, whereby conditions promoting signaling through one receptor simultaneously inhibit signaling through the other receptor, potentiating the difference in their relative signaling activity. Our results provide insight into how small but consistent changes in the ionic microenvironment of cells can significantly alter the balance between two signaling pathways, which may contribute to disease progression.

Acid-sensing ion channels 1a (ASIC1a) inhibit neuromuscular transmission in female mice.

Acid-sensing ion channels (ASIC) open in response to extracellular acidosis. ASIC1a, a particular subtype of these channels, has been described to have a postsynaptic distribution in the brain, being involved not only in ischemia and epilepsy, but also in fear and psychiatric pathologies. High-frequency stimulation of skeletal motor nerve terminals (MNTs) can induce presynaptic pH changes in combination with an acidification of the synaptic cleft, known to contribute to muscle fatigue. Here, we studied the role of ASIC1a channels on neuromuscular transmission. We combined a behavioral wire hanging test with electrophysiology, pharmacological, and immunofluorescence techniques to compare wild-type and ASIC1a lacking mice (ASIC1a (-/-) knockout). Our results showed that 1) ASIC1a (-/-) female mice were weaker than wild type, presenting shorter times during the wire hanging test; 2) spontaneous neurotransmitter release was reduced by ASIC1a activation, suggesting a presynaptic location of these channels at individual MNTs; 3) ASIC1a-mediated effects were emulated by extracellular local application of acid saline solutions (pH = 6.0; HEPES/MES-based solution); and 4) immunofluorescence techniques revealed the presence of ASIC1a antigens on MNTs. These results suggest that ASIC1a channels might be involved in controlling neuromuscular transmission, muscle contraction and fatigue in female mice.

Tumor Microenvironment Modulates Invadopodia Activity of Non-Selected and Acid-Selected Pancreatic Cancer Cells and Its Sensitivity to Gemcitabine and C18-Gemcitabine.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are favored by its extracellular matrix (ECM)-rich microenvironment, which is also highly hypoxic and acidic. Gemcitabine (GEM) is still the first-line therapy in PDAC. However, it is quickly deaminated to its inactive metabolite. Several GEM prodrugs have emerged to improve its cytotoxicity. Here, we analyzed how the acidic/hypoxic tumor microenvironment (TME) affects the response of PDAC cell death and invadopodia-mediated ECM proteolysis to both GEM and its C18 prodrug. METHODS: For this, two PDAC cell lines, PANC-1 and Mia PaCa-2 were adapted to pHe 6.6 or not for 1 month, grown as 3D organotypic cultures and exposed to either GEM or C18 in the presence and absence of acidosis and the hypoxia inducer, deferoxamine. RESULTS: We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments. CONCLUSIONS: We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.

Dual-Sensitive Fluorescent Nanoprobes for Simultaneously Monitoring In Situ Changes in pH and Matrix Metalloproteinase Expression in Stiffness-Tunable Three-Dimensional In Vitro Scaffolds.

A tumor microenvironment often presents altered physicochemical characteristics of the extracellular matrix (ECM) including changes in matrix composition, stiffness, protein expression, pH, temperature, or the presence of certain stromal and immune cells. Of these, overexpression of matrix metalloproteinases (MMPs) and extracellular acidosis are the two major hallmarks of cancer that can be exploited for tumor detection. The change in matrix stiffness and the release of certain cytokines (TNF-α) in the tumor microenvironment play major roles in inducing MMP-9 expression in cancerous cells. This study highlights the role of mechanical cues in upregulating MMP-9 expression in cancerous cells using stiffness-tunable matrix compositions and dual-sensitive fluorescent nanoprobes. Ionically cross-linked 3D alginate/gelatin (AG) scaffolds with three stiffnesses were chosen to reflect the ECM stiffnesses corresponding to healthy and pathological tissues. Moreover, a dual-sensitive nanoprobe, an MMP-sensitive peptide conjugated to carbon nanoparticles with intrinsic pH fluorescence properties, was utilized for in situ monitoring of the two cancer hallmarks in the 3D scaffolds. This platform was further utilized for designing a 3D core-shell platform for spatially mapping tumor margins and for visualizing TNF-α-induced MMP-9 expression in cancerous cells.

Acidosis Activates the Nrf2 Pathway in Renal Proximal Tubule-Derived Cells through a Crosstalk with Renal Fibroblasts.

Crosstalk of renal epithelial cells with interstitial fibroblasts plays an important role in kidney pathophysiology. A previous study showed that crosstalk between renal epithelial cells and renal fibroblasts protects against acidosis-induced damage. In order to gain further mechanistic insight into this crosstalk, we investigated the effect of acidosis on the transcriptome of renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) in co-culture by RNASeq, bioinformatics analysis and experimental validation. Cells were exposed to acidic media or control media for 48 h. RNA and protein from whole cell lysate were isolated. In addition, cells were fractionated into cytosol, nucleus and chromatin. RNASeq data were analyzed for differential expression and pathway enrichment (ingenuity pathway analysis, IPA, QIAGEN). Total and phosphorylated protein expression was assessed by Western blot (WB). Transcription factor activity was assessed by luciferase reporter assay. Bioinformatic analysis using differentially expressed genes according to RNASeq (7834 for NRK-52E and 3197 for NRK-49F) predicted the antioxidant and cell-protective Nrf2 pathway as acidosis-induced in NRK-52E and NRK-49F cells. Activation of Nrf2 comprises enhanced Nrf2 phosphorylation, nuclear translocation, DNA binding and initiation of a cell protective transcriptional program. Our data show that acidosis enhances chromatin-associated Nrf2 expression and the abundance of phosphorylated Nrf2 in the chromatin fraction of NRK-52E cells in co-culture but not in monoculture. Furthermore, acidosis enhances the activity of a reporter for Nrf2 (ARE-luciferase). Despite the bioinformatics prediction, NRK-49F cells did not respond with Nrf2 activation. Transketolase (TKT) is an important regulator of antioxidant and homeostatic responses in the kidney and a canonical Nrf2 target gene. We show that protein and mRNA expression of TKT is increased in NRK-52E cells under co-culture but not under monoculture conditions. In conclusion, our data show that extracellular acidosis activates the cytoprotective transcription factor Nrf2 in renal epithelial cells co-cultivated with renal fibroblasts, thereby enhancing the expression of cytoprotective TKT. This protective response is not observed in monoculture. Activation of the Nrf2 pathway represents a co-operative cellular strategy of protection against acidosis.

Extracellular Lactic Acidosis of the Tumor Microenvironment Drives Adipocyte-to-Myofibroblast Transition Fueling the Generation of Cancer-Associated Fibroblasts.

Lactic acidosis characterizes the tumor microenvironment (TME) and is involved in the mechanisms leading to cancer progression and dissemination through the reprogramming of tumor and local host cells (e.g., endothelial cells, fibroblasts, and immune cells). Adipose tissue also represents a crucial component of the TME which is receiving increasing attention due to its pro-tumoral activity, however, to date, it is not known whether it could be affected by the acidic TME. Now, emerging evidence from chronic inflammatory and fibrotic diseases underlines that adipocytes may give rise to pathogenic myofibroblast-like cells through the adipocyte-to-myofibroblast transition (AMT). Thus, our study aimed to investigate whether extracellular acidosis could affect the AMT process, sustaining the acquisition by adipocytes of a cancer-associated fibroblast (CAF)-like phenotype with a pro-tumoral activity. To this purpose, human subcutaneous adipose-derived stem cells committed to adipocytes (acADSCs) were cultured under basal (pH 7.4) or lactic acidic (pH 6.7, 10 mM lactate) conditions, and AMT was evaluated with quantitative PCR, immunoblotting, and immunofluorescence analyses. We observed that lactic acidosis significantly impaired the expression of adipocytic markers while inducing myofibroblastic, pro-fibrotic, and pro-inflammatory phenotypes in acADSCs, which are characteristic of AMT reprogramming. Interestingly, the conditioned medium of lactic acidosis-exposed acADSC cultures was able to induce myofibroblastic activation in normal fibroblasts and sustain the proliferation, migration, invasion, and therapy resistance of breast cancer cells in vitro. This study reveals a previously unrecognized relationship between lactic acidosis and the generation of a new CAF-like cell subpopulation from adipocytic precursor cells sustaining tumor malignancy.

Epithelial-Fibroblast Crosstalk Protects against Acidosis-Induced Inflammatory and Fibrotic Alterations.

Pathogenesis of chronic kidney disease (CKD) is accompanied by extracellular acidosis inflammation, fibrosis and epithelial-to-mesenchymal transition (EMT). The aim of this study was to assess the influence of acidosis on tubule epithelial cells (NRK-52E) and fibroblasts (NRK-49F) in dependence of cellular crosstalk. NRK-52E and NRK-49F were used in mono- and co-cultures, and were treated with acidic media (pH 6.0) for 48 h. The intracellular proteins were measured by Western blot. Secreted proteins were measured by ELISA. Distribution of E-cadherin was assessed by immunofluorescence and epithelial barrier function by FITC-dextran diffusion. Inflammation: Acidosis led to an increase in COX-2 in NRK-52E and TNF in NRK-49F in monoculture. In co-culture, this effect was reversed. EMT: Acidosis led to an increase in vimentin protein in both cell lines, whereas in co-culture, the effect was abolished. In NRK-52E, the E-cadherin expression was unchanged, but subcellular E-cadherin showed a disturbed distribution, and cellular barrier function was decreased. Fibrosis: Monoculture acidosis led to an increased secretion of collagen I and fibronectin in NRK-52E and collagen I in NRK-49F. In co-culture, the total collagen I secretion was unchanged, and fibronectin secretion was decreased. Intercellular crosstalk between epithelial cells and fibroblasts has a protective function regarding the development of acidosis-induced damage.

Motility and Mechanical Properties of Dendritic Cells Deteriorated by Extracellular Acidosis.

Dendritic cells (DCs) are the most powerful antigen-presenting cells known to date and play an important role in initiating and amplifying both innate and adaptive immune responses. Extracellular acidosis is an important hallmark of a variety of inflammatory processes and solid tumors. However, few studies have focused on the effect of extracellular acidosis on DCs and their functions. Cellular mechanical properties reflect the relationship between cell structure and function, including cytoskeleton (especially F-actin organization), membrane negative charges, membrane fluidity, and osmotic fragility. The study investigated the effects of extracellular acidosis on the DCs functions from the perspective of cellular migration and mechanical properties. The results showed that migration ability, F-actin contents, and membrane negative charges of DCs were reduced by extracellular acidosis no matter whether LPS stimulated its maturation or not. And these functions could not return to normal after removing acidic microenvironment, which revealed that the function impairment induced by extracellular acidosis might be irreversible. In addition, the proliferation capacity of stimulated allogeneic T cells was impaired by extracellular acidosis. Our results suggest extracellular acidosis may play an immunosuppressive role in DCs-mediated immune process.

Extracellular acidosis enhances Zika virus infection both in human cells and ex-vivo tissue cultures from female reproductive tract.

Zika virus (ZIKV) is a flavivirus transmitted by mosquitoes of the genus Aedes, but unlike other flaviviruses, ZIKV can be sexually transmitted by vaginal intercourse. The healthy vaginal pH ranges from 4.0 to 6.0, reaching values of 6.0-7.0 after semen deposition. Here, we report that low extracellular pH values (range 6.2-6.6) dramatically increase ZIKV infection on cell lines of different origin including some derived from the female genital tract and monocyte-derived macrophages. Furthermore, low pH significantly increased ZIKV infection of human ectocervix and endocervix cultured ex-vivo. Enhancement of infection by low pH was also observed using different ZIKV strains and distinct methods to evaluate viral infection, i.e. plaque assays, RT-PCR, flow cytometry, and fluorescence microscopy. Analysis of the mechanisms involved revealed that the enhancement of ZIKV infection induced by low pH was associated with increased binding of the viral particles to the heparan sulphate expressed on the target cell surface. Acidosis represents a critical but generally overlooked feature of the female genital tract, with major implications for sexual transmission diseases. Our results suggest that low vaginal pH might promote male-to-female transmission of ZIKV infection.

Acid Microenvironment in Bone Sarcomas.

In bone sarcomas, extracellular proton accumulation is an intrinsic driver of malignancy. Extracellular acidosis increases stemness, invasion, angiogenesis, metastasis, and resistance to therapy of cancer cells. It reprograms tumour-associated stroma into a protumour phenotype through the release of inflammatory cytokines. It affects bone homeostasis, as extracellular proton accumulation is perceived by acid-sensing ion channels located at the cell membrane of normal bone cells. In bone, acidosis results from the altered glycolytic metabolism of bone cancer cells and the resorption activity of tumour-induced osteoclasts that share the same ecosystem. Proton extrusion activity is mediated by extruders and transporters located at the cell membrane of normal and transformed cells, including vacuolar ATPase and carbonic anhydrase IX, or by the release of highly acidic lysosomes by exocytosis. To date, a number of investigations have focused on the effects of acidosis and its inhibition in bone sarcomas, including studies evaluating the use of photodynamic therapy. In this review, we will discuss the current status of all findings on extracellular acidosis in bone sarcomas, with a specific focus on the characteristics of the bone microenvironment and the acid-targeting therapeutic approaches that are currently being evaluated.

Extracellular acidosis triggers a senescence-like phenotype in human melanoma cells.

Acidosis of the tumor microenvironment is a characteristic of solid tumors such as malignant melanoma. Main causes of the extracellular acidification are metabolic alterations in cancer cells. While numerous studies showed that acidosis promotes tumor invasiveness, metastasis, and neoangiogenesis resulting in malignant progression, contrary data reported that acidosis induces cell apoptosis, inhibits cell proliferation, and mediates cell autophagy. Here, we show that low pH (pH 6.7) induces senescent/quiescent phenotype in melanoma cells after long-time treatment defined by induction of SA-ß-galactosidase, upregulation of p21, G1 /G0 cell cycle arrest, and reduction of proliferation. Moreover, we revealed that extracellular acidosis triggers the inhibition of eIF2α and subsequently the activation of ATF4 expression, a key component of the integrated stress response (ISR), indicating an acid-mediated translation reprogramming. Interestingly, we also demonstrated that acidosis represses microphthalmia-associated transcription factor (MITF) and activates the expression of the receptor tyrosine kinase AXL. This MITFlow /AXLhigh phenotype is correlated with drug resistance and therapeutic outcome in melanoma. Our results suggest that acidosis is an important microenvironmental factor triggering phenotypic plasticity and promoting tumor progression.

[Acid-sensing ion channels differentially affect ictal-like and non-ictal-like epileptic activities of mouse hippocampal pyramidal neurons in acidotic extracellular pH].

OBJECTIVE: To investigate the effects of acid-sensing ion channels (ASICs) on electrophysiological epileptic activities of mouse hippocampal pyramidal neurons in the extracellular acidotic condition. METHODS: We investigated effects of extracellular acidosis on epileptic activities induced by elevated extracellular K + concentration or the application of an antagonist of GABAA receptors in perfusate of mouse hippocampal slices under field potential recordings. We also tested the effects of extracellular acidosis on neuronal excitability under field potential recording and evaluated the changes in epileptic activities of the neurons in response to pharmacological inhibition of ASICs using a specific inhibitor of ASICs. RESULTS: Extracellular acidosis significantly suppressed epileptic activities of the hippocampal neurons by converting ictal-like epileptic activities to non-ictal-like epileptic activities in both high [K +]o and disinhibition models, and also suppressed the intrinsic excitability of the neurons. ASICs inhibitor did not antagonize the inhibitory effect of extracellular acidosis on ictal epileptic activities and intrinsic neuronal excitability, but exacerbated non-ictal epileptic activities of the neurons in extracellular acidotic condition in both high [K+]o and disinhibition models. CONCLUSIONS: ASICs can differentially modulate ictal-like and non-ictallike epileptic activities via its direct actions on excitatory neurons.

Acid sensing ion channels in rat cerebral arteries: Probing the expression pattern and vasomotor activity.

AIMS: The recent identification of acid sensing ion channels (ASICs) in vascular beds suggests their possible involvement in modulating vasomotor tone. Therefore, we investigated the gene expression profiles of ASIC subtypes in the middle cerebral artery (MCA) of Wistar rats and the functional implication of ASICs in acidosis-induced relaxation as well as maintenance of resting tension. MAIN METHODS: Real time PCR was employed to study the pattern of ASIC mRNA expression in the MCA wall in comparison with (i) matching brain tissue samples and (ii) arteries cultured for 24 h and 48 h. The functional implication regarding vasomotor response to acidosis and maintenance of resting tension was assessed using in vitro myography. KEY FINDINGS: A robust mRNA expression of ASIC-1, -2 and -4 was found in brain tissue samples and to a lower extent in freshly isolated MCA. In the MCA wall, short term culture induced a down-regulation of ASIC-1 and -2 expression without any remarkable change in ASIC-4 expression. Acidosis induced a pH-related relaxation of freshly isolated MCA ring segments, being more pronounced after short term culture. Incubation with the ASIC blocker amiloride moderately enhanced acidosis-induced relaxation, in cultured MCAs somewhat stronger than in freshly isolated vessels. In addition, amiloride resulted in a decrease of resting tension, albeit only in freshly isolated MCA. SIGNIFICANCE: Our results comprehensively describe ASIC subtype composition in the rat MCA in physiological and pathological conditions and strongly suggest the involvement of ASICs in the modulation of vasomotor responses under conditions of normal or decreased pH values.

Disturbances in H+ dynamics during environmental carcinogenesis.

Despite the improvement of diagnostic methods and anticancer therapeutics, the human population is still facing an increasing incidence of several types of cancers. According to the World Health Organization, this growing trend would be partly linked to our environment, with around 20% of cancers stemming from exposure to environmental contaminants, notably chemicals like polycyclic aromatic hydrocarbons (PAHs). PAHs are widespread pollutants in our environment resulting from incomplete combustion or pyrolysis of organic material, and thus produced by both natural and anthropic sources; notably benzo[a]pyrene (B[a]P), i.e. the prototypical molecule of this family, that can be detected in cigarette smoke, diesel exhaust particles, occupational-related fumes, and grilled food. This molecule is a well-recognized carcinogen belonging to group 1 carcinogens. Indeed, it can target the different steps of the carcinogenic process and all cancer hallmarks. Interestingly, H+ dynamics have been described as key parameters for the occurrence of several, if not all, of these hallmarks. However, information regarding the role of such parameters during environmental carcinogenesis is still very scarce. The present review will thus mainly give an overview of the impact of B[a]P on H+ dynamics in liver cells, and will show how such alterations might impact different aspects related to the finely-tuned balance between cell death and survival processes, thereby likely favoring environmental carcinogenesis. In total, the main objective of this review is to encourage further research in this poorly explored field of environmental molecular toxicology.