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COVID-19, a pandemic disease caused by a viral infection, is associated with a high mortality rate. Most of the signs and symptoms, e.g. cytokine storm, electrolytes imbalances, thromboembolism, etc., are related to mitochondrial dysfunction. Therefore, targeting mitochondrion will represent a more rational treatment of COVID-19. The current work outlines how COVID-19’s signs and symptoms are related to the mitochondrion. Proper understanding of the underlying causes might enhance the opportunity to treat COVID-19.

COVID-19, occurring due to SARS-COV-2 infection, is the most recent pandemic disease that has led to three million deaths at the time of writing. A great deal of effort has been directed towards altering the virus trajectory and/or managing the interactions of the virus with its subsequent targets in the human body; these interactions can lead to a chain reaction-like state manifested by a cytokine storm and progress to multiple organ failure. During cytokine storms the ratio of pro-inflammatory to anti-inflammatory mediators is generally increased, which contributes to the instigation of hyper-inflammation and confers advantages to the virus. Because cytokine expression patterns fluctuate from one person to another and even within the same person from one time to another, we suggest a road map of COVID-19 management using an individual approach instead of focusing on the blockbuster process (one treatment for most people, if not all). Here, we highlight the biology of the virus, study the interaction between the virus and humans, and present potential pharmacological and non-pharmacological modulators that might contribute to the global war against SARS-COV-2. We suggest an algorithmic roadmap to manage COVID-19.

In the present work, we investigated the potential of novel semi-interpenetrating polymer network (semi-IPN) cryogels, obtained through ultraviolet exposure of aqueous mixtures of poly(ethylene glycol) diacrylate and type I collagen, as tunable off-the-shelf platforms for 3D cancer cell research. We synthesized semi-IPN cryogels with variable collagen amounts (0.1% and 1% w/v) and assessed the effect of collagen on key cryogel properties for cell culture, for example, porosity, degradation rate and mechanical stiffness. Then, we investigated the ability of the cryogels to sustain the long-term growth of two pancreatic ductal adenocarcinoma (PDAC) cell populations, the parenchymal Panc1 cells and their derived cancer stem cells. Results revealed that both cell lines efficiently infiltrated, attached and expanded in the cryogels over a period of 14 days. However, only when grown in the cryogels with the highest collagen concentration, both cell lines reproduced their characteristic growth pattern previously observed in collagen-enriched organotypic cultures, biomimetic of the highly fibrotic PDAC stroma. Cellular preembedding in Matrigel, that is, the classical approach to develop/grow organoids, interfered with an efficient intra-scaffold migration and growth. Although preliminary, these findings highlight the potential of the proposed cryogels as reproducible and tunable cancer cell research platforms.

Cancer cells and tissues have an aberrant regulation of hydrogen ion dynamics driven by a combination of poor vascular perfusion, regional hypoxia, and increased the flux of carbons through fermentative glycolysis. This leads to extracellular acidosis and intracellular alkalinization. Dysregulated pH dynamics influence cancer cell biology, from cell transformation and tumorigenesis to proliferation, local growth, invasion, and metastasis. Moreover, this dysregulated intracellular pH (pHi) drives a metabolic shift to increased aerobic glycolysis and reduced mitochondrial oxidative phosphorylation, referred to as the Warburg effect, or Warburg metabolism, which is a selective feature of cancer. This metabolic reprogramming confers a thermodynamic advantage on cancer cells and tissues by protecting them against oxidative stress, enhancing their resistance to hypoxia, and allowing a rapid conversion of nutrients into biomass to enable cell proliferation. Indeed, most cancers have increased glucose uptake and lactic acid production. Furthermore, cancer cells have very dysregulated electrolyte balances, and in the interaction of the pH dynamics with electrolyte, dynamics is less well known. In this review, we highlight the interconnected roles of dysregulated pH dynamics and electrolytes imbalance in cancer initiation, progression, adaptation, and in determining the programming and reprogramming of tumor cell metabolism.

Low dose metronomic chemotherapy (MC) is becoming a mainstream treatment for cancer in veterinary medicine. Its mechanism of action is anti-angiogenesis by lowering vascular endothelial growth factor (VEGF) and increasing trombospondin-1 (TSP1). It has also been adopted as a compassionate treatment in very advanced human cancer. However, one of the main limitations of this therapy is its short-term effectiveness: 6 to 12 months, after which resistance develops. pH-centered cancer treatment (pHT) has been proposed as a complementary therapy in cancer, but it has not been adopted or tested as a mainstream protocol, in spite of existing evidence of its advantages and benefits. Many of the factors directly or indirectly involved in MC and anti-angiogenic treatment resistance are appropriately antagonized by pHT. This led to the testing of an association between these two treatments. Preliminary evidence indicates that the association of MC and pHT has the ability to reduce anti-angiogenic treatment limitations and develop synergistic anti-cancer effects. This review will describe each of these treatments and will analyze the fundamentals of their synergy.

Cancer cells have an unusual regulation of hydrogen ion dynamics that are driven by poor vascularity perfusion, regional hypoxia, and increased glycolysis. All these forces synergize/orchestrate together to create extracellular acidity and intracellular alkalinity. Precisely, they lead to extracellular pH (pHe) values as low as 6.2 and intracellular pH values as high as 8. This unique pH gradient (∆pHi to ∆pHe) across the cell membrane increases as the tumor progresses, and is markedly displaced from the electrochemical equilibrium of protons. These unusual pH dynamics influence cancer cell biology, including proliferation, metastasis, and metabolic adaptation. Warburg metabolism with increased glycolysis, even in the presence of Oxygen with the subsequent reduction in Krebs’ cycle, is a common feature of most cancers. This metabolic reprogramming confers evolutionary advantages to cancer cells by enhancing their resistance to hypoxia, to chemotherapy or radiotherapy, allowing rapid production of biological building blocks that support cellular proliferation, and shielding against damaging mitochondrial free radicals. In this article, we highlight the interconnected roles of dysregulated pH dynamics in cancer initiation, progression, adaptation, and in determining the programming and re-programming of tumor cell metabolism.

Helicobacter pylori (HP) is a facultative anaerobic bacterium. HP is a normal flora having immuno-modulating properties. This bacterium is an example of a microorganism inducing gastric cancer. Its carcinogenicity depends on bacteria-host related factors. The proper understanding of the biology of HP inducing gastric cancer offers the potential strategy in the managing of HP rather than eradicating it. In this article, we try to summarize the biology of HP-induced gastric cancer and discuss the current pharmacological approach to treat and prevent its carcinogenicity.

Background Information While enolase is a ubiquitous metalloenzyme involved in the glycolytic pathway, it is also known as a multifunctional protein, since enolases anchored on the outer surface of the plasma membrane are involved in tissue invasion. Results We have identified an extracellular enolase (Ae-ENO) produced by the teratocytes, embryonic cells of the insect parasitoid Aphidius ervi. We demonstrate that Ae-ENO, although lacking a signal peptide, accumulates in cytoplasmic vesicles oriented towards the cell membrane. Ae-ENO binds to and activates a plasminogen-like molecule inducing digestion of the host tissue and thereby ensuring successful parasitism. Conclusions These results support the hypothesis that plasminogen-like proteins exist in invertebrates. Interestingly the activation of a plasminogen-like protein is mediated by a mechanisms involving the surface enolase/fibrinolytic system considered, until now, exclusive of vertebrates, and that instead is conserved across species. Significance To our knowledge, this is the first example of enolase mediated Plg-like binding and activation in insect cells, demonstrating the existence of an ECM degradation process via a Plg-like protein in invertebrates.

While research previously focused extensively on the tumor cells, over the last two decades, the tumor microenvironment (TME) has received increasing attention with a particular emphasis in its role in tumor development, metabolism, progression, and treatment response [...]

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers principally because of early invasion and metastasis. The epidermal growth factor receptor (EGFR) is essential for PDAC development even in the presence of Kras, but its inhibition with erlotinib gives only a modest clinical response, making the discovery of novel EGFR targets of critical interest. Here, we revealed by mining a human pancreatic gene expression database that the metastasis promoter Na+/H+ exchanger (NHE1) associates with the EGFR in PDAC. In human PDAC cell lines, we confirmed that NHE1 drives both basal and EGF-stimulated three-dimensional growth and early invasion via invadopodial extracellular matrix digestion. EGF promoted the complexing of EGFR with NHE1 via the scaffolding protein Na+/H+ exchanger regulatory factor 1, engaging EGFR in a negative transregulatory loop that controls the extent and duration of EGFR oncogenic signaling and stimulates NHE1. The specificity of NHE1 for growth or invasion depends on the segregation of the transient EGFR/Na+/H+ exchanger regulatory factor 1/NHE1 signaling complex into dimeric subcomplexes in different lipid raftlike membrane domains. This signaling complex was also found in tumors developed in orthotopic mice. Importantly, the specific NHE1 inhibitor cariporide reduced both three-dimensional growth and invasion independently of PDAC subtype and synergistically sensitized these behaviors to low doses of erlotinib.

Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress the epidermal growth factor receptor (EGFR), but are resistant to tyrosine kinase inhibitors (TKIs) and anti-EGFR therapies. Mechanisms suggested for resistance to TKIs include EGFR independence, mutations and alterations in EGFR and in its downstream signalling pathways. Ligand-induced endocytosis and degradation of EGFR play important roles in the downregulation of the EGFR signal suggesting that its activity could be regulated by targeting its trafficking. Evidence in normal cells showing that the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) can associate with EGFR to regulate its trafficking, led us to hypothesize that NHERF1 expression levels could regulate EGFR trafficking and functional expression in TNBC cells and, in this way, modulate its role in progression and response to treatment. We investigated the subcellular localization of NHERF1 and its interaction with EGFR in a metastatic basal like TNBC cell model, MDA-MB‑231, and the role of forced NHERF1 overexpression and/or stimulation with EGF on the sensitivity to EGFR specific TKI treatment with gefitinib. Stimulation with EGF induces an interaction of NHERF1 with EGFR to regulate its localization, degradation and function. NHERF1 overexpression is sufficient to drive its interaction with EGFR in non-stimulated conditions, inhibits EGFR degradation and increases its retention time in the plasma membrane. Importantly, NHERF1 overexpression strongly sensitized the cell to the pharmacological inhibition by gefitinib of EGFR-driven growth, motility and invadopodia-dependent ECM proteolysis. The further determination of how the NHERF1‑EGFR interaction is regulated may improve our understanding of TNBC resistance to the action of existing anticancer drugs.

Cancer cells acquire an unusual glycolytic behavior relative, to a large extent, to their intracellular alkaline pH (pHi). This effect is part of the metabolic alterations found in most, if not all, cancer cells to deal with unfavorable conditions, mainly hypoxia and low nutrient supply, in order to preserve its evolutionary trajectory with the production of lactate after ten steps of glycolysis. Thus, cancer cells reprogram their cellular metabolism in a way that gives them their evolutionary and thermodynamic advantage. Tumors exist within a highly heterogeneous microenvironment and cancer cells survive within any of the different habitats that lie within tumors thanks to the overexpression of different membrane-bound proton transporters. This creates a highly abnormal and selective proton reversal in cancer cells and tissues that is involved in local cancer growth and in the metastatic process. Because of this environmental heterogeneity, cancer cells within one part of the tumor may have a different genotype and phenotype than within another part. This phenomenon has frustrated the potential of single-target therapy of this type of reductionist therapeutic approach over the last decades. Here, we present a detailed biochemical framework on every step of tumor glycolysis and then proposea new paradigm and therapeutic strategy based upon the dynamics of the hydrogen ion in cancer cells and tissues in order to overcome the old paradigm of one enzyme-one target approach to cancer treatment. Finally, a new and integral explanation of the Warburg effect is advanced.

Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease- dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the inva- dopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na + /H + exchanger 1 (NHE1)-driven extracellular acidifica - tion, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs pref- erentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1-dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidifi - cation and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.

Background information P2×7R is a member of the ionotropic family of purinergic receptors activated by millimolar concentrations of extracellular ATP such as induced by inflammatory stimuli. The receptor is widely expressed in cells of haematopoietic origin such as monocytes, macrophages and microglia. There is growing interest in anta-gonist compounds of the P2×7R since it has been demonstrated to be a viable therapeutic target for inflammatory diseases. Here, we tested the possible P2×7 antagonist effect of MED1101, a newly synthesised dialdehydic compound on U937 monocyte cells. Results Human U937 cells express the full-length P2×7A receptor isoform. Treatment with lipopolysaccharide (LPS), a potent inducer of inflammation, significantly increased the expression of the receptor in the plasma membrane. Importantly, MED1101 induced internalisation of the P2×7R already after 30 min incubation in both physiological conditions and in presence of the inflammatory stimulus (LPS) and this effect was observable for up to 12 h after its removal. Moreover, MED1101 induced an impairment of monocyte migration/transmigration through direct P2×7R antagonism and subsequent inhibition of the intracellular signal transduction processes of Ca2+ influx and MAPK phosphorylation. Conclusions Our results clearly demonstrate that in U937 monocyte cells MED1101 acts as a P2×7R antagonist through the induction of receptor internalisation and subsequent inhibition of down-stream signal transduction pathways that regulate monocyte migration/transmigration, thus playing a potential therapeutic role in inflammatory diseases.

During the last few years, the understanding of the dysregulated hydrogen ion dynamics and reversed proton gradient of cancer cells has resulted in a new and integral pH-centric paradigm in oncology, a translational model embracing from cancer etiopathogenesis to treatment. The abnormalities of intracellular alkalinization along with extracellular acidification of all types of solid tumors and leukemic cells have never been described in any other disease and now appear to be a specific hallmark of malignancy. As a consequence of this intracellular acid-base homeostatic failure, the attempt to induce cellular acidification using proton transport inhibitors and other intracellular acidifiers of different origins is becoming a new therapeutic concept and selective target of cancer treatment, both as a metabolic mediator of apoptosis and in the overcoming of multiple drug resistance (MDR). Importantly, there is increasing data showing that different ion channels contribute to mediate significant aspects of cancer pH regulation and etiopathogenesis. Finally, we discuss the extension of this new pH-centric oncological paradigm into the opposite metabolic and homeostatic acid-base situation found in human neurodegenerative diseases (HNDDs), which opens novel concepts in the prevention and treatment of HNDDs through the utilization of a cohort of neural and non-neural derived hormones and human growth factors.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease with a very poor prognosis, due to the influence of the tumor stroma, which promotes tumor growth, early invasion and chemoradiation resistance. Efforts to develop models for identifying novel anticancer therapeutic compounds have been hampered by the limited ability of in vitro models to mimic these in vivo tumor-stroma interactions. This has led to the development of various three-dimensional (3D) culture platforms recapitulating the in vivo tumor-stroma crosstalk and designed to better understand basic cancer processes and screen drug action. However, a consensus for different experimental 3D platforms is still missing in PDAC. We compared four PDAC cell lines of different malignancy grown in 2D monolayers to three of the more commonly used 3D techniques (ultralow adhesion concave microwells, Matrigel inclusion and organotypic systems) and to tumors derived from their orthotopic implantation in mice. In these 3D platforms, we observed that cells grow with very different tumor morphologies and the organotypic setting most closely resembles the tumor cytoarchitecture obtained by orthotopically implanting the four cell lines in mice. We then analyzed the molecular and cellular responses of one of these cell lines to epidermal growth factor receptor (EGFR) stimulation with EGF and inhibition with erlotinib and found that only in the 3D platforms, and especially the organotypic, cells: i) responded to EGF by changing the expression of signalling components underlying cell-stroma crosstalk and tissue architecture, growth, invasion and drug resistance (E-cadherin, EGFR, ezrin, β1 integrin, NHERF1 and HIF-1α) similar to those reported in vivo; ii) had stimulated growth and increased erlotinib sensitivity in response to EGF, more faithfully mimicking their known in vivo behaviour. Altogether, these results, indicate the organotypic as the most relevant physiological 3D system to study the complex tumor stroma interactions driving progression and determining chemio-resistance.

Bellizzi A, Mangia A, Malfettone A, Cardone R A, Simone G, Reshkin S J & Paradiso A (2011) Histopathology58, 1086–1095 Na+/H+exchanger regulatory factor 1 expression levels in blood and tissue predict breast tumour clinical behaviour Aims: Several studies have demonstrated that Na+/H+ exchanger regulatory factor 1 (NHERF1) protein, which is overexpressed and heterogeneously distributed in different stages of breast cancer, could be used as a tumour marker for prognosis in molecular detection strategies. We observed that tumour-infiltrated lymphocytes in the tumour tissue display a high level of NHERF1 staining, in contrast to those present in the contiguous non-involved tissue. Hypothesizing that cancer cells elicit a specific T-cell response associated with the characteristics of the solid tumour, our aim was to evaluate NHERF1 in peripheral lymphocytes from healthy donors and breast cancer patients. Methods and results: NHERF1 levels were analysed in 55 breast cancer patients and 40 healthy donors, and these levels were compared with clinical pathological features. NHERF1 was overexpressed in circulatory peripheral lymphocytes from patients as compared with those from healthy subjects. Furthermore, in both circulatory lymphocytes and tissues, NHERF1 was positively associated with tumour grade, Nottingham Prognostic Index and oestrogen receptor, whereas there was no association with other clinical parameters in either tissue. Conclusions: We propose that NHERF1 measurements in circulatory lymphocytes of breast cancer patients may be a valid method for the prediction of breast cancer occurrence and prognosis, and may have value in the management of cancer patients.

BACKGROUND INFORMATION: CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, DeltaF508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na(+)/H(+)-exchanger regulatory factor 1) in CF airway cells induced both a redistribution of DeltaF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of DeltaF508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o(-), with subsequent rescue of apical DeltaF508CFTR chloride transport activity. RESULTS: We found that CFBE41o(-) cells do express ERs (oestrogen receptors) in the nuclear fraction and that beta-oestradiol treatment was able to significantly rescue DeltaF508CFTR-dependent chloride secretion in CFBE41o(-) cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the DeltaF508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM beta-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the beta-oestradiol-dependent increase in DeltaF508CFTR protein expression levels and completely prevented the beta-oestradiol-dependent rescue of DeltaF508CFTR transport activity. CONCLUSIONS: These results demonstrate that beta-oestradiol-dependent up-regulation of NHERF1 significantly increases DeltaF508CFTR functional expression in CFBE41o(-) cells.

There is evidence that cystic fibrosis transmembrane conductance regulator (CFTR) interacting proteins play critical roles in the proper expression and function of CFTR. The Na(+)/H(+) exchanger regulatory factor isoform 1 (NHERF1) was the first identified CFTR-binding protein. Here we further clarify the role of NHERF1 in the regulation of CFTR activity in two human bronchial epithelial cell lines: the normal, 16HBE14o-, and the homozygous DeltaF508 CFTR, CFBE41o-. Confocal analysis in polarized cell monolayers demonstrated that NHERF1 distribution was associated with the apical membrane in 16HBE14o- cells while being primarily cytoplasmic in CFBE41o- cells. Transfection of 16HBE14o- monolayers with vectors encoding for wild-type (wt) NHERF1 increased both apical CFTR expression and apical protein kinase A (PKA)-dependent CFTR-mediated chloride efflux, whereas transfection with NHERF1 mutated in the binding groove of the PDZ domains or truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-dependent chloride efflux. These data led us to hypothesize an important role for NHERF1 in regulating CFTR localization and stability on the apical membrane of 16HBE14o- cell monolayers. Importantly, wt NHERF1 overexpression in confluent DeltaF508 CFBE41o- and DeltaF508 CFT1-C2 cell monolayers induced both a significant redistribution of CFTR from the cytoplasm to the apical membrane and a PKA-dependent activation of CFTR-dependent chloride secretion.

Recent research has highlighted the fundamental role of the tumour's extracellular metabolic microenvironment in malignant invasion. This microenvironment is acidified primarily by the tumour-cell Na(+)/H(+) exchanger NHE1 and the H(+)/lactate cotransporter, which are activated in cancer cells. NHE1 also regulates formation of invadopodia - cell structures that mediate tumour cell migration and invasion. How do these alterations of the metabolic microenvironment and cell invasiveness contribute to tumour formation and progression?

Background The aim of this study was to evaluate the effects of vitrification on morpho-functional parameters (blastomere/chromatin integrity and bioenergy/oxidative potential) of mouse preimplantation embryos. Methods In vivo produced mouse (4/16-cell, morulae and blastocyst-stage) embryos were randomly divided into vitrification and control groups. For vitrification, embryos were exposed to a 2-step loading of ethylene glycol and propylene glycol, before being placed in a small nylon loop and submerged into liquid nitrogen. After warming, the cryoprotectants were diluted by a 3-step procedure. Embryo morphology, chromatin integrity and energy/oxidative status were compared between groups. Results Vitrification induced low grade blastomere cytofragmentation (P

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires matrix degrading protrusions called invadopodia. The Na(+)/H(+) exchanger (NHE1) has recently been shown to be fundamental in the regulation of invadopodia actin cytoskeleton dynamics and activity. However, the structural link between the invadopodia cytoskeleton and NHE1 is still unknown. A candidate could be ezrin, a linker between the NHE1 and the actin cytoskeleton known to play a pivotal role in invasion and metastasis. However, the mechanistic basis for its role remains unknown. Here, we demonstrate that ezrin phosphorylated at T567 is highly overexpressed in the membrane of human breast tumors and positively associated with invasive growth and HER2 overexpression. Further, in the metastatic cell line, MDA-MB-231, p-ezrin was almost exclusively expressed in invadopodia lipid rafts where it co-localized in a functional complex with NHE1, EGFR, ß1-integrin and phosphorylated-NHERF1. Manipulation by mutation of ezrins T567 phosphorylation state and/or PIP2 binding capacity or of NHE1s binding to ezrin or PIP2 demonstrated that p-ezrin expression and binding to PIP2 are required for invadopodia-mediated ECM degradation and invasion and identified NHE1 as the membrane protein that p-ezrin regulates to induce invadopodia formation and activity.

Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression has been demonstrated to be altered in breast cancer, but its role in mammary cancerogenesis and progression remains still undefined. In this paper, we review what is known on the pathological role and the potential clinical application of NHERF1 protein in breast cancer. Recent evidence shows that an increased cytoplasmic expression of NHERF1 suggests a key role of its localization/compartmentalization in defining cancerogenesis, progression, and invasion. NHERF1 overexpression is associated with increasing tumor cytohistological grade, aggressive clinical behavior, unfavorable prognosis, and increased tumor hypoxia. Moreover, NHERF1 co-localizes with the oncogenic receptor HER2/neu in HER2/neu-overexpressing carcinoma and in distant metastases. These data make NHERF1 also a potential candidate of clinical relevance for anti-HER2/neu therapy.

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires membrane and released proteases focalized at membrane structures called invadopodia. While extracellular acidification is important in driving tumor invasion, the structure/function mechanisms underlying this regulation are still unknown. Invadopodia are similar in structure and function to osteoclast podosomes responsible for bone degradation, and extracellular acidification is central to podosome action, suggesting that it could also be for invadopodial function. Here, utilizing a novel system for in situ zymography in native matrices, we show that the Na(+)/H(+) exchanger (NHE1) and NHE1-generated extracellular acidification are localized at and necessary for invadopodial-dependent ECM degradation, thereby promoting tumor invasion. Stimulation with EGF increased both NHE1-dependent proton secretion and ECM degradation. Manipulation of the NHE1 expression by RNA interference or activity via either transport-deficient mutation or the specific inhibitor cariporide confirmed that NHE1 expression and activity are required for invadopodia-mediated ECM degradation. Taken together, our data show a concordance among NHE1 localization, the generation of a well-defined acidic extracellular pH in the nanospace surrounding invadopodia, and matrix-degrading activity at invadopodia of human malignant breast carcinoma cells, providing a structural basis for the role of NHE1 in invasion and identifying NHE1 as a strategic target for therapeutic intervention.

Background Endothelin-1 (ET-1) and Nitric Oxide (NO) are crucial mediators for establishing pulmonary artery hypertension (PAH). We tested the hypothesis that their imbalance might also occur in COPD patients with PAH. Methods The aims of the study were to measure exhaled breath condensate (EBC) and circulating levels of ET-1, as well as exhaled NO (FENO) levels by, respectively, a specific enzyme immunoassay kit, and by chemiluminescence analysis in 3 groups of subjects: COPD with PAH (12), COPD only (36), and healthy individuals (15). In order to evaluate pulmonary-artery systolic pressure (PaPs), all COPD patients underwent Echo-Doppler assessment. Results Significantly increased exhaled and circulating levels of ET-1 were found in COPD with PAH compared to both COPD (p < 0.0001) only, and healthy controls (p < 0.0001). In COPD with PAH, linear regression analysis showed good correlation between ET-1 in EBC and PaPs (r = 0.621; p = 0.031), and between arterial levels of ET-1 and PaPs (r = 0.648; p = 0.022), while arterial levels of ET-1 inversely correlated with FEV1%, (r = -0.59, p = 0.043), and PaPs negatively correlated to PaO2 (r = -0.618; p = 0.032). Significantly reduced levels of FENO were found in COPD associated with PAH, compared to COPD only (22.92 ± 11.38 vs.35.07 ± 17.53 ppb; p = 0.03). Thus, we observed an imbalanced output in the breath between ET-1 and NO, as expression of pulmonary endothelium and epithelium impairment, in COPD with PAH compared to COPD only. Whether this imbalance is an early cause or result of PAH due to COPD is still unknown and deserves further investigations.

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na+/H+exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1α expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na+/H+exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.

Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.

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 (ΔpHito 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.

We have previously demonstrated that in A(6) renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A(1) and A(2A) receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A(3) receptor-selective agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca(2+) influx. The agonist-induced rise in [Ca(2+)](i) was significantly inhibited by the selective adenosine A(3) receptor antagonists, 2,3-diethyl-4, 5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A(1) or A(2) receptors supporting the hypothesis that Cl-IB-MECA increases [Ca(2+)](i) by interacting exclusively with A(3) receptors. Cl-IB-MECA-elicited Ca(2+) entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca(2+)](i) response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A(3) receptor antagonist. Altogether, these data suggest that in A(6) cells a G(s)/protein kinase A pathway is involved in the A(3) receptor-dependent increase in calcium entry.

Accelerated osteoclastogenesis is the major event promoting the skeletal impairment in multiple myeloma (MM). Osteoclasts (OC) are directly activated by myeloma cells (MC), although these cells themselves may apparently undergo to OC-like morphologic transformation and produce bone erosion in vitro. Since OCs exert their function and promote osteoclastogenesis through activation of several adhesion molecules, including avb3, we investigated the role of this integrin expressed by MCs in their OC-like activity in vitro. Bone marrow MCs were purified from eight patients with severe skeletal disease (group A) and from two patients without bone lesions (group B). U266 and RPMI-8226 MC lines were the controls. Semi-nested PCR assessed the CDR3 immunoglobulin (Ig) gene rearrangement, whereas OC markers including TRAcP, cathepsin-k, calcitonin-receptor, carbonic anhydrase and vATPase were evaluated by RT-PCR. The cytoskeletal rearrangement of F-actin was analyzed by immunofluorescence. av and b3 expression on MCs was evaluated by flow-cytometry, whereas bone erosion on calcium phosphate discs and number of pits was measured by dedicated software. The effect of avb3 stimulation on the activation of osteoclastogenic function was investigated by exploring the phosphorilation of transcriptional kinases and downstream molecules, as ERK1/2 and cFos, respectively. The primary role of avb3 in OC-like functional transdifferentiation was explored in MCs by siRNA silencing for both chains. Ontogenetic derivation from the B-cell lineage was confirmed by the monoclonal CDR3 rearrangement, CD138/CD38 and Pax-5 expression. Cells from patients of group A expressed OC markers, in contrast with those of group B or U266 and RPMI-8226. Formation of the F-actin ring confirmed the differentiation of MCs toward the OC-like phenotype. Cells from group A expressed av and b3 (80±7% and 75±9%) similarly to U266 and RPMI-8226 (>90% in both instances), whereas a minimal expression was demonstrated in group B (av:6±2%; b3:8±3%). avb3+ cells produced a high number of erosive pits, at variance from avb3− cells (35±8 vs. 4±1 pits/cm2 ). The highest phosphorilation of ERK1/2 and expression of cFos was revealed in patients of group A as compared to B (840±110 OD and 905±210 OD vs. 270±35 OD and 315±80 OD, p

RhoA protein is over-expressed in breast cancer and other solid tumors and has been used in tumor biopsies as a quantitative tumor marker for progression, stage and prognosis in molecular detection strategies. Measuring protein markers in plasma or blood cells is preferred to tumor biopsies as it represents a minimally invasive, repeatable measurement that can be followed over time. In this study we evaluated the hypothesis that quantitative RhoA protein expression in circulatory lymphocytes is identically associated with the same tumor clinico-pathological features found in biopsies. RhoA protein levels were analyzed by Western blotting in circulating lymphocytes isolated from 52 consecutive patients with breast cancer and in 34 paired breast tumor biopsies from the same case study, and compared with the following clinico-pathological features of the patients: histological grade, tumor size, steroid receptor status, lymphonode status, proliferative activity and prognosis [Nottingham Prognostic Index (NPI)]. We observed that the level of circulatory, peripheral lymphocyte RhoA expression reflected that found in the matched biopsy of the same patient. Furthermore, similarly to previous reports regarding breast cancer tissue biopsies, the level of RhoA protein expression in both biopsies and in circulatory lymphocytes was positively associated with tumor size, grade, proliferative activity of the tumor biopsy and NPI, while there was no significant association of RhoA protein expression with either estrogen- or progesterone-receptor expression. Our study demonstrated that the association of lymphocyte RhoA protein expression with classical clinico-pathological parameters closely corresponded with that observed for RhoA protein expression in the tumor biopsies. We propose that measurement of RhoA expression in the circulatory lymphocytes of breast cancer patients can be used to predict breast cancer occurrence, progression and prognosis and may prove valuable in the management of cancer patients.

Background The ability to cryopreserve mammalian embryos has become an integral part of assisted reproduction, both in human and veterinary medicine. Despite differences in the size and physiological characteristics of embryos from various species, the embryos have been frozen by either of two procedures: slow freezing or vitrification. The aim of our study was to compare the effect of slow freezing and vitrification to the chromatin structure, energy status and reactive oxygen species production of mouse morulae and blastocysts. Methods Mouse morulae and blastocysts were randomly allocated into vitrification, slow freezing and control groups. For slow freezing, Dulbecco phosphate buffered saline based 10% glicerol solution was used. For vitrification, G-MOPS™ based solution supplemented with 16% ethylene glycol, 16% propylene glycol, Ficoll (10 mg/ml) and sucrose (0.65 mol/l) was used. After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups. Results Cryopreservation affected chromatin integrity at a greater extent at the morula than the blastocyst stage. Chromatin damage induced by slow freezing was more relevant compared to vitrification. Slow freezing and vitrification similarly affected mitochondrial distribution pattern. Greater damage was observed at the morula stage and it was associated with embryo grade. Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts. Effects induced by slow freezing were not related to embryo grade or mitochondrial pattern, as affected embryos were of all grades and with both mitochondrial patterns. However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status. Conclusions This study shows for the first time the joint assessment of chromatin damage and mitochondrial energy/redox potential in fresh and frozen mouse embryos at the morula and blastocyst stage, allowing the comparison of the effects of the two most commonly used cryopreservation procedures.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies. Present-day treatments have not shown real improvements in reducing the high mortality rate and the short survival of the disease. The average survival is less than 5% after 5 years. New innovative treatments are necessary to curtail the situation. The very dense pancreatic cancer stroma is a barrier that impedes the access of chemotherapeutic drugs and at the same time establishes a pro-proliferative symbiosis with the tumor, thus targeting the stroma has been suggested by many authors. No ideal drug or drug combination for this targeting has been found as yet. With this goal in mind, here we have explored a different complementary treatment based on abundant previous publications on repurposed drugs. The cell surface protein CD44 is the main receptor for hyaluronan binding. Many malignant tumors show over-expression/over-activity of both. This is particularly significant in pancreatic cancer. The independent inhibition of hyaluronan-producing cells, hyaluronan synthesis, and/or CD44 expression, has been found to decrease the tumor cell’s proliferation, motility, invasion, and metastatic abilities. Targeting the hyaluronan-CD44 pathway seems to have been bypassed by conventional mainstream oncological practice. There are existing drugs that decrease the activity/expression of hyaluronan and CD44: 4-methylumbelliferone and bromelain respectively. Some drugs inhibit hyaluronan-producing cells such as pirfenidone. The association of these three drugs has never been tested either in the laboratory or in the clinical setting. We present a hypothesis, sustained by hard experimental evidence, suggesting that the simultaneous use of these nontoxic drugs can achieve synergistic or added effects in reducing invasion and metastatic potential, in PDAC. A non-toxic, low-cost scheme for inhibiting this pathway may offer an additional weapon for treating pancreatic cancer.