The chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma. Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways. In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells.

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline-a specific inhibitor of large-conductance Ca2+-activated BK channels. The TEA-insensitive component was inhibited by senicapoc-a specific inhibitor of the Ca2+-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T. Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole-two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

There is a huge body of literature data on ZnOnanoparticles (ZnO NPs) toxicity. However, the reported results are seen to be increasingly discrepant, and deep comprehension of the ZnO NPs behaviour in relation to the different experimental conditions is still lacking. A recent literature overview emphasizes the screening of the ZnO NPs toxicity with more than one assay, checking the experimental reproducibility also versus time, which is a key factor for the robustness of the results. In this paper we compared high-throughput real-time measurements through Electric Cell-substrate Impedance-Sensing (ECIS®) with endpoint measurements of multiple independent assays.

The beneficial effect of Melatonin (Mel), recognized as an anti-inflammatory and antioxidant compound, has been already proven to prevent oxidative stress-induced damage associated to lipid peroxidation. As previous studies modeled the impact of oxidative stress on Band 3 protein, an anion exchanger that is essential to erythrocytes homeostasis, by applying H2O2 at not hemolytic concentrations and not producing lipid peroxidation, the aim of the present work was to evaluate the possible antioxidant effect of pharmacological doses of Mel on Band 3 protein anion exchange capability. The experiments have been performed on human erythrocytes exposed to 300 μM H2O2-induced oxidative stress. To this end, oxidative damage has been verified by monitoring the rate constant for SO4= uptake through Band 3 protein. Expression levels of this protein Mel doses lower than 100 µM have also been excluded due to lipid peroxidation, Band 3 protein expression levels, and cell shape alterations, confirming a pro-oxidant action of Mel at certain doses. On the other hand, 100 µM Mel, not provoking lipid peroxidation, restored the rate constant for SO4= uptake, Band 3 protein expression levels, and H2O2-induced cell shape alterations. Such an effect was confirmed by abolishing the endogenous erythrocytes antioxidant system. Therefore, the present findings show the antioxidant power of Mel at pharmacological concentrations in an in vitro model of oxidative stress not associated to lipid peroxidation, thereby confirming Band 3 protein anion exchange capability measurement as a suitable model to prove the beneficial effect of Mel and support the use of this compound in oxidative stress-related diseases affecting Band 3 protein.

O-GlcNAcylation is a post-translational modification of proteins that controls a variety of cellular processes, is chronically elevated in diabetes mellitus, and may contribute to the progression of diabetic complications, including diabetic nephropathy. Our previous work showed that increases in the O-GlcNAcylation of cellular proteins impair the homeostatic reaction of the regulatory volume decrease (RVD) after cell swelling by an unknown mechanism. The activation of the swelling-induced chloride current IClswell is a key step in RVD, and ICln, a ubiquitous protein involved in the activation of IClswell, is O-GlcNAcylated. Here, we show that experimentally increased O-GlcNAcylation of cellular proteins inhibited the endogenous as well as the ICln-induced IClswell current and prevented RVD in a human renal cell line, while decreases in O-GlcNAcylation augmented the current magnitude. In parallel, increases or decreases in O-GlcNAcylation, respectively, weakened or stabilized the binding of ICln to the intracellular domain of α-integrin, a process that is essential for the activation of IClswell. Mutation of the putative YinOYang site at Ser67 rendered the ICln-induced IClswell current unresponsive to O-GlcNAc variations, and the ICln interaction with α-integrin insensitive to O-GlcNAcylation. In addition, exposure of cells to a hypotonic solution reduced the O-GlcNAcylation of cellular proteins. Together, these findings show that O-GlcNAcylation affects RVD by influencing IClswell and further indicate that hypotonicity may activate IClswell by reducing the O-GlcNAcylation of ICln at Ser67, therefore permitting its binding to α-integrin. We propose that disturbances in the regulation of cellular volume may contribute to disease in settings of chronically elevated O-GlcNAcylation, including diabetic nephropathy.

Melatonin is a pleiotropic biofactor and an effective antioxidant and free radical scavenger and, as such, can be protective in oxidative stress-related brain conditions including epilepsy and aging. To test the potential protective effect of melatonin on brain homeostasis and identify the corresponding molecular targets, we established a new model of oxidative stress-related aging neuroglia represented by U-87 MG cells exposed to D-galactose (D-Gal). This model was characterized by a substantial elevation of markers of oxidative stress, lipid peroxidation, and protein oxidation. The function of the inward rectifying K+ channel Kir2.1, which was identified as the main Kir channel endogenously expressed in these cells, was dramatically impaired. Kir2.1 was unlikely a direct target of oxidative stress, but the loss of function resulted from a reduction of protein abundance, with no alterations in transcript levels and trafficking to the cell surface. Importantly, melatonin reverted these changes. All findings, including the melatonin antioxidant effect, were reproduced in heterologous expression systems. We conclude that the glial Kir2.1 can be a target of oxidative stress and further suggest that inhibition of its function might alter the extracellular K+ buffering in the brain, therefore contributing to neuronal hyperexcitability and epileptogenesis during aging. Melatonin can play a protective role in this context.

Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines.

d-Galactose (d-Gal), when abnormally accumulated in the plasma, results in oxidative stress production, and may alter the homeostasis of erythrocytes, which are particularly exposed to oxidants driven by the blood stream. In the present investigation, the effect of d-Gal (0.1 and 10 mM, for 3 and 24 h incubation), known to induce oxidative stress, has been assayed on human erythrocytes by determining the rate constant of SO42- uptake through the anion exchanger Band 3 protein (B3p), essential to erythrocytes homeostasis. Moreover, lipid peroxidation, membrane sulfhydryl groups oxidation, glycated hemoglobin (% A1c), methemoglobin levels (% MetHb), and expression levels of B3p have been verified. Our results show that d-Gal reduces anion exchange capability of B3p, involving neither lipid peroxidation, nor oxidation of sulfhydryl membrane groups, nor MetHb formation, nor altered expression levels of B3p. d-Gal-induced %A1c, known to crosslink with B3p, could be responsible for rate of anion exchange alteration. The present findings confirm that erythrocytes are a suitable model to study the impact of high sugar concentrations on cell homeostasis; show the first in vitro effect of d-Gal on B3p, contributing to the understanding of mechanisms underlying an in vitro model of aging; demonstrate that the first impact of d-Gal on B3p is mediated by early Hb glycation, rather than by oxidative stress, which may be involved on a later stage, possibly adding more knowledge about the consequences of d-Gal accumulation.

Aging, a time-dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d-galactose ( d-Gal)-induced aging model. Anion exchange capability, measured by the rate constant of SO₄²- uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d-Gal for 24 h. Our results show that: (i) in vitro application of d-Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d-Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age-related disturbances.

Cancer cell extravasation is a crucial step in cancer metastasis. However, many of the mechanisms involved in this process are only now being elucidated. Thus, in the present study we analysed the trans-endothelial invasion of melanoma cells by a high throughput label-free cell impedance assay applied to transwell chamber invasion assay. This technique monitors and quantifies in real-time the invasion of endothelial cells by malignant tumour cells, for a long time, avoiding artefacts due to preparation of the end point measurements. Results obtained by impedance analysis were compared with endpoint measurements. In this study, we used human melanoma M14 wild type (WT) cells and their drug resistant counterparts, M14 multidrug resistant (ADR) melanoma cells, selected by prolonged exposure to doxorubicin (DOX). Tumour cells were co-cultured with monolayers of human umbilical vein endothelial cells (HUVEC). Results herein reported demonstrated that: (i) the trans-endothelial migration of resistant melanoma cells was faster than sensitive ones; (ii) the endothelial cells appeared to be strongly affected by the transmigration of melanoma cells which showed the ability to degrade their cytoplasm; (iii) resistant cells preferentially adopted the transcellular invasion vs. the paracellular one; (iv) the endothelial damage mediated by tumour metalloproteinases seemed to be reversible.

The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study.

Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.

Acquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231.

Introduction: During their lifespan in the bloodstream, red blood cells (RBCs) are exposed to multiple stressors, including increased oxidative stress, which can affect their morphology and function, thereby contributing to disease. Aim: This investigation aimed to explore the cellular and molecular mechanisms related to oxidative stress underlying anion exchanger 1 activity (band 3, SLC4A1/AE1) in human RBCs. To achieve this aim, the relationship between RBC morphology and functional and metabolic activity has been explored. Moreover, the potential protective effect of an anthocyanin-enriched fraction extracted from Callistemon citrinus flowers was studied. Methods: Cellular morphology, parameters of oxidative stress, as well as the anion exchange capability of band 3 have been analyzed in RBCs treated for 1 h with 50 mM of the pro-oxidant 2,2'-azobis (2-methylpropionamide)-dihydrochloride (AAPH). Before or after the oxidative insult, subsets of cells were exposed to 0.01 μg/mL of an anthocyanin-enriched fraction for 1 h. Results: Exposure to AAPH caused oxidative stress, exhaustion of reduced glutathione, and over-activation of the endogenous antioxidant machinery, resulting in morphological alterations of RBCs, specifically the formation of acanthocytes, increased lipid peroxidation and oxidation of proteins, as well as abnormal distribution and hyper-phosphorylation of band 3. Expected, oxidative stress was also associated with a decreased band 3 ion transport activity and an increase of oxidized haemoglobin, which led to abnormal clustering of band 3. Exposure of cells to the anthocyanin-enriched fraction prior to, but not after, oxidative stress efficiently counteracted oxidative stress-related alterations. Importantly, protection of band3 function from oxidative stress could only be achieved in intact cells and not in RBC ghosts. Conclusion: These findings contribute a) to clarify oxidative stress-related physiological and biochemical alterations in human RBCs, b) propose anthocyanins as natural antioxidants to neutralize oxidative stress-related modifications, and 3) suggest that cell integrity, and therefore a cytosolic component, is required to reverse oxidative stress-related pathophysiological derangements in human mature RBCs.

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 μM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42- uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 μM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.

The volume regulated anion channel (VRAC) is known to be involved in different aspects of cancer cell behaviour and response to therapies. For this reason, we investigated the effect of DCPIB, a presumably specific blocker of VRAC, in two types of cancer: pancreatic duct adenocarcinoma (PDAC) and melanoma.

Cnidarians may negatively impact human activities and public health but concomitantly their venom represents a rich source of bioactive substances. Pelagia noctiluca is the most venomous and abundant jellyfish of the Mediterranean Sea and possesses a venom with hemolytic and cytolytic activity for which the mechanism is largely unknown. Here we show that exposure of mammalian cells to crude venom from the nematocysts of P. noctiluca profoundly alters the ion conductance of the plasma membrane, therefore affecting homeostatic functions such as the regulation and maintenance of cellular volume. Venom-treated cells exhibited a large, inwardly rectifying current mainly due to permeation of Na+ and Cl-, sensitive to amiloride and completely abrogated following harsh thermal treatment of crude venom extract. Curiously, the plasma membrane conductance of Ca2+ and K+ was not affected. Current-inducing activity was also observed following delivery of venom to the cytosolic side of the plasma membrane, consistent with a pore-forming mechanism. Venom-induced NaCl influx followed by water and consequent cell swelling most likely underlie the hemolytic and cytolytic activity of P. noctiluca venom. The present study underscores unique properties of P. noctiluca venom and provides essential information for a possible use of its active compounds and treatment of envenomation.