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The channels from the superfamily of transient receptor potential (TRP) activated by reactive oxygen species (ROS) are defined as redox channels. Those with the best exposure of the cysteine residues and hence the most sensitive to oxidative stress are TRPC4, TRPC5, TRPV1, TRPV4, and TRPA1, while others, such as TRPM2 and TRPM7 are indirectly activated by ROS. Activation by ROS has different effects on the tumorigenic process: some TRP channels may, upon activation, stimulate proliferation, apoptosis, or migration of cancer cells, while others inhibit these processes, depending on the cancer type, tumoral microenvironment and finally on the methods used for evaluation. Therefore, the use of these polymodal proteins as therapeutic targets is still an unmet need, despite their draggability and modulation by simple and mostly unharmful compounds. In this review, we intend to create some cellular models of the interaction between oxidative stress, TRP channels, and inflammation. Although the crosstalk between the three actors is rather theoretical, we intended to gather the recently published data and proposed pathways of cancer inhibition using modulators of TRP proteins, hoping that the experimental data corroborated with clinical information may finally bring the results from the bench to the bedside.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second most common cause of cancer-related death globally. This type of liver cancer is frequently detected at a late stage by current biomarkers because of the high clinical and biological heterogeneity of HCC tumours. From a plethora of molecules and cellular compounds, small nanoparticles with an endosomal origin are valuable cancer biomarkers or cargos for novel treatments. Despite their small sizes, in the range of 40–150 nm, these particles are delimited by a lipid bilayer membrane with a specific lipid composition and carry functional information—RNA, proteins, miRNAs, long non-coding RNAs (lncRNAs), or DNA fragments. This review summarizes the role of exosomal microRNA (miRNA) species as biomarkers in HCC therapy. After we briefly introduce the exosome biogenesis and the methods of isolation and characterization, we discuss miRNA’s correlation with the diagnosis and prognosis of HCC, either as single miRNA species, or as specific panels with greater clinical impact. We also review the role of exosomal miRNAs in the tumourigenic process and in the cell communication pathways through the delivery of cargos, including proteins or specific drugs.

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Background: Improving surgical outcomes in hepatocellular carcinoma (HCC) patients would greatly benefit from biomarkers. Angiogenesis and inflammation are hallmarks of HCC progression and therapeutic targets. Methods: We retrospectively evaluated preoperative clinical variables and circulating (plasma) biomarkers of angiogenesis and inflammation in a cohort of HCC patients who underwent liver resection (LR) or transplantation (LT). Biomarker correlation with outcomes&mdash, freedom of liver recurrence (FLR), disease-free survival (DFS) and overall survival (OS)&mdash, was tested using univariate and multivariate Cox regression analyses. Results: Survival outcomes associated with sVEGFR1, VEGF and VEGF-C in LT patients and with IL-10 in LR patients. Moreover, in LT patients within Milan criteria, higher plasma VEGF and sVEGFR1 were associated with worse outcomes, while in those outside Milan criteria lower plasma VEGF-C associated with better outcomes. Multivariate analysis indicated that adding plasma VEGF or VEGF-C to a predictive model including Milan criteria and AFP improved prediction of DFS and OS (all p &lt, 0.05). Conclusion: Survival outcomes after LR or LT differentially associated with angiogenic and inflammatory biomarkers. High plasma VEGF correlated with poorer prognosis within Milan criteria while low plasma VEGF-C associated with better prognosis outside Milan criteria. These candidate biomarkers should be further validated to improve patient stratification.

TRPM8 member of the TRP superfamily of membrane proteins participates to various cellular processes ranging from Ca2+ uptake and cold sensation to cellular proliferation and migration. TRPM8 is a large tetrameric protein with more than 70% of its residues located in the cytoplasm. TRPM8 is N-glycosylated, with a single site per subunit. This work focuses on the N-glycosylation of TRPM8 channel that was previously studied by our group in relation to proliferation and migration of tumoral cells. Here, experimental data performed with deglycosylating agents assess that the sole glycosylation site contains complex glycans with a molecular weight of 2.5 kDa. The glycosylation state of TRPM8 in cells untreated and treated with a deglycosylating agent was addressed with Terahertz (THz) spectroscopy. Results show a clear difference between cells comprising glycosylated and deglycosylated TRPM8, the first presenting an increased THz absorption. Human TRPM8 was modelled using as templates the available TRPM8 and other TRPM channels structures. Glycosylations were modelled by considering two glycan structures with molecular weight close to the experiment: shorter and branched at the first sugar unit (glc1) and longer and unbranched (glc2). Simulation of THz spectra based on the molecular dynamics of unglycosylated and the two glycosylated TRPM8 models in lipid membrane and solvation box showed that glycan structure strongly influences the THz spectrum of the channel and of other components from the simulation system. Only spectra of TRPM8 with glc1 glycans were in agreement with the experiment, leading to the validation of glc1 glycan structure.

Background The expression of microRNAs (miRNAs) is a promising prognostic and diagnostic tool in hepatocellular carcinoma (HCC). Here we performed small RNA sequencing (sRNA-seq) of tissue, serum and serum exosomes to investigate changes in miRNA expression between the different sample types and correlated the expression with clinical parameters. We also performed gene expression arrays on tumor and normal tissue. Results Paired tissue, serum and serum exosomes sequencing revealed consistent positive correlation of miR-21 between serum exosomes and tumor tissue, indicating that miR-21 could be exported from tissue to circulation via exosomes. We found that let-7 miRNAs are generally upregulated in serum exosomes compared to whole serum, indicating that these miRNAs could be preferentially loaded into exosomes. Comparing serum from HCC patients with serum from healthy individuals revealed a global increase of miRNAs in serum from HCC patients, including an almost 4-fold increase of several miRNAs, including the liver-specific miR-122. When correlating miRNA expression with clinical parameters we detected significant association between hepatitis B virus (HBV) infection and miR-122 in serum as well as several serum and tissue-miRNAs that correlated with surgery type. We found that miR-141 and miR-146 correlated with cirrhosis in tumor tissue and normal tissue, respectively. Finally, high expression of miR-21 in tumors were associated with poor survival. Focusing on gene expression we found several significant messenger RNAs (mRNAs) between tumor and normal tissue and a Gene Ontology (GO) analysis revealed that these changes were mainly related to cell cycle and metabolism. Further, we detected mRNAs that correlated with cirrhosis and HBV infection in tissue. Finally, GO analysis of predicted targets for miRNAs down-regulated in tumor found that these were enriched for functions related to collagen synthesis. Conclusions Our combined data point to altered miRNA and mRNA expression contributing to both generally impaired lipid metabolism and increased cell proliferation and a miRNA-driven increase in collagen synthesis in HCC. Our results further indicate a correlation in miRNA expression between exosomes, serum, and tissue samples suggesting export from tumors via exosomes. This correlation could provide a basis for a more tumor-specific miRNA profile in serum. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Background/Aims: The present study was conducted to investigate the renal tubular handling of inorganic cadmium (Cd2+) by exposing primary human tubular cell cultures to physiologically relevant doses of cadmium chloride (CdCl2). Furthermore, the cellular accumulation of Cd2+ was compared to that of metallothionein-1-bound Cd (Cd7MT-1). Finally, this study aimed to investigate the effect of the accumulation of Cd (both Cd2+ and Cd7MT-1) in renal cells on the expression of genes relevant to nephrotoxic processes. Methods: Cd concentration was measured using atomic absorption spectrometry. mRNA expression was evaluated by quantitative real-time RT-PCR. Results: Cd2+ accumulated into human tubular cells in a concentration- and time-dependent way. Furthermore, cellular accumulation of Cd2+ was different from the cellular accumulation of Cd7MT-1, indicative for different uptake routes. Finally, mRNA expression of the genes encoding the anti-oxidative proteins metallothionein-1 (MT-1) and heme-oxygenase-1 (HO-1) as well as the pro-apoptotic Bcl-2-associated X protein (Bax) were upregulated by CdCl2 and not by Cd7MT1. Conclusion: In the presence of physiologically relevant Cd concentrations, tubular accumulation of the element in its inorganic form is different from that of Cd7MT-1. Furthermore, the tubular accumulation of inorganic Cd induces mRNA expression of genes of which the protein products may play a role in Cd-associated renal toxicity.

Calcium oxalate is one of the main constituents of kidney stones and has a proved deleterious effect on renal cells that is mediated by oxidative stress. However, the subcellular source of this oxidative stress, and whether it is extending to the extracellular space or not, is still disputed. Therefore, an electrochemical superoxide biosensor was constructed, positioned above A6 renal cells, and used to measure in real-time the extracellular oxidative burst following addition of calcium oxalate crystals. It was observed that A6 cells do secrete superoxide into their extracellular space in few minutes after encountering calcium oxalate crystals. The amount of released superoxide peaks at about 20 min. Superoxide is cleared away from the extracellular space after approximately 3 h. Superoxide secretion depends on the presence of superoxide-scavenging enzyme superoxide dismutase, the age of the cells, the amount of calcium oxalate crystals, and the temperature. Moreover, the effect of calcium oxalate crystals was mimicked by phorbol 12-myristate 13-acetate. The developed sensing system can be a useful tool for biologists investigating nephrolithiasis at cellular level.

The function of the epithelial Na+ channel from the apical membrane of many Na+ transporting epithelia is modulated by various chemical compounds from the extracellular space, such as heavy metals, protons or chloride ions. We have studied the effect of extracellular Cd2+ on the function of the epithelial Na+ channel (ENaC) in heterologously expressed Xenopus laevis oocytes and Na+-transporting epithelia. We assayed channel function as the amiloride-sensitive sodium current (I(Na)). Cd2+ rapidly and voltage-independently inhibited INa in oocytes expressing αβγ Xenopus ENaC (xENaC). The extracellular Cd2+ inhibited Na+ transport and showed no influence on ENaC trafficking, as revealed by concomitant measurements of the transepithelial current, conductance and capacitance in Na+-transporting epithelia. Instead, amiloride inhibition was noticeably diminished in the presence of Cd2+ on the apical membrane. Using molecular modeling approaches, we describe the amiloride binding sites in rat and xENaC structures, and we present four putative binding sites for Cd2+. These results indicate that ENaC functions as a sensor for external Cd2+.

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes. Am J Physiol Cell Physiol 289: C946–C958, 2005. First published June 8, 2005; .—The epithelial Na+ channel (ENaC) is modulated by various extracellular factors, including Na+, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni2+ cation on ENaCs, we compared the Na+ permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis [wild-type (WT) xENaC] was stimulated by external Ni2+, whereas the divalent cation inhibited the channel cloned from the rat colon (WT rENaC). The kinetics of amiloride binding were determined using noise analysis of blocker-induced fluctuation in current adapted for the transoocyte voltage-clamp method, and Na+ conductance was assessed using the dual electrode voltage-clamp (TEVC) technique. The inhibitory effect of Ni2+ on amiloride binding is not species dependent, because Ni2+ decreased the affinity (mainly reducing the association rate constant) of the blocker in both species in competition with Na+. Importantly, using the TEVC method, we found a prominent difference in channel conductance at hyperpolarizing voltage pulses. In WT xENaCs, the initial ohmic current response was stimulated by Ni2+, whereas the secondary voltage-activated current component remained unaffected. In WT rENaCs, only a voltage-dependent block by Ni2+ was obtained. To further study the origin of the xENaC stimulation by Ni2+, and based on the rationale of the well-known high affinity of Ni2+ for histidine residues, we designed α-subunit mutants of xENaCs by substituting histidines that were expressed in oocytes, together with WT β- and γ-subunits. Changing His215 to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop between Na+ channel membrane segments M1 and M2 had no influence on the stimulatory effect of Ni2+, and neither did complete deletion of this segment. Next, we mutated His416 flanked by His411 and Cys417, a unique site for possible heavy metal ion chelation, and, with this quality, most proximal (∼100 amino acids upstream of the second putative amiloride binding site at the pore entrance), was found localized at M2. Replacing His416 with arginine, aspartate, tyrosine, and alanine clearly affected amiloride binding in all cases, as well as Na+ conductance, as expressed in the xENaC current-voltage relationship, especially with regard to aspartate and tyrosine. However, similarly to those obtained with the WYRFHY stretch, none of these mutations could either abolish the stimulating effect of Ni2+ or reverse it to an inhibitory type.

We developed a non-invasive technique for electrophysiological investigations of ion transport proteins endogenously or heterologously expressed in Xenopus laevis oocytes. We named this technique the transoocyte voltage clamp (TOVC). Whereas in the classical two-microelectrode voltage-clamp (TEVC) technique, the oocyte is impaled with two glass microelectrodes, we mount the egg in a modified Ussing chamber as used for transepithelial electrophysiological studies. The oocyte is introduced in a container that is positioned between the two chamber halves. Proper fixation of the oocyte in the aperture of the container is accomplished under a stereo binocular microscope and the electrical seal between the oocyte and the container is achieved with silicon grease. The new method allows measurement of transoocyte currents and conductances as well as the recording of membrane impedance and the fluctuation analysis of ion currents. We studied a K+ channel that resembles the inward rectifier K+ channel endogenously expressed in Xenopus laevis oocytes. K+ currents were obtained by exposing one side of the oocyte to K(+)-containing solutions and by the application of different voltages. Adding Cs+ and Ba2+ inhibited these currents. The analysis of the fluctuation in current demonstrated a Lorentzian component in the power density spectrum. With the transoocyte voltage clamped to zero, the corner frequency (fc) was 61+/-1.7 Hz. Imposed positive transoocyte potentials caused a downward shift of fc. These findings are consistent with previous data obtained using the TEVC technique, and extend the characterization of the channel with kinetic data obtained from noise analysis.

Transient receptor potential melastatin 8 (TRPM8), a membrane ion channel, is activated by thermal and chemical stimuli. In pancreatic ductal adenocarcinoma, TRPM8 is required for cell migration, proliferation, and senescence and is associated with tumor size and pancreatic ductal adenocarcinoma stages. Although the underlying mechanisms of these processes have yet to be described, this cation-permeable channel has been proposed as an oncological target. In this study, the glycosylation status of the TRPM8 channel was shown to affect cell proliferation, cell migration, and calcium uptake. TRPM8 expressed in the membrane of the Panc-1 pancreatic tumoral cell line is non-glycosylated, whereas human embryonic kidney cells transfected with human TRPM8 overexpress a glycosylated protein. Moreover, our data suggest that Ca 2+ uptake is modulated by the glycosylation status of the protein, thus affecting cell proliferation.

The electrophysiological characteristics of monolayers of cultured renal epithelial A6 cells were studied under short-circuit conditions. Replacing basolateral isosmotic (260 mOsm/kg H2O) media by hyposmotic (140 mOsm/kg H2O) solutions transiently increased the transepithelial capacitance (C(T)) by 57.3+/-2.3% after 16 min. The transepithelial Na+ current (I(Na)) increased concomitantly from 4.2+/-0.7 to 26.1+/-2.6 microA/cm2 with a time course that was noticeably slower, reaching its maximum after 60 min of hypotonicity. The transepithelial conductance (G(T)) increased synchronously with I(Na). Analysis of blocker-induced noise in I(Na), using the amiloride analogue 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), showed that the hypotonic shock increased Na+ channel density (N(T)) at the apical border. The presence of 10 mM Mg2+ on both sides of the epithelium suppressed the hypotonicity-induced C(T) increase to 14.3+/-0.5%, whereas the I(Na) increase was even larger than without Mg2+. Both effects of Mg2+ were located at an extracellular, basolateral site, because apical administration was without effect, whereas the acute basolateral addition of Mg2+ at the moment of the hypotonic shock was sufficient. Interaction between Mg2+ and Ca2+ influenced the behaviour of C(T). At constant osmolality (200 mOsm/kg H2O) 10 mM Mg2+ increased I(Na), leaving C(T) unaffected, whereas 10 mM Ca2+ stimulated both I(Na) and CT. In the presence of 1 mM Mg2+, however, the Ca(2+)-induced CT increase was abolished. The failure of CT to increase during stimulation of I(Na) by Mg2+ suggests that the divalent cation activates pre-existing channels in the apical membrane. Noise analysis showed that the natriferic effects of Mg2+ were also mediated by an increase in NT. The moderate initial increase in CT in the presence of Mg2+ under hypotonic conditions, occurring in parallel with increases in GT and I(Na), reflects most likely Na+ channel insertion induced by the hypotonic treatment. However, the large, transient, Mg(2+)-sensitive increase in CT, not correlated with increases in GT and I(Na), seems to be unrelated to Na+ channel recruitment.

Electrical potential oscillations were obtained across a liquid membrane composed of nitrobenzene/picric acid placed between two aqueous phases in the presence of various taste (i.e. salty, sweet and bitter) substances. The influence of these compounds on electrical oscillations was studied using Fourier analysis to establish a “fingerprint” of the substance that can be correlated with its taste index. Various concentrations of each substance were tested to obtain a Fourier spectrum with discrete peaks which can be further processed. The electrical oscillations consisted of a number of weak damped oscillators, and the Fourier spectra of these signals were found to have a number of discrete peaks of decreasing amplitude at low frequencies (0–0.5 Hz). A correlation of the frequency of the first peak of the Fourier spectrum with the taste index was found for bitter substances, whereas for salty substances the amplitude of the first two peaks of the spectrum was correlated with the taste index.

In cultured A6 monolayers from distal Xenopus kidney, external Ni2+ stimulated active Na+ uptake via the epithelial Na+ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni2+ effect. Na+ current noise analysis was performed using the neutral Na(+) -channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni2+ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni2+ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni2+ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na(+)--known to compete with amiloride and so producing the "self-inhibition" phenomenon--and Ni2+ exerted mutually exclusive analogous effects on amiloride kinetics. Na+ current kinetics revealed that Ni2+ prevents ENaC to be downregulated by self-inhibition. Co2+ behaved similarly to Ni2+, whereas Zn2+ did not. Attempts to disclose the chemical nature of the site reacting with Ni2+ suggested cysteine but not histidine as reaction partner.

Filters impregnated with soybean lecithin, placed between two identical aqueous alanine solutions, display spontaneous electric potential oscillations. Alanine solutions used in a large concentration range from 1 mM to 1 M produce damped oscillatory behaviour with an exponential decay in time. The dependence of decay time on concentration shows saturation behaviour which is well fitted with a sigmoidal curve. Power spectra obtained by Fourier transform show peaks specific for each concentration. When fitted with a Lorentzian curve in the peak domain, the centre of the peak height and width at half height could be extracted. All these parameters depend on alanine concentration in a saturating pattern.