Transient Receptor Potential Ankyrin 1 (TRPA1) Modulation by 4-Hydroxynonenal (4-HNE) in Pancreatic Adenocarcinoma Cell Lines: Putative Roles for Therapies.

BACKGROUND: Transient receptor potential channels (TRP) are overexpressed in some pancreatic adenocarcinoma (PDAC) patients and cell lines, settling them as putative therapeutic targets in this disease. Reactive oxygen species (ROS), with levels increased in PDAC, modulate some members of the TRP family renamed "redox channels". Here, we investigate the direct effects of 4-hydroxinonenal (4-HNE) on TRPA1, natively expressed in PDAC cell lines and in association with cell migration and cell cycle progression. METHODS: We performed microfluorimetry experiments, while the activation of resident membrane channels was investigated using confocal microscopy. We applied a prospective molecular docking of 4-HNE using Autodock and AutoDock Tools4. Also, we simulated the diffusion of 4-HNE through the membrane from the extracellular space with the Permeability of Molecules across Membranes (PerMM) web server. The analysis of cell migration was performed using the wound healing assay, and cell cycle progression was acquired using a Beckman Coulter CytoFlex flow cytometer. RESULTS: Our results show, for the first time in PDAC, that 4-HNE diffuses through the cell membrane and rapidly activates Ca2+ uptake in PDAC cells. This process depends on TRPA1 activation, as 4-HNE forms a covalent binding with a pocket-like region within the intracellular N-terminal of the channel, shaped by the cysteine residues 621, 641, and 665. The activation of TRPA1 by 4-HNE inhibits cell migration and induces cell cycle arrest in the G2/M phase. CONCLUSIONS: Our study brings new insights into the effects of 4-HNE, highlighting the activation of the TRPA1 channel, a druggable, putative target for PDAC-expressing tumors.

TRP Channels in Tumoral Processes Mediated by Oxidative Stress and Inflammation.

The channels from the superfamily of transient receptor potential (TRP) activated by reactive oxygen species (ROS) can be 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 TRPC3, TRPM2, and TRPM7, are indirectly activated by ROS. Furthermore, 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, using these polymodal proteins as therapeutic targets is still an unmet need, despite their draggability and modulation by simple and mostly unharmful compounds. This review intended to create some cellular models of the interaction between oxidative stress, TRP channels, and inflammation. Although somewhat crosstalk between the three actors was 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 clinical information may finally bring the results from the bench to the bedside.

The Association between TRP Channels Expression and Clinicopathological Characteristics of Patients with Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) has low survival rates worldwide due to its tendency to be detected late and its characteristic desmoplastic reaction, which slows the use of targeted therapies. As such, the discovery of new connections between genes and the clinicopathological parameters contribute to the search for new biomarkers or targets for therapy. Transient receptor potential (TRP) channels are promising tools for cancer therapy or markers for PDAC. Therefore, in this study, we selected several genes encoding TRP proteins previously reported in cellular models, namely, Transient Receptor Potential Cation Channel Subfamily V Member 6 (TRPV6), Transient receptor potential ankyrin 1 (TRPA1), and Transient receptor potential cation channel subfamily M (melastatin) member 8 (TRPM8), as well as the TRPM8 Channel Associated Factor 1 (TCAF1) and TRPM8 Channel Associated Factor 2 (TCAF2) proteins, as regulatory factors. We analyzed the expression levels of tumors from patients enrolled in public datasets and confirmed the results with a validation cohort of PDAC patients enrolled in the Clinical Institute Fundeni, Romania. We found significantly higher expression levels of TRPA1, TRPM8, and TCAF1/F2 in tumoral tissues compared to normal tissues, but lower expression levels of TRPV6, suggesting that TRP channels have either tumor-suppressive or oncogenic roles. The expression levels were correlated with the tumoral stages and are related to the genes involved in calcium homeostasis (Calbindin 1 or S100A4) or to proteins participating in metastasis (PTPN1). We conclude that the selected TRP proteins provide new insights in the search for targets and biomarkers needed for therapeutic strategies for PDAC treatment.

Exosomal microRNAs as Biomarkers and Therapeutic Targets for Hepatocellular Carcinoma.

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.

Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells.

The transient receptor potential ankyrin type 1 (TRPA1) channel belongs to the TRP superfamily of ion channels. TRPA1 is a membrane protein with multiple functions able to respond to noxious stimuli, reactive oxygen species, inflammatory cytokines or pungent substances, and it participates in pain signalling, taste, inflammation and various steps of the tumorigenic process. To date, no reports have addressed the expression and function of TRPA1 in pancreatic ductal adenocarcinoma (PDAC) cells. This work reports the endogenous expression of TRPA1 channels in human pancreatic adenocarcinoma cell lines and provides insights into the function of the TRPA1 protein in the Panc-1 cell line. This study reports that cell lines isolated from PDAC patients had different levels of TRPA1 expression. The channel activity in Panc-1 cells, as assessed with electrophysiological (whole-cell patch clamp) and microfluorimetry methods, showed that non-selective cationic currents were activated by allyl isothiocyanate (AITC) in Panc-1 cells and inhibited by the selective TRPA1 antagonist A-967079. The current elicited by the specific agonist was associated with a robust increase in intracellular Ca2+. Furthermore, siRNA-induced downregulation of TRPA1 enhanced cell migration in the wound healing assay, indicating a possible role of ion channels independent from pore function. Finally, TRPA1 activation changed the cell cycle progression. Taken together, these results support the idea of channel-dependent and independent role for TRPA1 in tumoral processes.

Potential Circulating Biomarkers of Recurrence after Hepatic Resection or Liver Transplantation in Hepatocellular Carcinoma Patients.

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-freedom of liver recurrence (FLR), disease-free survival (DFS) and overall survival (OS)-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 < 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.

N-glycosylation state of TRPM8 protein revealed by terahertz spectroscopy and molecular modelling.

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.

Comprehensive transcriptomic analyses of tissue, serum, and serum exosomes from hepatocellular carcinoma patients.

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.

N-glycosylation of the transient receptor potential melastatin 8 channel is altered in pancreatic cancer cells.

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 Ca2+ uptake is modulated by the glycosylation status of the protein, thus affecting cell proliferation.

Characterization of functional transient receptor potential melastatin 8 channels in human pancreatic ductal adenocarcinoma cells.

OBJECTIVE: Recently, the transient receptor potential melastatin 8 (TRPM8) channel has emerged as a putative biomarker for pancreatic ductal adenocarcinoma (PDA). This study aimed to evaluate the expression of TRPM8 and its modulation by specific agonists and antagonists in PDA cells. METHODS: We examined the protein expression of TRPM8 in 3 different PDA cell lines and compared it with a nontumoral epithelial cell line of human pancreatic origin using Western blotting and immunocytochemical analysis. To assess the function of TRPM8 channels, we measured the TRPM8 currents in whole-cell mode of the patch clamp technique. To explore the putative involvement of TRPM8 in cell migration, we investigated the motility of PDA cells using the scratch-wound assay. RESULTS: Pancreatic ductal adenocarcinoma cells express functional plasma membrane TRPM8 channels, which are responsive after exposure to agonists (menthol and icilin) and antagonists N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt. The silencing of TRPM8 expression by small interfering RNA augments the migration of PDA cells. Conversely, the activated form of TRPM8 inhibits PDA cell motility. CONCLUSIONS: An unglycosylated TRPM8 protein is expressed and is functional in the membrane of PDA cells. Transient receptor potential melastatin 8 inhibits the migration of PDA cells, suggesting a putative role as a biomarker or target for this channel for PDA therapy.

Low doses of cadmium chloride and methallothionein-1-bound cadmium display different accumulation kinetics and induce different genes in cells of the human nephron.

BACKGROUND/AIMS: The present study was conducted to investigate the renal tubular handling of inorganic cadmium (Cd(2+)) by exposing primary human tubular cell cultures to physiologically relevant doses of cadmium chloride (CdCl(2)). Furthermore, the cellular accumulation of Cd(2+) 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 Cd(2+) 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: Cd(2+) accumulated into human tubular cells in a concentration- and time-dependent way. Furthermore, cellular accumulation of Cd(2+) 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 CdCl(2) 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.

Rat ENaC expressed in Xenopus laevis oocytes is activated by cAMP and blocked by Ni(2+).

We used oocytes of the South African clawed toad Xenopus laevis to express the three subunits of the epithelial Na(+) channel from rat distal colon (rENaC). We combined conventional dual-microelectrode voltage-clamp with continuous capacitance (C(m)) measurements and noise analysis to evaluate the effects of cAMP and Ni(2+) on rENaC. Control oocytes or rENaC-expressing oocytes exhibited no spontaneous fluctuations in current. However, in rENaC-expressing oocytes amiloride induced a marked plateau-shaped rise of the power density spectra. Recordings using four different concentrations of amiloride revealed that the blocker-channel interactions were of the first order. A cocktail of the membrane permeant cAMP analogue chlorophenylthio-cAMP and IBMX (cAMP cocktail) increased amiloride-sensitive current (I(ami)) and conductance (G(ami)). Furthermore, C(m) was also increased following cAMP application, indicating an increase in plasma membrane surface area. Noise analysis showed that cAMP increased the number of active channels in the oocyte membrane while single-channel current decreased. From these data we conclude that cAMP triggered exocytotic delivery of preformed rENaCs to the plasma membrane. Ni(2+) (2.5 mM) inhibited about 60% of the rENaC current and conductance while C(m) remained unaffected. Noise analysis revealed that this inhibition could be attributed to a decrease in the apparent channel density, while single-channel current did not change significantly. These observations argue for direct effects of Ni(2+) on channel activity rather than induction of endocytotic removal of active channels from the plasma membrane.