The context-dependent role of the Na+/Ca2+-exchanger (NCX) in pancreatic stellate cell migration.

Pancreatic stellate cells (PSCs) that can co-metastasize with cancer cells shape the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) by producing an excessive amount of extracellular matrix. This leads to a TME characterized by increased tissue pressure, hypoxia, and acidity. Moreover, cells within the tumor secrete growth factors. The stimuli of the TME trigger Ca2+ signaling and cellular Na+ loading. The Na+/Ca2+ exchanger (NCX) connects the cellular Ca2+ and Na+ homeostasis. The NCX is an electrogenic transporter, which shuffles 1 Ca2+ against 3 Na+ ions over the plasma membrane in a forward or reverse mode. Here, we studied how the impact of NCX activity on PSC migration is modulated by cues from the TME. NCX expression was revealed with qPCR and Western blot. [Ca2+]i, [Na+]i, and the cell membrane potential were determined with the fluorescent indicators Fura-2, Asante NaTRIUM Green-2, and DiBAC4(3), respectively. PSC migration was quantified with live-cell imaging. To mimic the TME, PSCs were exposed to hypoxia, pressure, acidic pH (pH 6.6), and PDGF. NCX-dependent signaling was determined with Western blot analyses. PSCs express NCX1.3 and NCX1.9. [Ca2+]i, [Na+]i, and the cell membrane potential are 94.4 nmol/l, 7.4 mmol/l, and - 39.8 mV, respectively. Thus, NCX1 usually operates in the forward (Ca2+ export) mode. NCX1 plays a differential role in translating cues from the TME into an altered migratory behavior. When NCX1 is operating in the forward mode, its inhibition accelerates PSC migration. Thus, NCX1-mediated extrusion of Ca2+ contributes to a slow mode of migration of PSCs.

Ion Channel Profiling in Prostate Cancer: Toward Cell Population-Specific Screening.

In the last three decades, a growing number of studies have implicated ion channels in all essential processes of prostate carcinogenesis, including cell proliferation, apoptosis, migration, and angiogenesis. The changes in the expression of individual ion channels show a specific profile, making these proteins promising clinical biomarkers that may enable better molecular subtyping of the disease and lead to more rapid and accurate clinical decision-making. Expression profiles and channel function are mainly based on the tumoral tissue itself, in this case, the epithelial cancer cell population. To date, little data on the ion channel profile of the cancerous prostate stroma are available, even though tumor interactions with the microenvironment are crucial in carcinogenesis and each distinct population plays a specific role in tumor progression. In this review, we describe ion channel expression profiles specific for the distinct cell population of the tumor microenvironment (stromal, endothelial, neuronal, and neuroendocrine cell populations) and the technical approaches used for efficient separation and screening of these cell populations.

Involvement of ORAI1/SOCE in Human AML Cell Lines and Primary Cells According to ABCB1 Activity, LSC Compartment and Potential Resistance to Ara-C Exposure.

Acute myeloid leukemia (AML) is a hematological malignancy with a high risk of relapse. This issue is associated with the development of mechanisms leading to drug resistance that are not yet fully understood. In this context, we previously showed the clinical significance of the ATP binding cassette subfamily B-member 1 (ABCB1) in AML patients, namely its association with stemness markers and an overall worth prognosis. Calcium signaling dysregulations affect numerous cellular functions and are associated with the development of the hallmarks of cancer. However, in AML, calcium-dependent signaling pathways remain poorly investigated. With this study, we show the involvement of the ORAI1 calcium channel in store-operated calcium entry (SOCE), the main calcium entry pathway in non-excitable cells, in two representative human AML cell lines (KG1 and U937) and in primary cells isolated from patients. Moreover, our data suggest that in these models, SOCE varies according to the differentiation status, ABCB1 activity level and leukemic stem cell (LSC) proportion. Finally, we present evidence that ORAI1 expression and SOCE amplitude are modulated during the establishment of an apoptosis resistance phenotype elicited by the chemotherapeutic drug Ara-C. Our results therefore suggest ORAI1/SOCE as potential markers of AML progression and drug resistance apparition.

4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer.

Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5­triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca2+ release channels.

Calcium homeostasis in cancer: A focus on senescence.

Senescence is one of the primary responses to the activation of oncoproteins or down-regulation of tumor suppressors in normal cells and is therefore considered as being anti-tumorigenic but the mechanisms controlling this process are still much unknown. Calcium (Ca²âº) plays a major role in many cellular processes and calcium channels control many of the "hallmarks of cancer" but their involvement in tumor initiation is poorly understood and remains unclear. Therefore, in this article we review some striking senescence-associated characteristics and their potential regulation by Ca²âº. The main aim is to produce plausible hypothesis on how calcium homeostasis may participate in cancer-related senescence. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Cross-talk between alpha1D-adrenoceptors and transient receptor potential vanilloid type 1 triggers prostate cancer cell proliferation.

BACKGROUND: There is evidence that calcium (Ca(2+)) increases the proliferation of human advanced prostate cancer (PCa) cells but the ion channels involved are not fully understood. Here, we investigated the correlation between alpha(1D)-adrenergic receptor (alpha(1D)-AR) and the transient receptor potential vanilloid type 1 (TRPV1) expression levels in human PCa tissues and evaluated the ability of alpha(1D)-AR to cross-talk with TRPV1 in PCa cell lines. METHODS: The expression of alpha1D-AR and TRPV1 was examined in human PCa tissues by quantitative RT-PCR and in PCa cell lines (DU145, PC3 and LNCaP) by cytofluorimetry. Moreover, alpha(1D)-AR and TRPV1 colocalization was investigated by confocal microscopy in PCa cell lines and by fluorescence microscopy in benign prostate hyperplasia (BPH) and PCa tissues. Cell proliferation was assessed by BrdU incorporation. Alpha(1D)-AR/TRPV1 knockdown was obtained using siRNA transfection. Signalling pathways were evaluated by measurement of extracellular acidification rate, Ca(2+) flux, IP3 production, western blot and MTT assay. RESULTS: The levels of the alpha(1D)-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha(1D)-AR and TRPV1 expression levels was found. Moreover, alpha(1D)-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens. Noradrenaline (NA) induced an alpha(1D)-AR- and TRPV1-dependent protons release and Ca(2+) flux in PC3 cell lines; NA by triggering the activation of phospholipase C (PLC), protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways stimulated PC3 cell proliferation, that was completely inhibited by clopenphendioxan (WS433) and capsazepine (CPZ) combination or by alpha(1D)-AR/TRPV1 double knockdown. CONCLUSIONS: We demonstrate a cross-talk between alpha1D-AR and TRPV1, that is involved in the control of PC3 cell proliferation. These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha1D-AR and TRPV1 channels.

Converting cell death into senescence by PARP1 inhibition improves recovery from acute oxidative injury.

Excessive amounts of reactive oxygen species (ROS) lead to macromolecular damage and high levels of cell death with consequent pathological sequelae. We hypothesized that switching cell death to a tissue regenerative state could potentially improve the short-term and long-term detrimental effects of ROS-associated acute tissue injury, although the mechanisms regulating oxidative stress-induced cell fate decisions and their manipulation for improving repair are poorly understood. Here, we show that cells exposed to high oxidative stress enter a poly (ADP-ribose) polymerase 1 (PARP1)-mediated regulated cell death, and that blocking PARP1 activation promotes conversion of cell death into senescence (CODIS). We demonstrate that this conversion depends on reducing mitochondrial Ca2+ overload as a consequence of retaining the hexokinase II on mitochondria. In a mouse model of kidney ischemia-reperfusion damage, PARP inhibition reduces necrosis and increases transient senescence at the injury site, alongside improved recovery from damage. Together, these data provide evidence that converting cell death into transient senescence can therapeutically benefit tissue regeneration.

Onabotulinumtoxin-A intradetrusorial injections modulate bladder expression of NGF, TrkA, p75 and TRPV1 in patients with detrusor overactivity.

How onabotulinumtoxinA (onab/A) injected in the detrusor muscle improves detrusor overactivity (DO) is still a matter of debate. Nerve growth factor (NGF) seems to play a role in determining urgency and DO. Recent studies showed that NGF decreases in patients with DO who respond to onab/A treatment. We investigated onab/A-induced changes on gene expression of NGF, TRPV1, TrkA and p75 in bladder wall tissue of patients affected by neurogenic and idiopathic DO. Twenty-five patients (18 with neurogenic DO and 7 with idiopathic DO) received onab/A injections into the detrusor muscle. Urodynamic studies and cystoscopies with sampling of the bladder wall were performed before and 1 month after onab/A injections. Onab/A-induced changes in urodynamic variables (first volume and maximum pressure of uninhibited detrusor contractions and maximum cystometric capacity) and NGF, TRPV1, TRKA, p75 gene expression by means of quantitative Real Time-Polymerase Chain Reaction. NGF protein levels were assessed in tissue homogenates by enzyme-linked immunosorbent assay. Onab/A significantly improved urodynamic findings (as shown by the increase in maximum cystometric capacity), decreased the bladder tissue levels of NGF protein and significantly increased NGF, TrkA, p75 and TRPV1 gene expression independently from the etiology of DO. No significant correlation has been found between NGF down-regulation and the increase in MCC. Correlations between NGF gene expression and NGF receptors' gene expression were influenced by onab/A dosages. In the short time follow-up, onab/A decreases NGF protein levels and increases NGF and associated receptors' gene expression possibly by inhibiting NGF release. Further studies with longer follow-up will clarify time course of onab/A-induced modifications in NGF expression.

Brain activity of thioctic Acid enantiomers: in vitro and in vivo studies in an animal model of cerebrovascular injury.

Oxidative stress is an imbalance between the production of free radicals and antioxidant defense mechanisms, potentially leading to tissue damage. Oxidative stress has a key role in the development of cerebrovascular and/or neurodegenerative diseases. This phenomenon is mainly mediated by an enhanced superoxide production by the vascular endothelium with its consequent dysfunction. Thioctic, also known as alpha-lipoic acid (1,2-dithiolane-3-pentanoic acid), is a naturally occurring antioxidant that neutralizes free radicals in the fatty and watery regions of cells. Both the reduced and oxidized forms of the compound possess antioxidant ability. Thioctic acid has two optical isomers designated as (+)- and (-)-thioctic acid. Naturally occurring thioctic acid is the (+)-thioctic acid form, but the synthetic compound largely used in the market for stability reasons is a mixture of (+)- and (-)-thioctic acid. The present study was designed to compare the antioxidant activity of the two enantiomers versus the racemic form of thioctic acid on hydrogen peroxide-induced apoptosis in a rat pheochromocytoma PC12 cell line. Cell viability was evaluated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and free oxygen radical species (ROS) production was assessed by flow cytometry. Antioxidant activity of the two enantiomers and the racemic form of thioctic acid was also evaluated in spontaneously hypertensive rats (SHR) used as an in vivo model of increased oxidative stress. A 3-h exposure of PC12 cells to hydrogen peroxide (H(2)O(2)) significantly decreased cell viability and increased levels of intracellular ROS production. Pre-treatment with racemic thioctic acid or (+)-enantiomer significantly inhibited H(2)O(2)-induced decrease in cell viability from the concentration of 50 µmol/L and 20 µmol/L, respectively. Racemic thioctic acid and (+)-salt decreased levels of intracellular ROS, which were unaffected by (-)-thioctic acid. In the brain of SHR, the occurrence of astrogliosis and neuronal damage, with a decreased expression of neurofilament 200 kDa were observed. Treatment of SHR for 30 days with (+)-thioctic acid reduced the size of astrocytes and increased the neurofilament immunoreaction. The above findings could contribute to clarify the role played by thioctic acid in central nervous system injury related to oxidative stress. The more pronounced effect of (+)-thioctic acid observed in this study may have practical therapeutic implications worthy of being investigated in further preclinical and clinical studies.

Acidic Growth Conditions Promote Epithelial-to-Mesenchymal Transition to Select More Aggressive PDAC Cell Phenotypes In Vitro.

Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an acidic microenvironment, which contributes to therapeutic failure. So far there is a lack of knowledge with respect to the role of the acidic microenvironment in the invasive process. This work aimed to study the phenotypic and genetic response of PDAC cells to acidic stress along the different stages of selection. To this end, we subjected the cells to short- and long-term acidic pressure and recovery to pHe 7.4. This treatment aimed at mimicking PDAC edges and consequent cancer cell escape from the tumor. The impact of acidosis was assessed for cell morphology, proliferation, adhesion, migration, invasion, and epithelial-mesenchymal transition (EMT) via functional in vitro assays and RNA sequencing. Our results indicate that short acidic treatment limits growth, adhesion, invasion, and viability of PDAC cells. As the acid treatment progresses, it selects cancer cells with enhanced migration and invasion abilities induced by EMT, potentiating their metastatic potential when re-exposed to pHe 7.4. The RNA-seq analysis of PANC-1 cells exposed to short-term acidosis and pHe-selected recovered to pHe 7.4 revealed distinct transcriptome rewiring. We describe an enrichment of genes relevant to proliferation, migration, EMT, and invasion in acid-selected cells. Our work clearly demonstrates that upon acidosis stress, PDAC cells acquire more invasive cell phenotypes by promoting EMT and thus paving the way for more aggressive cell phenotypes.

The transient receptor potential vanilloid-2 cation channel impairs glioblastoma stem-like cell proliferation and promotes differentiation.

Malignant transformation of cells resulting from enhanced proliferation and aberrant differentiation is often accompanied by changes in transient receptor potential vanilloid (TRPV) channels expression. In gliomas, recent evidence indicates that TRPV type 2 (TRPV2) negatively controls glioma cell survival and proliferation. In addition, cannabinoids, the ligands of both cannabinoid and TRPV2 receptors, promote glioblastoma stem-like cells (GSCs) differentiation and inhibit gliomagenesis. Herein, we provide evidence on the expression of TRPV2 in human GSCs and that GSCs differentiation reduces nestin and progressively increases both the glial fibrillary acidic protein (GFAP) and TRPV2 expression. Therefore, we evaluated the role of TRPV2 cation channel in GSC lines differentiation. Treatment of GSC lines with the TRPV antagonist Ruthenium Red, with ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid or knockdown of TRPV2 gene during differentiation, decreases GFAP and class III beta-tubulin (ß(III)-tubulin) expression; conversely, phorbol-12-myristate-13-acetate stimulates GSCs proliferation, reduces TRPV2 expression and partially reverts astroglial differentiation. In addition, forced TRPV2 expression in GSC lines by stable TRPV2 transfection increases GFAP and ß(III)-tubulin expression and parallelly reduces proliferation. Finally, TRPV2 overexpression inhibits GSCs proliferation in a xenograft mouse model, as shown by reduced tumor diameter and mitotic index, and promotes the differentiation of GSCs toward a more mature glial phenotype. Overall, our results demonstrate that TRPV2 promotes in vitro and in vivo GSCs differentiation and inhibits their proliferation. Better understanding of the molecular mechanisms that regulate the balance between proliferation and differentiation of GSCs would lead to more specific and efficacious pharmacological approaches.

TRPC3 shapes the ER-mitochondria Ca2+ transfer characterizing tumour-promoting senescence.

Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca2+ load via negative regulation of IP3 receptor-mediated Ca2+ release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.

Capsaicin promotes a more aggressive gene expression phenotype and invasiveness in null-TRPV1 urothelial cancer cells.

Capsaicin (CPS) has been found to exhibit either tumor promoting or suppressing effects, many of which are mediated by the specific transient receptor potential vanilloid type-1 (TRPV1). Herein, we provide evidence that CPS treatment induced a more aggressive gene phenotype and invasiveness in 5637 cells-lacking TRPV1 receptor. CPS treatment of 5637 cells induced upregulation of pro-angiogenetic (angiopoietin 1, angiopoietin 2 and vascular endothelial growth factor), pro-invasive and pro-metastatic genes (MMP1, MMP9, TIMP1, TIMP3, granzyme A (GZMA), NM23A and S100A) with a downregulation of apoptotic genes (Fas/CD95 and tumor necrosis factor receptor superfamily member 1A). CPS increased the invasiveness of 5637 cells by triggering IGF (insulin-like growth factor)-1 release, GZMA and MMP9 activation, α-tubulin disassembly and cytoskeleton degradation. Finally, in order to evaluate the relationship between the lack of TRPV1 expression and increased CPS-induced invasiveness, we transfected 5637 cells with the TRPV1 complementary DNA (cDNA) sequence. We found that TRPV1-expressing cells show CPS-mediated calcium level increase, growth inhibition and apoptosis. Moreover, CPS-induced migration and MMP9 activation were reverted, suggesting an inhibitory role played by TRPV1 in urothelial cancer cell invasion and metastasis.

IL-22 mRNA in peripheral blood mononuclear cells from allergic rhinitic and asthmatic pediatric patients.

T helper (T(H) )-17 lymphocytes are characterized by the expression of many regulatory cytokines, including IL-17A and IL-22, but at present no clinical data are available on the expression of these cytokines in peripheral blood mononuclear cells (PBMC) from allergic asthmatic and rhinitic (AR) children. The aim of this study was to investigate a possible relationship between IL-22 and IL-17A mRNAs and clinical parameters in seroatopic, AR, and asthmatic children. The study, conducted during the pollen season, included 18 healthy and 18 allergic (n=9 asthmatic and n=9 rhinitic) children. Serum total and specific IgE, eosinophil count, and skin prick test were performed; in addition, IL-22 and IL-17A mRNA levels were detected in PBMC from healthy and allergic subjects by quantitative real-time PCR. Despite the absence of the mRNA for the IL-17A cytokine, IL-22 expression was found in PBMC from asthmatic patients, with increased IL-22 mRNA levels in patients with chronic severe respect to those with moderate asthma. A positive correlation between IL-22 mRNA and serum total IgE levels was found in asthmatic children. In addition, higher IL-22 and IL-17A mRNA levels were detected in both AR and asymptomatic seroatopic children, compared to healthy individuals, and a correlation between IL-22 and IL17A mRNA and serum total IgE levels was demonstrated. Moreover, the mRNA level of retinoic acid-related orphan receptor C, the T(H) 17 transcription factor, was found to be increased in AR but not in asthmatic patients. This study provides the first evidence that IL-22 mRNA might be expressed in chronic severe asthmatic and AR children. The expression of IL-22 and IL-17A mRNAs in asymptomatic monosensitized seroatopic children suggests a role of these cytokines in the early events involved in the development of these allergic diseases.

TRPV channels in tumor growth and progression.

Transient receptor potential (TRP) channels affect several physiological and pathological processes. In particular, TRP channels have been recently involved in the triggering of enhanced proliferation, aberrant differentiation, and resistance to apoptotic cell death leading to the uncontrolled tumor invasion. About thirty TRPs have been identified to date, and are classified in seven different families: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like). Among these channel families, the TRPC, TRPM, and TRPV families have been mainly correlated with malignant growth and progression. The aim of this review is to summarize data reported so far on the expression and the functional role of TRPV channels during cancer growth and progression. TRPV channels have been found to regulate cancer cell proliferation, apoptosis, angiogenesis, migration and invasion during tumor progression, and depending on the stage of the cancer, up- and down-regulation of TRPV mRNA and protein expression have been reported. These changes may have cancer promoting effects by increasing the expression of constitutively active TRPV channels in the plasma membrane of cancer cells by enhancing Ca(2+)-dependent proliferative response; in addition, an altered expression of TRPV channels may also offer a survival advantage, such as resistance of cancer cells to apoptotic-induced cell death. However, recently, a role of TRPV gene mutations in cancer development, and a relationship between the expression of specific TRPV gene single nucleotide polymorphisms and increased cancer risk have been reported. We are only at the beginning, a more deep studies on the physiopathology role of TRPV channels are required to understand the functional activity of these channels in cancer, to assess which TRPV proteins are associated with the development and progression of cancer and to develop further knowledge of TRPV proteins as valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting in cancer.

ORAI3 silencing alters cell proliferation and promotes mitotic catastrophe and apoptosis in pancreatic adenocarcinoma.

Changes in cytosolic free Ca2+ concentration play a central role in many fundamental cellular processes including muscle contraction, neurotransmission, cell proliferation, differentiation, gene transcription and cell death. Many of these processes are known to be regulated by store-operated calcium channels (SOCs), among which ORAI1 is the most studied in cancer cells, leaving the role of other ORAI channels yet inadequately addressed. Here we demonstrate that ORAI3 channels are expressed in both normal (HPDE) and pancreatic ductal adenocarcinoma (PDAC) cell lines, where they form functional channels, their knockdown affecting store operated calcium entry (SOCE). More specifically, ORAI3 silencing increased SOCE in PDAC cell lines, while decreasing SOCE in normal pancreatic cell line. We also show the role of ORAI3 in proliferation, cell cycle, viability, mitotic catastrophe and cell death. Finally, we demonstrate that ORAI3 silencing impairs pancreatic tumor growth and induces cell death in vivo, suggesting that ORAI3 could represent a potential therapeutic target in PDAC treatment.

Calcium channel ITPR2 and mitochondria-ER contacts promote cellular senescence and aging.

Cellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.

TRPV2 channel negatively controls glioma cell proliferation and resistance to Fas-induced apoptosis in ERK-dependent manner.

The aim of this study was to investigate the expression and function of the transient receptor potential vanilloid 2 (TRPV2) in human glioma cells. By Real-Time-PCR and western blot analysis, we found that TRPV2 messenger RNA (mRNA) and protein were expressed in benign astrocyte tissues, and its expression progressively declined in high-grade glioma tissues as histological grade increased (n = 49 cases), and in U87MG cells and in MZC, FCL and FSL primary glioma cells. To investigate the function of TRPV2 in glioma, small RNA interfering was used to silence TRPV2 expression in U87MG cells. As evaluated by RT-Profiler PCR array, siTRPV2-U87MG transfected cells displayed a marked downregulation of Fas and procaspase-8 mRNA expression, associated with upregulation of cyclin E1, cyclin-dependent kinase 2, E2F1 transcriptor factor 1, V-raf-1 murine leukemia viral oncogene homolog 1 and Bcl-2-associated X protein (Bcl-X(L)) mRNA expression. TRPV2 silencing increased U87MG cell proliferation as shown by the increased percentage of cells incorporating 5-bromo-2-deoxyuridine expressing beta(III)-tubulin and rescued glioma cells to Fas-induced apoptosis. These events were dependent on extracellular signal-regulated kinase (ERK) activation: indeed inhibition of ERK activation in siTRPV2-U87MG transfected cells by treatment with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, reduced Bcl-X(L) protein levels, promoted Fas expression, and restored Akt/protein kinase B pathway activation leading to reduced U87MG cell survival and proliferation, and increased sensitivity to Fas-induced apoptosis. In addition, transfection of TRPV2 in MZC glioma cells, by inducing Fas overexpression, resulted in a reduced viability and an increased spontaneous and Fas-induced apoptosis. Overall, our findings indicate that TRPV2 negatively controls glioma cell survival and proliferation, as well as resistance to Fas-induced apoptotic cell death in an ERK-dependent manner.

Expression of transient receptor potential vanilloid-1 (TRPV1) in urothelial cancers of human bladder: relation to clinicopathological and molecular parameters.

AIMS: To evaluate the expression of transient receptor potential vanilloid type-1 channel protein (TRPV1) in normal and neoplastic urothelial tissues and to correlate TRPV1 expression with clinicopathological parameters and disease-specific survival. METHODS AND RESULTS: TRPV1 expression was analysed in normal and neoplastic urothelial samples at both mRNA and protein levels by quantitative real time polymerase chain reaction (qPCR) and immunohistochemistry, respectively. TRPV1 downregulation was found in urothelial cancer (UC) specimens, which correlated with tumour progression. Moreover, TRPV1 mRNA levels were associated with clinicopathological parameters to assess the role of TRPV1 downregulation as a negative prognostic factor for survival. Kaplan-Meier survival analysis demonstrated a significantly shorter survival in patients showing TRPV1 mRNA downregulation. Multivariate Cox regression analysis indicated further that TRPV1 mRNA expression retained its significance as an independent risk factor. CONCLUSIONS: The progression of UC of human bladder is associated with a marked decrease in TRPV1 expression, with a progressive loss in high-grade muscle invasive UC. Downregulation of TRPV1 mRNA expression may represent an independent negative prognostic factor for bladder cancer patients.