Transient Receptor Potential Vanilloid Type-2 Research Articles

Protective role of TRPV2 in synaptic plasticity through the ERK1/2-CREB-BDNF pathway in chronic unpredictable mild stress rats

PurposeChronic stress is a significant risk factor for mood disorders such as depression, where synaptic plasticity plays a central role in pathogenesis. Transient Receptor Potential Vanilloid Type-2 (TRPV2) Ion Channels are implicated in hypothalamic-pituitary-adrenal axis disorders. Previous proteomic analysis indicated a reduction in TRPV2 levels in the chronic unpredictable mild stress (CUMS) rat model, yet its role in synaptic plasticity during depression remains to be elucidated. This study aims to investigate TRPV2's role in depression and its underlying mechanisms. MethodsIn vivo and in vitro experiments were conducted using the TRPV2-specific agonist probenecid and ERK1/2 inhibitors SCH772984. In vivo, rats underwent six weeks of CUMS before probenecid administration. Depressive-like behaviors were assessed through behavioral tests. ELISA kits measured 5-HT, DA, NE levels in rat hippocampal tissues. Hippocampal morphology was examined via Nissl staining. In vitro, rat hippocampal neuron cell lines were treated with ERK1/2 inhibitors SCH772984 and probenecid. Western blot, immunofluorescence, immunohistochemical staining, and RT-qPCR assessed TRPV2 expression, neurogenesis-related proteins, synaptic markers, and ERK1/2-CREB-BDNF signaling proteins. ResultsDecreased hippocampal TRPV2 levels were observed in CUMS rats. Probenecid treatment mitigated depressive-like behavior and enhanced hippocampal 5-HT, NE, and DA levels in CUMS rats. TRPV2 activation countered CUMS-induced synaptic plasticity inhibition. Probenecid activated the ERK1/2-CREB-BDNF pathway, suggesting TRPV2's involvement in this pathway via ERK1/2. ConclusionThese findings indicate that TRPV2 activation offers protective effects against depressive-like behaviors and enhances hippocampal synaptic plasticity in CUMS rats via the ERK1/2-CREB-BDNF pathway. TRPV2 emerges as a potential therapeutic target for depression.

Tetrandrine downregulates TRPV2 expression to ameliorate myocardial ischemia/reperfusion injury in rats via regulation of cardiomyocyte apoptosis, calcium homeostasis and mitochondrial function

Our previous study has indicated that tetrandrine (TET) can target miR-202-5p to repress the activation of transient receptor potential vanilloid type 2 (TRPV2), eventually ameliorating the progression of myocardial ischemia/reperfusion injury (MI/RI). This study is aimed to further ascertain the detailed mechanisms between TET and TRPV2 in MI/RI pathogenesis. Here, a myocardial I/R injury rat model and a hypoxia-reoxygenation (H/R) model in rat myocardial cell line (H9C2 cells) were established. We reported that pronounced upregulation of TRPV2 was observed in I/R rats and H/R-induced H9C2 cells. Silencing of TRPV2 could improve cardiac function and myocardial injury, reduced infarction size, and promoted cardiomyocyte proliferation in I/R rats. In I/R rats or H/R-induced H9C2 cells, cardiomyocyte apoptosis was inhibited by knocking-down TRPV2. Meanwhile, the silenced TRPV2 or TET treatment ameliorated the damaged mitochondrial structure, mitigated ROS generation, restored the impaired ΔΨM, inhibited mPTP opening and alleviated Ca2+ overload in H/R-induced H9C2 cells. The results obtained from the overexpression of TRPV2 were contrary to those depicted above. Moreover, TET could downregulate TRPV2 expression, while the overexpression of TRPV2 could reverse the above protective effects of TET in H/R-induced H9C2 cells. The results indicated that TET may function as a TRPV2 blocking agent, thereby attenuating the progression of MI/RI through modulation of cardiomyocyte apoptosis, calcium homeostasis and mitochondrial function. These findings offer a theoretical foundation for potential clinical application of TET therapy in patients with MI/RI.

Immunohistochemical characterization of transient receptor potential vanilloid types 2 and 1 in a trinitrobenzene sulfonic acid-induced rat colitis model with visceral hypersensitivity

Transient receptor potential vanilloid type 2 (TRPV2) and type 1 (TRPV1) are originally identified as heat-sensitive TRP channels. We compared the expression patterns of TRPV2 and TRPV1 in the rat distal colon and extrinsic primary afferent neurons, and investigated their roles in visceral hypersensitivity in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rats. Both TRPV2 and TRPV1 expressions in the colon, dorsal root ganglion (DRG), and nodose ganglion (NG) were significantly upregulated in the TNBS-induced colitis model. TRPV2 cell bodies co-localized with the intrinsic primary afferent marker NeuN and the inhibitory motor neuronal marker nNOS in the myenteric plexus. TRPV2 expressions were further detected in the resident macrophage marker ED2 in the mucosa. In contrast, no TRPV1-expressing cell bodies were detected in the myenteric plexus. Both TRPV2- and TRPV1-positive cell bodies in the DRG and NG were double-labeled with the neuronal retrograde tracer fluorescent fluorogold. Large- and medium-sized TRPV2-positive neurons were labeled with the A-fiber marker NF200, calcitonin gene-related peptide (CGRP), and substance P (SP) in the DRG while small-sized TRPV1-positive neurons were labeled with the C-fiber markers IB4, CGRP, and SP. TRPV2- and TRPV1-positive NG neurons were labeled with NF200 and IB4. TNBS treatment increased p-ERK1/2-positive cells in TRPV2 and TRPV1 neurons but did not affect the TRPV2 and TRPV1 subpopulations in the DRG and NG. Both TRPV2 and TRPV1 antagonists significantly alleviated visceral hypersensitivity in TNBS-induced colitis model rats. These findings suggest that intrinsic/extrinsic TRPV2- and extrinsic TRPV1-neurons contribute to visceral hypersensitivity in an experimental colitis model.

Synthetic alkyl-ether-lipid promotes TRPV2 channel trafficking trough PI3K/Akt-girdin axis in cancer cells and increases mammary tumour volume

The Transient Receptor Potential Vanilloid type 2 (TRPV2) channel is highly selective for Ca2+ and can be activated by lipids, such as LysoPhosphatidylCholine (LPC). LPC analogues, such as the synthetic alkyl-ether-lipid edelfosine or the endogenous alkyl-ether-lipid Platelet Activating Factor (PAF), modulates ion channels in cancer cells. This opens the way to develop alkyl-ether-lipids for the modulation of TRPV2 in cancer. Here, we investigated the role of 2-Acetamido-2-Deoxy-l-O-Hexadecyl-rac-Glycero-3-PhosphatidylCholine (AD-HGPC), a new alkyl-ether-lipid (LPC analogue), on TRPV2 trafficking and its impact on Ca2+ -dependent cell migration. The effect of AD-HGPC on the TRPV2 channel and tumour process was further investigated using calcium imaging and an in vivo mouse model. Using molecular and pharmacological approaches, we dissected the mechanism implicated in alkyl-ether-lipids sensitive TRPV2 trafficking. We found that TRPV2 promotes constitutive Ca2+ entry, leading to migration of highly metastatic breast cancer cell lines through the PI3K/Akt-Girdin axis. AD-HGPC addresses the functional TRPV2 channel in the plasma membrane through Golgi stimulation and PI3K/Akt/Rac-dependent cytoskeletal reorganization, leading to constitutive Ca2+ entry and breast cancer cell migration (without affecting the development of metastasis), in a mouse model. We describe, for the first time, the biological role of a new alkyl-ether-lipid on TRPV2 channel trafficking in breast cancer cells and highlight the potential modulation of TRPV2 by alkyl-ether-lipids as a novel avenue for research in the treatment of metastatic cancer.

The Transient Receptor Potential Vanilloid Type 2 (TRPV2) Channel-A New Druggable Ca2+ Pathway in Red Cells, Implications for Red Cell Ion Homeostasis.

The Transient Receptor Potential Vanilloid Type 2 (TRPV2) Channel-A New Druggable Ca2+ Pathway in Red Cells, Implications for Red Cell Ion Homeostasis.

TRPV2: A Cancer Biomarker and Potential Therapeutic Target.

The Transient Receptor Potential Vanilloid type-2 (TRPV2) channel exhibits oncogenicity in different types of cancers. TRPV2 is implicated in signaling pathways that mediate cell survival, proliferation, and metastasis. In leukemia and bladder cancer, the oncogenic activity of TRPV2 was linked to alteration of its expression profile. In multiple myeloma patients, TRPV2 overexpression correlated with bone tissue damage and poor prognosis. In prostate cancer, TRPV2 overexpression was associated with the castration-resistant phenotype and metastasis. Loss or inactivation of TRPV2 promoted glioblastoma cell proliferation and increased resistance to CD95-induced apoptotic cell death. TRPV2 overexpression was associated with high relapse-free survival in triple-negative breast cancer, whereas the opposite was found in patients with esophageal squamous cell carcinoma or gastric cancer. Another link was found between TRPV2 expression and either drug-induced cytotoxicity or stemness of liver cancer. Overall, these findings validate TRPV2 as a prime candidate for cancer biomarker and future therapeutic target.

The Transient Receptor Potential Vanilloid Type-2 (TRPV2) Ion Channels in Neurogenesis and Gliomagenesis: Cross-Talk between Transcription Factors and Signaling Molecules

Recently, the finding of cancer stem cells in brain tumors has increased the possibilities for advancing new therapeutic approaches with the aim to overcome the limits of current available treatments. In addition, a role for ion channels, particularly of TRP channels, in developing neurons as well as in brain cancer development and progression have been demonstrated. Herein, we focus on the latest advancements in understanding the role of TRPV2, a Ca2+ permeable channel belonging to the TRPV subfamily in neurogenesis and gliomagenesis. TRPV2 has been found to be expressed in both neural progenitor cells and glioblastoma stem/progenitor-like cells (GSCs). In developing neurons, post-translational modifications of TRPV2 (e.g., phosphorylation by ERK2) are required to stimulate Ca2+ signaling and nerve growth factor-mediated neurite outgrowth. TRPV2 overexpression also promotes GSC differentiation and reduces gliomagenesis in vitro and in vivo. In glioblastoma, TRPV2 inhibits survival and proliferation, and induces Fas/CD95-dependent apoptosis. Furthermore, by proteomic analysis, the identification of a TRPV2 interactome-based signature and its relation to glioblastoma progression/recurrence, high or low overall survival and drug resistance strongly suggest an important role of the TRPV2 channel as a potential biomarker in glioblastoma prognosis and therapy.

Structure-Based Discovery of a Subtype-Selective Inhibitor Targeting a Transient Receptor Potential Vanilloid Channel.

Discovery of potent selective inhibitors targeting a protein from a highly conserved family is challenging. Using a strategy combining structural and evolutionary information, we discovered transient receptor potential (TRP) subtype-selective inhibitors (transient receptor potential vanilloid type 2 (TRPV2) inhibitors). We unveiled three ligand-binding sites of TRPV2 and compounds that bind to these sites. Structural optimization of the best-hit compound provided a potent selective TRPV2 inhibitor, SET2. The molecular basis and subtype-selective inhibition mechanism were quantitatively characterized and experimentally verified. Then, as an effective chemical probe, SET2 was used to investigate the function role of TRPV2. SET2-induced inhibition of TRPV2 reduced prostate cancer migration, which indicated TRPV2 as an antimetastasis therapeutic target. In addition, functional assays suggested that TRPV2 was coupled to a validated metastasis mediator, LPAR1. The discovery of the potent selective inhibitor potentially leads to novel avenues for pharmacological applications and therapeutic development targeting the TRPV2 channel.

Elongation of the Hydrophobic Chain as a Molecular Switch: Discovery of Capsaicin Derivatives and Endogenous Lipids as Potent Transient Receptor Potential Vanilloid Channel 2 Antagonists.

The transient receptor potential vanilloid type-2 (TRPV2) protein is a nonselective Ca2+ permeable channel member of the TRPV subfamily, still considered an orphan TRP channel due to the scarcity of available selective and potent pharmacological tools and endogenous modulators. Here we describe the discovery of novel synthetic long-chain capsaicin derivatives as potent TRPV2 antagonists in comparison to the totally inactive capsaicin, the role of their hydrophobic chain, and how the structure-activity relationships of such derivatives led, through a ligand-based approach, to the identification of endogenous long-chain fatty acid ethanolamides or primary amides acting as TRPV2 antagonists. Both synthetic and endogenous antagonists exhibited differential inhibition against known TRPV2 agonists characterized by distinct kinetic profiles. These findings represent the first example of both synthetic and naturally occurring TRPV2 modulators with efficacy in the submicromolar/low-micromolar range, which will be useful for clarifying the physiopathological roles of this receptor, its regulation, and its targeting in pathological conditions.

TRPV2 is a novel biomarker and therapeutic target in triple negative breast cancer.

Transient receptor potential vanilloid type-2 (TRPV2) is an ion channel that is triggered by agonists like cannabidiol (CBD). Triple negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Chemotherapy is still the first line for the treatment of TNBC patients; however, TNBC usually gains rapid resistance and unresponsiveness to chemotherapeutic drugs. In this study, we found that TRPV2 protein is highly up-regulated in TNBC tissues compared to normal breast tissues. We also observed that TNBC and estrogen receptor alpha negative (ERβ-) patients with higher TRPV2 expression have significantly higher recurrence free survival compared to patients with lower TRPV2 expression especially those who were treated with chemotherapy. In addition, we showed that TRPV2 overexpression or activation by CBD significantly increased doxorubicin (DOX) uptake and apoptosis in TNBC cells. The induction of DOX uptake was abrogated by TRPV2 blocking or downregulation. In vivo mouse model studies showed that the TNBC tumors derived from CBD+DOX treated mice have significantly reduced weight and increased apoptosis compared to those treated with CBD or DOX alone. Overall, our studies for the first time revealed that TRPV2 might be a good prognostic marker for TNBC and ERβ- breast cancer patient especially for those who are treated with chemotherapy. In addition, TRPV2 activation could be a novel therapeutic strategy to enhance the uptake and efficacy of chemotherapy in TNBC patients.

Abstract 3487: A novel role of transient receptor potential vanilloid type 2 (TRPV2) for augmentation of chemotherapeutic drug efficacy in triple-negative breast cancer

Abstract Transient receptor potential vanilloid type 2 (TRPV2) is a non-selective cation channel that is triggered by agonists like cannabidiol (CBD). Chemotherapeutic drugs such as doxorubicin (DOX) and paclitaxel (PAC) are routinely used for treatment of Triple negative breast cancer (TNBC) patients however, these drugs are usually resisted and show limited efficacy and more side effects at higher doses. In the present study, we analyzed the ability of TRPV2 activation in enhancing the chemotherapeutic efficacy in TNBC cells. Interestingly, we found that TRPV2 is significantly up regulated in primary and metastatic breast tumor tissues compared to normal breast tissues using tissue microarray analysis. In addition, using publically available Kaplan Miere Plotter datasets, we found that estrogen receptor (ER) negative patients with higher TRPV2 expression levels and committed to anti-tumor chemotherapy have significantly higher survival than patients committed to anti-tumor chemotherapy but have lower TRPV2 expression levels. Our in vitro studies showed that TNBC cells subjected to combination treatment of CBD and DOX show enhanced proliferation inhibition compared to cells treated with DOX only . Analysis of molecular mechanisms showed higher levels of cleaved PARP and cleaved Caspase-3 in combination treatment compared to Dox alone. Further studies revealed that CBD activates more uptake of DOX into TNBC cells. This increased uptake was abrogated upon treatment with specific TRPV2 blocker. Furthermore, TRPV2 blocker inhibited the increased levels of cleaved PARP and cleaved caspase-3 in the combination treatment. By using two orthotopic mouse model, we showed that mice treated with CBD in combination with doxorubicin have significantly less tumor weight, fewer Ki67 and CD31 positive cells, and significantly higher level of cleaved caspase 3 and cleaved PARP compared to the mice treated with CBD or DOX alone. This study suggests the use of TRPV2 agonists as an adjuvant therapy to improve the anti-tumor chemotherapeutic efficacy especially in TNBC patients. Citation Format: Mohamad M. Elbaz, Mohd W. Nasser, Helong Zhao, Ramesh K. Ganju. A novel role of transient receptor potential vanilloid type 2 (TRPV2) for augmentation of chemotherapeutic drug efficacy in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3487. doi:10.1158/1538-7445.AM2015-3487

HIV-1-Tat excites cardiac parasympathetic neurons of nucleus ambiguus and triggers prolonged bradycardia in conscious rats

The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 transactivator of transcription (Tat, fragment 1-86) produced a concentration-dependent increase in cytosolic Ca(2+) in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using store-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca(2+) elevation: 1) lysosomal Ca(2+) mobilization, 2) Ca(2+) release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools, and 3) Ca(2+) influx via transient receptor potential vanilloid type 2 (TRPV2) channels. Activation of TRPV2, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca(2+) entry. In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV2 played a major role. Our results support previous studies, indicating that Tat promotes bradycardia and, consequently, may be involved in the QT interval prolongation reported in HIV-infected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy.

The effects of cannabidiol and its synergism with bortezomib in multiple myeloma cell lines. A role for transient receptor potential vanilloid type‐2

Multiple myeloma (MM) is a plasma cell (PC) malignancy characterised by the accumulation of a monoclonal PC population in the bone marrow (BM). Cannabidiol (CBD) is a non-psychoactive cannabinoid with antitumoural activities, and the transient receptor potential vanilloid type-2 (TRPV2) channel has been reported as a potential CBD receptor. TRPV2 activation by CBD decreases proliferation and increases susceptibility to drug-induced cell death in human cancer cells. However, no functional role has been ascribed to CBD and TRPV2 in MM. In this study, we identified the presence of heterogeneous CD138+TRPV2+ and CD138+TRPV2- PC populations in MM patients, whereas only the CD138+ TRPV2- population was present in RPMI8226 and U266 MM cell lines. Because bortezomib (BORT) is commonly used in MM treatment, we investigated the effects of CBD and BORT in CD138+TRPV2- MM cells and in MM cell lines transfected with TRPV2 (CD138+TRPV2+). These results showed that CBD by itself or in synergy with BORT strongly inhibited growth, arrested cell cycle progression and induced MM cells death by regulating the ERK, AKT and NF-κB pathways with major effects in TRPV2+ cells. These data provide a rationale for using CBD to increase the activity of proteasome inhibitors in MM.

Triggering of the TRPV2 channel by cannabidiol sensitizes glioblastoma cells to cytotoxic chemotherapeutic agents

The aggressive behavior of Glioblastoma multiforme (GBM) is mainly due to high invasiveness and proliferation rate as well as to high resistance to standard chemotherapy. Several chemotherapeutic agents like temozolomide (TMZ), carmustine (BCNU) or doxorubicin (DOXO) have been employed for treatment of GBM, but they display limited efficacy. Therefore, it is important to identify new treatment modalities to improve therapeutic effects and enhance GBM chemosensitivity. Recently, activation of the transient receptor potential vanilloid type 2 (TRPV2) has been found to inhibit human GBM cell proliferation and overcome BCNU resistance of GBM cells. Herein, we evaluated the involvement of cannabidiol (CBD)-induced TRPV2 activation, in the modulation of glioma cell chemosensitivity to TMZ, BCNU and DOXO. We found that CBD increases TRPV2 expression and activity. CBD by triggering TRPV2-dependent Ca(2+) influx increases drug uptake and synergizes with cytotoxic agents to induce apoptosis of glioma cells, whereas no effects were observed in normal human astrocytes. Moreover, as the pore region of transient receptor potential (TRP) channels is critical for ion channel permeation, we demonstrated that deletion of TRPV2 poredomain inhibits CBD-induced Ca(2+) influx, drug uptake and cytotoxic effects. Overall, we demonstrated that co-administration of cytotoxic agents together with the TRPV2 agonist CBD increases drug uptake and parallelly potentiates cytotoxic activity in human glioma cells.

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 (β(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 β(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.

TRPV2 Expression and Its Role in Proliferation of Human Multiple Myeloma Cell Lines

Abstract 5003Multiple myeloma (MM) is a disease typically characterized by repeated relapse that needs several new-drugs throughout its clinical course. Therefore, increasing knowledge of the pathways involved in the pathophysiology of MM will contribute to identified new therapeutic strategies. Specific signaling pathways as: nuclear factor kappa B (NFkB), farnelsyl-transferase (FTI), mitogenen-activated protein kinase (MAPK), proteosoma and others have been found to be disregulated in MM patients and cell lines. These findings have allowed the design of specific inhibitors that have been assessed in preclinical and clinical studies. Recent data have highlighted the contribute of ion channels (K+, Cl−, Na+ and Ca2+) in the regulation of cell proliferation, chemo-resistance, migration and invasion. Transient Receptor Potential Vanilloid type-2 (TRPV2) is a cation channel, member of the TRPV family, and its expression in human cancer cells and tissues has been reported for gliomas, prostate, bladder and pancreas, while no data were available in MM or in other hematological malignancies. TRPV2 expression has been found to influence cancer cell responses to chemo-therapeutic drugs as well as proliferation, migration and apoptosis, in part by increasing intracellular Ca2+ influx and/or by regulating specific signal pathways, like MAPK associated pathways. TRPV2 responds to noxious heat with an activation threshold of >52°C as well as to changes in osmolarity and membrane stretch; in addition, TRPV2 is activated by agonists such as 2-aminoethoxydiphenyl borate, as well as cannabinoids. The aim of this study was, firstly, to evaluate if TRPV2 was expressed in three human MM cell lines established models (RPMI, SKO-007, U266). Secondly we investigated on the role of TRPV2 activation in regulating the proteosoma inhibitor Bortezomib activity, in MM cell lines. By Real-Time PCR analysis we demonstrated that TRPV2 gene was expressed, with comparable relative levels, in the MM cell lines studied, and similar results were obtained at protein levels by Western blot analysis. Moreover, by immunofluorescence and FACS analysis we found that the percentage of TRPV2+ cells was 11% in RPMI, 3% in U266 and 2.6% in SKO-007. These data indicate that TRPV2 was expressed at low levels in MM cell lines. Since lost of TRPV2 expression was found to be associated with higher cell proliferation rate, in other tumor cell lines, and increasing of TRPV2 transcription and/or activity by specific agonists induced cell death and anti-proliferative effects, we evaluated the biological effects of TRPV2 activation in MM cell lines by MTT assay. The results showed that, after three incubation days, TRPV2 activation induced a decrease of cell viability of 70% in RPMI, 60% in U266 and 55% in SKO-007, compared to control. To evaluate if the reduced cell viability was dependent by an anti-proliferative and/or apoptotic process, the three cell lines treated with the TRPV2 agonist were analyzed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay for proliferation and by Annexin-V apoptosis assays. Data shown a decrease of BrdU+ cells in TRPV2 agonist treated cells (40% in RPMI, 30% in U266 and 28% in SKO-007) compared to control, while no significant differences were observed by Annexin-V analysis. Moreover, to evaluate a putative role of TRPV2 in influencing Bortezomib cytotoxicity, RPMI, SKO-007 and U266, were co-treated with the TRPV2 agonist in combination or not with Bortezomib, for three days. By a dose-response MTT analysis we determined that TRPV2 activation reduced MM cell lines viability more than 40% in RPMI, 20 % in U266 and 15% in SKO-007, compared to Bortezomib (5 ng/ml) alone. Summarizing, these preliminary data demonstrated that the TRPV2 cation channel was expressed in human MM cell lines and that activation of TRPV2 could play a role in increasing Bortezomib cytotoxicity, by inhibiting cell proliferation rather than increasing apoptosis of MM cells. These data may open new perspectives in combination therapy, albeit the molecular mechanisms undergone TRPV2 activation needs to be clarified. Disclosures:No relevant conflicts of interest to declare.

Transient Receptor Potential Vanilloid Type 2 (TRPV2) Expression in Normal Urothelium and in Urothelial Carcinoma of Human Bladder: Correlation with the Pathologic Stage

ObjectiveTo evaluate the expression of transient receptor potential vanilloid type 2 (TRPV2) in normal human bladder and urothelial carcinoma (UC) tissues. MethodsBladder specimens were obtained by transurethral resection or radical cystectomy. TRPV2 mRNA expression in normal human urothelial cells (NHUCs), UC cell lines, and formalin-fixed paraffin-embedded normal (n=6) and cancer bladder tissues (n=58) was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-PCR). TRPV2 protein expression was assessed by cytofluorimetric and confocal microscopy analyses in NHUCs and UC cells and by Western blotting and immunohistochemistry in normal and UC tissues. ResultsEnhanced TRPV2 mRNA and protein expression was found in high-grade and -stage UC specimens and UC cell lines. Both the full-length TRPV2 (hTRPV2) and a short splice-variant (s-TRPV2) were detected in NHUC and normal bladder specimens, whereas a progressive decline of s-TRPV2 in pTa, pT1, and pT2 stages was observed, up to a complete loss in pT3 and pT4 UC specimens. ConclusionsNormal human urothelial cells and bladder tissue specimens express TRPV2 at both the mRNA and protein levels. A progressive loss of s-TRPV2 accompanied by a marked increase of hTRPV2 expression was found in high-grade and -stage UC tissues.