Cannabidiol stimulates Aml-1a-dependent glial differentiation and inhibits glioma stem-like cells proliferation by inducing autophagy in a TRPV2-dependent manner.

Glioma stem-like cells (GSCs) correspond to a tumor cell subpopulation, involved in glioblastoma multiforme (GBM) tumor initiation and acquired chemoresistance. Currently, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population. Recently, the effect of cannabinoids (CBs) in promoting glial differentiation and inhibiting gliomagenesis has been evidenced. Herein, we demonstrated that cannabidiol (CBD) by activating transient receptor potential vanilloid-2 (TRPV2) triggers GSCs differentiation activating the autophagic process and inhibits GSCs proliferation and clonogenic capability. Above all, CBD and carmustine (BCNU) in combination overcome the high resistance of GSCs to BCNU treatment, by inducing apoptotic cell death. Acute myeloid leukemia (Aml-1) transcription factors play a pivotal role in GBM proliferation and differentiation and it is known that Aml-1 control the expression of several nociceptive receptors. So, we evaluated the expression levels of Aml-1 spliced variants (Aml-1a, b and c) in GSCs and during their differentiation. We found that Aml-1a is upregulated during GSCs differentiation, and its downregulation restores a stem cell phenotype in differentiated GSCs. Since it was demonstrated that CBD induces also TRPV2 expression and that TRPV2 is involved in GSCs differentiation, we evaluated if Aml-1a interacted directly with TRPV2 promoters. Herein, we found that Aml-1a binds TRPV2 promoters and that Aml-1a expression is upregulated by CBD treatment, in a TRPV2 and PI3K/AKT dependent manner. Altogether, these results support a novel mechanism by which CBD inducing TRPV2-dependent autophagic process stimulates Aml-1a-dependent GSCs differentiation, abrogating the BCNU chemoresistance in GSCs.

Resiniferatoxin induces death of bladder cancer cells associated with mitochondrial dysfunction and reduces tumor growth in a xenograft mouse model.

Bladder cancer (BC) is the fifth most common non-cutaneous malignancy and the most common form of BC in Western countries is transitional cell carcinoma. Resiniferatoxin (RTX) has found therapeutic usefulness for the treatment of bladder dysfunction but no data are available on its use as chemotherapeutic agent. The aim of this work is to evaluate the use of RTX as new anti-cancer drug in BC therapy. The effects of RTX on cell viability and cell death were evaluated on T24 and 5637 BC cell lines by MTT assay, cell cycle analysis, Annexin-V/PI staining and agarose gel electrophoresis of DNA. Mitochondrial depolarization and ROS production were assessed by flow cytometry. ADP/ATP ratio was measured by bioluminescence and caspase 3 cleavage by Western blot. For in vivo experiments, athymic nude mice, xenografted with T24 cells, received subcutaneous administrations of RTX. Tumor volumes were measured and immunohistochemistry was performed on tumor sections. Our data demonstrated that RTX influences cell cycle and induces necrotic cell death of BC cells by altering mitochondrial function, leading to depolarization, increase in ADP/ATP ratio and ROS production. Moreover, RTX is able to reduce tumor growth in a xenograft mouse model. Overall, we demonstrated that RTX induces necrotic cell death of BC cells and reduces tumor growth in a xenograft mouse model of BC, suggesting RTX as a new potential anti-cancer drug in BC chemotherapy.

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.

Advances in transient receptor potential vanilloid-2 channel expression and function in tumor growth and progression.

Aim of this review is to study the role of the TRPV2 channel, a member of the TRPV subfamily of TRP channels, in tumor progression. Physiologically, the triggering of TRPV2 by agonists/activators (e.g., growth factors, hormones and cannabinoids), by inducing TRPV2 translocation from the endosome to the plasmatic membrane, inhibit cell proliferation and induce necrosis and/or apoptosis. Thus, loss or alterations of TRPV2 proliferative and apoptotic signals, results in uncontrolled proliferation and augmented resistance to apoptotic stimuli. For example in prostate cancer cells, the TRPV2 activation following lysophospholipid or adrenomedullin stimulation enhances the invasiveness of cancer cells; furthermore, the increased malignancy of castration-resistant prostate cancer cells was associated with enhanced TRPV2 expression, mainly in metastatic prostate cancer cells. In addition, the TRPV2 cellular functions may also to be related to the presence of TRPV2 variants, able to interfere with the physiological functions of normal TRPV2 channels. In this regard, bladder cancer tumors show loss or reduction of a short TRPV2 variant during cancer progression, with increased malignancy and invasiveness. High expression of TRPV2 was also observed more frequently in esophageal squamous cell carcinoma patients with advanced pT stage, lymph node metastasis and advanced pathological stage.

Loss of TRPV2 Homeostatic Control of Cell Proliferation Drives Tumor Progression.

Herein we evaluate the involvement of the TRPV2 channel, belonging to the Transient Receptor Potential Vanilloid channel family (TRPVs), in development and progression of different tumor types. In normal cells, the activation of TRPV2 channels by growth factors, hormones, and endocannabinoids induces a translocation of the receptor from the endosomal compartment to the plasma membrane, which results in abrogation of cell proliferation and induction of cell death. Consequently, loss or inactivation of TRPV2 signaling (e.g., glioblastomas), induces unchecked proliferation, resistance to apoptotic signals and increased resistance to CD95-induced apoptotic cell death. On the other hand, in prostate cancer cells, Ca2+-dependent activation of TRPV2 induced by lysophospholipids increases the invasion of tumor cells. In addition, the progression of prostate cancer to the castration-resistant phenotype is characterized by de novo TRPV2 expression, with higher TRPV2 transcript levels in patients with metastatic cancer. Finally, TRPV2 functional expression in tumor cells can also depend on the presence of alternative splice variants of TRPV2 mRNA that act as dominant-negative mutant of wild-type TRPV2 channels, by inhibiting its trafficking and translocation to the plasma membrane. In conclusion, as TRP channels are altered in human cancers, and their blockage impair tumor progression, they appear to be a very promising targets for early diagnosis and chemotherapy.

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.

Expression and function of the transient receptor potential ion channel family in the hematologic malignancies.

Transient receptor potential (TRP) channels are important candidates mediating Ca(2+) influx in excitable and nonexcitable cells such as normal and neoplastic hematopoietic tissues. They are non selective cation channels implicated in Ca(2+) signaling in hematologic tumor cells. Here, we review the growing experimental evidence indicating that TRP channels should be included among the genes whose expression is altered in hematologic malignancies such as leukemias (AML, ALL, CML and CLL), B- and T-lymphomas and multiple myelomas (MM). These effects depend on the widespread roles played by the TRP channels in the modulation of the proliferation, differentiation and apoptosis of the hematopoietic cells. The analysis of the expression of the different TRP channels belonging to the TRPMs, TRPVs, TRPCs, TRPPs channel families expressed in different hematological malignacies, evidenced a widespread expression of TRPV2 channel in the myeloid and lymphoid leukemias, and a very peculiar expression of this channel in different types of B cell lymphomas and multiple myeloma, that is parallel to the restricted expression of TRPV2 in normal immune cells with respect to its presence in other human tissues. In vivo studies in children AML and ALL patients also evidenced the presence of a genetic polymorphism of the TRPM5 gene, that reduced the risk to develop leukemia in the children. Finally, the coexpression of TRPV5 and TRPV6 channels in lymphocytes, and their involvement in the radioresistance of K562 erythroleukemia cells to ionizing radiation exposure is of more interest. Thus in conclusion, the TRP channels represent promising targets for hematologic cancer therapy, and their exploitation may open to novel pharmaceutical and clinical approaches.

Caloric restriction increases the sensitivity to the hyperphagic effect of nociceptin/orphanin FQ limiting its ability to reduce binge eating in female rats.

RATIONALE: Nociceptin/orphanin FQ (N/OFQ) is a functional antagonist of corticotrophin-releasing factor, the main mediator of the stress response. Stress represents a key determinant of binge eating (BE) for highly palatable food (HPF). OBJECTIVES: In relation to the antistress properties of N/OFQ, we evaluated its effect on BE. After the observation that episodes of food restriction increase the sensitivity to its hyperphagic effects, the function of NOP receptor and N/OFQ was investigated after cycles of food restrictions. MATERIALS AND METHODS: In BE experiments, four groups were used: rats fed normally and not stressed or stressed, rats exposed to cycles of restriction/refeeding and then stressed, or not stressed. In the other experiments, two groups were used: rats exposed or not to food restriction. RESULTS: Only restricted and stressed rats exhibited BE for HPF (containing chocolate cream). Intracerebroventricular injections of N/OFQ of 0.5 nmol/rat significantly reduced BE. N/OFQ 1 nmol/rat did not reduce BE but significantly increased HPF intake following food restrictions. Cycles of food restriction increased animals' sensitivity to the hyperphagic effect of N/OFQ for HPF. In situ hybridization studies following food restrictions showed decreased ppN/OFQ mRNA expression in the bed nucleus of the stria terminalis and increased expression of ppN/OFQ and NOP receptor mRNA in the ventral tegmental area and in the ventromedial hypothalamus, respectively. CONCLUSIONS: These findings indicate that N/OFQ slightly reduces BE at low doses, while higher doses increase HPF intake, due to increased sensitivity to its hyperphagic effect following a history of caloric restrictions.

The role of transient receptor potential vanilloid type-2 ion channels in innate and adaptive immune responses.

The transient receptor potential vanilloid type-2 (TRPV2), belonging to the transient receptor potential channel family, is a specialized ion channel expressed in human and other mammalian immune cells. This channel has been found to be expressed in CD34(+) hematopoietic stem cells, where its cytosolic Ca(2) (+) activity is crucial for stem/progenitor cell cycle progression, growth, and differentiation. In innate immune cells, TRPV2 is expressed in granulocytes, macrophages, and monocytes where it stimulates fMet-Leu-Phe migration, zymosan-, immunoglobulin G-, and complement-mediated phagocytosis, and lipopolysaccharide-induced tumor necrosis factor-alpha and interleukin-6 production. In mast cells, activation of TRPV2 allows intracellular Ca(2) (+) ions flux, thus stimulating protein kinase A-dependent degranulation. In addition, TRPV2 is highly expressed in CD56(+) natural killer cells. TRPV2 orchestrates Ca(2) (+) signal in T cell activation, proliferation, and effector functions. Moreover, messenger RNA for TRPV2 are expressed in CD4(+) and CD8(+) T lymphocytes. Finally, TRPV2 is expressed in CD19(+) B lymphocytes where it regulates Ca(2) (+) release during B cell development and activation. Overall, the specific expression of TRPV2 in immune cells suggests a role in immune-mediated diseases and offers new potential targets for immunomodulation.

Oncogenic and anti-oncogenic effects of transient receptor potential channels.

Transient Receptor Potential (TRP) channels affect several inflammatory and neoplastic conditions. About thirty TRPs have been identified to date and divided into seven families: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPClike). Among these, 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 functional role of TRP channels in different types of cancers. TRP channels have been recently implicated in the triggering of enhanced proliferation, aberrant differentiation, and resistance to apoptotic cell death, leading to uncontrolled tumor growth and progression. Depending on cancer stage, up and down-regulation of TRP mRNAs and protein expression have been reported. These changes have been shown to exhibit cancer promoting (oncogenic) or inhibiting/delaying (tumor suppressor) effects. We are only at the beginning, and more detailed study on the physiopathologic role of TRP channels is required to understand how the deregulation of TRP channel expression and function contributes to tumor development and progression. It is hoped that greater knowledge about TRP biology will enable future development of new chemotherapeutic agents for specific TRP targets, and the use of TRP channels as evaluable markers in diagnostic and/or prognostic analysis.

Antioncogenic effects of transient receptor potential vanilloid 1 in the progression of transitional urothelial cancer of human bladder.

The progression of normal cells to a tumorigenic and metastatic state involves the accumulation of mutations in multiple key signaling proteins, encoded by oncogenes and tumor suppressor genes. Recently, members of the TRP channel family have been included in the oncogenic and tumor suppressor protein family. TRPM1, TRPM8, and TRPV6 are considered to be tumor suppressors and oncogenes in localized melanoma and prostate cancer, respectively. Herein, we focus our attention on the antioncogenic properties of TRPV1. Changes in TRPV1 expression occur during the development of transitional cell carcinoma (TCC) of human bladder. A progressive decrease in TRPV1 expression as the TCC stage increases triggers the development of a more aggressive gene phenotype and invasiveness. Finally, downregulation of TRPV1 represents a negative prognostic factor in TCC patients. The knowledge of the mechanism controlling TRPV1 expression might improve the diagnosis and new therapeutic strategies in bladder cancer.