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.

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 CD1381TRPV21 and CD1381TRPV22 PC populations in MM patients, whereas only the CD1381 TRPV22 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 CD1381TRPV22 MM cells and in MM cell lines transfected with TRPV2 (CD1381TRPV21). 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-jB pathways with major effects in TRPV21 cells. These data provide a rationale for using CBD to increase the activity of proteasome inhibitors in MM.Multiple myeloma (MM) is a haematological B cell malignancy characterised by clonal proliferation of plasma cells (PCs) and their accumulation in the bone marrow (BM). 1 MM displays enormous genomic instability and marked variation in clinical characteristics and patient survival. 1 In recent years, immunomodulatory drugs, proteasome inhibitors and other specific therapies have been developed to target myeloma cells and/or the BM microenvironment. 2 In addition, a number of new inhibitory agents targeting farnesyltransferase, mitogen-activated protein kinases (MAPKs), protein kinase B (AKT) and cell cycle proteins (e.g., cyclin D1 and D2) are currently under investigation for the treatment of relapsed/refractory MM in preclinical and clinical studies. 3 Bortezomib (BORT), a 26S proteasome inhibitor, is used to treat relapsed and refractory MM patients, but the molecular mechanisms responsible for the favourable outcome of this treatment remain unclear. 4,5 Although the initial overall rate of response to BORT is promising, the vast majority of patients who respond to this therapy develop resistance over time. 1,2 Key words: multiple myeloma, cannabidiol, transient receptor potential vanilloid type 2, bortezomib Abbreviations: Abs: antibodies; AKT: protein kinase B; BM: bone marrow; BORT: bortezomib; BrdU: 5-bromo-2-deoxyuridine; CBD: cannabidiol; Dw m : mitochondrial transmembrane potential; ERK: extracellular signal-related kinase; FACS: fluorescence activated cell sorting; FBS: foetal bovine serum; FISH: fluorescence in situ hybridisation; GAPDH: glyceraldehydes-3-phosphate dehydrogenase; MM: multiple myeloma; NAC: N-acetyl cysteine; pAKT: phospho protein kinase B; PC: plasma cell; pERK: phospho extracellular signalrelated kinase; ROS: reactive oxygen species; RP...

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 Ca2+ 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 Ca2+ ions flux, thus stimulating protein kinase A-dependent degranulation. In addition, TRPV2 is highly expressed in CD56+ natural killer cells. TRPV2 orchestrates Ca2+ 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 Ca2+ 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.

Background:Tyrosine kinase inhibitors (TKI) such as sunitinib and pazopanib display their efficacy in a variety of solid tumours. However, their use in therapy is limited by the lack of evidence about the ability to induce cell death in cancer cells. Our aim was to evaluate cytotoxic effects induced by sunitinib and pazopanib in 5637 and J82 bladder cancer cell lines.Methods:Cell viability was tested by MTT assay. Autophagy was evaluated by western blot using anti-LC3 and anti-p62 antibodies, acridine orange staining and FACS analysis. Oxygen radical generation and necrosis were determined by FACS analysis using DCFDA and PI staining. Cathepsin B activation was evaluated by western blot and fluorogenic Z-Arg-Arg-AMC peptide. Finally, gene expression was performed using RT–PCR Profiler array.Results:We found that sunitinib treatment for 24 h triggers incomplete autophagy, impairs cathepsin B activation and stimulates a lysosomal-dependent necrosis. By contrast, treatment for 48 h with pazopanib induces cathepsin B activation and autophagic cell death, markedly reversed by CA074-Me and 3-MA, cathepsin B and autophagic inhibitors, respectively. Finally, pazopanib upregulates the α-glucosidase and downregulates the TP73 mRNA expression.Conclusion:Our results showing distinct cell death mechanisms activated by different TKIs, provide the biological basis for novel molecularly targeted approaches.

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.

BackgroundThere is evidence that calcium (Ca2+) 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 alpha1D-adrenergic receptor (alpha1D-AR) and the transient receptor potential vanilloid type 1 (TRPV1) expression levels in human PCa tissues and evaluated the ability of alpha1D-AR to cross-talk with TRPV1 in PCa cell lines.MethodsThe 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, alpha1D-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. Alpha1D-AR/TRPV1 knockdown was obtained using siRNA transfection. Signalling pathways were evaluated by measurement of extracellular acidification rate, Ca2+ flux, IP3 production, western blot and MTT assay.ResultsThe levels of the alpha1D-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha1D-AR and TRPV1 expression levels was found. Moreover, alpha1D-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens. Noradrenaline (NA) induced an alpha1D-AR- and TRPV1-dependent protons release and Ca2+ 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 alpha1D-AR/TRPV1 double knockdown.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-921) contains supplementary material, which is available to authorized users.

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.

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.