Maria Beatrice Morelli

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.

Danger- and pathogen-associated molecular patterns recognition by pattern-recognition receptors and ion channels of the transient receptor potential family triggers the inflammasome activation in immune cells and sensory neurons

An increasing number of studies show that the activation of the innate immune system and inflammatory mechanisms play an important role in the pathogenesis of numerous diseases. The innate immune system is present in almost all multicellular organisms and its activation occurs in response to pathogens or tissue injury via pattern-recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Intracellular pathways, linking immune and inflammatory response to ion channel expression and function, have been recently identified. Among ion channels, the transient receptor potential (TRP) channels are a major family of non-selective cation-permeable channels that function as polymodal cellular sensors involved in many physiological and pathological processes.In this review, we summarize current knowledge of interactions between immune cells and PRRs and ion channels of TRP families with PAMPs and DAMPs to provide new insights into the pathogenesis of inflammatory diseases. TRP channels have been found to interfere with innate immunity via both nuclear factor-kB and procaspase-1 activation to generate the mature caspase-1 that cleaves pro-interleukin-1β cytokine into the mature interleukin-1β.Sensory neurons are also adapted to recognize dangers by virtue of their sensitivity to intense mechanical, thermal and irritant chemical stimuli. As immune cells, they possess many of the same molecular recognition pathways for danger. Thus, they express PRRs including Toll-like receptors 3, 4, 7, and 9, and stimulation by Toll-like receptor ligands leads to induction of inward currents and sensitization in TRPs. In addition, the expression of inflammasomes in neurons and the involvement of TRPs in central nervous system diseases strongly support a role of TRPs in inflammasome-mediated neurodegenerative pathologies. This field is still at its beginning and further studies may be required.Overall, these studies highlight the therapeutic potential of targeting the inflammasomes in proinflammatory, autoinflammatory and metabolic disorders associated with undesirable activation of the inflammasome by using specific TRP antagonists, anti-human TRP monoclonal antibody or different molecules able to abrogate the TRP channel-mediated inflammatory signals.

Pazopanib and sunitinib trigger autophagic and non-autophagic death of bladder tumour cells.

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.

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

Kalogris C, Caprodossi S, Amantini C, Lambertucci F, Nabissi M, Morelli M B, Farfariello V, Filosa A, Emiliozzi M C, Mammana G & Santoni G
(2010) Histopathology 57, 744–752
Expression of transient receptor potential vanilloid‐1 (TRPV1) in urothelial cancers of human bladder: relation to clinicopathological and molecular parameters

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.

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 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.

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 (βIII‐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 βIII‐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.

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.

Essential role of Gli proteins in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults. Despite several advances, little is known about GBM-specific aberrant signalling processes. The hedgehog (Hh) signalling pathway plays a central role in GBM pathogenesis and tumor progression. Its activation is mediated by sonic hedgehog (Shh), which binds to its receptor patched, PTCH, promoting Gli1 activation. Gli1 is a member of the Kruppel family of zinc finger transcription factors. Hh/Gli1 axis controls glioma stem cells (GSCs) behaviour, which is essential to GBM chemoand radioresistance. Thus, Gli1 modulates the expression of stemness genes and the self-renewal of CD133(+) GSCs. The activation of Hh/Gli1 in GSCs seems to be dependent on the insulin-like growth factor (IGF) signaling, which also contributes to intrinsic and acquired resistance of GSCs to temozolomide (TMZ). Beyond Hh signals, Gli1 activity is also regulated by several elements, including Ras, Myc, Akt, p53 and PTEN. Recently, a truncated variant of Gli1 (tGli1) has been demonstrated to gain the ability to regulate expression of genes that are not modulated by Gli1, such as the migration/invasion-associated CD24 or the human vascular endothelial growth factor-A (VEGF-A), leading to their upregulation. This review will summarize the role of Gli proteins in GBM tumorigenesis and their potential impact on GBM therapy and treatment resistance.

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.