Cannabigerol Induces Autophagic Cell Death by Inhibiting EGFR-RAS Pathways in Human Pancreatic Ductal Adenocarcinoma Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) is the most frequent infiltrating type of pancreatic cancer. The poor prognosis associated with this cancer is due to the absence of specific biomarkers, aggressiveness, and treatment resistance. PDAC is a deadly malignancy bearing distinct genetic alterations, the most common being those that result in cancer-causing versions of the KRAS gene. Cannabigerol (CBG) is a non-psychomimetic cannabinoid with anti-inflammatory properties. Regarding the anticancer effect of CBG, up to now, there is only limited evidence in human cancers. To fill this gap, we investigated the effects of CBG on the PDAC cell lines, PANC-1 and MIAPaCa-2. The effect of CBG activity on cell viability, cell death, and EGFR-RAS-associated signaling was investigated. Moreover, the potential synergistic effect of CBG in combination with gemcitabine (GEM) and paclitaxel (PTX) was investigated. MTT was applied to investigate the effect of CBG on PDAC cell line viabilities. Annexin-V and Acridine orange staining, followed by cytofluorimetric analysis and Western blotting, were used to evaluate CBG's effect on cell death. The modulation of EGFR-RAS-associated pathways was determined by Western blot analysis and a Milliplex multiplex assay. Moreover, by employing the MTT data and SynergyFinder Plus software analysis, the effect of the combination of CBG and chemotherapeutic drugs was determined.

The effects of cannabidiol via TRPV2 channel in chronic myeloid leukemia cells and its combination with imatinib.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by accumulation of immature cells in bone marrow and peripheral blood. Although successful results were obtained with tyrosine kinase inhibitors, several patients showed resistance. For this reason, the identification of new strategies and therapeutic biomarkers represents an attractive goal. The role of transient receptor potential (TRP) ion channels as possible drug targets has been elucidated in different types of cancer. Among natural compounds known to activate TRPs, cannabidiol (CBD) displays anticancer properties. By using FACS analysis, confocal microscopy, gene silencing, and cell growth assay, we demonstrated that CBD, through TRPV2, inhibits cell proliferation and cell cycle in CML cells. It promoted mitochondria dysfunction and mitophagy as shown by mitochondrial mass reduction and up-regulation of several mitophagy markers. These effects were associated with changes in the expression of octamer-binding transcription factor 4 and PU.1 markers regulated during cellular differentiation. Interestingly, a synergistic effect by combining CBD with the standard drug imatinib was found and imatinib-resistant cells remain susceptible to CBD effects. Therefore, the targeting of TRPV2 by using CBD, through the activation of mitophagy and the reduction in stemness, could be a promising strategy to enhance conventional therapy and improve the prognosis of CML patients.

Functional In Vitro Assessment of VEGFA/NOTCH2 Signaling Pathway and pRB Proteasomal Degradation and the Clinical Relevance of Mucolipin TRPML2 Overexpression in Glioblastoma Patients.

Glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high vascularization and its growth depends on the formation of new blood vessels. We have previously demonstrated that TRPML2 mucolipin channel expression increases with the glioma pathological grade. Herein by ddPCR and Western blot we found that the silencing of TRPML2 inhibits expression of the VEGFA/Notch2 angiogenic pathway. Moreover, the VEGFA/Notch2 expression increased in T98 and U251 cells stimulated with the TRPML2 agonist, ML2-SA1, or by enforced-TRPML2 levels. In addition, changes in TRPML2 expression or ML2-SA1-induced stimulation, affected Notch2 activation and VEGFA release. An increased invasion capability, associated with a reduced VEGF/VEGFR2 expression and increased vimentin and CD44 epithelial-mesenchymal transition markers in siTRPML2, but not in enforced-TRPML2 or ML2-SA1-stimulated glioma cells, was demonstrated. Furthermore, an increased sensitivity to Doxorubicin cytotoxicity was demonstrated in siTRPML2, whereas ML2-SA1-treated GBM cells were more resistant. The role of proteasome in Cathepsin B-dependent and -independent pRB degradation in siTRPML2 compared with siGLO cells was studied. Finally, through Kaplan-Meier analysis, we found that high TRPML2 mRNA expression strongly correlates with short survival in GBM patients, supporting TRPML2 as a negative prognostic factor in GBM patients.

TRPML2 Mucolipin Channels Drive the Response of Glioma Stem Cells to Temozolomide and Affect the Overall Survival in Glioblastoma Patients.

The survival of patients with glioblastoma (GBM) is poor. The main cause is the presence of glioma stem cells (GSCs), exceptionally resistant to temozolomide (TMZ) treatment. This last may be related to the heterogeneous expression of ion channels, among them TRPML2. Its mRNA expression was evaluated in two different neural stem cell (NS/PC) lines and sixteen GBM stem-like cells by qRT-PCR. The response to TMZ was evaluated in undifferentiated or differentiated GSCs, and in TRPML2-induced or silenced GSCs. The relationship between TRPML2 expression and responsiveness to TMZ treatment was evaluated by MTT assay showing that increased TRPML2 mRNA levels are associated with resistance to TMZ. This research was deepened by qRT-PCR and western blot analysis. PI3K/AKT and JAK/STAT pathways as well as ABC and SLC drug transporters were involved. Finally, the relationship between TRPML2 expression and overall survival (OS) and progression-free survival (PFS) in patient-derived GSCs was evaluated by Kaplan-Meier analysis. The expression of TRPML2 mRNA correlates with worse OS and PFS in GBM patients. Thus, the expression of TRPML2 in GSCs influences the responsiveness to TMZ in vitro and affects OS and PFS in GBM patients.

Coexpression of TRPML1 and TRPML2 Mucolipin Channels Affects the Survival of Glioblastoma Patients.

Among brain cancers, glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high cell heterogeneity, which can be linked to its high malignancy. We have previously demonstrated that TRPML1 channels affect the OS of GBM patients. Herein, by RT-PCR, FACS and Western blot, we demonstrated that TRPML1 and TRPML2 channels are differently expressed in GBM patients and cell lines. Moreover, these channels partially colocalized in ER and lysosomal compartments in GBM cell lines, as evaluated by confocal analysis. Interestingly, the silencing of TRPML1 or TRPML2 by RNA interference results in the decrease in the other receptor at protein level. Moreover, the double knockdown of TRPML1 and TRPML2 leads to increased GBM cell survival with respect to single-channel-silenced cells, and improves migration and invasion ability of U251 cells. Finally, the Kaplan-Meier survival analysis demonstrated that patients with high TRPML2 expression in absence of TRPML1 expression strongly correlates with short OS, whereas high TRPML1 associated with low TRPML2 mRNA expression correlates with longer OS in GBM patients. The worst OS in GBM patients is associated with the loss of both TRPML1 and TRPML2 channels.

The TRPV2 cation channels: from urothelial cancer invasiveness to glioblastoma multiforme interactome signature.

Changes in transient receptor potential (TRP) Ca2+ permeable channels are associated with development and progression of different types of cancer. Herein, we report data relative to the expression and function of TRP vanilloid 2 (TRPV2) channels in cancer. Overexpression of TRPV2 is observed in high-grade urothelial cancers and treatment with the TRPV2 agonist cannabidiol induces apoptosis. In prostate cancer, TRPV2 promotes migration and invasion, and TRPV2 overexpression characterizes the castration-resistant phenotype. In breast cancer cells, inhibition of TRPV2 by tranilast reduces the insulin-like growth factor-1 stimulated proliferation. TRPV2 overexpression in triple-negative breast cancer cells is associated with high recurrence-free survival. Increased TRPV2 overexpression is present in patients with esophageal squamous cell carcinoma associated with advanced disease, lymph node metastasis, and poor prognosis. Increased TRPV2 transcripts have been found both in benign hepatoma and in hepatocarcinomas, where TRPV2 expression is associated with portal vein invasion and reduction of cancer stem cell expression. TRPV2 expression and function has been also evaluated in gliomagenesis. This receptor negatively controls survival, proliferation, and resistance to CD95- or BCNU-induced apoptosis. In glioblastoma stem cells, TRPV2 activation promotes differentiation and inhibits the proliferation in vitro and in vivo. In glioblastoma, the TRPV2 is part of an interactome-based signature complex, which is negatively associated with survival, and it is expressed in high risk of recurrence and temozolomide-resistant patients. Finally, also in hematological malignancies, such as myeloma or acute myeloid leukemia, TRPV2 might represent a target for novel therapeutic approaches. Overall, these findings demonstrate that TRPV2 exhibits an oncogenic activity in different types of cancers, controlling survival, proliferation, migration, angiogenesis, and invasion signaling pathways. Thus, it prompts the pharmacological use of TRPV2 targeting in the control of cancer progression.

Exploring treatment with Ribociclib alone or in sequence/combination with Everolimus in ER+HER2-Rb wild-type and knock-down in breast cancer cell lines.

BACKGROUND: Breast cancer (BC) is the second most common type of cancer worldwide. Among targeted therapies for Hormone Receptor-positive (HR+) and Human Epidermal growth factor Receptor 2-negative (HER2-) BC, the Cyclin-Dependent Kinases (CDK4/6) are targeted by inhibitors such as Ribociclib (Rib); however, resistance to CDK4/6 inhibitors frequently develops. The aim of this work is to assess in vitro activity of Rib and Everolimus (Eve) in HR+HER2- MCF-7 and HR-HER2-BT-549 BC cell lines. METHODS: HR+HER2- MCF-7 and HR-HER2- BT-549 BC cell lines were treated with increasing concentration of Rib and Eve (up to 80 µg/mL) for 48-72 h. Subsequently, HR+HER2- MCF-7 cells were silenced for Retinoblastoma (Rb) gene, and thus, the effect of Rib in sequential or concurrent schedule with Eve for the treatment of both Rb wild type or Rb knock-down MCF-7 in vitro was evaluated. Cell viability of HR+HER2- MCF-7cells treated with sequential and concurrent dosing schedule was analyzed by MTT assay. Moreover, cell cycle phases, cell death and senescence were evaluated by cytofluorimetric analysis after treatment with Rib or Eve alone or in combination. RESULTS: The sequential treatment didn't produce a significant increase of cytotoxicity, compared to Rib alone. Instead, the cotreatment synergized to increase the cytotoxicity compared to Rib alone. The cotreatment reduced the percentage of cells in S and G2/M phases and induced apoptosis. Rib triggered senescence and Eve completely reversed this effect in Rb wild type BC cells. Rib also showed Rb-independent effects as shown by results in Rb knock-down MCF-7. CONCLUSION: Overall, the Rib/Eve concurrent therapy augmented the in vitro cytotoxic effect, compared to Rib/Eve sequential therapy or single treatments.

Calcium Signaling and the Regulation of Chemosensitivity in Cancer Cells: Role of the Transient Receptor Potential Channels.

Cancer cells acquire the ability to modify the calcium signaling network by altering the expression and functions of cation channels, pumps or transporters. Calcium signaling pathways are involved in proliferation, angiogenesis, invasion, immune evasion, disruption of cell death pathways, ECM remodelling, epithelial-mesenchymal transition (EMT) and drug resistance. Among cation channels, a pivotal role is played by the Transient Receptor Potential non-selective cation-permeable receptors localized in plasma membrane, endoplasmic reticulum, mitochondria and lysosomes. Several findings indicate that the dysregulation in calcium signaling induced by TRP channels is responsible for cancer growth, metastasis and chemoresistance. Drug resistance represents a major limitation in the application of current therapeutic regimens and several efforts are spent to overcome it. Here we describe the ability of Transient Receptor Potential Channels to modify, by altering the intracellular calcium influx, the cancer cell sensitivity to chemotherapeutic drugs.

Targeting Transient Receptor Potential Channels by MicroRNAs Drives Tumor Development and Progression.

Transient receptor potential (TRP) cation channel superfamily plays important roles in a variety of cellular processes such polymodal cellular sensing, adhesion, polarity, proliferation, differentiation and apoptosis. The expression of TRP channels is strictly regulated and their de-regulation can stimulate cancer development and progression.In human cancers, specific miRNAs are expressed in different tissues, and changes in the regulation of gene expression mediated by specific miRNAs have been associated with carcinogenesis. Several miRNAs/TRP channel pairs have been reported to play an important role in tumor biology. Thus, the TRPM1 gene regulates melanocyte/melanoma behaviour via TRPM1 and microRNA-211 transcripts. Both miR-211 and TRPM1 proteins are regulated through microphthalmia-associated transcription factor (MIFT) and the expression of miR-211 is decreased during melanoma progression. Melanocyte phenotype and melanoma behaviour strictly depend on dual TRPM1 activity, with loss of TRPM1 protein promoting melanoma aggressiveness and miR-211 expression supporting tumour suppressor. TRPM3 plays a major role in the development and progression of human clear cell renal cell carcinoma (ccRCC) with von Hippel-Lindau (VHL) loss. TRPM3, a direct target of miR-204, is enhanced in ccRCC with inactivated or deleted VHL. Loss of VHL inhibits miR-204 expression that lead to increased oncogenic autophagy. Therefore, the understanding of specific TRP channels/miRNAs molecular pathways in distinct tumors could provide a clinical rationale for target therapy in cancer.

The Effects of Cannabidiol and Prognostic Role of TRPV2 in Human Endometrial Cancer.

Several studies support, both in vitro and in vivo, the anti-cancer effects of cannabidiol (CBD), a transient receptor potential vanilloid 2 (TRPV2) ligand. TRPV2, often dysregulated in tumors, is associated with altered cell proliferation and aggressiveness. Endometrial cancer (EC) is historically divided in type I endometrioid EC and type II non-endometrioid EC, associated with poor prognosis. Treatment options with chemotherapy and combinations with radiation showed only limited efficacy. Since no data are reported concerning TRPV2 expression as well as CBD potential effects in EC, the aim of this study was to evaluate the expression of TRPV2 in biopsies and cell lines as well as the effects of CBD in in vitro models. Overall survival (OS), progression-free survival (PFS), cell viability, migration, and chemo-resistance have been evaluated. Results show that TRPV2 expression increased with the malignancy of the cancer tissue and correlated with shorter PFS (p = 0.0224). Moreover, in vitro TRPV2 over-expression in Ishikawa cell line increased migratory ability and response to cisplatin. CBD reduced cell viability, activating predominantly apoptosis in type I cells and autophagy in mixed type EC cells. The CBD improved chemotherapeutic drugs cytotoxic effects, enhanced by TRPV2 over-expression. Hence, TRPV2 could be considered as a marker for optimizing the therapy and CBD might be a useful therapeutic option as adjuvant therapy.

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.

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-1ß cytokine into the mature interleukin-1ß.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.

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.

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.

Anticancer effect of minor phytocannabinoids in preclinical models of multiple myeloma.

Multiple myeloma (MM) is a blood cancer caused by uncontrolled growth of clonal plasmacells. Bone disease is responsible for the severe complications of MM and is caused by myeloma cells infiltrating the bone marrow and inducing osteoclast activation. To date, no treatment for MM is truly curative since patients relapse and become refractory to all drug classes. Cannabinoids are already used as palliative in cancer patients. Furthermore, their proper anticancer effect was demonstrated in many cancer models in vitro, in vivo, and in clinical trials. Anyway, few information was reported on the effect of cannabinoids on MM and no data has been provided on minor phytocannabinoids such as cannabigerol (CBG), cannabichromene (CBC), cannabinol (CBN), and cannabidivarin (CBDV). Scientific literature also reported cannabinoids beneficial effect against bone disease. Here, we examined the cytotoxic activity of CBG, CBC, CBN, and CBDV in vitro in MM cell lines, their effect in modulating MM cells invasion toward bone cells and the bone resorption. Subsequently, according to the in vitro results, we selected CBN for in vivo study in a MM xenograft mice model. Results showed that the phytocannabinoids inhibited MM cell growth and induced necrotic cell death. Moreover, the phytocannabinoids reduced the invasion of MM cells toward osteoblast cells and bone resorption in vitro. Lastly, CBN reduced in vivo tumor mass. Together, our results suggest that CBG, CBC, CBN, and CBDV can be promising anticancer agents for MM.

Copper-Based Complexes with Adamantane Ring-Conjugated bis(3,5-Dimethyl-pyrazol-1-yl)acetate Ligand as Promising Agents for the Treatment of Glioblastoma.

The new ligand L2Ad, obtained by conjugating the bifunctional species bis(3,5-dimethylpyrazol-1-yl)-acetate and the drug amantadine, was used as a chelator for the synthesis of new Cu complexes 1-5. Their structures were investigated by synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS), near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and by combining X-ray absorption fine structure (XAFS) spectroscopy techniques and DFT modeling. The structure of complex 3 was determined by single-crystal X-ray diffraction analysis. Tested on U87, T98, and U251 glioma cells, Cu(II) complex 3 and Cu(I) complex 5 decreased cell viability with IC50 values significantly lower than cisplatin, affecting cell growth, proliferation, and death. Their effects were prevented by treatment with the Cu chelator tetrathiomolybdate, suggesting the involvement of copper in their cytotoxic activity. Both complexes were able to increase ROS production, leading to DNA damage and death. Interestingly, nontoxic doses of 3 or 5 enhanced the chemosensitivity to Temozolomide.

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.

Calcium influx, oxidative stress, and apoptosis induced by TRPV1 in chronic myeloid leukemia cells: Synergistic effects with imatinib.

Introduction: Calcium flux is the master second messenger that influences the proliferation-apoptosis balance. The ability of calcium flux alterations to reduce cell growth makes ion channels interesting targets for therapy. Among all, we focused on transient receptor potential vanilloid 1, a ligand-gated cation channel with selectivity for calcium. Its involvement in hematological malignancies is poorly investigated, especially in the field of chronic myeloid leukemia, a malignancy characterized by the accumulation of immature cells. Methods: FACS analysis, Western blot analysis, gene silencing, and cell viability assay were performed to investigate the activation of transient receptor potential vanilloid 1, by N-oleoyl-dopamine, in chronic myeloid leukemia cell lines. Results: We demonstrated that the triggering of transient receptor potential vanilloid 1 inhibits cell growth and promotes apoptosis of chronic myeloid leukemia cells. Its activation induced calcium influx, oxidative stress, ER stress, mitochondria dysfunction, and caspase activation. Interestingly, a synergistic effect exerted by N-oleoyl-dopamine and the standard drug imatinib was found. Conclusion: Overall, our results support that transient receptor potential vanilloid 1 activation could be a promising strategy to enhance conventional therapy and improve the management of chronic myeloid leukemia.

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.

Follicular fluid hormonal profile and cumulus cell gene expression in controlled ovarian hyperstimulation with recombinant FSH: effects of recombinant LH administration.

PURPOSE: Down-regulation with gonadodropin-releasing agonist (GnRH-a) protocol during IVF stimulation leads to a severe endogenous LH suppression, which may affect the follicular development. The aim of the study was to evaluate the effects of recombinant LH (r-LH) administration, during late follicular development stages, in recombinant FSH (r-FSH) stimulated cycles on follicular fluid (FF) parameters and on cumulus cell quality. METHODS: Twenty patients undergoing IVF were stimulated in a long GnRH agonist protocol with r-FSH alone or with r-LH supplementation when the leading follicle reached diameter of 14 mm. FF was collected at the time of oocyte retrieval from 32 follicles ≥ 18 mm. Serum FSH, LH, estradiol (E(2)), and progesterone (P(4)) were evaluated on the day of hCG administration. Intra-follicular E(2), P(4), AMH and TGF-ß were assayed. Total RNA from 18 individual cumuli was isolated for gene expression analyses. RESULTS: R-LH increased FF P(4) levels. FF TGF-ß levels and PTGS2 and HAS2 expression in cumulus cells (CCs) positively correlated with increased P(4) levels observed in FFs, while a negative correlation was found between P(4) and AMH levels. CONCLUSIONS: FF positive correlation between P(4) and TGF-ß levels and CC expression of PTGS2 and HAS2 suggest an association with a better follicle quality. In addition, our data suggest that late follicular phase r-LH supplementation leads to a more advanced stage of follicular maturation.