Giorgio Santoni

Capsaicin-induced apoptosis of glioma cells is mediated by TRPV1 vanilloid receptor and requires p38 MAPK activation.

We provide evidence on the expression of the transient receptor potential vanilloid type‐1 (TRPV1) by glioma cells, and its involvement in capsaicin (CPS)‐induced apoptosis. TRPV1 mRNA was identified by quantitative RT‐PCR in U373, U87, FC1 and FLS glioma cells, with U373 cells showing higher, and U87, FC1 and FLS cells lower TRPV1 expression as compared with normal human astrocytes. By flow cytometry we found that a substantial portion of both normal human astrocytes, and U87 and U373 glioma cells express TRPV1 protein. Moreover, we analyzed the expression of TRPV1 at mRNA and protein levels of glioma tissues with different grades. We found that TRPV1 gene and protein expression inversely correlated with glioma grading, with marked loss of TRPV1 expression in the majority of grade IV glioblastoma multiforme. We also described that CPS trigger apoptosis of U373, but not U87 cells. CPS‐induced apoptosis involved Ca2+ influx, p38 but not extracellular signal‐regulated mitogen‐activated protein kinase activation, phosphatidylserine exposure, mitochondrial permeability transmembrane pore opening and mitochondrial transmembrane potential dissipation, caspase 3 activation and oligonucleosomal DNA fragmentation. TRPV1 was functionally implicated in these events as they were markedly inhibited by the TRPV1 antagonist, capsazepine. Finally, p38 but not extracellular signal‐regulated protein kinase activation was required for TRPV1‐mediated CPS‐induced apoptosis of glioma cells.

Triggering of Transient Receptor Potential Vanilloid Type 1 (TRPV1) by Capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ATM-dependent manner.

Herein, we provide evidence on the expression of transient receptor potential vanilloid type 1 (TRPV1) on human urothelial cancer (UC) cells and its involvement in the apoptosis induced by the selective agonist capsaicin (CPS). We analyzed TRPV1 messenger RNA and protein expression on human UC cell lines demonstrating its progressive decrease in high-grade UC cells. Treatment of RT4 cells with CPS induced cell cycle arrest in G(0)/G(1) phase and apoptosis. These events were associated with rapid co-ordinated transcription of pro-apoptotic genes including Fas/CD95, Bcl-2 and caspase families and ataxia telangiectasia mutated (ATM)/CHK2/p53 DNA damage response pathway. CPS induced Fas/CD95 upregulation, but more importantly Fas/CD95 ligand independent, TRPV1-dependent death receptor clustering and triggering of both extrinsic and intrinsic mitochondrial-dependent pathways. Moreover, we observed that CPS activates ATM kinase that is involved in Ser15, Ser20 and Ser392 p53 phosphorylation as shown by the use of the specific inhibitor KU55933. Notably, ATM activation was also found to control upregulation of Fas/CD95 expression and its co-clustering with TRPV1 as well as RT4 cell growth and apoptosis. Altogether, we describe a novel connection between ATM DNA damage response pathway and Fas/CD95-mediated intrinsic and extrinsic apoptotic pathways triggered by TRPV1 stimulation on UC cells.

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.

Local Anticandidal Immune Responses in a Rat Model of Vaginal Infection by and Protection against Candida albicans

ABSTRACT Humoral (antibody [Ab]) and cellular Candida-specific immune responses in the vaginas of pseudoestrus rats were investigated during three successive infections by Candida albicans. After the first, protective infection, Abs against mannan and aspartyl proteinase antigens were present in the vaginal fluid, and their titers clearly increased during the two subsequent, rapidly healing infections. In all animals, about 65 and 10% of vaginal lymphocytes (VL) were CD3+ (T cells) and CD3−CD5+ (B cells), respectively. Two-thirds of the CD3+ T cells expressed the α/β and one-third expressed the γ/δ T-cell receptor (TCR). This proportion slightly fluctuated during the three rounds of C. albicans infection, but no significant differences between infected and noninfected rats were found. More relevant were the changes in the CD4+/CD8+ T-cell ratio, particularly for cells bearing the CD25 (interleukin-2 receptor α) marker. In fact, a progressively increased number of both CD4+ α/β TCR and CD4+ CD25+ VL was observed after the second and third Candida challenges, reversing the high initial CD8+ cell number of controls (estrogenized but uninfected rats). The CD3− CD5+ cells also almost doubled from the first to the third infection. Analysis of the cytokines secreted in the vaginal fluid of Candida-infected rats showed high levels of interleukin 12 (IL-12) during the first infection, followed by progressively increasing amounts of IL-2 and gamma interferon during the subsequent infections. No IL-4 or IL-5 was ever detected. During the third infection, VL with in vitro proliferative activity in response to an immunodominant mannoprotein antigen of C. albicans were present in the vaginal tissue. No response to this antigen by mitogen-responsive blood, lymph node, and spleen cells was found. In summary, the presence of protective Ab and T helper type 1 cytokines in the vaginal fluids, the in vitro proliferation of vaginal lymphocytes in response toCandida antigenic stimulation, and the increased number of activated CD4+ cells and some special B lymphocytes afterC. albicans challenge constitute good evidence for induction of locally expressed Candida-specific Ab and cellular responses which are potentially involved in anticandidal protection at the vaginal level.

Emerging role of tumor-associated macrophages as therapeutic targets in patients with metastatic renal cell carcinoma

Abstract Tumor-associated macrophages (TAMs) derived from peripheral blood monocytes recruited into the renal cell carcinoma (RCC) microenvironment. In response to inflammatory stimuli, macrophages undergo M1 (classical) or M2 (alternative) activation. M1 cells produce high levels of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12, IL-23 and IL-6, while M2 cells produce anti-inflammatory cytokines, such as IL-10, thus contributing to RCC-related immune dysfunction. The presence of extensive TAM infiltration in RCC microenvironment contributes to cancer progression and metastasis by stimulating angiogenesis, tumor growth, and cellular migration and invasion. Moreover, TAMs are involved in epithelial–mesenchymal transition of RCC cancer cells and in the development of tumor resistance to targeted agents. Interestingly, macrophage autophagy seems to play an important role in RCC. Based on this scenario, TAMs represent a promising and effective target for cancer therapy in RCC. Several strategies have been proposed to suppress TAM recruitment, to deplete their number, to switch M2 TAMs into antitumor M1 phenotype and to inhibit TAM-associated molecules. In this review, we summarize current data on the essential role of TAMs in RCC angiogenesis, invasion, impaired anti-tumor immune response and development of drug resistance, thus describing the emerging TAM-centered therapies for RCC patients.

Candida albicans expresses a fibronectin receptor antigenically related to α5β1 integrin

Cell adhesion molecules, by regulating host-micro-organism interaction, play major role in the pathogenesis of infectious diseases. The present study was undertaken to investigate the expression of the fibronectin (FN) receptor prototype, α5β1 integrin, on Candida albicans and its involvement in the adhesion to FN. By immunofluorescence and fluorescence activated cell sorter (FACS) analysis, several monoclonal antibodies (mAbs) directed against human α5 or β1 integrin subunits, or two different antisera to FN receptor positively stained C. albicans yeast and germ tube phases, this immunoreactivity increasing upon germ tube transition. Twenty-five to thirty per cent of [3H]glucose-labelled Candida yeasts specifically adhered to FN and this adhesion was increased upon germ tube transition. C. albicans yeast and gerr tube forms bound to an RGD-containing 120 kDa tryptic fragment of FN and adhesion to FN was markedly inhibited by GRGDSP, but not GRGESP peptides. Moreover, binding of both C. albicans phases to FN was strongly inhibited by anti-α5 SAM-1 mAb, or both anti-fibronectin receptor (FNr) antisera. Overall these results indicate that C. albicans yeast and germ tube phases express a receptor antigenically related to α5β1 integrin which mediates their adhesion to FN. The α5β1 integrin-like receptor expression on C. albicans could be relevant for fungus-host interaction and in the dissemination process of Candida infection.

TRP channels and cancer: new targets for diagnosis and chemotherapy.

The Transient Receptor Potential (TRP) channels family consists of seven different subfamilies, namely TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like) that are related to several physiological and pathological processes. Recent years have witnessed an increased interest of research into the connection between TRP channels and cancer, leading to the discovery of tumor-related functions such as regulation of proliferation, differentiation, apoptotis, angiogenesis, migration and invasion during cancer progression. Among the TRP families, TRPCs, TRPMs and TRPVs are mainly related to malignant growth and progression. Depending on the type and stage of the cancer, regulation of TRPs mRNA and protein expression have been reported; these changes may regulate ion-dependent cell proliferation and resistance of cancer cells to apoptotic-induced cell death with consequent cancer promoting effects and resistance to chemotherapic treatments. Considerable efforts have been made to fight cancer cells and targeted therapy seems to be the most promising strategy: in this regard, ion channels belonging to the TRP channel superfamily could play an important role. Aim of this review is to summarize data reported so far on the expression and the functional role of TRP channels during cancer growth and progression, and the relationship with clinico-pathological markers. Moreover, the feasibility of TRP channels as target of chemotherapy and the different approaches by which these channels can be targeted will be analyzed in detail. Deeper investigations are required to understand the role TRP channels in cancer in order to develop further knowledge of TRP proteins as valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting.

TRPV2 channel negatively controls glioma cell proliferation and resistance to Fas-induced apoptosis in ERK-dependent manner

The aim of this study was to investigate the expression and function of the transient receptor potential vanilloid 2 (TRPV2) in human glioma cells. By Real-Time-PCR and western blot analysis, we found that TRPV2 messenger RNA (mRNA) and protein were expressed in benign astrocyte tissues, and its expression progressively declined in high-grade glioma tissues as histological grade increased (n = 49 cases), and in U87MG cells and in MZC, FCL and FSL primary glioma cells. To investigate the function of TRPV2 in glioma, small RNA interfering was used to silence TRPV2 expression in U87MG cells. As evaluated by RT-Profiler PCR array, siTRPV2-U87MG transfected cells displayed a marked downregulation of Fas and procaspase-8 mRNA expression, associated with upregulation of cyclin E1, cyclin-dependent kinase 2, E2F1 transcriptor factor 1, V-raf-1 murine leukemia viral oncogene homolog 1 and Bcl-2-associated X protein (Bcl-X(L)) mRNA expression. TRPV2 silencing increased U87MG cell proliferation as shown by the increased percentage of cells incorporating 5-bromo-2-deoxyuridine expressing beta(III)-tubulin and rescued glioma cells to Fas-induced apoptosis. These events were dependent on extracellular signal-regulated kinase (ERK) activation: indeed inhibition of ERK activation in siTRPV2-U87MG transfected cells by treatment with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, reduced Bcl-X(L) protein levels, promoted Fas expression, and restored Akt/protein kinase B pathway activation leading to reduced U87MG cell survival and proliferation, and increased sensitivity to Fas-induced apoptosis. In addition, transfection of TRPV2 in MZC glioma cells, by inducing Fas overexpression, resulted in a reduced viability and an increased spontaneous and Fas-induced apoptosis. Overall, our findings indicate that TRPV2 negatively controls glioma cell survival and proliferation, as well as resistance to Fas-induced apoptotic cell death in an ERK-dependent manner.

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.

Effects of cadmium on lymphocyte activation.

The effects of cadmium (Cd) on phytohemoagglutinin or phorbol myristate acetate-induced lymphocyte activation were investigated and a dose-dependent inhibition of cell proliferation was found. Kinetic studies revealed that the Cd-sensitive step is an early event of T cell stimulation. Failure of IL2 secretion and reduction of IL2 receptor expression in the Cd-treated cells are also reported. Regardless of which mechanism is responsible for Cd effects, our studies show that the inhibition of lymphocyte activation is associated with reduced [3H]phorbol dibutyrate binding to Ca2+-phospholipid-dependent protein kinase and altered breakdown of phosphatidylinositols. Thus, Cd interferes with two biochemical events which play a critical role in lymphocyte signal transduction and activation.