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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.

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.

Capsaicin has been shown to induce apoptosis in various transformed cell types in vitro and in vivo. Apoptosis is triggered by an induction phase that is highly dependent on cell type and apoptogenic stimuli (e.g., death receptors, oxidative stress, DNA damage, ion fluctuations, and cytokines). This is followed by an effector phase where the cell undergo distinct biochemical changes as results of intrinsic mitochondrial-dependent and/or extrinsic death receptor-mediated apoptotic pathways. Depending on the concentration, duration of exposure and route of administration, CPS may either induce apoptosis in different cell types in a TRPV1-dependent and independent manner. It is generally accepted that CPS-mediated apoptosis is manifested by reactive oxygen species (ROS) generation, elevations in [Ca2+ ]i and mitochondrial transmembrane potential dissipation. However recent reports, indicate the ability of CPS to induce also up-regulation of Fas/CD95, TRAIL-R1/DR4, TRAIL-R2/DR5 and TNF-R1/DR1 death receptor expression, Fas Ligand-independent Fas-dependent TRPV1-mediated apoptosis of cancer cells, or to sensitize tumor cells to TRAIL-induced apoptosis. Unraveling the molecular mechanisms that underlie the CPS-induced targeting of death receptor in tumor cells and the death intracellular pathways activated by capsaicin, could provide the basis for novel therapeutic strategies in cancer patients.

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF expression in metastatic renal cell carcinoma (mRCC) is mostly regulated by hypoxia, predominantly via the hypoxia-induced factor (HIF)/Von Hippel-Lindau (VHL) pathway. Advances in our knowledge of VEGF role in tumor angiogenesis, growth, and progression have permitted development of new approaches for the treatment of mRCC, including several agents targeting VEGF and VEGF receptors: tyrosine kinase pathway, serine/threonine kinases,α5β1-integrin, deacetylase, CD70, mammalian target of rapamycin (mTOR), AKT, and phosphatidylinositol 3′-kinase (PI3K). Starting from sorafenib and sunitinib, several targeted therapies have been approved for mRCC treatment, with a long list of agents in course of evaluation, such as tivozanib, cediranib, and VEGF-Trap. Here we illustrate the main steps of tumor angiogenesis process, defining the pertinent therapeutic targets and the efficacy and toxicity profiles of these new promising agents.

5-HT(1A) receptor and α(1)-adrenoreceptor (α(1)-AR) binding sites recognized by the 1,4-dioxanes 2-4 display reversed stereochemical requirements. (S)-2 proved to be a potent 5-HT(1A) receptor agonist highly selective over α(1)-AR subtypes. Chirality influenced the anticancer activity of 3 and 4 in human prostate cancer cells (PC-3): (R)-4, eutomer at the α(1d)-AR subtype, was the most potent. The decreased effect of 4 and (R)-4 in α(1d)-AR silenced PC-3 cells confirmed that their anticancer activity was α(1d)-AR-dependent.

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.

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 (β(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 β(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.

TRPV1 activation and molecular mechanisms involved in the regulation of the CPS-induced autophagy in thymocytes. Autophagy is a highly conserved process involved in lymphocyte development and differentiation. Herein, we demonstrated for the first time that triggering of TRPV1 by the specific agonist CPS induces autophagy in mouse thymocytes. TRPV1-dependent autophagy required [Ca2+]i and ROS generation, resulting in AMPK activation. CPS specifically increased Atg4C mRNA expression and induced oxidation of Atg4C protein by ROS generation. TRPV1-triggered autophagy was Atg6/Beclin-1-dependent, as demonstrated by the use of Beclin-1+/− transgenic mice, and involved ROS- and AMPK-mediated up-regulation of Beclin-1 expression. Autophagy is activated as a prosurvival process, as its inhibition triggered apoptosis of thymocytes: this effect was accompanied by down-regulation of Atg4C, Bcl-XL, and Irgm1 mRNA expression, decreased Bcl-XL and Beclin-1 protein levels, and caspase-3 activation, suggesting the existence of a molecular interplay between autophagic and apoptotic programs. TRPV1 activation by CPS altered the expression of CD4 and CD8α antigens, inducing the development of DPdull. Interestingly, we found that CPS induces autophagy of DPdull cells, and inhibition of CPS-induced autophagy by the 3-MA autophagic inhibitor induces apoptosis of DPdull cells, suggesting the presence of an interplay between autophagic survival and apoptotic cell death. Overall, our findings suggest that DPdull cells constitute a distinct thymocyte subpopulation involved in the homeostatic control of cellularity and in the responses to chemical stress signals during thymocyte maturation, via regulating autophagy and apoptosis in a TRPV1-dependent manner.

Gliomas are primary brain tumours believed to arise from glial cells or their progenitors. They account for 78% of malignant brain tumours (Shwartzbaum et al., 2006). The vast majority of gliomas is high-grade glioblastoma multiforme (GBM), and is characterized by almost unrestrained growth. Consequently, the median survival of patients with GBM was approximately 12 months (Huncharek & Muscat, 1998). While research has generated abundant information regarding the growth characteristics of these cancers, clinical care remains palliative and the prognosis dismal (Butowski et al., 2006). Gliomagenesis and progression are complex processes only partly understood. At molecular level, tumor progression and the associated heterogeneity is likely the result of multiple mutations in certain key signaling proteins (Furnari et al., 2007). Among these proteins, the Transient Receptor Potential (TRP) channel family has been identified to profoundly affect a variety of physiological and pathological processes (Kiselyov et al., 2007; Nilius et al., 2007). Members of TRP channels control cellular homeostasis by regulating calcium flux, cell proliferation, differentiation and apoptosis; moreover, in the last years an additional role for TRP ion channel family in malignant cancer growth and progression has been recognized (Xu et al., 2001; Wisnoskey et al., 2003; Xin et al., 2005; Bidaux et al., 2007; Prevarskaya et al., 2007; Gkika & Prevarskaya, 2009). Approximately thirty TRPs have been identified to date, and are classified in seven different families: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like) (Montell, 2003) (Fig.1). The expression levels and activity of members of the TRPC, TRPM, and TRPV families have been correlated with malignant growth and progression (Duncan et al., 1998; Tsavaler et al., 2001; Wissenbach et al., 2001; Thebault et al., 2006; Amantini et al., 2007; Caprodossi et al., 2008; Nabissi et al., 2010). TRP channels may regulate glioma growth and progression at different levels by controlling cell proliferation, inhibiting apoptosis, stimulating angiogenesis and triggering the migration and the invasion during tumor progression (Table 1).

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.

Gene delivery to eukaryotic cells is the technique to study the regulation of gene expression. Human astrocytoma cell line 1321N1 could be useful to study G-protein-coupled receptors (GPCRs). Different transient transfection methods, namely calcium phosphate, Lipofectamine, FuGENE, Arrest-In, and microporation (Microporator), were investigated. Results were analyzed by fluorescence-activated cell sorting and fluorescence microscope using green fluorescent protein (GFP) as a reporter gene. To verify the transfection efficiency of these techniques, the expression of human GPR17 gene (hgpr17) was analyzed by transcription quantitative polymerase chain reaction. Microporation resulted in the best method to promote enriched hgpr17 delivery into the human astrocytoma cell line.

Transient receptor potential (TRP) channels affect several physiological and pathological processes. In particular, TRP channels have been recently involved in the triggering of enhanced proliferation, aberrant differentiation, and resistance to apoptotic cell death leading to the uncontrolled tumor invasion. About thirty TRPs have been identified to date, and are classified in seven different families: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like). Among these channel families, 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 the functional role of TRPV channels during cancer growth and progression. TRPV channels have been found to regulate cancer cell proliferation, apoptosis, angiogenesis, migration and invasion during tumor progression, and depending on the stage of the cancer, up- and down-regulation of TRPV mRNA and protein expression have been reported. These changes may have cancer promoting effects by increasing the expression of constitutively active TRPV channels in the plasma membrane of cancer cells by enhancing Ca(2+)-dependent proliferative response; in addition, an altered expression of TRPV channels may also offer a survival advantage, such as resistance of cancer cells to apoptotic-induced cell death. However, recently, a role of TRPV gene mutations in cancer development, and a relationship between the expression of specific TRPV gene single nucleotide polymorphisms and increased cancer risk have been reported. We are only at the beginning, a more deep studies on the physiopathology role of TRPV channels are required to understand the functional activity of these channels in cancer, to assess which TRPV proteins are associated with the development and progression of cancer and to develop further knowledge of TRPV proteins as valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting in cancer.

Capsaicin (CPS) has been found to exhibit either tumor promoting or suppressing effects, many of which are mediated by the specific transient receptor potential vanilloid type-1 (TRPV1). Herein, we provide evidence that CPS treatment induced a more aggressive gene phenotype and invasiveness in 5637 cells-lacking TRPV1 receptor. CPS treatment of 5637 cells induced upregulation of pro-angiogenetic (angiopoietin 1, angiopoietin 2 and vascular endothelial growth factor), pro-invasive and pro-metastatic genes (MMP1, MMP9, TIMP1, TIMP3, granzyme A (GZMA), NM23A and S100A) with a downregulation of apoptotic genes (Fas/CD95 and tumor necrosis factor receptor superfamily member 1A). CPS increased the invasiveness of 5637 cells by triggering IGF (insulin-like growth factor)-1 release, GZMA and MMP9 activation, α-tubulin disassembly and cytoskeleton degradation. Finally, in order to evaluate the relationship between the lack of TRPV1 expression and increased CPS-induced invasiveness, we transfected 5637 cells with the TRPV1 complementary DNA (cDNA) sequence. We found that TRPV1-expressing cells show CPS-mediated calcium level increase, growth inhibition and apoptosis. Moreover, CPS-induced migration and MMP9 activation were reverted, suggesting an inhibitory role played by TRPV1 in urothelial cancer cell invasion and metastasis.

Transient receptor potential (TRP) channels affect several physiological and pathological processes. In particular, TRP channels have been recently involved in the triggering of enhanced proliferation, aberrant differentiation, and resistance to apoptotic cell death leading to the uncontrolled tumor invasion. About thirty TRPs have been identified to date, and are classified in seven different families: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like). Among these channel families, 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 the functional role of TRPV channels during cancer growth and progression. TRPV channels have been found to regulate cancer cell proliferation, apoptosis, angiogenesis, migration and invasion during tumor progression, and depending on the stage of the cancer, up- and down-regulation of TRPV mRNA and protein expression have been reported. These changes may have cancer promoting effects by increasing the expression of constitutively active TRPV channels in the plasma membrane of cancer cells by enhancing Ca2+-dependent proliferative response; in addition, an altered expression of TRPV channels may also offer a survival advantage, such as resistance of cancer cells to apoptotic-induced cell death. However, recently, a role of TRPV gene mutations in cancer development, and a relationship between the expression of specific TRPV gene single nucleotide polymorphisms and increased cancer risk have been reported. We are only at the beginning, a more deep studies on the physiopathology role of TRPV channels are required to understand the functional activity of these channels in cancer, to assess which TRPV proteins are associated with the development and progression of cancer and to develop further knowledge of TRPV proteins as valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting in cancer.

Vulvovaginal candidiasis is a mucosal infection affecting many women, but the immune mechanisms operating against Candida albicans at the mucosal level remain unknown. A rat model was employed to further characterize the contribution of B and T cells to anti-Candida vaginal protection. Particularly, the protective role of vaginal B cells was studied by means of adoptive transfer of vaginal CD3(-) CD5(+) IgM(+) cells from Candida-immunized rats to naïve animals. This passive transfer of B cells resulted into a number of vaginal C. albicans CFU approximately 50% lower than their controls. Sorted CD3(-) CD5(+) IgM(+) vaginal B lymphocytes from Candida-infected rats proliferated in response to stimulation with an immunodominant mannoprotein (MP) antigen of the fungus. Importantly, anti-MP antibodies and antibody-secreting B cells were detected in the supernatant and cell cultures, respectively, of vaginal B lymphocytes from infected rats incubated in vitro with vaginal T cells and stimulated with MP. No such specific antibodies were found when using vaginal B cells from uninfected rats. Furthermore, inflammatory and anti-inflammatory cytokines, such as interleukin-2 (IL-2), IL-6 and IL-10, were found in the supernatant of vaginal B cells from infected rats. These data are evidence of a partial anti-Candida protective role of CD3(-) CD5(+) IgM(+) vaginal B lymphocytes in our experimental model.

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.

On the basis of continued interest in plant-derived natural products as anticancer agents, a shorter and more efficient synthesis of climacostol is reported. This compound showed an anticancer activity better than that of the natural product. The improved potency and selectivity can be due to the absence of traces of the undesired E-isomer present in the natural climacostol. Furthermore, the versatile strategy developed for this simple molecule such as climacostol could aid in the synthesis of other more complex natural products.

Conservative chemical modifications of the core structure of the lead spipethiane (1) afforded novel potent sigma(1) ligands. sigma(1) affinity and sigma(1/)sigma(2) selectivity proved to be favored by the introduction of polar functions (oxygen atom or carbonyl group) in position 3 or 4 (4-6) or by the elongation of the distance between the two hydrophobic portions of the molecule with the simultaneous presence of a carbonyl group in position 4 (8 and 9). The observed cytostatic effect against the human breast cancer cell line MCF-7/ADR, highly expressing sigma(1) receptors, and not against MCF-7, as well as the enhancement of morphine analgesia highlighted the sigma(1) antagonist profile of this series of compounds. In particular, due to its high sigma(1) affinity (pK(i) = 10.28) and sigma(1)/sigma(2) selectivity ratio (29510), compound 9 might be a novel valuable tool for sigma receptor characterization and a suitable template for the rational design of potential therapeutically useful sigma(1) antagonists.

Prostaglandin F2alpha (PGF2alpha) regulates fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor (FGFR) expression in osteoblasts. Here, the role of FGF-2 in PGF2alpha-induced proliferation and the signaling pathway involved, were determined in calvarial osteoblasts (COBs) from Fgf2+/+ and Fgf2-/- mice. The involvement of the exported FGF-2 isoform, was determined using the FGF-2 neutralizing antibody to alter its binding to FGFR1. PGF2alpha increased activity of Ras, and MAP-kinase cascade as well as Bcl-2 and c-Myc levels in Fgf2+/+ but not in Fgf2-/- COBs. Moreover, in Fgf2+/+ COBs, PGF2alpha-enhanced nuclear accumulation and co-localization of Bcl-2/c-Myc. Although up-regulation of multiple proliferative and survival signals were induced by PGF2alpha in Fgf2+/+ COBs, phospho-p53 was unmodified while p53 was increased. Increased phospho-p53 was, instead, found in Fgf2-/- COBs without up-regulation of oncogenic proteins. The lack of p53 activation in wild type osteoblasts could be due in part to the overexpression of MDM2 caused by PGF2alpha via FGF-2. PGF2alpha, also, increased cyclins D and E in Fgf2+/+ COBs and induced an expansion of Fgf2+/+ osteoblasts in G(2)/M phase. These data clearly show that PGF2alpha induces proliferation via endogenous FGF-2 and the exported isoform mediates PGF2alpha effects by acting in autocrine manner. Furthermore, silencing of FGFR1 in Fgf2+/+ COBs blocked PGF2alpha induced increase of phospho-MDM2 and cyclins.

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.

Climacostol (5-(Z)-non-2-enyl-benzene-1,3-diol) is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to the group of resorcinolic lipids, compounds that show antimicrobial, antiparasitic and antitumor activities. We investigated the cytotoxic activity of the chemically synthesized toxin on: (1) human tumor squamous carcinoma A431 cells, (2) human promyelocytic leukaemia HL60 cells, and (3) human non-tumor endothelial EA.hy926 cells. The results showed that climacostol effectively inhibited the growth of tumor cell lines in a dose-dependent manner by inducing programmed cell death, with non-tumor cells proving significantly more resistant to the toxin.

Objective To evaluate the expression of transient receptor potential vanilloid type 2 (TRPV2) in normal human bladder and urothelial carcinoma (UC) tissues. Methods Bladder specimens were obtained by transurethral resection or radical cystectomy. TRPV2 mRNA expression in normal human urothelial cells (NHUCs), UC cell lines, and formalin-fixed paraffin-embedded normal ( n =6) and cancer bladder tissues ( n =58) was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-PCR). TRPV2 protein expression was assessed by cytofluorimetric and confocal microscopy analyses in NHUCs and UC cells and by Western blotting and immunohistochemistry in normal and UC tissues. Results Enhanced TRPV2 mRNA and protein expression was found in high-grade and -stage UC specimens and UC cell lines. Both the full-length TRPV2 (hTRPV2) and a short splice-variant (s-TRPV2) were detected in NHUC and normal bladder specimens, whereas a progressive decline of s-TRPV2 in pTa, pT1, and pT2 stages was observed, up to a complete loss in pT3 and pT4 UC specimens. Conclusions Normal human urothelial cells and bladder tissue specimens express TRPV2 at both the mRNA and protein levels. A progressive loss of s-TRPV2 accompanied by a marked increase of hTRPV2 expression was found in high-grade and -stage UC tissues.

The good results obtained as potential antitumor drugs with aza-anthracenediones and aza-anthrapyrazoles, e.g. pixantrone, 1a, and 1b (Chart 1), prompted us to design and synthesize a series of symmetrical bis derivatives, compounds 7–10 (Chart 1). These compounds are dimers of different aza-anthracenedione and aza-anthrapyrazolone monomers connected by the linker found to be the most appropriate among potential bis intercalators synthesized by us. The DNA-binding properties of bis derivatives 7 and 8 have been examined using fluorometric techniques: these target compounds are excellent DNA ligands, with a clear binding site preference for AT-rich duplexes. In vitro cytotoxic activity of all target compounds 7–10 and of reference compound pixantrone toward human cancer adenocarcinoma cell line HT29 is also described. Two selected compounds have been investigated for their capacity of inducing early apoptosis.

This study analyzes the phenotype of vaginal dendritic cells (VDCs), their antigenic presentation and activation of T-cell cytokine secretion, and their protective role in a rat model ofCandidavaginitis. Histological observation demonstrated a significant accumulation of OX62+VDCs in the mucosal epithelium ofCandida albicans-infected rats at the third round of infection. We identified two subsets of OX62+VDCs differing in the expression of CD4 molecule in both noninfected andCandida-infected rats. The OX62+CD4+subset of VDCs displayed a lymphoid cell-like morphology and expressed the T-cell antigen CD5, whereas the OX62+CD4−VDC subset exhibited a myeloid morphology and was CD5 negative.Candidainfection resulted in VDC maturation with enhanced expression of CD80 and CD134L on both CD4+and CD4−VDC subsets at 2 and 6 weeks afterCandidainfection. CD5−CD4−CD86−CD80−CD134L+VDCs from infected, but not noninfected, rats spontaneously released large amounts of interleukin-12 (IL-12) and tumor necrosis factor alpha, whereas all VDC subsets released comparable levels of IL-10 and IL-2 cytokines. Furthermore, OX62+VDCs from infected rats primed naïve CD4+T-cell proliferation and release of cytokines, including gamma interferon, IL-2, IL-6, and IL-10, in response to staphylococcal enterotoxin B stimulation in vitro. Adoptive transfer of highly purified OX62+VDCs from infected rats induced a significant acceleration of fungal clearance compared with that in rats receiving naive VDCs, suggesting a protective role of VDCs in the anti-Candidamucosal immunity. Finally, VDC-mediated protection was associated with their ability to rapidly migrate to the vaginal mucosa and lymph nodes, as assessed by adoptive transfer of OX62+VDCs labeled with 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester.

A series of new alpha1-adrenoreceptor antagonists (5-18) was prepared by introducing various substituents (Topliss approach) into the ortho, meta, and para positions of the benzyloxy function of the phendioxan open analogue 4 ("openphendioxan"). All the compounds synthesized were potent antagonists and generally displayed, similarly to 4, the highest affinity values at alpha1D- with respect to alpha1A- and alpha1B-AR subtypes and 5-HT1A subtype. By sulforhodamine B (SRB) assay on human PC-3 prostate cancer cells, the new compounds showed antitumor activity (estimated on the basis of three parameters GI50, TGI, LC50), at low micromolar concentration, with 7 ("clopenphendioxan") exhibiting the highest efficacy. Moreover, this study highlighted for the first time alpha1D- and alpha1B-AR expression in PC3 cells and also demonstrated the involvement of these subtypes in the modulation of apoptosis and cell proliferation. A significant reduction of alpha1D- and alpha1B-AR expression in PC3 cells was associated with the apoptosis induced by 7. This depletion was completely reversed by norepinephrine.

Herein, we provide the first evidence on the capsaicin (CPS) receptor vanilloid receptor type-1 (VR1) by rat thymocytes, and its involvement in CPS-induced apoptosis. VR1 mRNA was identified by quantitative RT-PCR in CD5(+) thymocytes. By immunofluorescence and flow cytometry, we found that a substantial portion of CD5+ thymocytes, namely CD4+ and double negative (DN) cell subsets, express VR1 that was present on plasma membrane on discrete spots. By Western blot, VR1 protein was identified as a single band of 95 kDa. We also described that CPS could trigger two distinct pathways of thymocyte death, namely apoptosis and necrosis depending on the dose of CPS exposure. CPS-induced apoptosis involved intracellular free calcium (Ca2+) influx, phosphatidylserine exposure, mitochondrial permeability transmembrane pore (PTP) opening and mitochondrial transmembrane potential (Delta Psi m) dissipation leading to cytochrome c release, activation of caspase-9 and -3 and oligonucleosomal DNA fragmentation. VR1 was functionally implicated in these events as they were completely abrogated by the VR1 antagonist, capsazepine (CPZ). Finally, we demonstrated that VR1 expression on distinct thymocytes was associated with the selective ability of CPS to trigger DNA fragmentation in VR1+ CD4+ and DN thymocytes. Overall, our results suggest that the expression of VR1 on thymocytes may function as a sensor of harmful stimuli present in the thymic environment.

A series of potential DNA-binding antitumor agents, 2-[omega-(alkylamino)alkyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3-bc][1,5]naphthyridines (2a-f), 10-aza derivatives of PZA, has been prepared by condensation of 9-chloro-2-methoxy-6-nitro-5,10-dihydrobenzo[b][1,5]naphthyridin-10-one (6) with the appropriate (omega-aminoalkyl)hydrazine in tetrahydrofuran/methanol. Compound 6 was obtained by heating at 100 degrees C in H(2)SO(4)5, yielded by the condensation of 2,6-dichloro-3-nitrobenzoic acid (4) and 6-methoxy-3-pyridinamine (3). The non-covalent DNA-binding properties of 2 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives against human hormone-refractory prostate adenocarcinoma cell line (PC-3) are described and compared to that of parent drug PZA. We selected the most cytotoxic target derivatives 2c,d, the in vitro inactive 2f, and reference compound PZA to investigate whether in vitro treatment with these drugs was able to induce necrotic and/or apoptotic cell death. To this purpose, we evaluated the percentage of apoptotic cells in PC-3 treated with the target compounds 2c,d,f and reference compound PZA, by Annexin V staining and Propidium iodide (PI)/Annexin V, biparametric flow cytometric analysis and agarose gel electrophoresis.

The protective roles of different lymphocyte subsets were investigated in a rat vaginal candidiasis model by adoptive transfer of vaginal lymphocytes (VL) or sorted, purified CD3+T cells, CD4+or CD8+T cells, or CD3−CD5+B cells from the vaginas of naïve or immune rats following three rounds ofCandida albicansinfection. The adoptive transfer of total VL from nonimmune animals did not alter the course of vaginal candidiasis of the recipient rats. In contrast, the animals receiving total VL or CD3+T cells from immune rats showed a highly significant acceleration of fungus clearance compared with animals which received nonimmune VL. The animals with vaginal CD3−CD5+B cells transferred from immune rats also had fewerCandidaCFU than the controls, but fungal clearance was significantly retarded with respect to the animals administered immune T cells. Sorted, purified CD4+and CD8+vaginal T cells from immune rats were also adoptively transferred to naïve animals. Although both populations were seen to accelerate the clearance of the fungus from the vagina, CD4+T cells were much more effective than CD8+T cells. Overall, there was no difference between the antifungal effects of immune vaginal CD4+T cells and those achievable with the transfer of whole, immune VL. Histological observations of the vaginal tissues of rats with adoptively transferred immune T cells demonstrated a remarkable accumulation of lymphocytes in the subepithelial lamina propria and also infiltrating the mucosal epithelium. These results strongly suggest that distinct vaginal lymphocyte subsets participate in the adaptive anti-Candidaimmunity at the vaginal level, with the vaginal CD4+T cells probably playing a major role.

The present study was undertaken to investigate the expression of alpha(v)beta3 and alpha(v)beta5 integrin-like vitronectin receptors (VNRs) on Candida albicans germ tube and their involvement in its adhesion to vitronectin (VN) and human endothelial cells. By immunofluorescence and FACS analysis, several monoclonal antibodies directed against human alpha(v) or beta3 integrin subunit or alpha(v)beta3 and alpha(v)beta5 heterodimers, positively stained C. albicans germ tubes. C. albicans germ tubes specifically adhered (45-50%) to VN and this adhesion was markedly inhibited by RGD-, but not RGE-containing peptides. Adhesion of C. albicans germ tubes to VN was strongly inhibited by anti-alphav, anti-beta3 or anti-alpha(v)beta3, but not by alpha(v)beta5 monoclonal antibody. C. albicans germ tube adhesion to VN was also inhibited by glycosaminoglycans (GAGs) such as heparin or chondroitin sulphate. Finally, we show that C. albicans germ tubes adhere to the human EA.hy 926 endothelial cell line. This adhesion is markedly blocked by anti-beta3 monoclonal antibody, GRGDSP peptide or heparin, and is completely abolished by their combination. Overall these results indicate that C. albicans germ tube adherence to VN and to a human endothelial cell line is mediated by alpha(v)beta3, but not by alpha(v)beta5-like integrin, and depends on GAGs which may act by regulating alpha(v)beta3 integrin-like/VN adhesive interaction.

e15513 Background: Tyrosine kinase inhibitors (TKI), such as sunitinib, sorafenib and pazopanib, have replaced immunotherapy as the standard of care for metastatic renal cell carcinoma (mRCC). However, their use in sequential or combined strategies is limited by the lack of evidences on TKI-induced cell death in cancer cells. Aim of our study was to evaluate the different mechanisms responsible of the anti-proliferative and cytotoxic effects induced in vitro by µM doses of sunitinib, sorafenib and pazopanib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Necrotic cell death was evaluated by Annexin-V/PI staining and FACS analysis. The cathepsin B activation was evaluated by western blot using an anti-cathepsin B antibody; the cathepsin B proteolytic activity was determined using the fluorogenic Z-Arg-Arg-AMC peptide and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin was detected by a SpectraMax Gemini XPS microplate reader. Results: We found that sunitinib and pazopanib markedly reduced at mM dose the viability of BC cells. Treatment for 24h with 20µM of sunitinib, by triggering “Incomplete autophagy”, induced necrosis of BC cells. In addition, sunitinib as a lysosomotropic agent, entered free within the lysosomes, where by increasing lysosomal pH and impairing cathepsin B activity, induced lysosomal-dependent necrosis. By contrast, treatment of BC cells for 72h with 20µM of pazopanib induced autophagic cell death, which was markedly reversed in a dose-dependent manner by the autophagic inhibitor 3-MA. The pazopanib-induced autophagic cell death was associated with increased procathepsin B cleavage and enhanced cathepsin B activity. Conclusions: Overall, our results show different cathepsin B-dependent cancer cell death mechanisms induced by sunitinib or pazopanib, providing the biological basis for novel molecularly targeted approaches.

e15515 Background: Several tyrosine kinase inhibitors (TKIs), have been developed and approved for clinical use in multi-targeted cancer therapy. Among these, sorafenib is an orally available multikinase inhibitor approved for the treatment of the advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Aim of our study was to evaluate the mechanisms responsible for the cytotoxic effects induced by in vitro use of µM doses of sorafenib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with the anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Apoptosis, (ΔΨm) dissipation and ROS generation were determined by Annexin-V/PI, JC-1 and DCFDA staining, respectively and cytofluorimetric analysis. The cathepsin B activation was evaluated by western blot using an anti-cathepsin B antibody; the cathepsin B proteolytic activity was determined using the fluorogenic Z-Arg-Arg-AMC peptide and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin was detected by a SpectraMax Gemini XPS microplate reader. Results: We found that sorafenib markedly reduced at µM dose the viability of BC cells. Treatment for 24h with 20µM of sorafenib, triggered “Incomplete autophagy”, that induced apoptosis of BC cells. Sorafenib by inducing an increased cathepsin B activity and pro-apoptotic protein BID activation, triggered a ROS-mediated-mitochondrial-dependent apoptosis of BC cells. Moreover, the increase of cathepsin B activity induced by sorafenib was inhibited by a specific tyrosine phosphatase inhibitor (e.g., orthovanadate) strongly suggesting for a contribute of tyrosine-phosphatases in sorafenib-induced apoptosis. Conclusions: Sorafenib by triggering incomplete autophagy, stimulates a cathepsin B-induced-BID-mediated-ROS- and mitochondrial-dependent apoptosis of BC cells, which is likely regulated by tyrosine-phosphatases.

270 Background: Tyrosine kinase inhibitors (TKI), such as sunitinib, sorafenib and pazopanib, have replaced immunotherapy as the standard of care for metastatic renal cell carcinoma (mRCC). However, their use in sequential or combined strategies is limited by the lack of evidences on the ability of TKIs to induce cell death in cancer cells. Aim of our study was to evaluate the different mechanisms responsible of the cytotoxic effects induced in vitro by µM doses of sunitinib, sorafenib and pazopanib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with the anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Necrosis and apoptosis, (ΔΨm) dissipation and ROS generation were determined by Annexin-V/PI, JC-1 and DCFDA staining, respectively and cytofluorimetric analysis. The cathepsin B activity was evaluated by ELISA. Finally, by mRNA estraction and RT-PCR array the pazopanib-induced gene profile expression was evaluated. Results: We found that treatment of 5637 and J82 BC cells with the three TKI agents markedly reduced cell viability. Treatment for 24 h with sunitinib and sorafenib at 20 µM dose, triggers an incomplete autophagy of BC cells. In addition, inhibition of autophagy induced by sunitinib and sorafenib triggers cell death of BC cells. Thus, sunitinib by imparing the cathepsin B activity induces lysosomal-dependent necrosis. Similarly, sorafenib by defective lysosomial degradation triggers ROS- and mitochondrial-dependent apoptosis. As regard to pazopanib, we first demonstrate that treatment of BC cells for 72 hrs (20 µM) induces autophagic Type II cell death, which was markedly reversed in a dose-dependent manner by 3MA and chloroquine autophagic inhibitors. Finally, pazopanib upregulates the mRNA expression of α-glucosidase (GAA) and TP73 belonging to the p53 tumor suppressor genes. Conclusions: Overall, our results showing different TKI-induced cell death mechanisms provide the rationale for the sequential use of these agents and the biological basis for novel molecularly targeted approaches.

87 Background: Growing evidence supports the role of α1-ARs in the direct mitogenic effect of catecholamines on prostate cancer (PC) cell growth. The expression of α1D-AR on PC3 prostate cancer cells and the ability of noradrenalin (NA) to stimulate PC3 cell proliferation in a α1D-AR-dependent manner were reported (Quaglia et al., 2005). In addition, TRPV1 expression was also found in prostate cancers (Sanchez et al., 2006). Aim of this study was to investigate the relationship between α1D-AR and TRPV1 receptors and the involvement of TRPV1 in NA-induced proliferation in PC3 cells. Methods: By western blot analysis and confocal microscopy the expression α1D-AR and TRPV1 and localization in PC3 cells were evaluated. PC3 cells were incubated with NA alone or in combination with WS433 and capsazepine (CPZ), α1D-AR and TRPV1 antagonists. Proton release, calcium influx and cell proliferation were assessed in α1D-AR-, TRPV1- or α1D-AR/TRPV1 double-silenced PC3 cells by cytosensor and cytofluorymetric analyses. Finally, lysates from NA-treated PC3 cells alone or in combination with WS433 or CPZ were blotted with anti-phospho ERK, anti-ERK and anti-phospho-(Ser) PKC substrate Abs and Inositol-1,4,5-trisphosphate [3H] radioreceptor assay were performed. Results: α1D-AR and TRPV1 co-localize and are co-immunoprecipitated in PC3 cells. Treatment of PC3 cells with NA strongly stimulated proton release, calcium influx and cell proliferation that were reverted by α1D-AR WS433 and TRPV1 antagonist. NA-induced increase of survival and proliferation was totally abrogated in α1D-AR/TRPV1 silenced cells. In addition, NA stimulates ERK and PKC substrate phosphorylation that was inhibited by WS433 and CPZ. Finally, CPZ treatment inhibited NA-dependent PLC activation, while WS433 had no effect. Conclusions: A functional and structural cross-talk between α1D-AR and TRPV1 receptors control NA-induced proliferation of PC cells. These data strongly suggest the development of new pharmaceutical approaches based on bifunctional antibodies and molecules recognizing both α1D-AR and TRPV1 receptors.

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.

The present research examines the effects of 4-nonylphenol (4-NP) on mouse primary calvarial osteoblasts (COBs). Incubation of the cells with 4-NP at 10 −5 M and 10 −6 M striking decreased osteoblasts viability and phosphatidylserine (PS) exposure, measured by Annexin V, was greatly enhanced. In addition, an up-regulation of Bax/Bcl2 ratio with a drop in ΔΨm and an increase of cleaved caspase 9 and 3 was found, suggesting that the alkylphenol induced osteoblast death via the mitochondrial-dependent apoptotic pathway. Interestingly, treatment with 4-NP was also able to increase cleaved caspase 8 in parallel with the truncated active Bid (t-Bid) suggesting that 4-NP-mediated apoptosis depends on cross talk between the extrinsic and intrinsic pathways. It is of relevance, that the apoptotic effects of 4-NP overcame 17-β-Estradiol (17-β E 2 ) induced-survival on osteoblasts. Also, the alkylphenol interfered with 17-β E 2 regulated estrogen receptors expression.

Background Wickerhamomyces anomalus is a yeast associated with different insects including mosquitoes, where it is proposed to be involved in symbiotic relationships with hosts. Different symbiotic strains of W. anomalus display a killer phenotype mediated by protein toxins with broad-spectrum antimicrobial activities. In particular, a killer toxin purified from a W. anomalus strain (WaF17.12), previously isolated from the malaria vector mosquito Anopheles stephensi, has shown strong in vitro anti-plasmodial activity against early sporogonic stages of the murine malaria parasite Plasmodium berghei. Results Here, we provide evidence that WaF17.12 cultures, properly stimulated to induce the expression of the killer toxin, can directly affect in vitro P. berghei early sporogonic stages, causing membrane damage and parasite death. Moreover, we demonstrated by in vivo studies that mosquito dietary supplementation with activated WaF17.12 cells interfere with ookinete development in the midgut of An. stephensi. Besides the anti-sporogonic action of WaF17.12, an inhibitory effect of purified WaF17.12-killer toxin was observed on erythrocytic stages of P. berghei, with a consequent reduction of parasitaemia in mice. The preliminary safety tests on murine cell lines showed no side effects. Conclusions Our findings demonstrate the anti-plasmodial activity of WaF17.12 against different developmental stages of P. berghei. New studies on P. falciparum are needed to evaluate the use of killer yeasts as innovative tools in the symbiotic control of malaria. Electronic supplementary material The online version of this article (10.1186/s13071-019-3587-4) contains supplementary material, which is available to authorized users.