Valerio Farfariello

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.

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

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

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

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

Brain Activity of Thioctic Acid Enantiomers: In Vitro and in Vivo Studies in an Animal Model of Cerebrovascular Injury

Oxidative stress is an imbalance between the production of free radicals and antioxidant defense mechanisms, potentially leading to tissue damage. Oxidative stress has a key role in the development of cerebrovascular and/or neurodegenerative diseases. This phenomenon is mainly mediated by an enhanced superoxide production by the vascular endothelium with its consequent dysfunction. Thioctic, also known as alpha-lipoic acid (1,2-dithiolane-3-pentanoic acid), is a naturally occurring antioxidant that neutralizes free radicals in the fatty and watery regions of cells. Both the reduced and oxidized forms of the compound possess antioxidant ability. Thioctic acid has two optical isomers designated as (+)- and (−)-thioctic acid. Naturally occurring thioctic acid is the (+)-thioctic acid form, but the synthetic compound largely used in the market for stability reasons is a mixture of (+)- and (−)-thioctic acid. The present study was designed to compare the antioxidant activity of the two enantiomers versus the racemic form of thioctic acid on hydrogen peroxide-induced apoptosis in a rat pheochromocytoma PC12 cell line. Cell viability was evaluated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and free oxygen radical species (ROS) production was assessed by flow cytometry. Antioxidant activity of the two enantiomers and the racemic form of thioctic acid was also evaluated in spontaneously hypertensive rats (SHR) used as an in vivo model of increased oxidative stress. A 3-h exposure of PC12 cells to hydrogen peroxide (H2O2) significantly decreased cell viability and increased levels of intracellular ROS production. Pre-treatment with racemic thioctic acid or (+)-enantiomer significantly inhibited H2O2-induced decrease in cell viability from the concentration of 50 μmol/L and 20 μmol/L, respectively. Racemic thioctic acid and (+)-salt decreased levels of intracellular ROS, which were unaffected by (−)-thioctic acid. In the brain of SHR, the occurrence of astrogliosis and neuronal damage, with a decreased expression of neurofilament 200 kDa were observed. Treatment of SHR for 30 days with (+)-thioctic acid reduced the size of astrocytes and increased the neurofilament immunoreaction. The above findings could contribute to clarify the role played by thioctic acid in central nervous system injury related to oxidative stress. The more pronounced effect of (+)-thioctic acid observed in this study may have practical therapeutic implications worthy of being investigated in further preclinical and clinical studies.

Antioncogenic Effects of Transient Receptor Potential Vanilloid 1 in the Progression of Transitional Urothelial Cancer of Human Bladder

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.

Cross-talk between alpha1D-adrenoceptors and transient receptor potential vanilloid type 1 triggers prostate cancer cell proliferation.

BackgroundThere is evidence that calcium (Ca2+) increases the proliferation of human advanced prostate cancer (PCa) cells but the ion channels involved are not fully understood. Here, we investigated the correlation between alpha1D-adrenergic receptor (alpha1D-AR) and the transient receptor potential vanilloid type 1 (TRPV1) expression levels in human PCa tissues and evaluated the ability of alpha1D-AR to cross-talk with TRPV1 in PCa cell lines.MethodsThe expression of alpha1D-AR and TRPV1 was examined in human PCa tissues by quantitative RT-PCR and in PCa cell lines (DU145, PC3 and LNCaP) by cytofluorimetry. Moreover, alpha1D-AR and TRPV1 colocalization was investigated by confocal microscopy in PCa cell lines and by fluorescence microscopy in benign prostate hyperplasia (BPH) and PCa tissues. Cell proliferation was assessed by BrdU incorporation. Alpha1D-AR/TRPV1 knockdown was obtained using siRNA transfection. Signalling pathways were evaluated by measurement of extracellular acidification rate, Ca2+ flux, IP3 production, western blot and MTT assay.ResultsThe levels of the alpha1D-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha1D-AR and TRPV1 expression levels was found. Moreover, alpha1D-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens. Noradrenaline (NA) induced an alpha1D-AR- and TRPV1-dependent protons release and Ca2+ flux in PC3 cell lines; NA by triggering the activation of phospholipase C (PLC), protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways stimulated PC3 cell proliferation, that was completely inhibited by clopenphendioxan (WS433) and capsazepine (CPZ) combination or by alpha1D-AR/TRPV1 double knockdown.ConclusionsWe demonstrate a cross-talk between alpha1D-AR and TRPV1, that is involved in the control of PC3 cell proliferation. These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha1D-AR and TRPV1 channels.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 11. (1) reversed enantioselectivity of 1,4-dioxane derivatives in α1-adrenergic and 5-HT1A receptor binding sites recognition.

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.

4-nonylphenol triggers apoptosis and affects 17-β-estradiol receptors in calvarial osteoblasts.

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.

Expression and Function of the Transient Receptor Potential Ion Channel Family in the Hematologic Malignancies

Transient receptor potential (TRP) channels are important candidates mediating Ca(2+) influx in excitable and nonexcitable cells such as normal and neoplastic hematopoietic tissues. They are non selective cation channels implicated in Ca(2+) signaling in hematologic tumor cells. Here, we review the growing experimental evidence indicating that TRP channels should be included among the genes whose expression is altered in hematologic malignancies such as leukemias (AML, ALL, CML and CLL), B- and T-lymphomas and multiple myelomas (MM). These effects depend on the widespread roles played by the TRP channels in the modulation of the proliferation, differentiation and apoptosis of the hematopoietic cells. The analysis of the expression of the different TRP channels belonging to the TRPMs, TRPVs, TRPCs, TRPPs channel families expressed in different hematological malignacies, evidenced a widespread expression of TRPV2 channel in the myeloid and lymphoid leukemias, and a very peculiar expression of this channel in different types of B cell lymphomas and multiple myeloma, that is parallel to the restricted expression of TRPV2 in normal immune cells with respect to its presence in other human tissues. In vivo studies in children AML and ALL patients also evidenced the presence of a genetic polymorphism of the TRPM5 gene, that reduced the risk to develop leukemia in the children. Finally, the coexpression of TRPV5 and TRPV6 channels in lymphocytes, and their involvement in the radioresistance of K562 erythroleukemia cells to ionizing radiation exposure is of more interest. Thus in conclusion, the TRP channels represent promising targets for hematologic cancer therapy, and their exploitation may open to novel pharmaceutical and clinical approaches.

Resiniferatoxin induces death of bladder cancer cells associated with mitochondrial dysfunction and reduces tumor growth in a xenograft mouse model.

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.