Claudio Cardinali

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.

Capsaicin triggers autophagic cell survival which drives epithelial mesenchymal transition and chemoresistance in bladder cancer cells in an Hedgehog-dependent manner

Bladder cancer (BC) is a common urologic tumor characterized by high risk of recurrence and mortality. Capsaicin (CPS), used as an intravesical drug for overactive bladder, was demonstrated to induce cell death in different cancer cells including BC cells. Here we found that treatment of high-grade BC cells with high dose of CPS triggers autophagy. Infact, the CPS treatment alters the redox homeostasis by inducing production of radicals, mitochondrial depolarization, alterations of ADP/ATP ratio and activation of AMPK pathway stimulating the autophagic process in BC cells. The inhibition of autophagy, by using the specific inhibitor bafilomycin A or Beclin 1 knock-down, enhanced the CPS-induced cell death, demonstrating that CPS-induced autophagy acts as a pro-survival process in BC cells. By using PCR arrays and FACS analysis, we found that the CPS-treated BC cells displayed typical mesenchymal features of the epithelial mesenchymal transition (EMT) as elongated shape and over-expression of vimentin, α5 and β1 integrin subunits, integrin-like kinase and the anti-apoptotic Bcl-2 proteins. Moreover, we demonstrated that CPS treatment stimulates upregulation of Dhh/Ptch2/Zeb2 members of the Hedgehog signaling pathway, increases CD24, VEGFA and TIMP1 and decreases CD44 and ALCAM mRNA expression levels. By PTCH2 knock-down we found that the Hedgehog signaling pathway is involved in the CPS-induced autophagy and EMT phenotype. Finally, we also showed that the CPS-resistant EMT-positive BC cells displayed an increased drug-resistance to the cytotoxic effects of mitomycin C, gemcitabine and doxorubicine drugs commonly used in BC therapy.

Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells

Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC. Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy. On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner. Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity.

Overexpression of transient receptor potential mucolipin-2 ion channels in gliomas: role in tumor growth and progression

The Transient Receptor Potential (TRP) superfamily consists of cation-selective and non-selective ion channels playing an important role both in sensory physiology and in physiopathology in several complex diseases including cancers. Among TRP family, the mucolipin (TRPML1, −2, and −3) channels represent a distinct subfamily of endosome/lysosome Ca2+ channel proteins. Loss-of-function mutations in human TRPML-1 gene cause a neurodegenerative disease, Mucolipidosis Type IV, whereas at present no pathology has been associated to human TRPML-2 channels. Herein we found that human TRPML-2 is expressed both in normal astrocytes and neural stem/progenitor cells. By quantitative RT-PCR, western blot, cytofluorimetric and immunohistochemistry analysis we also demonstrated that TRPML-2 mRNA and protein are expressed at different levels in glioma tissues and high-grade glioma cell lines of astrocytic origin. TRPML-2 mRNA and protein levels increased with the pathological grade, starting from pylocitic astrocytoma (grade I) to glioblastoma (grade IV). Moreover, by RNA interference, we demonstrated a role played by TRPML-2 in survival and proliferation of glioma cell lines. In fact, knock-down of TRPML-2 inhibited the viability, altered the cell cycle, reduced the proliferation and induced apoptotic cell death in glioma cell lines. The DNA damage and apoptosis induced by TRPML-2 loss increased Ser139 H2AX phosphorylation and induced caspase-3 activation; furthermore, knock-down of TRPML-2 in T98 and U251 glioma cell lines completely abrogated Akt and Erk1/2 phosphorylation, as compared to untreated cells. Overall, the high TRPML-2 expression in glioma cells resulted in increased survival and proliferation signaling, suggesting a pro-tumorigenic role played by TRPML-2 in glioma progression.

Axitinib induces senescence-associated cell death and necrosis in glioma cell lines: The proteasome inhibitor, bortezomib, potentiates axitinib-induced cytotoxicity in a p21(Waf/Cip1) dependent manner

Glioblastoma is associated with a poor overall survival despite new treatment advances. Antiangiogenic strategies targeting VEGF based on tyrosine kinase inhibitors (TKIs) are currently undergoing extensive research for the treatment of glioma. Herein we demonstrated that the TKI axitinib induces DNA damage response (DDR) characterized by γ-H2AX phosphorylation and Chk1 kinase activation leading to G2/M cell cycle arrest and mitotic catastrophe in U87, T98 and U251 glioma cell lines. Moreover, we found that p21(Waf1/Cip1) increased levels correlates with induction of ROS and senescence-associated cell death in U87 and T98 cell lines, which are reverted by N-acetyl cysteine pretreatment. Conversely, U251 cell line showed a resistant phenotype in response to axitinib treatment, as evidenced by cell cycle arrest but no sign of cell death. The combinatorial use of axitinib with other therapies, with the aim of inhibiting multiple signaling pathways involved in tumor growth, can increase the efficiency of this TKI. Thus, we addressed the combined effects of axitinib with no toxic doses of the proteasome inhibitor bortezomib on the growth of U87 and T98 axitinib-sensitive and axitinib-resistant U251 cell lines. Compared to single treatments, combined exposure was more effective in inhibiting cell viability of all glioma cell lines, although with different cell death modalities. The regulation of key DDR and cell cycle proteins, including Chk1, γ-H2AX and p21(Waf1/Cip1) was also studied in glioma cell lines. Collectively, these findings provide new perspectives for the use of axitinib in combination with Bortezomib to overcome the therapy resistance in gliomas.

Cannabinoids synergize with carfilzomib, reducing multiple myeloma cells viability and migration

Several studies showed a potential anti-tumor role for cannabinoids, by modulating cell signaling pathways involved in cancer cell proliferation, chemo-resistance and migration. Cannabidiol (CBD) was previously noted in multiple myeloma (MM), both alone and in synergy with the proteasome inhibitor bortezomib, to induce cell death. In other type of human cancers, the combination of CBD with Δ9-tetrahydrocannabinol (THC) was found to act synergistically with other chemotherapeutic drugs suggesting their use in combination therapy. In the current study, we evaluated the effects of THC alone and in combination with CBD in MM cell lines. We found that CBD and THC, mainly in combination, were able to reduce cell viability by inducing autophagic-dependent necrosis. Moreover, we showed that the CBD-THC combination was able to reduce MM cells migration by down-regulating expression of the chemokine receptor CXCR4 and of the CD147 plasma membrane glycoprotein. Furthermore, since the immuno-proteasome is considered a new target in MM and also since carfilzomib (CFZ) is a new promising immuno-proteasome inhibitor that creates irreversible adducts with the β5i subunit of immuno-proteasome, we evaluated the effect of CBD and THC in regulating the expression of the β5i subunit and their effect in combination with CFZ. Herein, we also found that the CBD and THC combination is able to reduce expression of the β5i subunit as well as to act in synergy with CFZ to increase MM cell death and inhibits cell migration. In summary, these results proved that this combination exerts strong anti-myeloma activities.

Advances in Transient Receptor Potential Vanilloid-2 Channel Expression and Function in Tumor Growth and Progression

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.

The TRPV1 ion channel regulates thymocyte differentiation by modulating autophagy and proteasome activity

Autophagy and the ubiquitin-proteasome system (UPS) control thymus cell homeostasis under resting and endoplasmic reticulum (ER) stress conditions. Several evidence support a cross-talk between UPS and autophagy; abrogation of UPS responses stimulates autophagy, and vice versa the inhibition of autophagy alters the UPS functions. Herein, we found that TRPV1 activation induces ER stress, proteasome dysfunction and autophagy in thymocytes by modulating the expression of UPR-related genes. The TRPV1-mediated autophagy prevents the UPR activation by inhibiting BiP, Grp94 and ERp57 chaperone protein expression. Thymocytes from TRPV1 KO mice display both autophagy and proteasome dysfunctions, resulting in increased apoptotic cells and reduced total DP thymocyte number. In addition, positive selection of thymocytes triggered by anti-TCRβ/CD2 Ab-mediated costimulation induces apoptosis in thymocytes from TRPV1 KO as compared with WT mice. Stimulation of TRPV1 KO thymocytes with anti-TCRβ/CD2 mAbs modulates the expression of CD4 antigen on purified DP thymocytes, with reduced number of mature, single positive (SP) CD4 and increased number of immature SP CD4low and DP CD4lowCD8+ thymocytes, further supporting the intrinsic role of TRPV1 in T cell maturation. Finally, a reduction in CD8+ and CD4+ T cells is evidenced in the peripheral blood and spleen of TRPV1 KO, as compared with WT mice. Therapeutic strategy by restraining or stimulating the TRPV1 expression and functions in thymocytes might represent a new pharmacological tool in the regulation of different inflammatory T cell responses.

Cooperative Interaction between the Alpha1-Adrenoceptors (ñ1-AR) andTransient Receptor Potential (TRP) Triggers a Proliferative Cell Signal inProstate Cancer Cell Lines

Calcium (Ca2+) increases the proliferation of human advanced prostate cancer (PCa) cells but the ion channels involved are unknown. Santoni and Quaglia groups investigated the correlation between alpha1D-adrenergic receptor (α1D-AR) and the transient receptor potential vanilloid type 1 (TRPV1) ion channel expression in PCa cells. The α1D-AR and TRPV1 mRNAs are increased in PCa compared to BPH. α1D-AR and TRPV1 are co-expressed in PCa cells. Norepinephrine (NE) induced α1D-AR- and TRPV1-dependent protons release, Ca2+ flux in PC3 cells and activation of PLC, PKC and ERK path-ways that stimulated PC3 cell proliferation. Similarly, a role for TRPC6 or GPR55 in α1-AR-dependent proliferation of mesangial cells and PCa cells was reported. Overall, a crosstalk between α1-AR and TRPs in PCa cells, involved in the control of cell proliferation has been demonstrated. These data strongly promote a putative novel pharmacological approach in the treatment of PCa by targeting both α1-AR and TRP channels.

High CTLA-4 expression correlates with poor prognosis in thymoma patients

Thymomas, tumors that arise from epithelial cells of the thymus gland, are the most common neoplasms of the anterior mediastinum, with an incidence rate of approximately 2.5 per million/year. Cytotoxic T Lymphocyte Antigen 4 (CTLA-4 or CD152) exerts inhibitory activity on T cells, and since its oncogenic role in the progression of different types of tumors, it has emerged as a potential therapeutic target in cancer patients. In this study, we assessed the expression of CTLA-4 both at mRNA and protein levels in paraffin embedded-tissues from patients with thymomas. Furthermore, we evaluated the relationship between CTLA-4 expression and the clinical-pathologic characteristics and prognosis in patients with thymomas. Sixty-eight patients with median age corresponding to 62 years were included in this analysis. Thymomas were classified accordingly to the WHO and Masaoka-Koga for histochemical analysis and for prognostic significance. A statistical difference was found between CTLA-4 mRNA levels in human normal thymus compared with thymoma specimens. CTLA-4 expression was statistically found to progressively increase in A, B1, B2, AB and it was maximal in B3 thymomas. According to Masaoka-Koga pathological classification, CTLA-4 expression was lower in I, IIA and IIB, and higher in invasive III and IV stages. By confocal microscopy analysis we identified the expression of CTLA-4 both in tumor cells and in CD45+ tumor-infiltrating leukocytes, mainly in B3 and AB thymomas. Finally, CTLA-4 overexpression significantly correlates with reduced overall survival in thymoma patients and in atypical thymoma subgroup, suggesting that it represents a negative prognostic factor.