Carcinogenesis Advance Access originally published online on June 5, 2009
Carcinogenesis 2009 30(8):1320-1329; doi:10.1093/carcin/bgp138
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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
1 Department of Experimental Medicine and Public Health
2 Department of Molecular, Cellular and Animal Biology, University of Camerino, via Madonna delle Carceri 9, 62032 Camerino (MC), Italy
3 Department of Experimental Medicine, University "La Sapienza", 00161 Rome, Italy
4 Pathology and Cytodiagnostic Unit
5 Urology Operative Unit, ASUR 9, 62100 Macerata, Italy
* To whom correspondence should be addressed. Tel: +39 0737 403319; Fax: +39 0737 403325; Email: giorgio.santoni{at}unicam.it
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 G0/G1 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.
Abbreviations: Ab, antibody; ATM, ataxia telangiectasia mutated; CPS, capsaicin; CPZ, capsazepine; ER, endoplasmic reticulum; FACS, fluorescent activated cell sorting; FITC, fluorescein isothiocyanate; GAM, goat anti-mouse; HRP, horseradish peroxidase; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide; mAb, monoclonal antibody; mRNA, messenger RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NHUC, normal human urothelial cells; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RAG, rabbit anti-goat; RT, reverse transcription; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TRPV1, transient receptor potential vanilloid type 1; UC, urothelial cancer
Received February 2, 2009; revised May 26, 2009; accepted May 28, 2009.