ABSTRACT
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.
Subject(s)
Apoptosis/drug effects , Capsaicin/pharmacology , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , TRPV Cation Channels/metabolism , Tumor Suppressor Proteins/metabolism , Urinary Bladder Neoplasms/pathology , fas Receptor/metabolism , Ataxia Telangiectasia Mutated Proteins , Blotting, Western , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Flow Cytometry , Fluorescent Antibody Technique , Humans , Membrane Potential, Mitochondrial/drug effects , Neoplasm Invasiveness , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sensory System Agents/pharmacology , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/metabolism , Urothelium/drug effects , Urothelium/metabolismABSTRACT
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.
Subject(s)
Carcinoma, Transitional Cell/genetics , Carcinoma, Transitional Cell/pathology , TRPV Cation Channels/metabolism , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , Urothelium/metabolism , Urothelium/pathology , Analysis of Variance , Biomarkers, Tumor/analysis , Blotting, Western , Carcinoma, Transitional Cell/metabolism , Chi-Square Distribution , Flow Cytometry , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Microscopy, Confocal , Neoplasm Staging , Prognosis , Reverse Transcriptase Polymerase Chain Reaction , Urinary Bladder Neoplasms/metabolismABSTRACT
Archival pathologic specimens are a rich source for the studies of hereditary diseases, cancer genetics, and identification cases in forensic science. In this study, the intraindividual consistency of eight identifying microsatellite polymorphisms (i.e., HMTH01, vWFA31, F13A, MITMH26, FES-FPS, CD4, TPOX, CSF1PO)in a cohort of 40 patients with invasive breast carcinoma were analyzed. Nests of cancer and adjacent morphologically normal ductal-lobular structures (TDLUs) were microdissected as discrete regions from hematoxylin-eosin-stained slides. As controls for each case, DNA templates were prepared from TDLUs located in nontumor quadrants and from unaffected breast skin. Over 1,400 carefully controlled PCR reactions were reviewed, and no evidence was found for microsatellite mismatches among intraindividual cancer and control DNAs. The negative results, supported by validation experiments, strongly argue that alterations of simple repeats are rare somatic events during the onset and progression of breast cancer. This study suggests that PCR artifacts may be a relevant cause of misdiagnosis of microsatellite instability in human sporadic cancer.
