title: Vanilloids induce oral cancer apoptosis independent of TRPV1.
abstract: OBJECTIVE
  To investigate the mechanisms of vanilloid cytotoxicity and anti-tumor effects in oral squamous cell carcinoma (OSCC).
  
  
  METHODS
  Immunohistochemistry and qPCR analyses demonstrated expression of the TRP vanilloid type 1 (TRPV1) receptor in OSCC. Using cell proliferation assays, calcium imaging, and three mouse xenograft models, prototypical vanilloid agonist (capsaicin) and antagonist (capsazepine) were evaluated for cytotoxic and anti-tumor effects in OSCC.
  
  
  RESULTS
  OSCC cell lines treated with capsaicin displayed significantly reduced cell viability. Pre-treatment with capsazepine failed to reverse these effects. Moreover, capsazepine alone was significantly cytotoxic to tumor cells, suggesting the mechanism-of-action is independent of TRPV1 activation. This was further confirmed by calcium imaging indicating that TRPV1 channels are not functional in the cell lines tested. We then examined whether the observed vanilloid cytotoxicity was due to the generation of reactive oxygen species (ROS) and subsequent apoptosis. Induction of ROS was confirmed by flow cytometry and reversed by co-treatment with the antioxidant N-acetyl-cysteine (NAC). NAC also significantly reversed vanilloid cytotoxicity in cell proliferation assays. Dose-dependent induction of apoptosis with capsazepine treatment was demonstrated by FACS analyses and c-PARP expression in treated cells. Our in vivo xenograft studies showed that intra-tumoral injections of capsazepine exhibited high effectiveness in suppressing tumor growth with no identifiable toxicities.
  
  
  CONCLUSIONS
  These findings confirm TRPV1 channel expression in OSCC. However anti-tumor effects of vanilloids are independent of TRPV1 activation and are most likely due to ROS induction and subsequent apoptosis. Importantly, these studies demonstrate capsazepine is a potential therapeutic candidate for OSCC.
authors.affiliation: Cancer Therapy and Research Center, UTHSCSA, San Antonio, TX 78229, USA; Comprehensive Dentistry, UTHSCSA Dental School, San Antonio, TX 78229, USA. Electronic address: gonzalesc5@uthscsa.edu.
authors.forename: Cara B
authors.initials: CB
authors.lastname: Gonzales
authors.affiliation: Cancer Therapy and Research Center, UTHSCSA, San Antonio, TX 78229, USA; Molecular Medicine, UTHSCSA, San Antonio, TX 78229, USA.
authors.forename: Nameer B
authors.initials: NB
authors.lastname: Kirma
authors.affiliation: Comprehensive Dentistry, UTHSCSA Dental School, San Antonio, TX 78229, USA.
authors.forename: Jorge J
authors.initials: JJ
authors.lastname: De La Chapa
authors.affiliation: Comprehensive Dentistry, UTHSCSA Dental School, San Antonio, TX 78229, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Chen
authors.affiliation: Endodontics, UTHSCSA Dental School, San Antonio, TX 78229, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Henry
authors.affiliation: Comprehensive Dentistry, UTHSCSA Dental School, San Antonio, TX 78229, USA.
authors.forename: Songjiang
authors.initials: S
authors.lastname: Luo
authors.affiliation: Endodontics, UTHSCSA Dental School, San Antonio, TX 78229, USA; Pharmacology, UTHSCSA Dental School, San Antonio, TX 78229, USA.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Hargreaves
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002211:Capsaicin / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D004224:Diterpenes / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D009062:Mouth Neoplasms / Q000378:metabolism / Q000473:pathology*; D016133:Polymerase Chain Reaction; D050916:TRPV Cation Channels / Q000502:physiology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D004224:Diterpenes; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D002211:Capsaicin
keywords: Apoptosis; Capsaicin; Capsazepine; Oral cancer; TRPV1