title: High-throughput screening identifies cardiac glycosides as potent inhibitors of human tissue kallikrein expression: implications for cancer therapies.
abstract: OBJECTIVE
  Human tissue kallikreins (KLK) comprise a subgroup of 15 homologous secreted serine proteases. Primarily known for their clinical use as cancer biomarkers (e.g., PSA), KLKs have recently been directly implicated in cancer-related processes, including invasion, angiogenesis, and tumor growth regulation. Therefore, the identification of compounds that would modulate expression of KLKs might be of considerable therapeutic value.
  
  
  METHODS
  A cell-based high-throughput screening (HTS) of three small molecule libraries ( approximately 4,500 compounds) was undertaken; KLK expression in the breast cancer cell line MDA-MB-468 was assessed with sensitive ELISAs.
  
  
  RESULTS
  The initial screening resulted in 66 "putative hits" that decreased KLK5 expression by at least 50% over control. Secondary screening and mini-dose-response assays resulted in 21 "validated hits." These 21 compounds were clustered in only three distinct functional families and were further analyzed in vitro to determine their effectiveness (IC(50)s). Hits that failed to show dose-responsiveness or interfered with the viability of the cells were excluded. Multiple members of the cardiac glycoside family were found to be novel inhibitors of KLK expression, acting at low concentrations (10-50 nmol/L). Furthermore, members of the same family induced marked decreases in c-MYC and c-FOS expression, in a dose-dependent manner that correlated the KLK inhibition, suggesting a transcriptional mechanism of regulation of KLK expression.
  
  
  CONCLUSIONS
  We conclude that cardiac glycosides can dramatically suppress the transcription of KLKs and that these effects may be linked to proto-oncogene (c-myc/fos) expression. These findings may partially explain the recently realized antineoplastic actions of cardiac glycosides.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Prassas
authors.forename: Miltiadis
authors.initials: M
authors.lastname: Paliouras
authors.forename: Alessandro
authors.initials: A
authors.lastname: Datti
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D002301:Cardiac Glycosides / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D006801:Humans; D000090063:Proto-Oncogene Mas; D020840:Tissue Kallikreins / Q000037:antagonists & inhibitors*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D002301:Cardiac Glycosides; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D020840:Tissue Kallikreins