title: Digitoxin induces apoptosis in cancer cells by inhibiting nuclear factor of activated T-cells-driven c-MYC expression.
abstract: BACKGROUND
  Cardiac glycosides such as digitoxin have been shown to directly cause apoptotic death of cancer cells both in vitro, and in vivo. However, the mechanism connecting cardiac glycoside action to apoptosis is not known. It has been reported that compounds resembling digitoxin are able to reduce c-MYC expression. Furthermore, it has been previously shown that the transcription of c-MYC depends on nuclear factor of activated T-cells (NFAT) binding sites in the c-MYC promoter. We have therefore hypothesized that NFAT might mediate digitoxin effects on c-MYC mRNA message.
  
  
  METHODS
  We have chosen to study this process in HeLa cells where structurally intact c-MYC genes in 8q24 co-localize with human papilloma virus 18 at all integration sites.
  
  
  RESULTS
  Here we show that within the 1(st) h following treatment with digitoxin, a significant reduction in c-MYC mRNA occurs. This is followed by a precipitous loss of c-MYC protein, activation of caspase 3, and subsequent apoptotic cell death. To test the NFAT-dependence mechanism, we analyzed the effects of digitoxin on NFAT isoform-dependent auto-activation of a NFAT-luciferase expression system. Drug dependent effects on expression varied according to each of the four canonical NFAT isoforms (1, 2, 3 or 4). The most digitoxin-sensitive NFAT isoform was NFAT1. Using c-MYC chromatin immune precipitation, we find that digitoxin inhibits interaction of NFAT1 with the proximal c-MYC promoter.
  
  
  CONCLUSIONS
  These results suggest that the carcinotoxic activity of digitoxin includes suppression of NFAT-driven c-MYC expression.
authors.affiliation: Department of Anatomy, Physiology, and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
authors.forename: Qing Feng
authors.initials: QF
authors.lastname: Yang
authors.forename: Clifton L
authors.initials: CL
authors.lastname: Dalgard
authors.forename: Ofer
authors.initials: O
authors.lastname: Eidelman
authors.forename: Catherine
authors.initials: C
authors.lastname: Jozwik
authors.forename: Bette S
authors.initials: BS
authors.lastname: Pollard
authors.forename: Meera
authors.initials: M
authors.lastname: Srivastava
authors.forename: Harvey B
authors.initials: HB
authors.lastname: Pollard
keywords: Cardiac glycosides; c-MYC; nuclear factor of activated T-cells