title: Induction of Epigenetic Alteration by CPUK02, An Ent- kaurenoid Derivative of Stevioside.
abstract: BACKGROUND
  Dietary polyphenols, such as those found in green tea and red wine, are linked to antitumor activity. They are known to influence many signaling pathways epigenetically within the human body. In this regard, CPUK02 (15-Oxosteviol benzyl ester) is a new ent-kaurenoid derivative of stevioside and exhibits strong anti-cancer activityin vitroandin vivo. Nowadays, the role of epigenetics in cancer has been the subject of intensive study and DNA methylation targeting represents a relevant strategy for cancer treatment. There are no reports regarding the effects of CPUK02 on epigenetic alterations in colorectal cancer cell line. This study was an attempt to compare CPUK02 with 5-AZA as DNMT inhibitor agent and evaluate whether it can induce its anti-cancer effects via altering the level of DNMT3b mRNA, MGMT and SFRP2 methylation pattern in HCT 116 cell line.
  
  
  METHODS
  To evaluate DNMT3b expression, DNMT3B mRNA levels in HCT116 CRC cell line were quantified by real-time reverse-transcriptase Polymerase Chain Reaction (PCR) assay after 24hrof incubation time with CPUK02 and 5-AZA. In addition, the methylation patterns of 2 CpG islands in this cell line were examined by methylation-specific PCR methods.
  
  
  RESULTS
  CPUK02 surprisingly, decreased the DNMT3b mRNA level. The average expression levels of DNMT3b in HCT116 treated with CPUK02 and 5-AZA relative to the GAPDH expression level in control were 0.16 and 0.5%, respectively. Furthermore, CPUK02 could decrease the methylated allele of MGMT and SFRP2 genes in HCT 116 after 24hr.
  
  
  CONCLUSIONS
  In this study, positive correlation was found between mRNA expression of DNMT3b and gene promoter hypermethylation after treatment with CPUK02 and 5-AZA. Our data confirmed that CPUK02 like 5-AZA exhibits demethylating properties.
authors.affiliation: Gasteroenterohepatology Research Center, Nemazee Hospital, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Pooneh
authors.initials: P
authors.lastname: Mokarram
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Zeinab
authors.initials: Z
authors.lastname: Mohammadi
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Saeid
authors.initials: S
authors.lastname: Khazayel
authors.affiliation: Drug Research Institute, China Pharmaceutical University, Jiangsu, China.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Dayong
keywords: 5-AZA; Colorectal neoplasm; DNMT; Epigenetic; Methylation