title: Emodin enhances the demethylation by 5-Aza-CdR of pancreatic cancer cell tumor-suppressor genes P16, RASSF1A and ppENK.
abstract: 5-Aza-2'-deoxycytidine (5-Aza-CdR) is currently acknowledged as a demethylation drug, and causes a certain degree of demethylation in a variety of cancer cells, including pancreatic cancer cells. Emodin, a traditional Chinese medicine (TCM), is an effective monomer extracted from rhubarb and has been reported to exhibit antitumor activity in different manners in pancreatic cancer. In the present study, we examined whether emodin caused demethylation and increased the demethylation of three tumor-suppressor genes P16, RASSF1A and ppENK with a high degree of methylation in pancreatic cancer when combined with 5-Aza-CdR. Our research showed that emodin inhibited the growth of pancreatic cancer Panc-1 cells in a dose- and time-dependent manner. Dot-blot results showed that emodin combined with 5-Aza-CdR significantly suppressed the expression of genome 5mC in PANC-1 cells. In order to verify the effect of methylation, methylation-specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP) combined with TA were selected for the cloning and sequencing. Results of MSP and BSP confirmed that emodin caused faint demethylation, and 5-Aza-CdR had a certain degree of demethylation. When emodin was combined with 5-Aza-CdR, the demethylation was more significant. At the same time, fluorescent quantitative PCR and western blot analysis results confirmed that when emodin was combined with 5-Aza-CdR, the expression levels of P16, RASSF1A and ppENK were increased more significantly compared to either treatment alone. In contrast, the expression levels of DNA methyltransferase 1 (DNMT1) and DNMT3a were more significantly reduced with the combination treatment than the control or either agent alone, further proving that emodin in combination with 5-Aza-CdR enhanced the demethylation effect of 5-Aza-CdR by reducing the expression of methyltransferases. In conclusion, the present study confirmed that emodin in combination with 5-Aza-CdR enhanced the demethylation by 5-Aza-CdR of tumor-suppressor genes p16, RASSF1A and ppENK by reducing the expression of methyltransferases DNMT1 and DNMT3a.
authors.affiliation: Department of Surgical Oncology, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Feng-Ping
authors.initials: FP
authors.lastname: Pan
authors.affiliation: Department of Hepatobiliary Surgery, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Hong-Kun
authors.initials: HK
authors.lastname: Zhou
authors.affiliation: Department of Coloproctological Surgery, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang 310012, P.R. China.
authors.forename: He-Qi
authors.initials: HQ
authors.lastname: Bu
authors.affiliation: Department of Surgical Oncology, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Zi-Qiang
authors.initials: ZQ
authors.lastname: Chen
authors.affiliation: Department of Hepatobiliary Surgery, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Hepatobiliary Surgery, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Lu-Ping
authors.initials: LP
authors.lastname: Xu
authors.affiliation: Department of Surgical Oncology, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Jian
authors.initials: J
authors.lastname: Tang
authors.affiliation: Department of Hepatobiliary Surgery, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Qing-Jiang
authors.initials: QJ
authors.lastname: Yu
authors.affiliation: Department of Surgical Oncology, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Yong-Quan
authors.initials: YQ
authors.lastname: Chu
authors.affiliation: Department of Neurology, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Jie
authors.initials: J
authors.lastname: Pan
authors.affiliation: Department of Anesthesiology and Pain Medicine, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Fei
authors.affiliation: Department of Hepatobiliary-Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: Sheng-Zhang
authors.initials: SZ
authors.lastname: Lin
authors.affiliation: Department of Surgery, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang, P.R. China.
authors.forename: Dian-Lei
authors.initials: DL
authors.lastname: Liu
authors.affiliation: Department of Surgical Oncology, First Hospital of Jiaxing, Jiaxing, Zhejiang 314000, P.R. China.
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019941:Cyclin-Dependent Kinase Inhibitor p16; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D004357:Drug Synergism; D004642:Emodin / Q000494:pharmacology*; D004745:Enkephalins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011498:Protein Precursors / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004745:Enkephalins; D009363:Neoplasm Proteins; D011498:Protein Precursors; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; C029992:proenkephalin; D000077209:Decitabine; D004642:Emodin; D001374:Azacitidine