Yu-Mei Gu1, Yi-Hui Ma, Wu-Gan Zhao, Jie Chen. 1. Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100730, China.
Abstract
AIM: To elucidate the role of dickkopf3 (Dkk3) in human pancreatic cancer cell growth. METHODS: Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by real-time reverse transcription polymerase chain reaction (real-time RT-PCR), Western blotting and immunofluorescence. Methylation of the Dkk3 promoter sequence was examined by methylation-specific polymerase chain reaction (MSP) and Dkk3 mRNA expression was determined by real-time RT-PCR after 5-aza-2'-deoxycytidine (5-aza-dC) treatment. The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells. The expression of β-catenin, phosphorylated extracellular signal-regulated protein kinases (pERK) and extracellular signal-regulated protein kinases (ERK) was also examined by real-time RT-PCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells. RESULTS: The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested. The Dkk3 promoter sequence was methylated in the MIA PaCa-2 and AsPC-1 cell lines, which showed reduced Dkk3 expression. These two cell lines, which initially had a methylated Dkk3 promoter, showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent, 5-aza-dC, treatment (P < 0.05 or P < 0.01). When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa-2 cells, the ability of cells to proliferate decreased (P < 0.01), and the expression of β-catenin and pERK was downregulated (P < 0.01). Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmid-transfected cells. CONCLUSION: Our findings, for the first time, implicate Dkk3 as a tumor suppressor in human pancreatic cancer, through the downregulation of β-catenin expression via the ERK-mediated pathway.
AIM: To elucidate the role of dickkopf3 (Dkk3) in humanpancreatic cancer cell growth. METHODS:Dkk3 mRNA and protein expression in humanpancreatic cancer cell lines were detected by real-time reverse transcription polymerase chain reaction (real-time RT-PCR), Western blotting and immunofluorescence. Methylation of the Dkk3 promoter sequence was examined by methylation-specific polymerase chain reaction (MSP) and Dkk3 mRNA expression was determined by real-time RT-PCR after 5-aza-2'-deoxycytidine (5-aza-dC) treatment. The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) after transfecting the Dkk3 expression plasmid into humanpancreatic cancer cells. The expression of β-catenin, phosphorylated extracellular signal-regulated protein kinases (pERK) and extracellular signal-regulated protein kinases (ERK) was also examined by real-time RT-PCR and Western blotting after upregulating Dkk3 expression in humanpancreatic cancer cells. RESULTS: The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested. The Dkk3 promoter sequence was methylated in the MIA PaCa-2 and AsPC-1 cell lines, which showed reduced Dkk3 expression. These two cell lines, which initially had a methylated Dkk3 promoter, showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent, 5-aza-dC, treatment (P < 0.05 or P < 0.01). When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa-2 cells, the ability of cells to proliferate decreased (P < 0.01), and the expression of β-catenin and pERK was downregulated (P < 0.01). Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmid-transfected cells. CONCLUSION: Our findings, for the first time, implicate Dkk3 as a tumor suppressor in humanpancreatic cancer, through the downregulation of β-catenin expression via the ERK-mediated pathway.
Entities:
Keywords:
Cell growth; Dickkopf3; In vitro; Overexpression; Pancreatic cancer
Authors: T Tsuji; I Nozaki; M Miyazaki; M Sakaguchi; H Pu; Y Hamazaki; O Iijima; M Namba Journal: Biochem Biophys Res Commun Date: 2001-11-23 Impact factor: 3.575
Authors: Bingyu Mao; Wei Wu; Gary Davidson; Joachim Marhold; Mingfa Li; Bernard M Mechler; Hajo Delius; Dana Hoppe; Peter Stannek; Carmen Walter; Andrei Glinka; Christof Niehrs Journal: Nature Date: 2002-05-26 Impact factor: 49.962
Authors: J Roman-Gomez; A Jimenez-Velasco; X Agirre; J A Castillejo; G Navarro; M Barrios; E J Andreu; F Prosper; A Heiniger; A Torres Journal: Br J Cancer Date: 2004-08-16 Impact factor: 7.640
Authors: Jack L Leonard; Deborah M Leonard; Scot A Wolfe; Jilin Liu; Jaime Rivera; Michelle Yang; Ryan T Leonard; Jacob P S Johnson; Prashant Kumar; Kate L Liebmann; Amanda A Tutto; Zhongming Mou; Karl J Simin Journal: PLoS One Date: 2017-07-24 Impact factor: 3.240