Please wait a minute...
Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (2) : 153-157     DOI: 10.1007/s11684-009-0034-5
Effects of hydralazine and valproate on the expression of E-cadherin gene and the invasiveness of QBC939 Cells
Hong LI, Shaoqin CHEN, Yi SHU, Yongjun CHEN, Ying SU, Xin WANG, Shengquan ZOU()
Department of General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
Download: PDF(166 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

To clarify the effect of DNA methylation and histone deacetylase inhibitors on the expression of the E-cadherin gene and the invasiveness of the QBC939 cells, the QBC939 cells were separately treated with hydralazine, valproate, or combination of the two drugs. The mRNA expression of E-cadherin was examined with reverse transcription-polymerase chain reaction (RT-PCR), the protein of the gene with Western blotting. The methylation status of the promoter region of the gene was detected with methylation-specific PCR (MSP). The invasiveness of QBC939 cells was detected with transwell assay. It was found that the promoter region of the E-cadherin gene of QBC939 cells was hypermethylated. Valproate alone could not contribute to demethylation of the gene, whereas hydralazine could make them to be partly demethylated. However, the methylation status of the gene could be thoroughly reversed by using valproate and hydralazine in combination. What’s more, it was confirmed that the E-cadherin gene of QBC939 cells could not be transcriptionally reactivated by Valproate alone, whereas hydralazine alone could induce moderate reexpression of the gene. However, using valproate and hydralazine in combination could result in robust reexpression of the E-cadherin gene (P=0.000). Likewise, the invasiveness of the QBC939 cells was sharply decreased by treatment with two drugs in combination and slightly decreased with one drug alone. It could be concluded that the two drugs have synergistic effect on the demethylation and reexpression of the E-cadherin gene of QBC939 cells, and also on the reduction of the invasiveness of the QBC939 cells.

Keywords DNA methylation inhibitor      histone deacetylase inhibitor      bile duct carcinoma      E-cadherin     
Corresponding Authors: ZOU Shengquan,   
Issue Date: 05 June 2009
URL:     OR
primer setsequencelengthanneal temperature
E-cadherin-M (antisense) 5'-CATAACTAACCGAAAACGCCG-3'204 bp57°C
E-cadherin-U (antisense)5'-ACCCATAACTAACCAAAAACACCA-3'211 bp57°C
E-cadherin (sense) 5'-AGCCTTCGGCTGACTGGCTGG-3'
E-cadherin (antisense) 5'-CTGCCCATCATCATGACCTGG-3'524 bp59°C
β-actin (sense) 5'-GGCGTCGTGCGCAAAGGCC-3'
β-actin (antisense) 5'-GAACCTTGATGAAGCCTGTG-3'156 bp58°C
Tab.1  Primers used for MS-PCR and RT-PCR
Fig.1  Detection of methylation status in the promoter region of E-cadherin by MS-PCR. M: marker; 1, 2: control group (untreated cells); 3, 4: valproate group (treated with valproate alone); 5, 6: hydralazine group (treated with hydralazine alone); 7, 8: combined drug group (treated with valproate and hydralazine). M: product using methylated primers; U: product of unmethylated primers; methylated band 204 bp, unmethylated band 211 bp. MS-PCR: methylation-specific polymerase chain reaction.
Fig.2  Detection of mRNA expression of RASSF1A by RT-PCR. M: marker; C: control group (untreated cells); V: valproate group (treated with valproate alone); H: hydralazine group (treated with hydralazine alone); H+V: combined drug group (treated with valproate and hydralazine together).
Fig.3  Detection of protein expression of E-cadherin by Western blotting. C: control group (untreated cells); V: valproate group (treated with valproate); H: hydralazine group (treated with hydralazine); H+V: combined drug group (treated with valproate and hydralazine).
Fig.4  Detection of invasiveness of QBC-939 cells by transwell. (a) Control group (untreated cells); (b) valproate group (treated with valproate alone); (c) hydralazine group (treated with hydralazine alone); (d) combined drug group (treated with valproate and hydralazine).
1 Birchmeiev W, Behrens J. Cadherin expression in carcinomas: role of the formation of cell junction and the prevention of invasiveness. Biochim Biophys Acta , 1994, 1198(1): 11-26
2 Liotta L A, Steeg P S, Stetler-Stevenson W G. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell , 1991, 64(2): 327-336
doi: 10.1016/0092-8674(91)90642-C
3 Mareel M, Bracke M, Van Roy F. Cancer metastasis: negative regulation by an invasion-suppressor complex. Cancer Detect Prev , 1995, 19(5): 451-464
4 Berx G, Van Roy F. The E-cenhaoadherin/catenin complex: an important gatekeeper in breast cancer tumorigenesis and malignant progression. Breast Cancer Res , 2001, 3(5): 289-293
doi: 10.1186/bcr309
5 Li Q, Wang J M, Liu C, Xiao B L, Su Y, Zou S Q. Expression and significance of aPKC-ι and E-cadherin in Cholangiocarcinoma. Ai Zheng , 2007, 26(7): 715-718 (in Chinese)
6 Tamura G, Yin J, Wang S, Fleisher A S, Zou T, Abraham J M, Kong D, Smolinski K N, Wilson K T, James S P, Silverberg S G, Nishizuka S, Terashima M, Motoyama T, Meltzer S J. E-Cadherin gene promoter hypermethylation in primary human gastric carcinomas. J Natl Cancer Inst , 2000, 92(7): 569-573
doi: 10.1093/jnci/92.7.569
7 Z?chbauer-Müller S, Fong K M, Virmani A K, Geradts J, Gazdar A F, Minna J D. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res , 2001, 61(1): 249-255
8 Yang B, House M G, Guo M, Herman J G, Clark D P. Promoter methylation profiles of tumor suppressor genes in intrahepatic and extrahepatic cholangiocarcinoma. Mod Pathol , 2005, 18(3): 412-420
doi: 10.1038/modpathol.3800287
9 Jones P A, Baylin S B. The fundamental role of epigenetic events in cancer. Nat Rev Genet , 2002, 3(6): 415-428
10 Zhu W G, Otterson G A. The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells. Curr Med Chem Anti-Cancer Agents , 2003, 3(3): 187-199
doi: 10.2174/1568011033482440
11 de la Cruz-Hernández E, Pérez-Cárdenas E, Contreras-Paredes A, Cantú D, Mohar A, Lizano M, Due?as-González A. The effects of DNA methylation and histone deacetylase inhibitors on human papillomavirus early gene expression in cervical cancer, an in vitro and clinical study. Virol J , 2007, 4: 18
doi: 10.1186/1743-422X-4-18
12 Herman J G, Graff J R, Myohanen S, Nelkin B D, Baylin S B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A , 1996, 93(18): 9821-9826
doi: 10.1073/pnas.93.18.9821
13 Yap A S, Brieher W M, Gumbiner B M. Molecular and functional analysis of cadherin-based adherens junctions. Annu Rev Cell Dev Biol , 1997, 13: 119-146
doi: 10.1146/annurev.cellbio.13.1.119
14 Mikami T, Saegusa M, Mitomi H, Yanaqisawa N, Ichinoe M, Okayasu I. Significant Correlations of E-Cadherin, Catenin, and CD44 Variant Form Expression With Carcinoma Cell Differentiation and Prognosis of Extrahepatic Bile Duct Carcinomas. Am J Clin Pathol , 2001, 116(3): 369-376
doi: 10.1309/VV6D-3GAH-VEJM-DUJT
15 Joo Y E, Rew J S, Park C S, Kim S J. Expression of E-cadherin, alpha- and beta-catenins in patients with pancreatic adenocarcinoma. Pancreatology , 2002, 2(2): 129-137
doi: 10.1159/000055903
16 Berx G, Cleton-Jansen A M, Nollet F, de Leeuw W J, van de Vijver M, Cornelisse, C, van Roy F. E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. EMBO J , 1995, 14(24): 6107-6115
17 Vleminckx K, Vakaet L Jr, Mareel M, Fiers W, van Roy F. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell , 1991, 66(1): 107-119
doi: 10.1016/0092-8674(91)90143-M
18 Asayama Y, Taguchi Ki K, Aishima Si S, Nishi H, Masuda K, Tsuneyoshi M. The mode of tumour progression in combined hepatocellular carcinoma and cholangiocarcinoma: an immunohistochemical analysis of E-cadherin, alpha-catenin and beta-catenin. Liver , 2002, 22(1): 43-50
doi: 10.1046/j.0106-9543.2001.01580.x
19 Sharma D, Blum J, Yang X, Beaulieu N, Macleod A R, Davidson N E. Release of methyl CpG binding proteins and histone deacetylase 1 from the Estrogen receptor alpha (ER) promoter upon reactivation in ER-negative human breast cancer cells. Mol Endocrinol , 2005, 19(7): 1740-1751
doi: 10.1210/me.2004-0011
[1] Lei CHEN, Liang HU, Liang LI, Yuan LIU, Qian-Qian TU, Yan-Xin CHANG, He-Xin YAN, Meng-Chao WU, Hong-Yang WANG, . Dysregulation of β-catenin by hepatitis B virus X protein in HBV-infected human hepatocellular carcinomas[J]. Front. Med., 2010, 4(4): 399-411.
[2] Bin YANG MS , Xianglin YUAN , Yanmei ZOU , Qingsong XI , Guoxian LONG , Qiang FU , Guangyuan HU MM , . Effects of hypoxia inducible factor-1alpha siRNA on the invasion of human Hela cells and expression of related proteins[J]. Front. Med., 2009, 3(3): 303-308.
Full text