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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
RESEARCH ARTICLE |
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
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Abstract  

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,Email:sqzou05@yahoo.com.cn   
Issue Date: 05 June 2009
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0034-5     OR     http://academic.hep.com.cn/fmd/EN/Y2009/V3/I2/153
primer setsequencelengthanneal temperature
MS-PCR
E-cadherin-M (sense)5'-TGAATTTTTAGTTAATTAGCGGTAC-3'
E-cadherin-M (antisense) 5'-CATAACTAACCGAAAACGCCG-3'204 bp57°C
E-cadherin-U (sense)5'-GGTAGGTGAATTTTTAGTTAATTAGTGGTA-3'
E-cadherin-U (antisense)5'-ACCCATAACTAACCAAAAACACCA-3'211 bp57°C
RT-PCR
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).
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