Effects of hydralazine and valproate on the expression of E-cadherin gene and the invasiveness of QBC₉₃₉ Cells

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

Introduction

Researches demonstrated that cell-cell adhesion participates in histogenesis and plays a critical role in the establishment and maintenance of cell polarity and society and that the mutual adhesiveness of cancer cells is significantly weaker than that of the corresponding normal cells [1]. One of the most characteristic features of cancer cells is loss of “contact inhibition,” which reflects disordered signal transduction from cell-cell adhesion to cell growth [2]. Invasion and metastasis are thought to be critically important in carcinogenetic steps. Because of the poor diagnosis, invasion and metastasis always have already taken place before the bile duct carcinoma is diagnosed. Enhancing cell-cell adhesion to reduce the invasiveness of tumor cells is significant for the cure of the disease.

E-cadherin has been considered to be an important molecule in invasion and metastasis, largely because its expression in some cases inversely correlated with tumor aggressiveness. It is proved that the decreased expression of E-cadherin is common in a variety of human tumors [3, 4]. In our previous studies, we also found that E-cadherin expression was sharply reduced and negatively correlated to the differentiation and invasion of cholangiocarcinoma, so E-cadherin might reflect the differentiation and invasive potential of cholangiocarcinoma [5]. It was reported that aberrant methylation of the promoter regions of E-cadherin gene, which resulted in the gene silence, was observed in many cancers [6, 7]. In the Johns Hopkins’s study, the frequency of E-cadherin gene methylation in cholangiocarcinoma was 65% [8], which means that aberrant methylation of the promoter regions of E-cadherin is a frequent event in bile duct carcinoma. At the same time, recent research demonstrated that CpG island hypermethylation is just one facet of an integrated change in chromatin structure and histone modifications [9], whereas DNA hypermethylation and histone deacetylation were critical in determining a closed chromatin structure responsible for or related with aberrant gene transcription in malignancy [10]. In this research, we explored the effect of DNA methylation inhibitors (hydralazine) and histone deacetylase inhibitors (valproate) on the methylation status of the E-cadherin gene promoter region, the expression of the E-cadherin genes and the invasiveness of QBC₉₃₉ cells.

Materials and methods

Materials

Human biliary tract carcinoma cell line QBC₉₃₉ was a generous gift from Dr. Shu-Guang Wang (Third Military Medical University, China). Hydralazine (a DNA methyltransferase inhibitor) and valproate (a histone deacetylase inhibitor) [11] were purchased from Sigma Co. (USA). Monoclonal antibody to E-cadherin (mouse IgG1) was procured from ABcom Co. (USA). Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG(H+L) was purchased from BD Co. (USA). All the primers in this study were synthesized by Takara Co. Ltd (Japan).

Methods

Cell culture

The human biliary tract carcinoma cell line QBC₉₃₉ cells were cultured in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum and incubated in a humidified atmosphere with 5% CO₂ at 37°C.

Chemical intervention with hydralazine and valproate

The hydralazine and valproate were dissolved in RPMI1640 medium, and the final concentration of hydralazine and valproate were adjusted to be both 10 μmol/mL. The QBC₉₃₉ cells were divided into 4 groups. One group was added with hydralazine and valproate, and then, the final concentration was adjusted to 10 μmol/mL. One group treated with the same volume of RPMI1640 medium was taken as the control group. The other two groups were added with either hydralazine or valproate, at a final concentration of 10 μmol/mL. The cells in the 4 groups were harvested 48 h later for further analysis.

Extraction of genomic DNA and bisulfite modification

Total genomic DNA was extracted from the collected cells of the experimental group and the control group according to the instruction of the DNA extraction kit. The bisulfite modification of genomic DNA was carried out according to the method of Herman [12].

Methylation-specific polymerase chain reaction

The methylation status in the promoter region of the E-cadherin gene was detected by methylation-specific polymerase chain reaction (MS-PCR). The methylated and unmethylated primers to amplify the bisulfite-converted E-cadherin promoter sequences were described in Table 1.

Reverse transcription-polymerase chain reaction analysis

Total RNA was extracted from the cells of experimental group and control group using Trizol reagent. The first strand of cDNA taken as PCR template was synthesized by reverse transcription (RT) according to the instruction of the RT kit. The sequences of primers of E-cadherin gene and β-actin gene were described in Table 1. β-actin was used as an internal control. The PCR reaction was performed according to the parameters as follows: 94°C for 5 min, 32 cycles at 94°C for 1 min, 58°C for 1 min, and 74°C for 1 min. Then, it was extended at 74°C for 5 min. The PCR products were isolated on a 1.5% agarose gel containing 0.5% ethidium bromide and observed under UV illumination. The optical density was automatically measured and integrated by gel data acquisition AlphaImager software (Alpha Innotech Corp., San Leandro, CA, USA). The relative level of E-cadherin expression was presented by the ratio of E-cadherin and β-actin.

Western blot analysis

The total protein of the cells were extracted and separated on 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel followed by electroblotting onto PVDF membranes. Membranes were blocked in Tris-buffered saline with 5% nonfat dry milk and then incubated with 1∶1000 dilution of the mouse monoclonal antibody against human E-cadherin overnight at 4°C. After several washes in TBS-T, goat anti-mouse secondary antibody labeled with horseradish peroxidase was applied to bind the primary antibody at room temperature for 2-3 h, and detection was performed by chemiluminescence system as directed by the manufacturer.

Transwell

The assays were conducted using 8.0-μm pore size and 6.5-mm diameter transwell filters (Costar, USA). The undersurface of the polycarbonate membrane of the chambers was coated with Matrigel (BD, USA), 50 μg per chamber. The membrane was washed in phosphate buffered saline (PBS) to remove excess ligands, and the lower chamber was filled with 500 μL of 10% FBS-containing medium. Control group cells and treated cells that were diluted to 1.5×10⁵/mL with serum-free medium were added to the upper chambers. After 48 h at 37°C in 5% CO₂, cells were fixed with 95% alcohol for 15 min at room temperature and stained with 0.5% methylene blue. The cells on the upper surface of the membrane were removed using cotton buds. The number of migrated cells on the underside of the membrane was counted microscopically at magnification of 400 times (cells/mm²).

Results

Detection of methylation status in the promoter region of E-cadherin

The result of MS-PCR showed that a 204 bp DNA band was amplified by using methylated primers, and no corresponding DNA band was detected by using unmethylated primers in the control group and valproate group. In the hydralazine group, a 204 bp DNA band was amplified using methylated primers, and a 211 bp corresponding DNA band was also amplified. However, the 211 bp DNA band was amplified using unmethylated primers, but no DNA band was observed using methylated primers in combination of both drugs group. This result suggested that the promoter region of E-cadherin gene was methylated in QBC-939 cells. Treatment of valproate alone could not induce the promoter of E-cadherin gene demethylation, and hydralazine could partly demethylate the promoter of E-cadherin gene. However, methylation status in the promoter region of E-cadherin gene was thoroughly reversed from methylation to unmethylation when both drugs were applied (Fig. 1).

Detection of E-cadherin mRNA expression

As shown in Fig. 2, the fragments of E-cadherin and β-actin (internal control) cDNA were 524 bp and 156 bp, respectively. There was no fragment of E-cadherin in the control group, suggesting that no E-cadherin was expressed in pretreated QBC-939 cells. Likewise, there was no fragment of E-cadherin in the valproate group, indicating that valproate could not reactivate E-cadherin gene alone. However, E-cadherin mRNA was detected in the hydralazine group and both drug groups. The level of E-cadherin expression induced by combined drugs was much higher than that of the hydralazine group (P<0.01) (Fig. 2).

Detection of E-cadherin protein expression

The result of Western blot showed that four specific protein bands that represented β-actin were detected both in the experimental and control groups on β-actin-detection membrane. Meanwhile, two 97-kD protein bands that represented the E-cadherin expression at protein level was observed on the E-cadherin detection membrane of the hydralazine and combined drug groups. There was no corresponding protein band in the control and valproate groups. This result demonstrated that human biliary tract carcinoma cell line QBC₉₃₉ did not express E-cadherin protein. Valproate alone could not contribute to the reexpression of E-cadherin protein, whereas hydralazine alone could induce moderate reexpression of the E-cadherin genes. However, using valproate and hydralazine in combination could result in robust reexpression of the gene (P=0.000) (Fig. 3).

Detection of invasiveness of QBC-939 cells

Compared with control cells, the invasiveness of the QBC₉₃₉ cells treated with different drugs was decreased in various degrees. The amount of cells treated with combined drugs migrated to the bottom chamber was significantly less than the amount of cells in any other group in this experiment (P=0.000); the amount of cells migrated to the bottom chamber treated with hydralazine was less than the amount of cells treated with valproate (P=0.000).

Discussion

E-cadherin, a classic member of the cadherin superfamily, expresses on epithelial cells and mediates Ca²⁺-dependent homophilic cell-cell adhesion [13]. The reduction or absence of E-cadherin expression is considered to be a hallmark of malignancy in a variety of cancers including pancreatic and bile duct carcinoma [14, 15]. Down-regulation of E-cadherin has been reported in many different cancers and was often associated with increased carcinoma invasion, metastasis, and poor prognosis [16]. A direct role of E-cadherin in the suppression of tumor invasion has been demonstrated by the reversion of the invasive phenotype in malignant epithelial tumor cells following transfection with E-cadherin cDNA. Invasiveness of the transfected cells could be restored by treatment with E-cadherin antibodies or by reducing E-cadherin expression with an E-cadherin antisense RNA [17]. Thus, loss of function of E-cadherin tumor suppressor protein correlates with increased invasiveness and metastasis of tumors. Meanwhile, some research reported that recovered E-cadherin expression may be a beneficial effect on reestablishing the tissue architecture at the metastatic site in cholangiocarcinoma [18]. All these suggest that the E-cadherin gene expression is critically important for the treatment of bile duct carcinoma. However, in our study, we detected that E-cadherin gene was silent as revealed by RT-PCR and Western blot assays.

After being treated with drugs, from the methylation-specific PCR (MSP) results, we found that the E-cadherin promoter region of the pretreated and the valproate-treated QBC₉₃₉ cells was hypermethylated, whereas the E-cadherin promoter region of the cells treated with hydralazine alone was partly demethylated. However, the E-cadherin gene promoter region methylation status of the cells treated with combination of drugs was completely reversed to be unmethylation. From the RT-PCR and Western blot results, we noticed that the combination usage of drugs could induce more E-cadherin mRNA and protein reexpression than the usage of either one drug alone (P=0.000). Given the correlation between E-cadherin gene expression and cell invasiveness, the invasiveness of corresponding cells was detected. The result of transwell assay indicated that the combination usage of drugs could significantly reduce the invasiveness of QBC₉₃₉ cells than either of the two drugs alone. Thus, we could draw a conclusion from the study that the hypermethylation of the promoter region of E-cadherin might be the main reason for the silence of the gene in QBC₉₃₉ cells and that the DNA methyltransferase inhibitor and histone deacetylase inhibitor had a synergy effect on reexpression and demethylation of E-cadherin. The mechanism may be related to the removal of the methyl-binding proteins from the demethylated promoters and the recovery of the chromatin structure from dense to relax, which allows the accessibility of the transcription factors to initiate gene expression [19]. Then, reexpressed E-cadherin gene reduced the invasiveness of QBC₉₃₉ cells. With the reexpression of E-cadherin, the invasiveness of the treated QBC₉₃₉ cells was reduced correspondingly. However, valproate could not reactivate the E-cadherin; the inhibitory effect of valproate on invasiveness of QBC₉₃₉ cells might be related to cytotoxic effect. DNA methylation and histone deacetylase inhibitors are promising drugs for the treatment of bile duct carcinoma.

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