Bile acids repress E-cadherin through the induction of Snail and increase cancer invasiveness in human hepatobiliary carcinoma - PubMed (original) (raw)

. 2008 Sep;99(9):1785-92.

doi: 10.1111/j.1349-7006.2008.00898.x. Epub 2008 Aug 7.

Hideo Ohtsuka, Tohru Onogawa, Hiroshi Oshio, Takayuki Ii, Mitsuhisa Mutoh, Yu Katayose, Toshiki Rikiyama, Masaya Oikawa, Fuyuhiko Motoi, Shinichi Egawa, Takaaki Abe, Michiaki Unno

Affiliations

Bile acids repress E-cadherin through the induction of Snail and increase cancer invasiveness in human hepatobiliary carcinoma

Koji Fukase et al. Cancer Sci. 2008 Sep.

Abstract

Although some kinds of bile acids have been implicated in colorectal cancer development, the mechanism of cancer progression remains unexplored in hepatobiliary cancer. From our personal results using complementary DNA microarray, we found that chenodeoxycholic acid (CDCA) induced Snail expression in human carcinoma cell lines derived from hepatocellular carcinoma and cholangiocarcinoma. Snail expression plays an important role in the regulation of E-cadherin and in the acquisition of invasive potential in many types of human cancers including hepatocellular carcinoma. We found that CDCA and lithocholic acid (LCA) induced Snail expression in a concentration-dependent manner and down-regulated E-cadherin expression in hepatocellular carcinoma and cholangiocarcinoma cell lines. Moreover, Snail short interference RNA (siRNA) treatment reduced the down-regulation of E-cadherin by CDCA or LCA. Luciferase analysis demonstrated that the promoter region from -111 to -24 relative to the transcriptional start site was necessary for this induction and, at least in part, nuclear factor Y (NF-Y) and stimulating protein 1 (Sp1) might be an inducer of Snail expression in response to bile acids. In addition, using an in vitro wound healing assay and invasion assay, we observed that CDCA and LCA induced cell migration and invasion. These results suggest that bile acids repress E-cadherin through the induction of transcription factor Snail and increase cancer invasiveness in human hepatocellular carcinoma and cholangiocarcinoma. Inhibition of this bile acid-stimulated pathway may prove useful as an adjuvant in the therapy of hepatocellular carcinoma.

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Figures

Figure 1

Figure 1

Chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) induced expression of Snail mRNA and reduced expression of E‐cadherin mRNA. Cells were incubated with 100 µM of CDCA or LCA for 24 h. Then, total RNA was isolated and quantitative real‐time reverse transcription–polymerase chain reaction (RT‐PCR) was performed. (a) Hep3B cells were treated with 100 µM of CDCA or LCA. (b) HuCCT‐1 cells were treated with 100 µM of CDCA. Values for each gene were normalized to values obtained for GAPDH. _Y_‐axis represents a ratio for control (dimethyl sulfoxide [DMSO]). The data show the mean ± SD. *Significant difference (P < 0.01) from the respective control value.

Figure 2

Figure 2

Chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) increased Snail promoter activities in a concentration‐dependent manner. To investigate the effects of bile acids on the transcription of the Snail gene, 0.3 µg of the –1824/66 Snail pGL3 constructs and 10 ng of pRL‐TK vector were cotransfected into Hep3B cells. After 24 h, the bile acid was added to the cell culture media and the cells were incubated for 24 h. All reported firefly luciferase values were normalized for transfection efficiency using the pRL‐TK, Renilla‐luciferase value. _Y_‐axis represents the ratio for control (dimethyl sulfoxide [DMSO]). (a) The transfected cells treated with the various bile acids. The transfected cells treated with increasing amounts of (b) The transfected cells treated with CDCA or LCA at the concentration of 10–200 µM. The data show the mean ± SD of quadruplicate assay.

Figure 3

Figure 3

Deletion and mutagenesis analyzes of Snail promoter. 0.3 µg of each construct and 10 ng of pRL‐TK vector were cotransfected into Hep3B cells. After 24 h, the bile acid was added to the cell culture media and the cells were incubated for 24 h. All reported firefly luciferase values were normalized for transfection efficiency using the pRL‐TK, Renilla‐luciferase value activity and are shown as the relative activity compared to that for –1824/66 Snail pGL3 constructs treated with dimethyl sulfoxide (DMSO). The data show the mean ± SD of quadruplicate assay. (a) Deletion analysis of Snail promoter for the induction by LCA. (b) Effect of mutagenesis in NF‐Y, AP‐1 and Sp1 binding site on Snail promoter activity. The mutant promoter constructs used are schematically drawn.

Figure 4

Figure 4

Effect of chenodeoxycholic acid (CDCA) or lithocholic acid (LCA) on E‐cadherin protein expression and subcellular distribution. Hep3B cells were incubated with bile acids or dimethyl sulfoxide (DMSO) for 24 h (a) Hep3B cells were fixed and probed with an anti‐E‐cadherin antibody followed by Alexa Fluor 488‐conjugated antimouse secondary antibody. Immunofluorescence showed the localization of E‐cadherin proteins (green fluorescence). Panel (left); DMSO (middle); CDCA 100 µM (right); LCA 100 µM treatmrnt. Bar, 20 µm (b) Protein levels were determined by Western blotting of whole cell extracts using mouse monoclonal anti‐E‐cadherin antibody. The expression of actin was analyzed in the same samples as a control for the amount of protein present in each sample.

Figure 5

Figure 5

Snail short interference RNA (siRNA) treatment reduces down‐regulation of E‐cadherin by chenodeoxycholic acid (CDCA) or lithocholic acid (LCA). 200 ng of siRNA (final concentration: 100 nM) were transfected into Hep3B cells. After 72 h total RNA was isolated and quantitative real‐time reverse transcription–polymerase chain reaction (RT‐PCR) was performed. (a) The mRNA expression level of Snail. (b) The mRNA expression level of E‐cadherin. Mock: treated with only transfection reagent and N.C.: Non‐Targeting siRNA used as negative control. *Significant difference (P < 0.01) from N.C. (c) After transfection, Hep3B cells were incubated for 48 h and then cells were incubated with CDCA or LCA for 24 h. Total RNA was isolated. As a control, the same volume of dimethyl sulfoxide (DMSO) was used. The data show the mean ± SD. *Significant difference (P < 0.01) from the respective control value.

Figure 6

Figure 6

Chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) induce cell migration. (a) The motility behavior of bile acid treated cells was analyzed in an in vitro wound model. Confluent cultures of Hep3B cells treated with bile acids were gently scratched with a pipette tip to produce a wound. Quantitative analysis was performed as described in Materials and methods. Data are mean ± SD. *Significant different (P < 0.01) from the respective control (dimethyl sulfoxide [DMSO]). (right panels) Photographs of cell migration were taken immediately after the incision and after 12 h. (b) The cell invasion assays were performed as described in Materials and methods. The invading cells were counted under the microscope in 6 randomly selected fields for each membrane filter (×200). Each sample was assayed in duplicate in at least two independent experiments. Data are mean ± SD. *Significant difference (P < 0.01) from the respective control (DMSO). (c) The cell invasion assays were performed using Snail knocked down cells. Before plating, 200 ng of short interference RNA (siRNA) (final concentration: 100 nM) were transfected into Hep3B cells and incubated for 72 h. N.C.: Non‐Targeting siRNA used as negative control.

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