Cancer Res Treat.  2007 Mar;39(1):30-36.

Analysis of Cellular Changes Resulting from Forced Expression of Dickkopf-1 in Hepatocellular Carcinoma Cells

• Kwack MH
• Hwang SY
• Jang IS
• Im SU
• Kim JO
• Kim MK
• Kim JC
• Sung YK

Abstract

PURPOSE: Recent studies have shown that Dickkopf-1 (DKK-1) is overexpressed in some tumors, including hepatocellular carcinoma. However, the role of increased DKK-1 in these tumors is not known. In this study, the DKK-1 expression in hepatocellular carcinoma (HCC) cell lines was evaluated and the effect of DKK-1 overexpression in HCC cell lines was studied.
MATERIALS AND METHODS
The expression of DKK-1 in hepatocellular carcinoma cell lines was evaluated by RT-PCR. Stable cell lines that overexpressed DKK-1 were established. Cell growth, adhesion, migration and invasion assays were performed.
RESULTS
RT-PCR analysis showed that 5 out of 8 HCC cell lines expressed DKK-1. The forced expression of DKK-1 suppressed the growth of cells and increased the population of cells in the sub-G1 phase. In addition, DKK- 1 reduced the cellular adhesion capacity to collagen type I and fibronectin, and it increased migratory capacity. However, overexpression of DKK-1 did not increase the invasion capacity of the HCC cell line.
CONCLUSION
Collectively, our data suggest that overexpression of DKK-1 affects the biology of HCC cells.

Keyword

Apoptosis; Cell adhesion; Cell line; Dickkopf-1; Hepatocellular carcinoma

MeSH Terms

Apoptosis
Biology
Carcinoma, Hepatocellular*
Cell Adhesion
Cell Line
Collagen Type I
Fibronectins
Collagen Type I
Fibronectins

Figure

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  Fig. 1 DKK-1 expression in the HCC cell lines. (A) RT-PCR analysis of the DKK-1 transcript. (B) The relative DKK-1 expression levels as measured from the RT-PCR analysis by densitometry.

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  Fig. 2 Forced expression of DKK-1 reduces the β-catenin level in SNU 475 cells. (A) RT-PCR analysis of the DKK-1 transcript. (B) Immunoblot analysis of the DKK-1/Myc-His fusion protein in the culture medium. (C) Immunoblot analysis of the β-catenin expression. Control: SNU 475 cells transfected with empty vector, Clones #19 and #22: clones transfected with vector pDKK-1/Myc-His.

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  Fig. 3 Growth suppression by DKK-1. (A) Direct counting. Values represent the means±standard error of 2 determinations per experiment from 3 independent experiments over 4 days of culture (*p<0.05 compared to the control). (B) A representative DNA histogram. Cells were stained with propidium iodide and analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is shown.

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  Fig. 4 DKK-1 reduces cell-to-matrix adhesion. The cells were seeded on wells coated with collagen type I or fibronectin. The attached cells were stained with crystal violet and the optical density was measured at 630 nm (A) and the cells were photographed under a microscope (B). Values represent the means±standard error of 5 determinations per experiment from 3 independent experiments (*p<0.05 compared to the control). (C) Immunoblot analysis of β1-integrin, a transmembrane receptor that binds to fibronectin and collagen. Probing with anti-actin antibody confirmed equal loading and transfer.

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  Fig. 5 DKK-1 increases the cells' migratory capacity. The cells were grown on transwells and the cells that had migrated to the underside of the transwells were stained and counted (A), and then photographed under a microscope (B). The values represent the means±standard error of 3 determinations.

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  Fig. 6 DKK-1 does not increase the cells' invasion capacity. The cells were grown on Matrigel-coated transwells and the cells that had invaded to the underside of the transwells were stained and counted (A), and then photographed under a microscope (B). The values represent the means±standard error of 2 determinations.

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