Diverse effect of WWOX overexpression in HT29 and SW480 colon cancer cell lines

Abstract

WW-domain-containing oxidoreductase (WWOX) is the tumour suppressor gene from the common fragile site FRA16D, whose altered expression has been observed in tumours of various origins. Its suppressive role and influence on basic cellular processes such as proliferation and apoptosis have been confirmed in many in vitro and in vivo studies. Moreover, its protein is thought to take part in the regulation of tissue morphogenesis and cell differentiation. However, its role in colon cancer formation remains unclear. The aim of this study was to characterize the influence of WWOX on the process of colon cancerogenesis, the basic features of the cancer cell and its expression profiles. Multiple biological tests, microarray experiments and quantitative reverse transcriptase (RT)-PCR were performed on two colon cancer cell lines, HT29 and SW480, which differ in morphology, expression of differentiation markers, migratory characteristics and metastasis potential and which represent negative (HT29) and low (SW480) WWOX expression levels. The cell lines were subjected to retroviral transfection, inducting WWOX overexpression. WWOX was found to have diverse effects on proliferation, apoptosis and the adhesion potential of modified cell lines. Our observations suggest that in the HT29 colon cancer cell line, increased expression of WWOX may result in the transition of cancer cells into a more normal colon epithelium phenotype, while in SW480, WWOX demonstrated well-known tumour suppressor properties. Our results also suggest that WWOX does not behave as classical tumour suppressor gene, and its influence on cell functioning is more global and complicated.

Fig. 1

Confirmation of SW480 cell line transfection on the protein level and calculated relative protein expression. The values are mean ± SD. *p < 0.05. Lines 1–3 SW480/vector, 4–6 SW480/WWOX

Fig. 2

Confirmation of HT29 cell line transfection on the protein level and calculated relative protein expression. The values are mean ± SD. *p < 0.05. Lines 1–3 HT29/WWOX, 4–6 HT29/vector

Fig. 3

Changes in the viability of a SW480 and b HT29 cell line variants. Fluorescence is expressed in relative reference units (RFU). The values are mean ± SD. *p < 0.05

Fig. 4

The effect of WWOX overexpression on a proliferation and b apoptosis in the SW480 cell line. Fluorescence is expressed in relative reference units (RFU). Eu europium used as antibody label in proliferation assay, Sm samarium used as antibody label in apoptosis assay. The values are mean ± SD. *p < 0.05

Fig. 5

The effect of WWOX overexpression on a proliferation and b apoptosis in the HT29 cell line. Fluorescence is expressed in relative reference units (RFU). Eu europium used as antibody label in proliferation assay, Sm samarium used as antibody label in apoptosis assay. The values are mean ± SD. *p < 0.05

Fig. 6

Adhesion to ECM proteins for the SW480 transfected cell line. The values are mean ± SD. *p < 0.05

Fig. 7

Adhesion to ECM proteins for the HT29 transfected cell line. The values are mean ± SD. *p < 0.05

Fig. 8

Invasion assay and calculated difference in migration for both transfected colon cancer cell lines. The values are mean ± SD. *p < 0.05

Fig. 9

Growth in soft agar and calculated number of colonies for both cell lines. The values are mean ± SD. *p < 0.05

Fig. 10

Validation of microarray experiments. The values are mean ± SD;*p < 0.05; Lines 1–4 HT29/vector, 5–8 HT29/WWOX