Tight junction protein, claudin-6, downregulates the malignant phenotype of breast carcinoma

Abstract

Claudin-6 is a protein component of tight junctions and its expression has been found to be undetectable or at low levels in some human and rat breast cancer cells. Here we investigated the effect of claudin-6 upregulation on the malignant phenotype of human MCF-7 breast cancer cells. MCF-7 sublines with stable claudin-6 expression were established by transfection with a pcDNA3.1-claudin-6 expressing vector. The expression of claudin-6 on mRNA and protein levels was confirmed by reverse transcription-PCR, western blot and immunofluorescent assays. Then the effects of claudin-6 on cell proliferation and cell cycle were examined by 5-diphenyl terazolium-bromide assay and flow cytometry, respectively. Colony-forming assays were used to examine two-dimensional and three-dimensional colony forming ability. Invasive and migratory traits of claudin-6 expressing cells were determined by matrigel-based Boyden chamber invasion assay and monolayer wound-healing assay. The structure and function of tight junctions in both parental and claudin-6 expression MCF-7 cells were evaluated by measuring transepithelial electrical resistance. Immunofluorescent assays showed that transfected cells expressed claudin-6 on their membranes. Cells with high level expression of claudin-6 grew slowly and had a higher rate of death than control cells. Anchorage-independent growth, invasive and migratory traits were also substantially decreased in cells with claudin-6 expression; whereas the transepithelial electrical resistance was increased in the claudin-6 transfected cells. In conclusion, these results suggest that claudin-6 may function as a cancer suppressor; its downregulation may contribute to the malignant progression of certain types of breast cancers.