Effects of stable up-regulation of tight junction protein claudin-6 upon biological phenotypes of breast cancer cell MCF-7

Abstract

To explore the effect of over-expression of tight junction protein claudin-6 upon the biological characters of breast cancer cell line MCF-7. The MCF-7 subline expressing a high level of claudin-6 was established by transfection with a pcDNA3.1-claudin-6 expression vector. The expression of claudin-6 in mRNA and its protein level were confirmed by RT-PCR, Western blot and immunofluorescent assays. Then the effect of claudin-6 upon cell proliferation was examined by MTT assay. Colony-forming assays were used to examine 2-D and 3-D colony-forming capacities. Invasive and migratory traits of claudin-6 expressing cells were determined by 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 membrane. Cells with a high level expression of claudin-6 grew slowly than control cells. Anchorage-independent growth, invasive and migratory traits also decreased substantially in cells with claudin-6 expression; whereas the transepithelial electrical resistance increased in the claudin-6 transfected cells. The up-regulation of claudin-6 expression in MCF-7 breast cancer cells suppresses their malignant phenotypes with a correlation with the restoration of tight junction integrity. Claudin-6 may function as a cancer suppressor whose down-regulation contributes to the malignant progression of certain types of breast cancers.