The Effect Of K~+ Channels Blocker On The Proliferation Of Breast Epithelial Cells Associated With Caveolin-1

Breast cancer is the most common kind of malignant tumor and a kind of multi-factor influenced cancer. Both of these factors combine to cause gene mutation or the over-expression of oncogene. Varieties of natural genetic and environmental oncogene are involved in the process. However, the molecular mechanism of malignant transformation for breast epithelial cells is still unclear.Caveolin-1(Cav-1) is the main structural component of non-clathrin, flask-shaped invaginations named Caveolae. Cav-1 is very important in a variety of cellular functions, such as; the endocytic process, homeostasis (both cholesterol and lipid), signal transduction and tumor suppression. Caveolae are thought to serve as sites for the gathering of signaling complexes, thereby facilitating the initiation and cross-talk of signaling events. In previous research, Cav-1 haploinsufficiency cell lines were obtained by using a gene trapping approach , mRNA and protein levels were reduced; furthermore, Cav-1 haploinsufficiencies induce early transformation of human breast epithelial cells by upregulated MAPK signaling pathways. However, the relationship with other signals in Caveolae is still unclear.Potassium channels (K+ channels) are major signaling molecules expressed in a wide range of tissues where they have significant involvement in determining a variety of cellular functions, including; proliferation, solute transport, volume control, enzyme activity, secretion, invasion, gene expression, excitation-contraction coupling and intercellular communication. Previous reports show that K+ channels are linked to the physical activity of breast cancer cells. A current study has confirmed that a variety of important K+ channels localize on the caveolae and are modulated by caveolae.In this study, we used MCF0A, MCF10A-ST1 and MCF7 cells which have expressed different levels of Cav-1. First, an MTT assay was used to observe the inhibitory rate of MCF10A, MCF10A-ST1 and MCF7 cells treated with 4-AP and TEA. Second, RT-PCR and western blot techniques were used to analyze the expression of K+ channels. Co-immunoprecipitation was then used to investigate the link between K+ and Caveolin-1. Third, the western blot was again used to detect MAPK signal pathways of MCF10A, MCF10A-ST1, MCF7 cells after being treated with 4-AP. The object of this study was to investigate the effect of K+ channels during the transformation of breast cells and the mechanism of signaling pathways associated with Cav-1; while the aim was to investigate the mechanism of breast cancer's occurrence and development. Furthermore, another aim was to provide a new method for diagnosis and therapy in breast cancer.Results:1. The effects of TEA (K+ channel blocker) and 4-AP (Kv channel blocker) on proliferation of the human breast epithelial cells MCF10A, MCF-10A-ST1 and breast cancer cells MCF7 are dose-time-dependent. Results indicated that many K+ channels and Kv channels distribute on breast cell membranes and they are important in cell growth.2. The expression of Kv channels of breast cells are different. Expression of Kv1.5, Kv1.2 in MCF10A, MCF10A-ST1, MCF7 indicates that expression of Kv 1.5 (but not Kv1.2) is reduced with breast cancer progression. Expression of Cav-1 is reduced with breast cancer progression and immuno-precipitation studies show that the Kv1.5 is associated with caveolin-1. It suggested that effect of K+ channels in the proliferation of breast cells relate to the role of Cav -1.3. p-ERK1/2 protein level was not effected by incubation with 4-AP (no serum) after serum starvation. However, p-ERK1 /2 protein levels were increased significantly after incubation with 4-AP (added serum after 4-AP 10min). It indicated that 4-AP might effect the phosphorylation of MAPK in human breast epithelial cells.Conclusion:1. The present results demonstrated that a variety of Kv distribute on the breast cell membrane and Kv channels play important role during the transformation and proliferation of human breast epithelial cells.2. We hypothesize that Kv1.5 might be involved in the early transformation of breast epithelial cells. Its activity and expression were modulated by the signal pathway associated Cav-1.