The Role Of E Protein In Drug Resistance Of Breast Cancer

Objectives: E protein is a new membrane protein. Our previous results showed that E protein binds to P-gp. We will further evaluate the expression of E protein and P-gp in breast cancer tissue, and analyze the correlation between E protein and drug resistance protein. Besides, we will study the relationship between E protein localization and the sensitivity to drugs in primary breast cancer cells. In addition, we will further study the drug sensitivity of primary breast cancer cells in three-dimensional culture condition.Methods: 1. Immunohistochemical method was used to detect the expression levels and localization of E protein and P-gp in breast cancer for analyzing the relationship between the target proteins. 2. The method of primary culture of breast cancer cells was built to obtain a high-purity of primary breast cancer cells. 3. MTT assay was used to detect drug sensitivity to ADR and DDP of different sources of primary breast cancer cells. 4. Western Blot was applied to detect the expression levels of primary breast cancer cells. We further analyzed the relationship between the overall expression of E protein in primary breast cancer cells and drug sensitivity to ADR and DDP. 5. Immunofluorescence was used to observed the localization of endogenous E protein, P-gp and BCRP, and the co-localization of E protein and P-gp in primary breast cancer cells. 6. Matrigel 3D culture system was built to compare the survival outcome of primary breast cancer cells in different dimension.Results: 1. E protein can express in the membrane, cytoplasm and nucleus in breast cancer, a more significant correlation was observed between the of E protein in the cytoplasmic membrane and P-gp. 2. Primary breast cancer cells were successfully cultured in 28 cases, with 20 out of 28 cases obtaining enough cells for subsequent experiments. 3. The expression levels of overall E protein among the 20 patients were measured using Western Blot. 4. Drug sensitivity to ADR and DDP was measured among 16 and 12 patients-derived primary breast cancer cells, respectively. The IC50 value was calculated separately. In the E protein high expression group, reduced sensitivity to DDP was observed. As for drug sensitivity to ADR, the two groups had no significant difference. 5. E protein expression was enriched and co-localization on the relatively resistant cells surface membrane with P-gp. 6. In the three-dimensional environment, the sensitivity of primary breast cancer cells to ADR decreased.Conclusions: 1. E protein can express in the membrane, cytoplasm and nucleus in breast cancer, and the expression of E protein in the cytoplasmic membrane and P-gp was more significant correlation. The patients of Luminal type were more popular in the E protein high expression group. 2. In primary breast cancer cells, no significant relationship was showed between the overall level of E protein and sensitivity to drugs. E protein which express on the membrane affect the sensitivity to drugs. 3. Three-dimensional culture method better reflects the primary tumor cell sensitivity to drugs. The results in two-dimensional culture systerm were conformed in the three-dimensional systerm.