Correlation Between Expression Of P38MAPK And Integrin Beta3 In Breast Cancer

ObjectiveThe invasion and metastasis of cancer cell is a complex process, involving multi-factors. The adhering of cell and ECM is the first step of malignant tumor invasion. The adhering of cancer cell and ECM is mediated by idio-receptor of cell surface. Among all, the integrin family is the most important adhesion molecule. According to recent studies, tumor cell's liberation out or traversing basilemma which generated by the adhering of tumor cell surface integrin receptor and ECM is the beginning step to the infiltrating growth and distant metastasis of malignant tumor. Tenascin-c is a kind of Oligosaccharide protein with unique sexi-brachi structure in ECM. Besides, Tenascin-c is one of the most important ligands of integin. p38MAPK is one important member of mitogen-activated protein kinases (MAPKs). Recently, attention has been paid that p38MAPK signaling pathway is involved in the process of tumor invasion and metastasis by positively regulating mechanism. Therefore, we investigated the expression of p-p38,integrinβ3 and TN-c protein in breast cancer tissues and cells and the effect of p38MAPK signaling pathway on the expression by integrinβ3 and TN-c in breast cancer cells. Our studies suggested that p38MAPK might be a potential therapeutic target for anti-breast cancer treatment.Material and method1. SamplesA total of 80 paraffin-embedded breast cancer tissues obtained from the surgical resection at the pathological department of the First Affiliated Hospital of China Medical University between the year 2001 to 2005. According to the World Health Organization breast carcinoma histological classification criteria(2003), All specimens were infitrating ductal carcinoma. Human breast cancer cells MDA-MB-231 and MCF-7 were obtained from Chinese Peking Union University.2. ReagentsThe first antibodies were murine monoclonal antibody to TN-c and rabbit polyclonal antibody to integrinβ3 and p-p38 was purchased from Sigma and Cell signaling.DMEM and RPMI 1640 were purchased from Gibco.3. MethodsImmunohistochemistry (S-P): IHC was performed according to the indirect streptavidin-biotin-hyperoxidase method, as manufacture protocol. For the negative controls, the primary antibody was omitted by PBS, but all incubation steps were identical. Previously identified strongly staining tumor tissue sections were used as positive controls. We used an intensity-adjusted scoring system to evaluate immunostaining indices. The sections were scanned by light microscope. Five fields were randomly selected and 100 tumor cells in each field were counted. The staining correspond to negative and positive, respectively. integrinβ3 and p-p38:With respect to the invasive cell count,＜50% positive cells or no color were negetive,＞50% were positive. TN-c: no color or less yellow were negative, yellow or strong yellow were positive.Western blot: Protein concentration was measured by Bradford method. Tissue lysates were electrophoresed in polyacrylamide SDS gel and transferred onto polyvinylidene difluride membranes, Protein bands were blocked and incubated with the first and second antibody and visualized with DAB kit. Protein contents were calculated by densitometry.4. Statistical analysisThe results were compared using chi-squared test and Pearson test. P＜0.05 was considered statistically significant.Results1. Correlation between p-p38,integrinβ3 and Tenascin-c expression in breast cancer tissuesImmunohistochemical results showed that p-p38 protein was observed in nucleus of breast cancer cells and stained darkly and its positive rate is 57.50% (46/80).integrinβ3 was observed in cytoplasm of breast cancer cells and stained darkly and its positive rate is 56.25% (45/80). Tenascin-c was in ECM of breast cancer cells and stained darkly and its positive rate is 86.25% (69/80). Statistical analysis suggested a positive relationship between expression of p-p38,integrinβ3 and Tenascin-c(r=0.422, P＜0.05; r=0.355, P＜0.05; r=0.303, P＜0.05).2. Relationship between p-p38,integrinβ3 and Tenascin-c expression and clinicalpathological characteristicsThere was significant correlation between the level of p-p38,Tenascin-c, integrinβ3 expression and lymph node status and clinical stage (P＜0.05). and it was not related to patients age and tumor size were not significantly related to the respective level of p-p38,integrinβ3 and Tenascin-c (P＞0.05).3. Expression of p-p38,integrinβ3 and Tenascin-c in two differently metastatic breast cancer cellsWestern blot analysis indicated that the expression of p-p38,integrinβ3 and Tenascin-c in the highly metastatic MDA-MB-231 cells was higher than that in lowly metastatic MCF-7 cells. In MCF-7 cells, Tenascin-c is not expression.4. Change of p-p38 protein expression after blocked by anti-integrinβ3 and anti-Tenascin-cThe expression level of p-p38 protein in breast cancer MDA-MB-231 cells decreased after anti-integrinβ3 and anti-Tenascin-c was added into the culture fluid of MDA-MB-231 cells.DiscussionThe mitogen-activated protein kinases (MAPKs) are serine/ threonine kinases, which generally exist in various cells. MAPKs have been shown to transduce extracellular signals into endocells and be involved in cell proliferation, differentiation and malignant transformation and play important roles in tumor generation and development. p38MAPK is one important member of mitogen-activated protein kinase(MAPK) family. The p38MAPK undergoes phosphorylation at both tyrosine and threonine sites and can be activated by a wide spectrum of stimuli, including inflammatory cytokines, growth factors and cellular stress. p38MAPK signaling pathway has been implicated in cell growth, apoptosis, motion and invasive phaenotype and mediate cell migration, tumor invasion and metastasis. Integrin is a kind of 2-adicity ion-dependence surface glucoprotein. The functions of integrin are mediating the adhering of cell and basilemma, also controlling the existence and apoptosis of cell by delivering special signals or inducing genes.TN is a kind of oligosaccharide protein in ECM. Recently, TN has been found a kind of important ligand with unique sexi-brachi structure.By analyzing the expressions of p-p38,integrinβ3 and TN-c in bread cancer organism and their relationship with clinical pathology signs, we found that the expressions of p-p38 integrinβ3 and TN-c are significantly higher in breast cancer organism than that in normal mammary gland. Combining patho-index analytic results of breast cancer, we found that as the degree of tumor malignant increases, the expressions of p-p38,TN-c and integrinβ3 increase, but the expressions are independent with the diameter and age of the tumor. Besides, the three proteins relate to each other. In further study, we investigated the relationship between p-p38 expression and integrinβ3 and TN-c. Western blot analysis showed that anti-integrinβ3 and anti-TN-c decreased p-p38 protein expression in breast cancer MDA-MB-231 cells. Thereby demonstrating that p38MAPK signaling pathway might be involved in integrinβ3 expression. Conclusion1. There was correlation between the level of integrinβ3, Tenascin-c and p-p38 expression and lymph node status and clinical stage.2. There was a positive relationship between expression of integrinβ3,Tenascin-c and p-p38.3. With the decrease of integrinβ3 and Tenascin-c,so the p38.