Correlation Between Expression Of P38MAPK Signaling Molecule And UPA In Breast Cancer

INTRODUCTIONBreast cancer is the most common malignancy in women and its incidence rate is increasing year by year. Breast cancer invasion and metastasis related -factor has currently become one of investigation emphases, because breast cancer often generate invasion and metastasis. Up to now, a growing body of evidence has showed that the overexpression of urokinase - type plasminogen activator (uPA) is detected in various malignant tumors and plays a key role in tumor invasion and metastasis. However, less studies demonstrated the molecular regulation mechanisms for uPA expression. 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 and uPA protein in breast cancer tissues and cells and the effect of p38MAPK signaling pathway on the expression of uPA in breast cancer cells. Our studies suggested that the inhibitor of p38MAPK might be a potential therapeutic target for anti - breast cancer treatment.METERAL AND METHODS1. SamplesA total of 60 paraffin - embedded breast cancer tissues were obtained fromthe surgical resection at the pathological department of the First Affiliated Hospital of China Medical University between the year 2001 to 2003. According to the World Health Organization breast carcinoma histological classification criteria (2003) , All specimens were invasive ductal carcinomas. Human breast cancer cells MDA - MB - 231 and MCF - 7 were obtained from pathological department of China Medical University.2. ReagentsDMEM and RPMI 1640 were purchased from Gibco . The first antibodies were mouse monoclonal antibody to phosphorylated - p38 and rabbit polyclonal antibody to uPA. SB203580, an specific inhibitor of p38MAPK was purchased from Calbiochem.3. Methods3.1 Immunohistochemistry (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 intensity was graded 0 — 2, corresponding to weak, moderately strong, and intense staining, respectively. With respect to the invasive cell count, < 10% positive cells were scored as 0, 10-50% were scored as 1, and >50% were scored as 2. By multiplying these two factors, an immu-noreactive score ^2 was positive.3.2 Western blot: Cells were lysed in RIPA buffer, then were ultrasounded and centrifuged at low temperature. Protein concentration was measured by Bradford method. Cell lysates were electrophoresed in 12% poly aery lamide 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 analysisAll statistical calculations were carried out using the SPSS 10. 0 statisticalsoftware. The results were compared using chi - squared test and Pearson test. P <0.05 was considered statistically significant.RESULTS1. Correlation between p - p38 and uPA protein expression in breast cancer tissues:Immunohistochemical results showed that p — p38 protein was observed in nucleus of breast cancer cells and stained darkly and its positive rate is 56.7% ( 34/60). uPA protein was in cytoplasm of breast cancer cells and stained darkly and its positive rate is 60.0% (36/60). Twenty - five uPA protein expressed positively in 34 cases of breast cancers which were positively expressed of p -p38 protein , and 15 cases were negatively expressed of uPA protein in 26 cases of breast cancers with p - p38 negative expression . Statistical analysis suggested a positive relationship between expression of p - p38 and uPA( r=0.316,P<0. 05).2. Relationship between p - p38 and uPA protein expression and clinical-pathological characteristics:There was significant correlation between the level of uPA N p - p38 expression and lymph node status and clinical stage( P <0. 05). The level of uPA and p -p38 was not significantly related to patients'age and tumor size(P >0. 05).3. Expression of p - p38 and uPA protein in two differently metastatic breast cancer cells:Western blot analysis indicated that the expression of p - p38 and uPA in the highly metastatic MDA - MB - 231 cells was higher than that in lowly metastatic MCF-7 cells.4. Change of uPA protein expression after p38MAPK signaling pathway was blocked by SB203580:The expression level of uPA protein in breast cancer MDA - MB - 231 cells decreased after SB203580,an specific inhibitor of p38MAPK was added into the culture fluid of MDA - MB -231 cells . SB203580 concentration - dependencyreduced uPA protein expression.DISCUSSIONThe mitogen - activated protein kinases ( MAPKs ) are serine/ threonine ki-nases, 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 phosphoryla-tion 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 mediates cell migration, tumor invasion and metastasis. Urokinase plasmitogen activator (uPA) is a serine protease and initiates the conversion of plasminogen to plasmin. These proteases confer on the cells the ability to degrade the extracellular matrix . uPA has been clearly demonstrated to be essential in the maintenance of invasive and metastatic pheno-types.At present, less studies demonstrated the relationship between uPA expression and signal transduction pathway. Recent researches have indicated that p38MAPK signaling pathway participates in regulating uPA expression of gastric, lung, leukoma and breast cancer cells. Our studies suggested that there was significant correlation between the level of uPA N p - p38 expression and lymph node status and clinical stage of breast cancer and the expression of p -p38 and uPA in the highly metastatic MDA - MB -231 cells was higher than that in lowly metastatic MCF -7 cells. Our results hinted that overexpression of p - p38 and uPA might be related to malignant progression, invasion and metastasis of breast cancer. Immunohistochemical results showed an positive relationship between expression of p - p38 and uPA and suggested uPA protein expression might be regulated by p38MAPK signaling pathway. In further study, we investigated the relationship between uPA expression and p38MAPK signalingpathway. Western blot analysis showed that SB203580 decreased uPA protein expression in a concentration - dependent manner in breast cancer MDA - MB -231 cells. Thereby demonstrating that p38MAPK signaling pathway might be involved in regulating uPA expression.Together, p38MAPK signaling pathway promoted breast cancer malignant progression , invasion and metastasis. However, the precise mechanisms remain unknown. Currently, an inhibitor of MAPK signaling pathway has represented a novel target for cancer intervention strategies. Our study suggests that p38MAPK signaling pathway may be potential therapeutic target for breast cancer treatment.CONCLUSION1. There was correlation between the level of uPA ^ p - p38 expression and lymph node status and clinical stage, and the expression of p -p38 and uPA in the highly metastatic MDA - MB -231 cells was higher than that in lowly me-tastatic MCF-7 cells.2. There was a positive relationship between expression of p - p38 and uPA3. SB203580 , an specific inhibitor of p38MAPK concentration - depend-ently reduced uPA protein expression.