The Relationship Between The Expression Of P38 Protein And Lymph Nodes Metastasis In Human Breast Carcinomas

OBJECTIVEBreast cancer is a common malignant tumor of women. There are many clinical patients who have metastasis in lymph nodes. And the metastasis in lymph nodes is one of the important indictors of prognosis evaluation. So the study of mechanism and prognosis of metastasis may play an important role in instruction therapy and prediction the consequence. Metastasis of tumors is a complex multiple - steps and multiple factors participated process, which including the degradation of extracellular matrix, the formation of newly born blood vessels and lymphatic vessels. In fact, the invasion and metastasis of tumors are based on the abnormality of signal transduction between cells.Some study indicated the upregulation of MAPK pathways played an important role in the development of breast cancer. Recently, some tests in vitro found that the mainly member, namely, p38MAPK was involved in the enhancement of cellular movements and the expression of MMP uPA and VEGF. p38 transmitted signals by its tris - grade kinase cascade, which was similar to other members of MAPK. Inflammatory factors and stresses made p38 biophosphory-lated in tyrosine and threonine residue thus p38 activated. Activated p38 can activate many transcription factors, and influence the transcription of cells the synthesis of proteins the expression of receptors on cellular surface and alternation of cellular structure.Nevertheless the tests in vitro have demonstrated sufficiently that p38 could promote the invasion and metastasis of breast cancer cell lines, the studies about p38 in human was relatively few. The objective of our study was to detect the change of p38 protein expression and investigate the relationship of p38 andlymph nodes metastasis of breast cancer. Our study would indicate further the significance of p38 in breast cancer metastasis and provide beneficial indicator for prognosis in breast cancer.MATERIAL AND METHODS1. SamplesA total of 60 breast tissues achieved from the center of pathological diagnosis in the First Affiliated hospital of China Medical University, which were surgical resection between the year 2001. 6 to 2003. 11. All samples were fixed in 10% formalin and embedded in paraffin. 30 samples used in western blot analysis including breast cancer tissues and adjacent normal tissues were achieved from surgical resection in the first affiliated hospital of China Medical University between the year 2003.11 to 2004.4. Each samples allowed one part to be snap -frozen in liquid N2 for later western blot analysis and one part to be fixed in formalin. The patients had not undergone any chemo - or radiotherapy before surgical resection.2. ReagentsRabbit anti - p38 polyclonal antibody and mouse anti - p - p38 monoclonal antibody were purchased from Cell Signaling Inc. USA. Goat anti - rabbit and anti - mouse secondary antibody marked by horseradish peroxidase were purchased from Zhong Shan Inc in Beijing. S - P ultrasensitive immunohistochemi-cal kit and DAB kit were purchased from Hua Mei Inc.3. Methods3.1 IHC was performed according to the indirect streptavidin - biotin -hyperoxidase method. Each case was observed in 5 fields and counted 100 tu-morous cells in every microscopic field. No staining was scored 0; light yellow and yellow staining was 1 and brown staining was 2. The amount and positive cells <10% was scored 0; 10% -50% was 1 and >50% was 2. The product of multiplication of the above two items scored 0 was negative; scored 1-2 was weak positive and scored 4 was positive.3.2 Western Blot: 3mm tissues were lysed, grinded, centrifuged, elec-trophoresed and electrophoretic transferred. The transferred samples were incubated with the first antibody and visualized with DAB kit. p38 and p - p38 protein contents were calculated by densitometry.4. Statistical analysisAll statistical calculations were carried out using the SPSS11.5 statistical software. The results were compared using chi - squared test and t - test. P < 0.05 was considered statistically significant.RESULTS1. The relationship between p38 p - p38 protein expression and lymph nodes metastasisImmunohistochemical results showed p38 protein was observed in breast cancer and normal cytoplasm and stained darkly in breast cancer cells while lightly in normal cells, p - p38 was positive in nucleus in breast cancer. 29 p38 protein expressed positively in 39 patients who had lymph nodes metastasis, and the positive rate was 76. 3%. 9 p38 protein expressed positively in 22 patients who had no lymph nodes metastasis, the positive rate was 40. 9%. And there was a significant difference between these two groups ( P < 0.01 ). The positive rate of p - p38 in patients who had lymph nodes metastasis was 68.4% , and the positive rate in patients who had no metastasis was 36.4% , and there was a significant difference between these two groups ( P < 0.05 ).The results of western blot were similar to the results of immunohistochemis-try, the protein content of p38 p - p38 in metastasis group was higher than no metastasis group( P < 0.05 ).2. The relationship between p38 p - p38 protein expression and other clinical pathological characteristic. Our study found p38 and p - p38 protein expression had relation with clinical pathological grades in breast cancer which in grade III were higher than grade I II(P<0.05) while had no relation with patients' age and tumor size( P >0.05 ).DISCUSSIONMAPK ( Mitogen - Activated Protein Kinase) is a superfamily of serine/ threonine kinase that is existed extensively in cells and is an important substance that transmits extracellular signals to nucleus and changes the function of nucleus. MAPK pathways regulates proliferation differentiation ,mitosis ,death and the synchronization of cellular functions. Recently, the important member -p38MAPK has been the hotspot of study in tumorous invasion and metastasis. p38 has four isoforms in which p38α and p38β express in almost all tissues; p38γ exists in muscle tissues and p38δ exists in kidney and lung tissues. The relative molecular weight of p38 is 38000 and p38 contains 360 amino acid residues. The process of invasion and metastasis of tumors is composed a series of complicated and continuous steps, including the enhancement of the cellular movement, the degradation of ECM and the proliferation of tumorous blood vessels and lymphatic vessels. Many tests in vitro suggested that p38MAPK pathway takes part in the process by promoting the enhancement of tumorous cellular movement, regulating the expression of uPA MMP VEGF and accelerating the invasion and metastasis of tumors.Through the analysis of p38 and activated p38 protein expression and the metastasis of axillary lymph nodes in breast cancer, we found they had relationship. The levels of p38 protein expression and activation in axillary lymph nodes metastasis positively were higher than in axillary lymph nodes metastasis negatively. The above - mentioned results suggested excessive p38 expression and activation promoted the metastasis of breast cancer. Furthermore, we investigated the relationship between p38 p - p38 protein expression and other clinical indictors and found there was no correlation patients' age and tumor size but there was correlation between p38,p - p38 protein expression and clinical pathological grades. p38 p -p38 protein expression in grade III was higher than in grade I and II , which suggested that the levels of p38 p - p38 protein expression were rising along with the development of breast cancer. The above - mentioned results suggested patients with advanced levels of p38 p - p38 protein ex-...