Expression And Significance Of GSK3β And P38MAPK In The Breast Cancer Tissues

IntroductionGlycogen synthase kinase-3β(GSK3β), is a serine/threonine protein kinase, it involved in tumor glucose synthesis, cell proliferation and apoptosis, angiogensis, microtubule dynamics and cell motility. Oncogenic transcription factors and proto-oncoproteins are putative GSK3βsubstrates for phosphorylation dependent inactivation,inhibition of GSK3βactivity or expression results in EMT, indicating that GSK3P play important role in tumorigenesis and tumor invasion. P38MAPK is one important member of mitogen-activated protein kinase(MAPK) family mediates cell migration,tumor invasion and metastasis. GSK3βactivity is regulated by site-specific phosphorylation. phosphorylation at Ser9 inhibits GSK3βactivity. There is relationship between GSK3βand p38MAPK. In Mouse F9 teratocarcinoma, p38MAPK regulates canonical Wnt signaling by induced GSK3βSer9 phosphorylation inhibiting GSK3βactivity. In the brain and thymocytes, p38MAPK inactivates GSK3βby direct phosphorylation at its C terminus, and this inactivation can lead to an accumulation ofβ-catenin.GSK3βmay regulated by p38MAPK.The tests about GSK3βin vitro breast cancer cell line were relatively many, inhibiting GSK3P activity inhibit breast cancer cell apoptosis, activating Wnt signal pathyway, indicating inactivation GSK3βcontribute to cellular malignant transformation and tumor development., but the studies about GSK3βin human breast cancer tissue were few. p38MAPK may phosphorylation GSK3βinto inactive phosphorylated GSK3β-ser9. In the present study, p-GSK3β,p-p38 and GSK3βexpressions were examined in breast carcinoma tissues and compared with the clinicopathological parameters of the tumors, Our study would describe the significance of GSK3βin breast cancer.Material and Methods 1. Samples(1) A total of 80 paraffin-embedded breast cancer tissues were obtained from the surgical resection at the pathological department of the First Affiliated Hospital of China Medical University between the year 2005to 2008. According to the World Health Organization breast carcinoma histological classification criteria(2003),All specimens were invasive ductal carcinomas.50 fresh breast tissues(30 IDC and 20 breast fibroadenoma)were obtained from the First Affiliated Hospital of China Medical University.(2) Rabbit anti-p-GSK3β-ser9 (sc-11757-R)polyclonal antibody was purchased from Santa Cruz company, mouse anti-p-p38(#9216) monoclonal antibody was purchased from Cell Signaling company, Rabbit anti-GSK3β(BS1402) polyclonal antibody was purchased from Bioworld Technology company. S-P ultrasensitive immunohistochemical kit, DAB kit and Matrigel invasion chambers were purchased from Zhong Shan Inc.2. Methods(1) Immunohistochemistry (S-P) IHC was performed according to the indirect streptavidin-biotin-hyperoxidase method. 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.(2)Western Blot 3mm3 tissues were lysed, grinded, centrifuged, electrophoresed and electrophoretic transferred. The transferred samples were incubated with the first antibody (p-p381:200, p-GSK3β1:300) and corresponging secondary antibody.blots were incubated in enhanced chemiluminescence kits and exposed to photographicfilm.p-GSK3βand p-p38 protein contents were calculated by densitometry.3. Statistical analysisAll statistical calculations were carried out using the SPSS 11.5 statistical software. The results were compared using chi-squared test, t-test and Pearson test. P<0.05 was considered statistically significant.Results1. Relationship between p-p38,p-GSK3β,GSK3βprotein expression and clinical pathological characteristics Immunohistochemical results showed that p-p38 protein was observed in nucleus of breast cancer cells and its positive rate is 60%(48/80).p-GSK3βprotein was in cytoplasm of breast cancer cells and its positive rate is 67.5%(54/80). p-GSK3βand p-p38 expressions were significant correlation with lymph node status and clinical stage (P<0.05). But were not significantly related to patients age and tumor size (P>0.05). p-GSK3P protein had relations with histological grades. Loss/or reduced level of cytoplasmic GSK3βexpression was observed in 45/80 (56%) breast carcinomas,26/80 (32.5%) of breast carcinomas showed nuclear accumulation of GSK3β, Nuclear GSK3βexpression was associated with tumor stage and ER.2. Correlation between p-p38 and p-GSK3βprotein expression in breast cancer tissues36 cases p-GSK3βprotein expressed positively in 48 cases of breast cancers which were positively expressed of p-p38 protein, and 14 cases were negatively expressed of p-GSK3βprotein in 32 cases of breast cancers with p-p38 negative expression.Statistical analysis suggested a positive relationship between expression of p-p38 and p-GSK3β(r=0.316, P＜0.05).DiscussionGSK3βrepress the Wnt/β-catenin pathway and regulate the balance between cellular proliferation and apoptosis, play important role in tumorigenesis. In vitro breast cancer cell line, inhibiting GSK3βactivity inhibit breast cancer cell apoptosis. Marganit Farago found expression kinase-inactive GSK3p in a dominant-negative fashion promoting Wnt Signaling and breast cancer development. Inhibition of GSK3P activity results in EMT. So its inactivation may interfere with tumor development. The tests about GSK3βin vitro breast cancer cell line were relatively many, but the studies about GSK3βin human breast cancer tissues were few. phosphorylated GSK3βexpression was examined in breast carcinoma tissues and we found p-GSK3βwas significantly higher expression in breast cancer tissues, this result was similar with expression of p-GSK3βin liver cancer and lung cancer, its expression was significant correlation with clinical stage of breast cancer, indicating p-GSK3βpromote to breast cancer development. Western blot analysis showed there was significantly increase in expression of p-GSK3βprotein in breast cancer tissues compared with the breast fibroadenoma tissues. We observed loss expression of GSK3βprotein in breast cancer tissues, suggesting that tumor development require repression of GSK3β.Recently, some research had showed GSK3βand P38MAPK have certain relationship, In Mouse F9 teratocarcinoma, p38MAPK induced GSK3βSer9 phosphorylation inhibiting GSK3βactivity, contributes to accumulation ofβ-catenin. p-p38 may as upstream kinase regulates p-GSK3β.expression, In our research,we found there was a positive correlation between p-p38 and p-GSK3βprotein expression, p-p38 may inactive GSK3βcontribute to tumor development.p38MAPK may as upstream kinase regulates GSK3βexpression, upregulated p-GSK3βexpression, further regulated GSK3βrelated oncogenic genes results in breast cancer malignant development. p38MAPK signaling pathway inhibit GSK3βexpression. Our study suggests that p38MAPK signaling pathway may be potential therapeutic target for breast cancer treatment.Conclusions1. The over-expression of p-GSK3βand p-p38 protein were correlated with clinical TNM staging and lymphatic metastasis in IDC, p-GSK3βprotein had relations with histological grades. Nuclear GSK3βexpression was associated with tumor stage and ER.2. There was a positive relationship between expression of p-GSK3βand p-p38 protein.