| INTRODUCTIONProapoptotic protein BAD ( Bcl-xl/Bcl-2-Associated Death promoter) belongs to the Bcl-2 family, whose activated form, namely, dephosphorylated BAD, blocks anitapoptotic protein Bcl-xl, thus cell apoptosis takes place. On the other hand, as the meeting of multiple signal pathways, BAD is phosphoryla-ted by many kinases such as those induced by cell growth factor. Antiapoptotic protein ERK ( Extracellular signal-Regulated Kinase) is the one of the upstream kinases of BAD and studied extensively in breast cancer. The activity of ERK is regulated by phosphorylation, too. Nevertheless, the activity of phos-pho-ERK can be elevated by 100-1000 times. p-ERK phosphorylates/inactivates BAD at serinell2 site. Phosphorylated BAD is sequestered away from its site of action in the mitochondria by binding to cytosolic 14-3-3 protein, thus the antiapoptotic effect of BAD is inhibited. It has been reported that phosphorylation of BAD serll2 by ERK is essential for inhibition of the proapoptotic effect of BAD.We have examined the expression of BAD, p-BAD and p-ERK protein in breast normal tissues, intraductal hyperplasia tissues and invasive ductal carcinomas by methods of immunohistochemistry and western blot analysis. The objectives of our study were to investigate the significance of BAD, p-BAD and p-ERK protein in the breast cancer progression.MATERIAL AND METHODS1. Tissue samplesA total of 131 archived formalin fixed, paraffin-embedded tissues of breast obtained from surgical resection at the First Affiliated Hospital of China Medical University between the year 2001 to 2003 were studied. According to the World Health Organization breast carcinoma histological classification criteria (2003) , all cases were reevaluated for grade and histological type, including 9 normal breast tissues, 15 breast usual intraductal hyperplasis tissues, 10 mild-middle a-typical hyperplasia, 20 severe atypical hyperplasis and intraductal carcinoma in situ and 77 invasive ductal carcinomas. 77 invasive ductal carcinoma samples were scored according to the Scarff-Bloom-Richardson criteria.27 breast invasive ductal carcinoma samples were received fresh allowing one part to be fixed in formalin and one part to be snap-frozen in liquid N2 for later western blot analysis. The part for western blot analysis included tumor are-a and surrouding apparently normal tissues.2. Methods1) Immunohistochemistry for BAD, p-BAD and p-ERK proteinIHC was performed according to the indirect streptavidin-biotin-hyperoxi-dase method, as manufacture protocol. Controls: Human hepatocellular carcinoma with intense staining for BAD, p-BAD and p-ERK were as positive controls. Primary antibodies were omitted as negative controls. Evaluation: The cells with brown-colored cytoplasmic staining were considered BAD and p-BAD positive cells and nuclear staining were considered p-ERK positive. >20% of the cells stained were considered proteins expression positively.2) Western blotting for BAD, p-BAD and p-ERK proteinWestern blot analysis was made as previously described with some modification. The transferred samples were incubated with the primary antibody (dilution; BAD1:1000 p-BADl: 500 p-ERK: 1: 1000) overnight at 4℃. β-Actin levels were shown as protein loading control. Changes of protein content in invasive ductal carcinoma compared with normal tissue were calculated by densitom-etry.3. Statistical analysisThe result of immunohistochemistry was compared using chi-squared test. The result of western blotting was compared using two-tailed unpaired t-test and Pearson test. P <0.05 was considered statistically significant. All statistical calculations were carried out using the SPSS 11.5 statistical software.RESULTS1. The expression of BAD, p-BAD and p-ERK protein in groups of breast tissuesThere was a statistically significant difference of BAD, p-BAD and p-ERK protein between the usual ductal hyperplasis and severe ductal atypical hyperpla-sia and intraductal carcinoma in situ (P = 0.022 0.034 and 0.011). And there was statistically significant difference of p-BAD protein expression positive rate between the severe ductal atypical hyperplasia and intraductal carcinoma and invasive ductal carcinomas ( P <0.001).2. The relationship between the expression of BAD, p-BAD and p-ERK protein in breast cancer and the clinical pathological dataThere was no relationship between the three protein expression and patients'age, clinical stage and tumor size. However, p-ERK protein expression was related to metastasis of armpit lymph node (P =0.041). Three proteins had relations with histological grade (P = 0. 026 0. 039 and 0. 026, respectively) , and the low the histological grade was, the high the positive rates of the three protein expression were (the positive rates of BAD in the three histological grades were 80% , 97.2% and 100% ; p-BAD were 80% , 91.7% and 100% ; p-ERK were 80% , 97.2% and 100% ).3. Western blot analysis of BAD p-BAD and p-ERK protein content in breast invasive ductal carcinomaThe results showed that detectable expression of BAD in the adjacent normal tissue was observed while p-BAD and p-ERK did not. There was a significant increase of the three proteins expression in breast cancer tissues comparedwith the adjacent normal tissues (P <0.001, respectively) , which agreed with the immunohistochemical results. There was a positive correlation between p-BAD and p-ERK protein content.DISCUSSIONPhosphorylation event of BAD, as the gatekeepers of conversion of function , let BAD regulate the status of cell survival rapidly responding to the extracellular environment. It is the center status of BAD between various growth factor signal pathways and mitochondrial apoptotic pathway that let it might be the promising target of biological therapy.Our studies showed that BAD expressed in normal tissue and the positive rates of protein expression were rising in intraductal hyperplasia and invasive ductal hyperplasia. The expression of p-BAD was 0 (0/9) in normal tissues but was markedly enhanced in invasive ductal carcinoma (92. 9% ). As the upstream kinase, the change tendency of p-ERK resembled p-BAD and the results of western blotting showed there was positive correlation of protein content between p-BAD and p-ERK (r = 0.721 ,P =0.032) in cancer tissues. The above-mentioned results raised the possibility that in normal tissues, BAD protein comprised only dephosphorylated form and exerted antiproliferative and apoptosis-inducing activities. However, the expression level of BAD protein was rising in the development of breast cancer and since the activation of upstream kinases in growth factor signal pathways such as p-ERK, antiapoptotic protein p-BAD expression was enhanced, too. Maybe we can make BAD dephosphorylated by blocking kinases such as p-ERK, thus increase the apoptotic ratio of tumor cells and provide new opportunities for the treatment of tumors. On the other hand, the total of BAD protein was gradually increased in the development of breast cancer, which suggested there were other factors regulate BAD in the level of transcription or translation. In further study, we will launch on finding what the factor regulating BAD gene is.There was a statistically significant difference of BAD, p-BAD and p-ERK protein between the usual ductal hyperplasis and severe ductal atypical hyperpla-... |
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