Expression And Significance Of P-BAD,p-AKT Protein In The Breast Cancer Progression

INTRODUCTIONProapoptotic protein BAD ( Bcl - xl/Bcl - 2 - Associated Death promoter) belongs to the Bcl - 2 family, whose mechanism interacts with prosurvival protein Bcl - 2 and/or Bcl - xl to suppress their function of survival, thus cell ap-optosis takes place. On the other hand, as the meeting of multiple signal pathways, BAD is phosphorylated by many kinases such as those induced by cell growth factors. Antiapoptotic protein AKT/PKB (protein kinase B) is the one of the up - stream kinases of BAD and studied extensively in breast cancer. The activity of AKT is regulated by phosphorylation, too. p - AKT phosphorylates/ inactivates BAD at serinel36 site. Phosphorylated BAD is sequestered away from its site of action in the mitochondria by binding to cytosolic 14 - 3 - 3 protein, thus the proapoptotic effect of BAD is inhibited. It has been reported that phosphorylation of BAD s136 by AKT is essential for inhibition of the proapoptotic effect of BAD.We have examined the expression of p - BAD and p - AKT 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 p - BAD and p - AKT protein in the breast cancer progression.MATERIAL AND METHODS1. Tissue samplesA total of 161 archived formalin fixed, paraffin - embedded tissues ofbreast 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 10 normal breast tissues, 13 breast usual intraductal hyperplasis tissues, 23 mild - middle atypical hyperplasia, 20 severe atypical hyperplasis and intraductal carcinoma in situ and 95 invasive ductal carcinomas. 95 invasive ductal carcinoma samples were scored according to the new Bloom - Richardson criteria. 30 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 area and surrouding apparently normal tissues. 2. Methods1) Immunohistochemistry for p - BAD and p - AKT proteinIHC was performed according to the indirect streptavidin - biotin - hyperox-idase method, as manufacture protocol. Controls-. Human hepatocellular carcinoma with intense staining for p - BAD and p - AKT were as positive controls. Primary antibodies were omitted as negative controls. Evaluation;The cells with brown — colored cytoplasmic staining were considered p — BAD positive cells, cytoplasmic and nuclear staining were considered p - AKT positive. The degree of positive staining for all antibodies was evaluated by scoring on a scale of 0 -2 for percentage of positive cells and on a scale of 0 - 2 for strength of intensity of staining. The percentage of positive cells was evaluated using the following scale :0, ^10% of the epithelium stain positive;1 + ,10% -50% of the epithelium stain positive;2 + , >50%of the epithelium stain positive;As for the evaluation of strength of intensity of staining, it was evaluated using the following scale;0,no staining of epithelial cells;1 + ,mild staining;2 + ,moderate staining and intense staining. The final total score was generated by multiplying the score for percentage of positive cells and maximum score for an area were 0 and 4, respectively. 1 -2was weakly positive cases, >2was strongly positive cases.2) Western blotting for p - BAD and p - AKT proteinWestern blot analysis was made as previously described with some modifica-tion. The transferred samples were incubated with the primary antibody ( dilution : p - BAD: 1:300 p - AKT: 1:400) overnight at 4°C. p - Actin levels were shown as protein loading control. Changes of protein content in invasive ductal carcinoma compared with normal tissue were calculated by densitometry.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 , Pearson test and Ridit analysis. P < 0. 05 was considered statistically significant. All statistical calculations were carried out using the SPSS11.5 statistical software.RESULTS1. The expression of p - BAD and p - AKT protein in groups of breast tissues;There was a rising tendency in positive rate of protein expression ranging from the usual ductal hyperplasis, mild - middle atypical hyperplasia, severe ductal atypical hyperplasia and intraductal carcinoma in situ to invasive ductal carcinoma. There was a statistically significant difference of p - BAD and p -AKT protein between the usual ductal hyperplasis and severe ductal atypical hyperplasia and intraductal carcinoma in situ (P <0. 001). 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) . With the progression of hyperplasis and cancer, the expression of p - BAD and p - AKT were also obvious. There was a statistically significant difference of p - BAD and p - AKT protein between breast cancer group anql breast usual ductal hyperplasis, between invasive ductal carcinomas and severe ductal atypical hyperplasia and intraductal carcinoma ( P <0. 05 ) . the expression of p - BAD and p - AKT in severe ductal atypical hyperplasia and intraductal carcinoma were higher than mild - middle atypical hyperplasia. The expression of p - BAD and p - AKT (-----? +++ ) and histologicalgrade were positive correlation by Spearman test.2. The relationship between the expression of p - BAD and p - AKT protein in breast cancer and the clinical pathological dataThere was no relationship between the two protein expression and patients' age ^clinical stage and tumor size. However, p - AKT protein expression was related to metastasis of armpit lymph node (P =0.003). Two proteins had relations with histological grade (P=0.019^0.013), and the low the histological grade was, the high the positive rates of the two protein expression were (the positive rates of p - BAD in the three histological grades were were 80% ,91. 1% and 100%;p -ERK were 80% , 95.6% and 100% ).3. Western blot analysis of p - BAD and p - AKT protein content in breast invasive ductal carcinomaThe results showed that p - BAD and p - AKT were not expressed in the adjacent normal tissue. There was a significant increase of the two proteins expression in breast cancer tissues compared with the adjacent normal tissues (P = 0.001^0.003) , which agreed with the immunohistochemical results. There was a positive correlation between p - BAD and p - AKT protein content ( P = 0. 025).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. BAD was also usual target of lots of stimulations. These extra stimulation includes growth factor>,cell factor^Ca2+ et al. As we all known that many survival signaling pathway act on BAD, for instance PI3K/AKTA MAPK/ERK signaling pathways et al. Through extracellular stimulation, the residue of BAD including serine 112Nserinel36Nserinel55 were phosphorylated, therefore, the destiny of cells was changed. 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 p - BAD was not expressed in normal tissues but was enhanced in intraductal hyperplasis and invasive ductal carcinoma, in latterpositive rate markedly enhanced (94.70% ). As the upstream kinase,there was a statistically significant difference of p - BAD and p - AKT protein between the usual ductal hyperplasis and severe ductal atypical hyperplasia and intraductal carcinoma in situ, which showed these proteins might play roles in monitoring early malignant transformation of breast lesions. And there was statistically significant difference of p - BAD and p - AKT protein expression positive rate between the severe ductal atypical hyperplasia and intraductal carcinoma and invasive ductal carcinomas, which suggested p - BAD and p - AKT protein might serve as a marker used as a diagnostic indicator of the differential diagnosis.The change tendency of p - AKT resembled p - BAD and the results of western blotting showed that p - BAD and p - AKT did not expression in the adjacent normal tissue. There was a significant increase of the two proteins expression in breast cancer tissues, compared with the adjacent normal tissues (P =0. 001 J). 003 ). There was a positive correlation between p - BAD and p - AKT protein content(r = 0. 291 NP = 0. 025 ) . According to above - mentioned results, there was rising tendency for expression of p - BAD and p - AKT in breast cancer progression. Thus, it suggests PI3K/AKT/BAD signaling pathway participates breast cancer progression.To further assess the relationship of p - BAD and p - AKT protein and malignant potency of breast cancer, we explored the difference of two proteins expression between different clinical pathological data. Similar results were observed in Khor' study: they reported there was a difference of p - BAD expression between cancer tissue and adjacent normal tissue in human colon cancer, and p - BAD was not related to patients'age and tumor size, p - AKT was related to metastasis, which had been reported in several tumors such as pancreatic cancer and melanoma. The positive rate was rising as the demotion of histologi-cal grade, which suggested prosurvival protein p - BAD and p - AKT were all related to the malignant degree of breast cancer.CONCLUSIONS1. p - BAD and p - AKT protein expression showed a rising tendency inthe development of breast cancer.2. Three proteins had relations with histological grade, and the low the histological grade was, the high the positive rates of the three protein expression were.3. Expression of p - BAD and p - AKT in breast invasive ductal carcinoma showed a positive linear correlation.