Expression Of Bim In Breast Cancer And The Relationship With PI3K/Akt Signaling Cascade

INTRODUCTIONBim is a pro-apoptotic member of the Bcl-2 protein family, which express extensively in normal tissues, including hematopoietic, epithelial, neuronal, and germ cell. PI3K/Akt signal cascade participate a series of physiological activity including apoptosis, hyperplasia, differentiation and metabolism. In present, many researches indicate that the abnormity of signal cascade have closed relationship with the occurrence and progression of tumor. It is confirmed that after the stimulus of different growth factor, PKB/Akt phosphorylated forkhead transcription factor, which changed their localization. Without PI3K/Akt, forkhead located in nuclear, combinated with specifically cis acting element to promote the transcription of apoptosis related gene such as Fas-1, IGBFP1 and Bim. It is reported recently that in Ba/F3 cell, activated Akt can directly phosphorylate the Ser87 of BimEL, which promote its ubiquitin and degradation. Our department's researches found that PI3K-Akt-Bad signal cascade play an important role in the occurrence and progression of breast cancer. Bim belonged to BH3-only subfamily as same as Bad, whether or not having an equal effect in breast cancer have an important significance to instruct the clinic diagnosis and therapy.MATERIAL AND METHODS1. Samples(1) Tissue samples: A total of 101 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 2005 were studied. According to the World Health Organization breast carcinoma histological classification criteria (2003), all cases were reevaluated for grade and histological type, including 15 normal breast tissues, 12 breast usual intraductal hyperplasis tissues, 10 mild-middle atypical hyperplasia, 17 severe atypical hyperplasis and intraductal carcinoma in situ and 47 invasive ductal carcinomas.(2) Cell lines: MCF-7 and MDA-MB-231 were purchased from the Cell Center of Peking Union Medical College.2. ReagentsThe first antibodies were rabbit polyclonal antibody to Bim and p-Akt 1/2/3 (Ser473) which purchased from Neomarkers and Santa Cruz. LY294002, a special PI3K/Akt inhibitor, purchased from cell signaling.3. Methods(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 tissue sections were used as positive controls. The percentage of positive cells was evaluated using the following scale: 0, no epithelium stain positive; 1+,＜30% of the epithelium stain positive; 2+, 30-70% of the epithelium stain positive; +3,＞70% 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+, less yellow staining; 2+, moderate yellow staining; +3, strong yellow staining. The final total score was generated by multiplying the score for average of positive cells and maximum score for an area were 0 and 4, respectively.≥2+ was positive cases.(2) Western blot: Protein concentration was measured by Bradford method. Cell lysates were electrophoresed in 12% polyacrylamide SDS gel and transferred onto polyvinylidene difluride membranes, Protein bands were blocked and incubated with the first (dilution: Bim: 1:200, p-Akt: 1:300) 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 11.5 statistical software. P＜0.05 was considered statistically significant.RESULTS1. The expression of Bim protein in groups of breast tissues:There was a decreasing tendency in positive rate of protein expression ranging from the usual ductal hyperplasia, mild-middle atypical hyperplasia, severe ductal atypical hyperplasia and intraductal carcinoma in situ to invasive ductal carcinoma. There was a statistically significant difference of Bim protein between the usual ductal hyperplasia and severe ductal atypical hyperplasia and intraductal carcinoma in situ (P=0.026). And there was statistically significant difference of Bim protein expression positive rate between the severe ductal atypical hyperplasia and intraductal carcinoma and invasive ductal carcinomas (P=0.014).2. The relationship between the expression of Bim protein in breast cancer and the clinical pathological data:The expression of Bim had relation with histological grade (P=0.033), and the low the histological grade was, the low the positive rate of the protein expression was.3. The expression of Bim in two breast cancer cell lines:Western Blotting showed that the expression of Bim in MCF-7 which having low potential malignancy was higher than MDA-MB-231 which having prominent potential malignancy.4. The expression of p-Akt and Bim protein after PI3K/Akt signal cascade inhibited by LY294002:Different density of LY294002 (0,10,25,50μmol/L) was added in culture fluid of MCF-7, we showed that the expression of p-Akt in decreasing, but Bim was increasing.DISCUSSIONBcl-2 protein family, the critical element to accommodate apoptosis, was the principal regulatory factor for chondriosome and the releasing of apoptotic factor. Bim is a pro-apoptotic member of the Bcl-2 protein family, which express extensively in normal tissues, including hematopoietic, epithelial, neuronal, and germ cell. The pro-apoptotic activity of Bim protein received strict control including transcriptional level regulation and post-translational modification. Absence of Bim could induce many tumors which always came from hematopoietic systerm, it certificated forcefully that Bim was a tumor inhibiting factor. Bim is a pro-apoptotic member of the Bcl-2 protein family, which express extensively in normal tissues, including hematopoietic, epithelial, neuronal, and germ cell.Our studies found that the positive rate of Bim protein expression showed a decreasing tendency ranging from the intraductal hyperplasia to invasive ductal carcinoma and the expression of Bim in MCF-7 having low potential malignancy was higher than MDA-MB-231 having prominent potential malignancy, which suggested that the change of Bim expression might have some effect in the progression of breast cancer. Our department's researches found that with the progression of hyperplasia and cancer, the expression of p-Akt was also obvious. In order to identify the change of Bim expression whether concerned with the PI3K/Akt signaling cascade, we reseached two breast cancer lines in vitro. We found that in breast cancer cell MCF-7, LY294002 blocked the PBK/Akt signaling cascade, induced the expression of p-Akt and increased the expression of Bim, which suggested that the change of Bim expression might controlled by PI3K/Akt signaling cascade in breast cancer.Our studies showed that the change of Bim expression might concerned with the occurrence and progression of breast cancer and suggested that PI3K/Akt signaling cascade have an important effect in breast cancer. Currently, an inhibitor of PBK/Akt signaling cascade pathway have represented a novel target for cancer intervention strategies. Our studies suggested that PI3K/Akt signaling cascade pathway might be potential therapeutic target for breast cancer treatment.CONCLUSION1. The expression of Bim protein showed a decreasing tendency in the normal breast tissues, ductal intraepithelial neoplasia and breast cancer.2. Bim protein had relation with histological grade, and the low the histological grade was, the low the positive rate of the Bim protein expression was.3. In breast cancer cell MCF-7, LY294002 blocked the PI3K/Akt signaling cascade, induced the expression of p-Akt and increased the expression of Bim.