| Background and PurposeBreast cancer is a malignant tumor from the terminal duct lobular or ductal epithelial cells. In the past 50 years, incidence rates showing a slow upward trend, exceeding the incidence of cervical cancer ranked first in female malignant tumor. The incidence of breast cancer increased by 47% in the past 10 years, In China, it shows a trend of getting younger and younger age of patients with breast cancer in Compared with Europe and the United States. In China, survey shows that 40,000 people died from breast cancer each year, which were called "the first killer" in urban women. Currently, the pathogenesis of breast cancer has not yet fully elucidated, which is associated with long-term effects of estrogen, family genetic predisposition, environmental factors and time factors such as high-dose radiation exposure etc.The development of breast cancer is similar to other malignant tumors, a complex multi-stage and multi-step process. An important characteristic of malignant tumor is invasion and metastasis, which is also a complex, multi-step process. It's widely accepted that extracellular matrix and basement membrane degradation is an important step of tumor invasion and metastasis. Many studies have indicated that the degradation of extracellular matrix and basement membrane remodeling is a key aspect of the process of cancer cells invasion and metastasis, which requires the help of protein-degrading enzyme expression and activation. Now known Proteolytic enzymes contain cysteine protease, serine proteases, aspartic proteases and matrix metalloproteinases (MMPs). MMPs are a group of important extracellular matrix degrading enzyme, play an important role in tumor invasion and metastasis, by degradating different components of extracellular matrix. MMP-9, an important member of MMPs family, can degrade the major components of extracellular matrix, such as the type IV collagen, fibronectin and laminin, some studies suggest that which are associated with tumor invasion and metastasis.Tumor invasion is a multi-factors involved, multi-steps process:receptors of tumor cells membrane surface adhere to proteins laminin ofextracellular matrix (ECM); then the release and activation of protease and precursor; the degradation of ECM (mainly collagen IV) by proteolytic enzymes; and the migration of tumor cells through the basement membrane. In the formation of two major components of ECM (basement membrane and interstitial), the basement membrane form a block barrier, which is composed of IV collagen of the basement membrane, so its degradation is a key step of tumor invasion and transfer. Numerous studies have demonstrated the ability of tumor cell invasion is closely related to the capacity induction and production of protease, which is used to degrade its extracellular matrix and basement membrane. Moreover, two types of enzyme systems involved in this process: Plasminogen system and matrix metalloproteinases (MMPs) system. MMP-9 is the most important protease in MMPs. Tissue inhibitor of matrix metalloproteinases (TIMPs) are natural inhibitors of MMPs, the balance of TIMPs and MMPs plays an important role in keeping the steady-state of ECM. As extracellular matrix metalloproteinase inducer (EMMPRIN), CD147 is an immunoglobulin superfamily glycoprotein, which widely present in various tissues and organs of human, plays an important role in the regulation of cell-cell and cell- stroma adhesion as a new adhesion molecule, and involves a variety of physiological processes in the body. Recent studies have reported 108 cases of bladder cancer by immunohistochemistry about the expression of CD147 protein, which showed that the expression of CD147 protein had significant correlation with lymph node metastasis, clinical stage, histological grade, and prognosis of bladder cancer patients. By multivariate analysis, other statistics showed that over-expression of CD147 protein can serve as an independent risk factor for prognosis, so CD147 protein may be as a potential therapeutic target. Some scholars demonstrate CD147 protein can lead to lung cancer cell invasion, proliferation and metastasis by upregulating the classic wnt/beta-catenin signal path in the mouse xenograft model. Some scholars detected CD147 protein, MMP-9 and MMP-2 protein expression by immunohistochemistry in 48 laryngeal cancer patients and 15 patients with laryngitis, it showed high expression of these three proteins in laryngeal cancer patients, and they were associated with clinical stage and lymph node metastasis. Some scholars detected expression of CD147 protein and MMP-9 protein by immunohistochemistry in children non-Hodgkin's lymphoma, which showed that both proteins were associated with clinical stage, tumor size, bone marrow transfer, immune-related phenotype and poor prognosis. And high expressions of these two proteins are associated with poor prognosis..Recent studies have shown that fibroblasts usually produce only a small amount of MMPs, but can produce large amounts of that in the presence of tumor cells. It has been confirmed that the stimulating factor is CD 147, which is adhesion molecule and ECM & metalloproteinase inducer present in a surface of tumor cells. CD 147, which involves in cell-cell, cell-matrix interactions, stimulate the adjacent stromal cells to produce matrix metalloproteinase (MMP-9, MMP-2), and degrades the ECM, available to the tumor metastasis.Annexins protein is a class of calcium-binding protein family, which is regulated by calcium, can bind to membrane phospholipids negatively, involved in a series of calcium-related biological activities. Since calcium ions involved in a wide range of human life activities, the biological role of Annexins have also been more attention. Recent studies showed that AnnexinⅡprotein has a variety of functions in the cytoplasm and the nucleus, and its abnormal expression are closely related to tumor development. Annexinll protein plays an important role in the membrane domain of the establishment or stability, membrane transport, ion channel formation, DNA synthesis and cell proliferation etc. and Annexinll extracellular protein as a receptor, is also involved in dissolution of the fiber protein, cells adhesion, ligand mediated signal transduction and viral infections etc. abnormal expression of Annexinll protein is involved in human diseases.For expression of CD 147, MMP-9 and Annexinll in breast cancer and breast fibroadenoma are rarely reported at home and broad. In this paper, the expression and their interrelation of CD 147, MMP-9 and AnnexinⅡprotein in human breast fibroadenoma and invasive ductal carcinoma were detected by immunohistochemical method. To further clarify the significance and the interrelation of CD147, MMP-9 and AnnexinⅡprotein in breast cancer; to explore the occurrence, development and metastasis mechanisms of breast cancer and provide a theoretical basis for more effective prevention and treatment of breast cancer.Materials and methods 1. Experimental materialsarchived paraffin blocks of 60 cases of breast invasive ductal carcinoma and 60 cases of breast fibroadenoma tissue from the Third Affiliated Hospital of Zhengzhou University, Department of Pathology. All is surgical specimens for the January 2008-January 2010, from female patients, age 26-70 years, and mean age was 45.9 years. No preoperative chemotherapy, immune and radiotherapy. All specimens were confirmed by histopathological examination for infiltrating ductal carcinoma. All surgical resection specimens were fixed in 4% neutral formaldehyde, embedded in paraffin after the consecutive sections for histopathological and immunohistochemical examination. HE biopsies of breast invasive ductal carcinoma were scored and classified according to the grading standard made by WHO in 2003.Rating criteria:(1) luminal formation:accounts for the majority (>75%) for 1 point, moderate (10-75%) for 2 points and no less (<10%) 3 points; (2)nuclear polymorphism:small and rules consistent cells for 1 point, medium size and pleomorphism for 2 points, obviously abnormalfor 3 points, (3) for every 10 at high magnification mitotic figures count:0-5 for 1 point,6-10 for 2 points,> 11 for 3 points. Sum of the above three by the histological grade,3-5 score in gradeⅠ,6-7 score in gradeⅡ,8-9 score in gradeⅢ.2. reagentsGoat anti-human CD147 polyclonal antibody (sc-25531) was purchased from Santa Cruz company; MMP9 polyclonal antibody was purchased from Beijing Boao Sen Biotechnology Co., Ltd.; AnnexinⅡpolyclonal antibody (sc-9061), SP sensitivity kit (SP-9001) and DAB chromogenic enzyme substrate kit (DABZLI-9032) were purchased from Beijing Zhongshan Golden Bridge Biotechnology Development Company.3.methods Immunohistochemistry (S-P) was used to detect the expression of CD 147, MMP-9 protein and AnnexinⅡin 60 cases of breast fibroadenoma and 60 cases of breast invasive ductal carcinoma tissue.1).Step:The Streptomyces element-peroxidase (SP). PBS instead of primary antibody as negative control was negative, known positive result as a positive control was positive.2).Criteria:The cell cytoplasm appeared brown particles,which shows AnnexinⅡprotein and MMP-9 positive; Cell membrane and cytoplasmic particles appear brown, which shows CD 147 positive. The judge:to evaluate in a double-blind manner according to Fromowitz the dyeing standards:CD 147 expression positive means it appears the brown particles in the tumor cell membrane and the cytoplasm; MMP-9 expression positive means it appeared brown particles in tumor cell cytoplasm. Color intensity is divided into:colorless (-) as 0, light yellow (+) for 1 point, yellow (++) for 2 points, brown (+++) for 3 points. Percentage of positive cells:< 30%for 0,31%-70% for 1 point,≥71% for 3 points. The evaluation by the sum of the two above:0 for negative, 1～2 scores for weakly positive (+),3～4 scores for moderately positive (++),5～6 scores for strongly positive (+++).4.Statistical analysisStatistical software SPSS for windows 13.0, theχ2 test and Spearman correlation analysis were applied, testing levelα=0.05.Results1. CD 147, MMP-9 and AnnexinⅡprotein showed a negative expression or only weakly positive in breast fibroadenoma tissues and strong expression in breast cancer tissues. The contrasts of three proteins expression in breast fibroadenoma and invasive ductal carcinoma tissue were statistically significant (P=0.000). Invasive ductal carcinomas were significantly higher than that of fibroadenoma tissue.2. CD147 protein expression rate was 91.67%(55/60). CD147 protein expression in breast cancer tissue were not related to the patient's age(χ2=0.024,df=1, P=0.876>0.05), family history (χ2=0.000,df=1, P=1.000>0.05), tumor size (χ2=1.185,df=2,P=0.553>0.05) and clinical stage(χ2=2.937,df=1,P=0.086>0.05), no statistically significant. But the CD147 protein in breast cancer tissue and tumor histological grade, i.e. the different histological grading rate of CD147 protein expression was statistically significant difference between the (χ2= 8.601, P=0.006<0.01), and further histological grade wereⅠto do with theⅡ-class level, I level and CD147 protein expression levelⅢpositive rate comparisons of results are as follows:histological gradeⅠandⅡgrade level (χ2= 4.773, P= 0.029<0.05/ 2=0.025), I-level and gradeⅢ(P=0.018<0.05/2=0.025) in patients with positive expression of CD147 between the rate differences were statistically significant lower histological grade, CD147 protein was the higher the rate expression. In addition, the rate of CD147 protein expression was statistically significant difference between lymph node metastasis and patients without (χ2= 7.253, df=1,P=0.007<0.05).3. MMP-9 protein in invasive ductal carcinoma positive expression rate was 86.67% (52/60). MMP-9 protein in breast invasive ductal carcinoma with age (χ2=0.341, df=1,P=0.559> 0.05), family history (χ2=0.192, df=1, P=0.661> 0.05), tumor size(χ2=0.574, df=2,P= 0.750> 0.05) and clinical stage (χ2=1.737, df= 1,P=0.188> 0.05) were independent, that is, there was no significant difference between the different ages, with or without family history, tumor size and different patients with clinical stage of the MMP-9 protein expression rate. However, MMP-9 protein in breast cancer tissue and tumor histological grade, i.e. MMP-9 protein expression rate was statistically significant difference between the different histological gradings (χ2=6.438, P=0.026<0.05), and further the comparisons of the positive rate of MMP-9 protein expression histological gradeⅠandⅡ,ⅠandⅢshowed there was no significant difference for MMP-9 protein level between the histological gradeⅠandⅡ(χ2=1.116, df=1, P=0.291> 0.05/ 2=0.025), I grade and histological grade III were significant differences (P= 0.018<0.05/2=0.025), and lower histological grade, MMP-9 protein expression rate. In addition, patients with lymph node metastasis positive for MMP-9 protein expression were statistically significant differences in the rate (χ2 =8.679, df=1, P=0.003<0.01).4. The positive expression rate of AnnexinⅡprotein in breast invasive ductal carcinoma was 90.00%(54/60). AnnexinⅡprotein in breast invasive ductal carcinoma tissues with age (χ2= 0.365, df=1, P=0.546> 0.05), family history (χ2=0.000, df=1, P=1.000>0.05), tumor size (χ2=1.069, df=2, P= 0.586> 0.05) and clinical stage(χ2=1.350, df=1,P=0.245>0.05) were independent, that is, the expression of different positive rate of Annexinll was no significant difference between the different ages, with or without family history, tumor size and clinical stage. However, AnnexinⅡprotein in breast cancer tissue and tumor histological grade, i.e. the different histological grade Annexinll protein expression rate was statistically significant difference between the (χ2= 7.356, P = 0.017<0.05), and furtherⅠandⅡ, the comparison of positive rate of AnnexinⅡprotein showed the positive rate of expression of AnnexinⅡwere not statistically significant differences between the histological gradeⅠandⅡ(χ2= 3.308, df=1, P=0.081> 0.05/2= 0.025), were significant differences between gradeⅠandⅢ(P=0.018<0.05/2=0.025), and the lower histological grade, the higher positive expression rate of AnnexinⅡ. Another the difference of AnnexinⅡ positive rate of expression was statistically significant between lymph node metastasis and patients without (χ2= 5.192, df=1, P=0.023<0.05).5. CD 147, MMP-9 and AnnexinⅡprotein expression in breast cancer tissues showed positive correlation between two. Application of statistical methods of Spearman correlation test, it showed CD 147 was correlated significantly with MMP-9, r value for 0.769, P value for 0.000; CD 147 was also significantly correlated AnnexinⅡ, r values for 0.905, P value for 0.000; In addition, MMP-9 was also significantly correlated AnnexinⅡ, r value for 0.850, P value for 0.000.Conclusions1. CD147, MMP-9 and AnnexinⅡprotein have no expression or only a slight expression in breast fibroadenoma tissue; but strong expression in breast cancer tissues, it suggests its high expression is related to the development of breast invasive ductal cancer.2. CD 147, MMP-9 and AnnexinⅡprotein are related to histological grade of breast cancer and axillary metastasis, which suggests that their expression promotes breast cancer progression and lymph node metastasis3. CD 147, MMP-9 and AnnexinⅡprotein expression are positive correlation between two in breast carcinoma, which suggests they play a synergistic role in the development of breast cancer.4. The expression rate of CD 147, MMP-9 and AnnexinⅡprotein expression in invasive ductal breast cancer tissues was significantly higher, and relate to histological grade and lymph node metastasis, which suggests that these three proteins may play an important role in the development and invasion and transfer process of breast cancer. |
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