| Breast cancer is the highest worldwide incidence of malignant tumors of women in recent years, and its incidence is rising every year. According to statistics, about 1.4 million women worldwide develop breast cancer each year,0.4 million women died of it. In China, the incidence of breast cancer of women is the first in developed cities as Beijing and Shanghai. The original single-treatment model of breast cancer is replaced by the integrated treatment model composed of a set of surgery, chemotherapy, radiotherapy, endocrine therapy and targeted therapy, so, if we can accurately predict the prognosis of patients with breast cancer, guided adjuvant-targeted treatment is to promote the therapeutic effectiveness.It is known that tumor cells escaping immune cell recognition is associated with glucose-based anomaly, and then spread in the body. Tumor-associated carbohydrate-binding protein Galectin-3 is a galactose-binding protein, widely expressed in normal tissue and tumor tissue, involved in various physiological and pathological processes. Galectin-3 N-terminal domain of 12 amino acids, including serine phosphorylation sites, can regulate the cell targeting; the class a-collagen sequence of 110 amino acids, can be used as a substrate for matrix metalloproteinase; its carboxyl terminal domain consists of 130 amino acids, including carbohydrate recognition domain (CRD),whereβ-galactose residues is on a special affinity.Galectin-3 plays an important role in mediating cell growth and differentiation, immune, cell adhesion, inflammation, apoptosis, vascular invasion, and many other areas. Recently the expression of Galectin-3 has been found in many malignant tumors and Galectin-3 expression increased along with tumor progression. Shalom et al. found that human breast cancer cells was inhibited by Galectin-3 through the transfection of antisense oligonucleotides or antisense cDNA into cells. Miyaaki et al. found that the expression of Galectin-3 was very high in gastric cancer cells with peritoneal metastasis, but that was very low in the absence of peritoneal metastasis of gastric cancer cell; Jonkman et al. found the expression of Galectin-3 in the head and neck adenoid cystic carcinoma was significantly correlated local metastasis and distant metastasis.It is not completely known that Galectin-3 regulates tumor cell growth through interaction with specific signaling molecules. It is found that high expression of Galectin-3 activate PI3K/AKT signal transduction pathway, which promotes cell movement and then resist to TRAIL-induced apoptosis. The two may also be coordinated in Ras activation in different ways such as signaling pathways.Studies showed that the PI3K/AKT signal transduction regulation imbalance plays an important role in the pathogenesis of a variety of tumors, and research about phosphorylation of serine/threonine protein kinase (p-AKT) in breast cancer is gained in attention, but Galectin-3 and p-AKT expression and correlation of the breast cancer has not been reported. In this study, immunohistochemistry was performed to detect Galectin-3 and p-AKT protein expression in 97 cases of breast invasive ductal carcinoma(IDC), and to explore the significance of their expression in breast invasive ductal carcinoma. Materials and Methods:The samples had been collected from the pathology department of the Second Affiliated Hospital College of Medical Science Zhejiang University. Specimens of invasive carcinoma and mammary adenosis tissue were obtained from excision biopsy procedures or mastectomies. All specimens were fixed in neutral 4%buffered formaldehyde. None of the patients with invasive breast cancer had received any preoperative therapy. Mammary adenosis tissue(from 20 patients) and invasive ductal carcinoma(from 97 patients) were examined by two pathologists. Grading of invasive cancer was done according to the procedure of Elston and Ellis. The two respective grading observers were blinded in scoring all slides for Galectin-3 and p-AKT staining.Immunohistochemistry:The expression of Galectin-3 and p-AKT was detected immunohistochemically in paraffin-embedded specimens fixed in 4% buffered formalin. Histological slides,4μm in thickness, were deparaffined in xylene. Slides were heated in 0.01 M citrate buffer for 15 min in a microwave oven, then 3% hydrogen peroxide for 10 minutes to eliminate endogenous peroxidase. For immunohistochemical detection of Galectin-3 and p-AKT,specimens were incubated 1 hour at 37℃with a monoclonal antibody respectively(Galectin-3 rabbit anti-human polyclonal antibody was purchased from Beijing Zhong Shan biotechnology company,in a dilution of 1:100, p-AKT (THr308) rabbit anti-human polyclonal antibody was purchased from Santa Cruz company, in a dilution of 1:200). Visualization of bound antibodies was performed by using Envision two-step method. As chromogen,diaminobenzidine was used in all stainings.Quantification:For the staining, only cells with completely and darkly stained were regarded positive. Galectin-3 mainly expressed in the cytoplasm, a small amount in nuclei, while p-AKT mainly expressed in cellular nuclei. Two observers estimated the result using IP (Intensity X Percentages) score method, composing of staining intensity and positive cells number:(1) The staining intensity score:0 represented no dyed or colored with the same non-specific background,1 represented light yellow and brown staining,2 represented darkly brown staining; (2) positive cells score:0 represented no positive cell,1 represented positive cells less than 50%,2 represented positive cells more than 50%. According to the two score multiplication, the positive grades were made.O meant negative,1,2,4 points meant positive (1 point:low expression; 2 points:moderate expression; 4:high expression).Statistical Methods:SPSS 17.0 statistical software package was used and X2 test performed to evaluate whether the levels of Galectin-3 and p-AKT were increased during breast carcinogenesis. P values of less than 0.05 was regarded statistically significant. Correlation of Galectin-3 and p-AKT was analyzed by Spearman correlation.Results:In IDC of breast, positive rates of Galectin-3 and p-AKT were significantly higher than those in mammary adenosis tissues(84.5% vs 25%,77.3% vs 30%,respectively), and all adenosis tissues staining were low expression. In the death group, the positive rate of Galectin-3 was 97.1%(34/35), while it was 77.4%(48/62) in the survival group; The positive rate of p-AKT was 91.4%(32/35) in the death group,while it was 69.4% (43/62) in the survival group.the difference was statistically significant(P<0.05). The positive rates of Galectin-3 and p-AKT were higher in lymph node positive group than that in lymph node negative group (86.7% vs 81.1%,88.3% vs 59.6%, respectively). Galectin-3 and p-AKT expressions were correlated with patients's age, while they were unrelated with histological grade, TNM stages. There was a positive relationship between the expression of Galectin-3 and p-AKT in IDC(r=0.606, P<0.01). Conclusions:1,The level of Galectin-3 and p-AKT protein was significantly increased in breast IDC, suggesting they might play a role during breast carcinogenesis.2,The enhanced expression of Galectin-3 and p-AKT was associated with the lymph node metastasis, suggesting that they might be involved in metastasizing process of breast IDC.3,The level of Galectin-3 and p-AKT protein was significantly increased in the death group than that in the survival group, suggesting they might be associated with poor prognosis of breast IDC.4,There was a positive relationship between the expression of Galectin-3 and p-AKT in IDC, suggesting they could be involved in the occurrence and progress of breast IDC in coordinated ways. |
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