[Abstract] Objective To investigate the HIF  1α, Glut  1 expression in breast cancer and its relationship with microvascular density. Method for immunohistochemical assay of 20 paracancerous organizations in 60 cases of breast cancer, HIF  1α, Glut  1 expression in breast cancer vascular endothelial cells labeled with CD34 antibody, microvessel density (MVD). HIF  1α intensity of Glut  1 in breast cancer positive expression were 68.3%, 63.3%, were higher, the difference was statistically significant (P <0.01) the both expression was positively correlated (r = 0.461, P <0.001). HIF  1α, Glut  1-positive breast cancer MVD was higher than the negative, the difference was statistically significant (P <0.01), HIF  1α, Glut-1 were positive MVD (P <0.01), HIF  1α , Glut  1, MVD with clinical stage, lymph node metastasis (P <0.01). Conclusion breast cancer tissue HIF  1α, Glut  1 were overexpressed, both in its occurrence, may have a synergistic effect in the development process, and may have an important role in tumor angiogenesis.

Key words breast cancer; by hypoxia-inducible factor-1α; glucose transporter proteins  1; MVD; immunohistochemistry

  Relationship of Expression of HIF  1α and Glut  1 with Angiogenesis in Breast Carcinoma

    MA Zhao  sheng, ZHOU Shi  fu, JIN Lin  fang, CAI Feng  lin, SHI Wei  feng

    The Fourth Hospital Affiliated of Soochow University, Wuxi 214062, ChinaAbstract: Objective To study the relationship of the expression of HIF  1αand Glut  1 with Angiogenesis in breast carcinoma, and to analyze the correlation of them. Methods HIF  1α and Glut  1 protein expression were detected by immunohistochemical methods in 60 cases of breast carcinoma and 20 cases of tumor adjacent normal breast tissue.CD34 marking vascular endothelialcell was used to measure the microvascular density (MVD). Results The positive expression rates of HIF  1α and Glut1 in breast carcinoma was 68.3% and 63.3%, showing a significantly difference in the expression of HIF  1αand Glut  1 between breast carcinoma and control group (P <0.01), Expression of HIF  1αwas positively correlated with expression of Glut  1 (r = 0.461, P <0.001). MVD in HIF  1α (+) or Glut  1 (+) group was significantly higher than that in HIF  1α (-) or Glut  1 (-) group (P <0.01). The MVD reached the maximum in patients whose HIF  1α and Glut  1 were both positive (P <0.01). The expression of HIF  1α, Glut  1 and MVD were significantly correlated with lymphatic metastasis and surgical  pathologic staging (P < 0.01). Conclusion There have overexpresion of HIF  1α and Glut  1 in breast carcinoma, they may have a synergistic effect in the genesis and development of breast carcinoma, and may play an important role in angiogenesis of breast carcinoma.

    Key words: Breast carcinoma; HIF  1α; Glut  1; MVD; Immunohistochemistry

The rapid proliferation of tumor cells, hypoxia is a common phenomenon, the tumor cells to adapt to in hypoxic microenvironment major mechanisms include improving the energy metabolism of tumor tissue and induce the formation of new blood vessels. Hypoxia-inducible factor  1α (hypoxia inducible factor  1α, HIF  1α) plays a key role. Glucose transporter protein  1 (glucose transporter, Glut  1 was) widespread human tissues, has an important role in the regulation of glucose uptake and participate in the body’s energy metabolism, the study examined breast tissue HIF  1α, Glut  1 and microvascular density (microvasular density, MVD), HIF  1α and intensity of Glut  1 expression and its correlation with angiogenesis, the relationship between clinicopathological factors.

    1 Materials and Methods

    1.1 General Information Select hospital in September 2006 to 2007 menstrual pathologically confirmed 60 cases of modified radical mastectomy for primary breast cancer specimens were without preoperative anti-cancer therapy. All specimens are invasive breast cancer. Axillary lymph node metastasis in 32 cases of positive and negative in 28 cases. Another 20 cases of cancer adjacent tissues as control.

    1.2 Reagents rabbit anti-human HIF  1α polyclonal antibody, mouse anti-human CD34 monoclonal antibody was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., rabbit anti-human polyclonal intensity of Glut-1 antibody was purchased from Fuzhou Maixin Biotechnology Development Co., Ltd..

    1.3 Experimental methods organizations by 10% formalin-fixed, paraffin-embedded, 4 μm thick slices were HE staining and immunohistochemical staining HIF  1α polyclonal rabbit anti-human antibody working concentration of 1:40, CD34 antibody, The intensity of Glut  1 antibody for ready-to-use, using immunohistochemistry Envision two-step, The positive photo by Reagent Company, as a negative control antibody was replaced with PBS.

    1.4 staining results to determine the the high magnification randomly selected five fields, counting 200 cells per field, a total of 000. HIF  1α expression of the positive judgment standard reference Zhong et al [1] reported: (-): no staining or <1% nuclear staining; (+): 1% to 10% of the nuclear chromatin, and (or) weak 50%的细胞核染色,和(或)较强的胞">cytoplasmic staining; (+ +): 11% to 50% nuclear staining, and (or) a clear cytoplasmic staining; (+ + +):> 50% nuclear staining, and (or) a strong intracellular pulp dyeing. Intensity of Glut the positive criteria  1 expression in reference to Cooper et al [2] reported: cytoplasm and (or) membrane stained brownish yellow Glut  1-positive cells (-): no cancer cells staining (+): 1 % ~ 10% carcinoma cells staining, (+ +): 11% to 50% carcinoma cells staining, (+ + +):> 50% carcinoma cells staining. MVD determination reference bovine Xinjie, etc. [3] each slice pick vascular distribution of the highest area 100 times the light microscope and light microscope at 200 times the number of blood vessels in mind the five horizons within CD34 stained brown, and the mean value for the The value of the MVD of the tumors, the results to ± s. The positive staining clearly separated near microvascular endothelial cells or cell clusters are considered independent microvascular diameter greater than 8 red blood cells of the blood vessels do not count.

    1.5 Statistical Methods SPSS13.0 software for statistical analysis with the χ2 test, Spearman rank correlation test, analysis of variance analysis of relevant data.

    2 Results

    2.1 HIF  1α and intensity of Glut  1 expression in breast tissue HIF  1α and intensity of Glut  1 in breast cancer and high expression in low expression in the cancer tissue, the difference was significant (P <0.01), as shown in Table 1.

    2.2 expression of HIF  1α, the intensity of Glut  1 and MVD expression with clinicopathological factors by the relationship between expression of HIF-1α, Glut  1 and MVD expression and clinical stage, lymph node metastasis (P <0.05), as shown in Table 2. Table 1 HIF  1α, Glut  1 expression in breast tissue

    2.3 HIF  1α and intensity of Glut  correlation by Spearman test HIF  1α and the intensity of Glut  1 expression intensity was positively correlated, r = 0.461, P <0.001, Table 3. Table 3 HIF  1α and Glut  1 in the relationship between breast cancer expression

    HIF  1αGlut  1 – + ~ + + + nP-11819 + ~ + + +1130410.000

    2.4 expression of HIF  1α, Glut  1 and MVD relationship between HIF  1α intensity of Glut  1 expression was positive breast cancer MVD was significantly higher than its corresponding expression negative, P <0.01, as shown in Table 4. Table 4 HIF  1α, Glut  1 expression and MVD relationship

    Expression of the case number of cases of MVD (± s) PHIF  1α -1922.35 ± 4.09 + + + +4127.53 ± 4.86 <0.01Glut  1 -2222.47 ± 3.53 + ~~ + + +3825.89 ± 5.20 <0.01

    2.5 HIF  1α/Glut  1 expression and MVD relationship HIF  1α/Glut  1 (+ / +) MVD was significantly higher than HIF  1α/Glut,  1 (- / +) (+ / -) and (- / -) group, the difference was statistically significant, P <0.05. And HIF  1α/Glut  1 ( / +) (+ / -) MVD was also significantly higher than HIF  1α/Glut,  1 (- / -) group, P <0.05, Table 5. Table 5 HIF  1α/Glut  1 expression and MVD in relations

    HIF  1α/Glut,  1 expression group nMVD (± s) P (- / -) * 1120.60 ± 3.51 (- / +) (+ / -) ** 1924.53 ± 2.99 <0.01 (+ / +) *** 3028.70 ± 5.20

    q test: * compared with **: P = 0.017; ** and ***: P = 0.001; * comparison: P = 0.000 and ***

    Table 2 expression of HIF  1α, the intensity of Glut  1 and MVD expression and clinicopathological factors, the relationship between clinical and pathological

    3 Discussion

    Hypoxia inducible factor (HIF  1) cells of the body to adapt to hypoxic stress transcription factor [4], constituted by HIF  1α and HIF  1β subunit, the transcriptional activity of HIF  1α expression levels and stability. HIF  1α and hypoxia response genes combine to promote these gene transcription, promote the formation of new tumor blood vessels, to maintain the energy metabolism of tumor cells to provide the material basis for tumor cell proliferation and metastasis. In a range of human tumor expression of HIF  1α highly expressed in some precancerous lesions can also be detected by the presence of HIF  1α, and no expression in normal tissues, the study was similar conclusions, suggesting that hypoxia occurs before the cancer, continued the whole process of tumorigenesis, HIF  1α abnormal expression may be the early behavior of the carcinogenesis process. At the same time, we found that HIF  1α in breast cancer lymph node metastasis, clinical stage, HIF  1α may be highly invasive biomarker, possible mechanisms: high expression of HIF  1α (1) induced angiogenesis neovascularization of tumor tissue oxygen supply can also provide access for the transfer of cancer cells; (2) HIF  1α expression, activation of cathepsin D, matrix metalloproteinase-2 and urokinase-type plasminogen activator receptor invasion-related gene expression [5], the basement membrane of tumor cells, extracellular matrix degradation, cell  cells, cells  matrix adhesion, fibronectin, vimentin, transforming growth factor alpha autocrine mobile factor force lowering, invasive increases, eventually leading to the separation and transfer of the cells.

    Exuberant malignant cell metabolism, glucose utilization is increased significantly, while the sugar is a water-soluble substance, can not freely pass through the membrane lipid bilayer, Glut (Glucose transporter) is distributed in the membrane of a class of transmembrane glycoprotein is glucose transmembrane transporter carrier. So far, it has been found there are 14 kinds of Glut Glut  1 of the most widely distributed and its main function is to provide the cells to provide a basic supply of glucose, the ATP generated by glycolysis survive. Therefore, Glut  1 abnormal expression is a marker of tumor hypoxia, it may be a pathological cell adaptation process. Glut  1 are overexpressed reported in the literature in a variety of malignancies such as breast cancer, bladder cancer, kidney cancer, colorectal cancer, this study Glut  1 expression in breast cancer was 63.3%, in the low expression of adjacent tissues , expression and axillary lymph node metastasis, TNM stage, suggesting that Glut  1 involved in the occurrence of malignant tumors, may be an early indicator with tumor invasion and poor prognosis.

    Possible mechanisms of hypoxia-inducible expression of Glut  1 in the absence of oxygen, HIF  1α expression increased dramatically, activation intensity of Glut-1 5 ‘end of the enhancer sequence, causing the intensity of Glut  expression of [6], which in turn increase glucose transporter and sugar degradation contribute to tissue cells to ischemia, hypoxia tolerance. This study shows that HIF  1α positive in 41 cases, 38 cases of Glut  1 positive expression was positively correlated (r = 0.461, P <0.001). HIF  1α and Glut  1 expression in breast cancer tissue, this correlation further supports the the Glut  1 regulated by HIF  1α doctrine, prompted in tumor tissue hypoxia, HIF  1α is involved in regulating tumor tissue an important role in glucose metabolism associated protein gene expression.

    We found that HIF  1α, Glut  1 positive expression specimens MVD was significantly higher than the negative expression, suggesting that HIF  1α, Glut  1 high expression of breast cancer angiogenesis, HIF  1α, Glut-1 were positive MVD was highest, suggesting that both may have a synergistic effect. Currently, VEGF is HIF  1α target genes [7], the lack of oxygen conditions, HIF  1α and VEGF hypoxia response element (hypoxia response element, HRE) sequences combined increase VEGFmRNA stability, as well as VEGF the transcriptional activity of VEGF expression, thereby inducing angiogenesis. The process of tumor growth, the blood supply often presents hysteresis state, tumor Glut  1 is highly expressed by glycolysis to meet the growth needs to maintain a high metabolic state, to promote. HIF  1α, Glut  1 co-expression of costimulatory angiogenesis, the exact mechanism needs further research.

    The hypoxic microenvironment, tumor cells will produce a series of physiological and biochemical changes, increased resistance to radiotherapy and chemotherapy, and the tumor cells more aggressive, prone to distant metastases, leading to treatment failure. HIF  1α, Glut  1 in the role of which is gradually becoming a hotspot HIF  1α With further research, targeted inhibition of gene expression to curb energy metabolism and angiogenesis of tumor cells may become cancer treatment and prevention one important means.