[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  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  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.  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.
2.1 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
Hypoxia inducible factor (HIF 1) cells of the body to adapt to hypoxic stress transcription factor , 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 , 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 , 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 , 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.