Hypoxia-Inducible Factor-1α, Semaphorin4D And Tumor-associated Macrophages Are Related To The Aggressiveness Of Colorectal Cancer

Background and objective:Colorectal cancer ranks among the most frequent malignancies. Ahmedin and colleagues showed that the morbidity and mortality of colorectal cancer were in the third place and fourth place of malignant tumors respectively in2008on the worldwide cancer data analysis. With upgrade of living standards and the way people live more westernized, an increasing trend in the morbidity and mortality of this cancer has been noticed in Asian countries, particuLarly China. The reason that it causes the high death rate lies in the metastasis depending on angiogenesis. Hypoxia in the tumor microenvironment is a common pathological status, hypoxia-inducible factor-1(HIF-1) is a key transcription factor reguLating hypoxia adaptive response. HIF-1is composed of an oxygen-sensitive HIF-1α and a constitutive HIF-1β subunit. HIF-1a is the oxygen-reguLated subunit that determines HIF-1activity. HIF-1β activity is primarily reguLated by hypoxic conditions and the inflammatory cytokines, such as interleukin1β (IL-1β) and tumor necrosis factor-a (TNF-a) and so on. Under hypoxic inflammatory microenvironment, HIF-1a is stabilized, translocates into the nucleus where it dimerizes with HIF-1β, and binds to hypoxia response elements (HRE), which can activate expression of a range of genes that mediate many of the adaptive responses to decreased oxygen concentration, such as enhanced glucose uptake and formation of new blood vessels via proliferation and migration of endothelial cells toward the developing tumor. Angiogenesis is essential for the growth, invasion, and metastasis of tumors.HIF-1is able to influence the angiogenesis by increased production of pro-angiogenic proteins, such as vascuLar endothelial growth factor (VEGF) and Semaphorin4D (Sema4D). Sema4D (also known as CD100), a homologous dimeric glycoprotein, originally discovered in the immune system in1992. Sema4D plays an important role in immunological reaction and neural system development. Recently, Sema4D has been shown to promote angiogenesis and enhance invasive growth and proliferation upon binding its receptor Plexin-Bl by Rho/Rho-kinase pathway. Sema4D is identified as a new pro-angiogenic protein and over-expressed in many malignant solid tumors, such as Head and neck squamous cell cancers, breast cancer and lung cancer. Studies show that Tumor-associated macrophages (TAMs) make up a significant part of the tumor-infiltrating inflammatory cells, and they can influence HIF-1α and Sema4D expression in tumor and subsequently affect proliferation, invasion,migration and angiogenesis. Right now, although there is some data concerning HIF-la and Sema4D, however, the biological functions of HIF-1α and Sema4D in the development of colorectal cancer have not been clear. In this study, we examine the expression of Sema4D and HIF-la by immunohistochemistry and establish the co-cuLture system of LoVo cell and TAMs, to explore clinical significance of HIF-la and Sema4D in colorectal cancer. In addition, we also evaluate the effect of Tumor-associated macrophages on the expression of Sema4D and HIF-la and the impact of biologic behavior in colon cancer cells. Along with the deepening of research of correlative mechanism, HIF-la and Sema4D will inject vitality in the prophylactic-therapeutics of tumor.Methods:The expressions of HIF-la and Sema4D were examined in86cases of colorectal cancer and52normal colorectal tissues by SP immunohistochemical staining. The relationship between the expressions and clinical pathological characters were analysied. Establish the co-cuLture system of LoVo cell and TAMs, The blank control group is only LoVo cell, the co-cuLture system of LoVo cell and M2 macrophage is the experimental group. Scratch healing, migration and invasion assays were used to examine the effect of Tumor-associated macrophages on migration and invasion in LoVo cell. Western Blotting analysis was used to examine the expression changes of HIF-1α and Sema4D between the blank control group and the experimental group. Tube formation was performed to study the inhibitory effect of co-cuLture system supernatant on angiogenesis by endothelial cells. ELISA assay was used to detect the variation of Sema4D in the co-cuLture system supernatant. Statistical analysis was performed using SPSS18.0statistical software.ResuLts:1. The positive expressions of HIF-la and Sema4D were58%and60%in colorectal cancer, respectively. It had statistical significant differences from8%and12%in normal mucosa,and HIF-1α and Sema4D were closely correlated with histological types, TNM stages and lymphatic metastasis (P<0.05), but there was no correlation with age, sex and the tumor size (P>0.05). A significant correlation was observed between the expressions of HIF-la and Sema4D in colorectal cancer with the Spearman rank correlation (r=0.567, P<0.01).2. In the Transwell co-cuLture system, compared with the blank control group, LoVo cells co-cuLtured with M2macrophages, a striking feature of LoVo cells communicating with M2macrophages was a morphologic switch, the LoVo cells become long spindle, increase pseudopodia and low cell confluence to a variable extent. Compared with the control group, these LoVo cells co-cuLtured with M2macrophages displayed enhanced migration and invasion (P<0.01). Consistent significant up-reguLation of HIF-1α and Sema4D expression was observed in the LoVo cells after co-cuLture with M2macrophages (P<0.05)3. The endothelial tube formation experiment showed:the blank control group and the experimental group had tubuLar number of29.80±1.14and55.80±1.95respectively per average view (P<.05). The ELISA assay indicated:compared with control group, the co-cuLture system supernatant showed a significant increase of secreted Sema4D (1301.13±51.18pg/mL VS685.15±29.56pg/mL, P<.05). Conclusions:1. A significant correlation was observed between the expression of HIF-la and Sema4D proteins and HIF-la and Sema4D were closely correlated with histological types, TNM stages and lymphatic metastasis in colorectal cancer.2. Tumor-associated macrophages (TAMs) promoted the LoVo cells migration, invasion and endothelial tube formation.3. The induced migration, invasion and angiogenesis couLd be due to the up-reguLation of HIF-1α and Sema4D.