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Introduction
Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer and is also the third leading cause of cancer-associated mortality worldwide, with ~700,000 mortalities reported annually (1,2). The incidence of HCC has demonstrated a notable increase worldwide, particularly in developing countries within Asia and sub-Saharan Africa, where hepatitis B and C viral infections are endemic, and in regions where food contaminated with Aflatoxin B1 is consumed (3). HCC is usually diagnosed at an advanced, incurable and metastatic stage due to the usual absence of specific symptoms and history of cirrhosis. Thus, the five-year overall survival rate is <10%; the effects of surgical and chemo-radiation therapies are poor for advanced and metastatic stages of HCC (4).
HCC is a solid tumor with numerous blood vessels, and the most important characteristic of HCC is the uncontrollable cell proliferation, which leads to a novel hypoxic microenvironment with increased oxygen consumption. Therefore, it is important to establish a vascular and nutrient supply system to meet the oxygen demands (5,6). Hypoxia is one of the fundamental biological phenomena that are strongly associated with the development and aggressiveness of a wide variety of solid tumors, including HCC. It has been reported that hypoxia inducible factor-1 (HIF-1α), the key to mediating hypoxia-responsive genes, and insulin-like growth factor I (IGF-1), which is secreted in the liver, may potentially be synergistic in regulating the vascular endothelial growth factor (VEGF) expression in type 2 diabetes (7). The present study focused on whether VEGF expression in HCC is also regulated by HIF-1α and IGF-1.
In the current study, a model of hypoxia was created using cobalt chloride (CoCl2) and an MTT assay was used to observe the influence of hypoxia on the proliferation of HepG2 cells. The effect of hypoxia on HepG2 cell invasion and angiogenesis was examined, as well as the expression and correlation of HIF-1α, IGF-1 and VEGF under hypoxic conditions.
Materials and methods
Drug and reagents
Dulbecco’s modified Eagle’s medium (DMEM), CoCl2 dissolved in DMEM, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit, RIPA buffer, goat anti-rabbit fluorescein isothiocyanate immunoglobulin (Ig)G and tetramethylrhodamine isothiocyanate IgG antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA). Goat anti-rabbit HIF-1α, IGF-1R and VEGF monoclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA,...