Hypoxia-inducible factor-1 alpha mediates the toll-like receptor 4 signaling pathway leading to anti-tumor effects in human hepatocellular carcinoma cells under hypoxic conditions

摘要

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) and toll-like receptor 4 (TLR4) are involved in numerous mechanisms of cancer biology, including cell proliferation and survival; however the interaction of the two factors under hypoxic conditions remains unclear. The present study investigated the in vitro mechanism that results in the suppression of tumor cell growth and cellular functions when HIF-1 alpha is silenced. In the present study, the human hepatocellular carcinoma HepG2 cell line was transfected with short hairpin RNA (shRNA) against HIF-1 alpha and cultured under hypoxic conditions (1% O-2 for 24 h). The expression of HIF-1 alpha and various growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), were examined using quantitative polymerase chain reaction and immunoblotting. Tumor growth was measured using a Cell Counting Kit-8 assay and tumor activity was measured using tumor cell invasion and migration assays. Lipopolysaccharide and TAK-242 were used to activate and inhibit TLR4, respectively, to observe the role of TLR4 in the HIF-1 alpha silenced tumor cells. The expression of TLR4 signaling pathway associates, including myeloid differentiation primary response gene 88 (MyD88), apoptosis signal-regulating kinase 1 (ASK1), p38 mitogen-activated protein kinases and HIF-1 alpha, were analyzed by western blot assay. Under hypoxic conditions, silencing of HIF-1 alpha expression suppressed tumor cell growth and regulated the expression of tumor growth-associated genes, including EGF, HGF, VEGF and FG2. Suppression of tumor cell invasion and migration was also observed in the HIF-1 alpha silenced HepG2 cell line. In addition, TLR4 was identified to be involved in HIF-1 alpha and MyD88 accumulation, and activation of ASK1 and p38 were demonstrated to be critical for TLR4-mediated HIF-1 alpha pathway. In conclusion, silencing of HIF-1 alpha expression may induce anti-tumor effects under hypoxic conditions in HepG2 cells via the TLR4 mediated pathway, suggesting that the HIF-1 alpha/TLR4 signaling cohort may act as a novel therapeutic target for the treatment of hepatocellular cancer.