Effects Of EGCG On Trans-endothelial Migration Of HepG2 Liver Cancer Cells And The Underlying Mechanisms

BACKGROUNDHepatocellular carcinoma (HCC) is a common worldwide malignancy, affecting especially high in Asia and Africa. Although the diagnosis and treatment methods of HCC have been developing over the last 20 years, the recurrence and mortality of HCC is still high because of the extensive characteristics of HCC including intrahepatic metastasis, venous invasion, and distant metastasis. Adhesion of cancer cell to endothelial cells and the subsequent trans-endothelial migration are key steps in cancer metastasis.The lectin-like oxidized low-density lipoprotein receptor-1, LOX-1 (the type D scavenger receptor) was initially cloned from bovine aortic endothelial cells in 1997 by Sawamura et al. It is the major receptor responsible for uptake and metabolism of ox-LDL in vascular endothelial cells, and plays an important role in the pro-inflammatory signaling, endothelial dysfunction and atherosclerosis. Studies have shown that LOX-1 may act as adhesion molecules mediating the interaction between cancer cells and endothelial cells. EGCG, the main ingredient of Catechin, has powerful treatment and prevention for liver cancer. However, the anti-cancer molecular mechanisms and the targets that EGCG directly act have not yet been sufficiently studied.METHODSAfter cultured with the serum-free medium for 12 h, the HUVEC-C cells were pretreated with various concentrations (5,10 or 20μM) of EGCG for 2 h prior to exposure to TNF-a (50 ng/ml). Cancer cell adhesion ability was analyzed by using adhesion assay. Migration ability of the cancer cells was evaluated by trans-endothelial migration assay. The level of LOX-1 mRNA was detected by real time PCR. The level of LOX-1 protein was determined by Western Blot.RESULTS1. Real time PCR showed that LOX-1 mRNA expression was up-regulated by TNF-a in a concentration-dependent manner. Incubation of HUVEC-C with TNF-a at 50 ng/ml cells for 8 h led to about 6.67 fold increase of LOX-1 mRNA expression.2. The protein level of LOX-1 was up-regulated by TNF-a in a concentration-dependent manner.3. Incubation of HUVEC-C cells with TNF-a (50 ng/mL) for 8 h led to a significant increase adhesion of HepG2 liver cancer cells and endothelial cells. These effects of TNF-a were reversed by EGCG in a concentration-dependent manner. Similarly, anti-LOX-1 anbody (TS 20) also inhibited these effects of TNF-a.4. Incubation of HUVEC-C cells with TNF-a (50 ng/mL) for 8 h significantly promoted trans-endothelial migration of HepG2 liver cancer cells. These effects of TNF-a were reversed by EGCG in a concentration-dependent manner. Similarly, anti-LOX-1 anbody (TS 20) also inhibited these effects of TNF-a.5. Real time PCR results showed that TNF-a up-regulated LOX-1 mRNA expression. EGCG significantly reduced the increase in LOX-1 mRNA expression induced by TNF-a in a concentration-dependent manner.6. Western Blot results showed that TNF-a up-regulated LOX-1 protein expression. EGCG significantly reduced the increase in LOX-1 protein expression induced by TNF-αin a concentration-dependent manner.CONCLUSIONEGCG could inhibit the adhesion of liver cancer cell to endothelial cells and the subsequent trans-endothelial migration induced by TNF-a via reducing the expression of LOX-1. LOX-1 may act as an adhesion molecule to mediate liver cancer cell/endothelial cell interactions.