Expression of the lysyl oxidase propeptide in hepatocellular carcinoma and its clinical relevance.

Lysyl oxidase is an important extracellular matrix remodeling enzyme and plays critical roles in tumor progression and development. Its tumor-suppressor activity has been shown to depend on the propeptide region. Previous studies have reported that the expression levels of lysyl oxidase propeptide (LOX-PP) are associated with cancer of the breast, pancreas, lung, prostate and gastrointestinal system. However, to date, the exact effects and molecular mechanisms of LOX-PP in hepatocellular carcinoma progression are still unclear. The present study aimed to investigate the expression and clinical significance of LOX-PP in human hepatocellular carcinoma. First, 42 cases of hepatocellular carcinoma and corresponding adjacent non-cancerous tissues (ANCTs) were collected, and the expression of LOX-PP in these samples was assessed by immunohistochemistry (IHC). The clinicopathological characteristics of all patients were recorded. Next, in in vitro studies, recombinant adenovirus LOX (ad-LOX-PP) was used to infect hepatocellular carcinoma cell lines to determine the function of LOX-PP. To determine whether ad-LOX-PP affects hepatocellular carcinoma cell survival, cell viability was examined by CCK-8 assay, and cell cycle progression was assessed by flow cytometry. We also investigated the effects of LOX-PP on the expression of cell cycle regulators (cyclin D1 and cyclin E) by western blot analysis. The migration and invasion capacities of hepatocellular carcinoma cells were observed by wound-healing and tranwell invasion assays. To further investigate how LOX-PP affects migration levels of matrix metallopeptidase (MMP)-2 and MMP-9 were assessed by western blot analysis. Additionally, markers of the PI3K and MAPK signaling pathway were detected to further confirm the mechanisms of LOX-PP. As a result, reduced expression of LOX-PP was found in hepatocellular carcinoma tissues, when compared with that in the ANCTs (15 vs. 83%, P<0.01), and its expression was associated with tumor stage and distant metastasis (each P<0.05). Proliferation in hepatocellular carcinoma cells was significantly decreased in the ad-LOX-PP group as indicated by CCK-8 assay. LOX-PP significantly reduced the expression of Ki-67, while prominent increases in the rate of apoptosis and cell cycle arrest were observed. Similarly, cell migration was significantly inhibited in the ad-LOX-PP group as evidenced by transwell invasion and wound-healing assays. The expression levels of MMP-2 and MMP-9 were attenuated in the ad-LOX-PP group, suggesting that LOX-PP inhibits hepatocellular carcinoma cell migration via down-regulation of MMPs expression. When LOX-PP expression was potentiated by an adenovirus containing LOX-PP, the expression of p-ERK was significantly downregulated, indicating that LOX-PP inhibits hepatocellular carcinoma cell proliferation and induces its apoptosis probably through downregulation of the MAPK/ERK pathway.