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.