Effect Of Inflammation On Hepatocellular Carcinoma Progression And Matastasis In Vivo And Vitro

Background:Inflammation and tumors are closely related. Inflammation is one of the most important factors in tumors development. HCC is the typical inflammation-related cancer. Chronic liver injury caused by chronic viral hepatitis, alcoholic liver and cirrhosis promotes the hepatocarcinogenesis and development, but the mechanism has not yet entirely clear. In this study, we research the effect of inflammation on HCC development and metastasis in molecules, cells, tissues and patients, from liver cancer cell lines, animal models to HCC tissues.Method:In vitro research. Three human hepatoma carcinoma cells HepG2, Hep3B, Huh7 were given lipopolysaccharide (LPS, inflammatory factors) and Celecoxib (inhibiting inflammatory factors). Study the effect of inflammation on hepatoma carcinoma cells morphous, proliferation, cell cycle, apoptosis, angiogenesis, invasion and metastasis with a microscope, MTT, flow cytometry, western blot. Further, we investigated the signaling pathway of inflammation on hepatoma carcinoma cells proliferation and metastasis by testing the expressions of key proteins in Wnt/β-catenin, EGFR signaling pathway.Animal study.Nude mice HCC xenograft model was first established by cell suspension inoculation. Then nude mice were divided into 4 groups and given saline, LPS, LPS+Celecoxib and Celecoxib by intraperitoneal injection every other day. We regularly observed the mice and tumor growth, measured the tumor long diameter (a), the short diameter (b), and calculated the tumor volume according to the formula V=ab2/2. Mice were killed 35 days later. Effect of inflammation on HCC development in vivo was confirmed by observing tumor growth and metastasis.HCC tissue study.Hepatocellular carcinoma tissues from patients receiving liver resection were detected by immunohistochemical expression of COX-2, VEGF, NF-κB and Ki-67, and established the standard of inflammation grade in HCC tissues microenvironment, according to the proportion of inflammatory cells. We analysised the relationships between inflammation COX-2, NF-κB and VEGF, Ki-67 expressions, and the relationships between inflammatory grade, COX-2 expression and clinical pathological characteristics.Results:Part 1. Hepatoma carcinoma cells number was increased and strong growth by treated LPS, and decreased and weak growth by Celecoxib under microscopic and by MTT assay. Flow cytometry showed that cells after treatment, the number of S stage is LPS group> control group> Celecoxib group. Western blot analysis discovered that HepG2 cells proteins of COX-2, NF-κB, MMP-9, VEGF,β-catenin, EGFR in LPS group were significantly increased, while the Celecoxib group were lower than the control group.Part 2.Nude mice experiments demonstrated that HCC subcutaneous xenograft model could be successfully established by inoculating subcutaneously 5×106/0.2ml of HepG2 cells into nude mice. The tumor growth velocity, size and quality in LPS group were significantly higher, but Celecoxib group were lower than control group.Part 3.The COX-2 expression in HCC was positive association with inflammation grade of tissue microenvironment. Inflammation grade and COX-2 expression were related with AFP, but no relationship with patient age, gender, tumor size, tumor differentiation, tumor number, viral infection and tumor thrombus. The COX-2 expression was positive association with expression levels of VEGF, NF-κB, and NF-κB expression was positive association with expression of VEGF, Ki-67.Conclusion:1. Inflammation could promote hepatoma carcinoma cells proliferation, growth and angiogenesis, affect cell cycle, enhance invasion and metastasis and activate NF-κB signaling pathway by promoting NF-κB translocation expression, while inhibition of inflammation could suppress the malignant behaviors.2.Inflammation could promote HCC growth and metastasis by activating the Wnt/β-catenin, EGFR signaling pathways.3. In nude mice, inflammation can promote tumor growth, but inhibition of inflammation could suppress tumor growth.4. Inflammation plays an important role in the hepatocarcinogenesis, development and metastasis, which may be related to angiogenesis and NF-κB signaling pathway.