Enhanced migration of tissue inhibitor of metalloproteinase overexpressing hepatoma cells is attributed to gelatinases: Relevance to intracellular signaling pathways

AIM: To study the effect of gelatinases (especially MMP-9)on migration of tissue inhibitor of metalloproteinase (TIMP-1) overexpressing hepatoma cells.METHODS: Wild type HepG2 cells, cells stably transfected with TIMP-1 and TIMP-1 antagonist (MMP-9-H401A, a catalytically inactive matrix metalloproteinase (MMP) which still binds and neutralizes TIMP-1) were incubated in Boyden chambers either with or without Galardin (a synthetic inhibitor of MMP-1, -2, -3, -8, -9) or a specific inhibitor of gelatinases.RESULTS: Compared to wild type HepG2 cells, the cells overexpressing TIMP-1 showed 115% migration (P＜0.05)and the cells overexpressing MMP-9-H401A showed 62% migration (P＜0.01). Galardin reduced cell migration dose dependently in all cases. The gelatinase inhibitor reduced migration in TIMP-1 overexpressing cells predominantly.Furthermore, we examined intracellular signal transduction pathways of TIMP-1-dependent HepG2 cells. TIMP-1deactivates cell signaling pathways of MMP-2 and MMP-9involving p38 mitogen-activated protein kinase. Specific blockade of the ERK pathway suppresses gelatinase expression either in the presence or absence of TIMP-1.CONCLUSION: Overexpressing functional TIMP-1-enhanced migration of HepG2-TIMP-1 cells depends on enhanced MMP-activity, especially MMP-9.

Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC

AIM:To investigate the proteolytic contribution of tumor-associated macrophages(TAM)in tumor invasion,we analyzed whether TAM at the invasive front of small HCC in Abcb4-/--mice show an enhanced expression of MMP-9. METHODS:Liver cryosections of the hepatocellular carcinoma(HCC)invasive front from 12 mo old Abcb4-/--mice were stained for collagen typeⅠand MMP-9 using Alexa488 and Alexa568 labeled secondary antibodies.Afterwards,the Alexa568 dye was bleached and the macrophage marker F4/80 was visualized using Alexa568 labeled secondary antibodies.Finally, photographs of the invasive tumor front were digitally overlaid and analyzed. RESULTS:After complete bleaching of the primary dye,specific fluorescence staining of a third antigen, here F4/80,was successfully performed on the same histological section.With this method,we were able to identify conglomerates of matrix metalloproteinase (MMP-9)expressing macrophages within the tumor capsule of HCC. CONCLUSION:MMP-9 expressing macrophages are involved in matrix remodelling at the invasive tumor front of HCC.The described staining protocol provides a simple yet powerful extension of conventional immuno-histochemistry,facilitating visualization of at least three different antigens plus nuclei in one single histological section.