Figure 7

HCC cell expression of MMP-2, MMP-9, MT1-MMP, and TIMP-2. Chang, HLE, and HLF secrete detectable levels of inactive and active MMP-2, but not of MMP-9 in serum-free medium. On the contrary, Sk-Hep1 secretes MMP-9 but not MMP-2, whereas Hep3B, Alexander, HepG2, and HepG2.2.15 do not secrete MMP-2 or MMP-9, as shown in Panel a. To confirm the presence of MMP-2 in HCC concentrated media, samples were analyzed by Western blotting and the membrane was probed with a monoclonal antibody against MMP-2, as reported in Panel b. In conditioned media preparations, the expression of TIMP-2 was also investigated by Western blot using a monoclonal antibody. As seen in Panel c, TIMP-2 is present in all HCC cell lines. MT1-MMP was investigated in plasma membrane by western blot using a polyclonal antibody. As shown in Panel d, MT1-MMP was expressed on the cell surface of all of the HCC cell lines. Plasma membranes were also evaluated for MMP expression because ECM proteolysis occurs at the cellular advancing edge, in a focused manner. Consistent with the data found in the zymogram, in Panel e, MMP-2 is present in Chang, HLE, and HLF preparations, MMP-9 is present only in SK-Hep1, and no MMP are detectable in the Hep3B, HepG2, HepG2.2.15, or Alexander samples.