Purification of an angiogenesis inhibitor from culture medium conditioned by a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8

Abstract

We previously reported that a novel human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, produced an anti-tumor angiogenesis factor and secreted it into the culture medium [Takigawa et al.: Anticancer Res., 10, 311–316, 1990]. In the present study, we purified the inhibitor by monitoring gelatinase inhibitory activity from the conditioned medium (CM) of HCS-2/8 cells. By a simple three-step procedure, gel filtration chromatography, ion-exchange chromatography, and reverse-phase HPLC, 200 μg of the inhibitor was obtained from 6 liters of CM with 239-fold enrichment. Purified inhibitor, named hCHIAMP (human chondrocyte-derived inhibitor of angiogenesis and metalloproteinase activity), showed a single protein band with a molecular mass ( M r) of 24 000 (24K) under reducing conditions and M r 22K under nonreducing conditions on SDS-PAGE. On reverse-zymography, purified hCHIAMP showed a single band of 22K M r under nonreducing conditions. Its NH 2-terminal amino acid sequence determined up to the 11th amino acid residue was identical with that of the tissue inhibitor of metalloproteinases-2 (TIMP-2). On Western blotting, anti-human TIMP-2 antibody cross-reacted with hCHIAMP. hCHIAMP at a dose of as little as 0.45 μg significantly inhibited angiogenesis in the yolk sac of chick embryos induced by 0.25 μmol of spermine. Because HCS-2/8 is a clonal cell line, these findings clearly showed for the first time that chondrocytes themselves produce a potent inhibitor of angiogenesis, which is also an inhibitor of gelatinase. The findings also indicate that hCHIAMP is a TIMP-2-like molecule. Because the HCS-2/8 cells are an immortal cell line and of human origin, hCHIAMP could be useful for therapy of angiogenic diseases including solid tumors.