Purification of an angiogenesis inhibitor from culture medium conditioned by a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8.
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 chromtography, and reverse-phase HPLC, 200 micrograms 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 NH2-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 microgram significantly inhibited angiogenesis in the yolk sac of chick embryos induced by 0.25 mumol 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.
Ohba
Y
Y
Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Japan.
Goto
Y
Y
Kimura
Y
Y
Suzuki
F
F
Hisa
T
T
Takahashi
K
K
Takigawa
M
M
D002813:Chondrosarcoma / Q000737:chemistry*; D017077:Culture Media, Conditioned / Q000737:chemistry; D018093:Gelatinases / Q000037:antagonists & inhibitors; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D009389:Neovascularization, Pathologic; D011506:Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D011506:Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases