Cystatin C and cathepsin B in human colon carcinoma: expression by cell lines and matrix degradation.
Expression of the cysteine proteinase cathepsin B and its physiological inhibitor cystatin C was analyzed in vitro in 1 human fibrosarcoma and 4 human colon carcinoma cell lines. Cystatin C antigen as well as cathepsin B activity were detected in the conditioned media of the 5 cell lines. The corresponding cell extracts expressed high levels of cathepsin B activity, whereas only trace amounts of cystatin C antigen could be found. Northern-blot analysis revealed the presence in the 5 cell lines of a 0.8-kb cystatin C mRNA transcript and 2 cathepsin B transcripts of 2.3 and 4.3 kb. Pepsin treatment of tumor-cell-released cathepsin B induced an average 7.3-fold increase in activity, indicating that the enzyme was mainly present as a latent form in conditioned medium. The pepsin-activated cathepsin B from one colon carcinoma cell line was further characterized using the cysteine proteinase inhibitors E-64, recombinant cystatin C, a cystatin-C-derived peptidyl inhibitor (Z-LVG-CHN2), and cathepsin-B-specific diazomethyl ketone inhibitors (Z-FT(OBzl)-CHN2, Z-FS(OBzl)-CHN2). This activity was totally neutralized by recombinant cystatin C, suggesting a potential for interaction between released extracellular cathepsin B and cystatin C. In vitro assays of degradation of extracellular matrix showed that cysteine proteinase inhibitors could decrease matrix degradation induced by pepsin-activated conditioned media. With colon cells, this inhibition was not observed, indicating a requirement for an extracellular activation of latent cathepsin B. Our data provide evidence that cystatin C and latent cathepsin B are both released extracellularly by colon carcinoma cells in vitro. They suggest that cystatin C and cathepsin B interactions may participate, in an as yet unelucidated way, in the modulation of the invasive phenotype of human colonic tumors.
Corticchiato
O
O
Swiss Institute for Experimental Cancer Research (ISREC), Epalinges.
Cajot
J F
JF
Abrahamson
M
M
Chan
S J
SJ
Keppler
D
D
Sordat
B
B
D002277:Carcinoma / Q000201:enzymology*; D002401:Cathepsin B / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000502:physiology; D003110:Colonic Neoplasms / Q000201:enzymology*; D017077:Culture Media, Conditioned; D055316:Cystatin C; D015891:Cystatins / Q000032:analysis*; D015853:Cysteine Proteinase Inhibitors / Q000032:analysis*; D004789:Enzyme Activation; D005109:Extracellular Matrix / Q000378:metabolism*; D005354:Fibrosarcoma / Q000201:enzymology; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C525030:CST3 protein, human; D017077:Culture Media, Conditioned; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011480:Protease Inhibitors; D002401:Cathepsin B