Zymographic analysis of circulating and tissue forms of colon carcinoma gelatinase A (MMP-2) and B (MMP-9) separated by mono- and two-dimensional electrophoresis.
Gelatinase A (MMP-2) and gelatinase B (MMP-9) play a key role in the proteolytic cascade leading to ECM degradation during invasion and metastasis. The enzyme activity is regulated both at the intra- and extra-cellular level. Extracellular regulation is achieved mainly through the balance between proenzyme activation and inhibition, which appears to be altered in cancer patients. One of the mechanisms of MMP inhibition is the binding of the enzymes to appropriate tissue inhibitors (TIMP). In the recent literature, it has been suggested that MMP-2 and/or MMP-9 are indeed over-produced in many carcinomas, while the identity of the various enzymatic forms (latent, activated and enzyme/inhibitor complexes) remains to be elucidated. In this study we have analyzed the circulating forms of MMP-9 and MMP-2 in serum samples of patients with colon carcinoma, as well as the enzymatic activities present in tissue extracts from surgical fragments (primary tumor and its paired healthy tissue). Proteins were separated by means of mono-dimensional or bidimensional electrophoresis, and the enzymes detected by gelatin zymography and immunological assays. The results of densitometric analyses demonstrate that proMMP-9, but not proMMP-2, is significantly higher in the oncologic sera vs. the normal sera. In addition, several oligomeric circulating and tissue forms of MMP-9 are preferentially found in the oncologic samples, both in mono- and second-dimension zymograms. The activated forms of MMP-2 and MMP-9 are uniquely present in the primary tumor extracts, thus confirming the involvement of the tissue microenvironment in gelatinase activation and function.
Pucci-Minafra
I
I
Center of Experimental Oncobiology, University of Palermo, Viale delle Scienze, 90128 Palermo, Italy. idapucci@unipa.it
Minafra
S
S
La Rocca
G
G
Barranca
M
M
Fontana
S
S
Alaimo
G
G
Okada
Y
Y
D000328:Adult; D003110:Colonic Neoplasms / Q000097:blood / Q000201:enzymology* / Q000473:pathology; D003720:Densitometry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004792:Enzyme Precursors / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*
D004792:Enzyme Precursors; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9