Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer

Abstract

The bioactivity of matrix metalloproteinases was studied in tissues from colorectal cancer patients by means of both quantitative gelatin zymography and a fluorometric activity assay. Next to paired samples of tumour tissue and distant normal mucosa (n=73), transitional tissue was analysed from a limited (n=33) number of patients. Broad-spectrum matrix metalloproteinase activity and both the active and latent forms of the gelatinases matrix metalloproteinase-2 and -9 were higher in tumour than in normal mucosa. The ratio's between active and latent forms of matrix metalloproteinase-2 and -9 were highest in tumour tissue and normal mucosa, respectively. Matrix metalloproteinase-2 levels, both active and latent forms, correlated inversely with stage of disease, the tumours without synchronous distant metastases containing significantly (P=0.005) more active matrix metalloproteinase-2 than the others. At much lower levels of activity, the same trend was observed in distant normal mucosa. The level of latent form of matrix metalloproteinase-9 in tumour depended on tumour location. Neither the active form of matrix metalloproteinase-9 nor broad-spectrum matrix metalloproteinase activity in tumour tissue did correlate with any of the clinicopathological parameters investigated. The results demonstrate explicit differences between the activity of matrix metalloproteinase-2 and -9, indicating different roles for both gelatinases in tumour progression. Such data are necessary in order to develop rational anti-cancer therapies based on inhibition of specific matrix metalloproteinases.

Figure 1

Proteolytic MMP activity in human CRC tissue extracts detected by quantitative gelatin zymography. MMP-2 (−), gelatinolytic activity of recombinant proMMP-2; MMP-2 (+), APMA-activated recMMP-2; MMP-9 (−), recombinant proMMP-9; MMP-9 (+), APMA activated recMMP-9; T, tumour; T–N, transitional (tumour to normal) tissue; N, distant normal mucosa; 1–3, case numbers of three representative patients. Positions of active MMP-2 (62 kDa), proMMP-2 (72 kDa), active MMP-9 (82 kDa) and proMMP-9 (92 kDa) are indicated by arrows. Recombinant MMP-2 and -9 were applied in doses of 1 and 0.5 ng per lane, respectively.

Figure 2

A comparison of MMP activity in CRC tumour (n=73), transitional tissue (T–N, n=33) and normal mucosa (n=73). Data are shown for active MMP-2 (A), proMMP-2 (B), active MMP-9 (C) and proMMP-9 (D). Horizontal bars represent the median values, boxes represent the interquartile range, vertical lines represent the 10 to 90% range of the observations. Enzyme activity, as measured by quantitative zymography, is expressed as specific activity in arbitrary units per milligram protein. Significant differences with normal mucosa: **P<0.001; *P<0.01.

Figure 3

Ratio between active enzyme and proenzyme for both MMP-2 and -9. Data were obtained by quantitative gelatin zymography. (A) CRC tumour tissue; (B) transitional tissue (T–N); (C) normal mucosa. Horizontal bars represent median values, boxes represent the interquartile range, vertical lines represent the 10 to 90% range of the observations. P values represent the statistical differences between matched data on the MMP-2 and MMP-9 ratios.

Figure 4

Active MMP-2 in primary CRC tissue extracts of stage I (n=15), II (n=23), III (n=16) and IV (n=17) tumours determined by quantitative gelatin zymography. Horizontal bars represent median values. Stage II versus stage IV: P<0.05.

Figure 5

Broad-spectrum MMP activity in CRC tumour (n=71), transitional (T–N; n=29) and normal tissue (n=71) determined by quenched fluorogenic substrate hydrolysis. Activity is expressed as the rate of substrate conversion on basis of protein. Horizontal bars represent median values, boxes represent the interquartile range, vertical lines represent the 10 to 90% range of the observations. *P<0.01, **P<0.001 versus normal mucosa.