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Inhibition by Retinoic Acid of Type IV Collagenolysis and Invasion through Reconstituted Basement Membrane by Metastatic Rat Mammary Adenocarcinoma Cells¹

Department of Tumor Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [M. N., D. L., M. M. B., R. M. C., R. L.], and Department of Anatomy [W. R. W., M. J. C. H.], University of Arizona, College of Medicine, Tucson, Arizona 85724

The activity of type IV collagenase, which enables tumor cells to degrade collagen type IV found in the subendothelial basement membrane, has been correlated with the metastatic potential in several tumor types, including the rat 13762NF mammary adenocarcinoma cell line and its clones. In this study, we examined whether all-trans-retinoic acid (all-trans-RA) and other retinoids, which exhibit antitumor activity in vitro and in vivo, affect the collagenolytic activity of metastatic rat 13762NF mammary adenocarcinoma cells. Cells of the highly metastatic lung-colonizing clone MTF7.T35.3, derived from the 13762NF cell line, were treated for 3 days with 0.1, 1, or 10 µM all-trans-RA, harvested, and seeded on [³H]proline-labeled extracellular matrix deposited by cultured rat lung endothelial cells or on a film of purified [³H]proline-labeled type IV collagen. The amount of radioactivity released into the medium during the subsequent 24 to 72 h was measured, and it was found that all-trans-RA treatment inhibited degradation of extracellular matrix and type IV collagen by 50 to 60%. This effect was observed whether the cells had been treated with all-trans-RA in serum-free medium or in medium supplemented with heat-inactivated or acid-treated fetal bovine serum. The growth of the cells was not inhibited under these conditions, except after treatment with 10 µM all-trans-RA in serum-free medium. The reduction in collagenolytic activity was observed in viable cells as well as in conditioned medium. A 24-h exposure of cells to all-trans-RA was sufficient to cause a 30% decrease in the collagenolytic activity, and this inhibitory effect was reversible. The direct addition of all-trans-RA to conditioned medium had no effect on secreted collagenase activity. The apparent molecular weights of the collagenolytic enzymes were determined by electrophoresis of cell extracts and concentrated conditioned medium in type IV collagen-embedded polyacrylamide gels followed by renaturation and activation of the enzymes within the gels. Two major type IV collagenolytic metalloproteinases exhibiting molecular weights of 64,000 and 88,000, respectively, were detected by this method. These two enzymes were also found to have specificity for gelatin. The M_r 64,000 enzyme could be extracted from viable cells (presumably from the cell membrane) by 2% 1-butanol. Treatment with all-trans-RA decreased the level of these enzymes in the cellular, cell membrane, and conditioned medium compartments. Other retinoids, including 13-cis-RA, retinol, trimethylmethoxyphenyl analogue of RA, tetrahydrotetramethyl naphthalenyl propenylbenzoic acid, and 3,5-di-tert-butyl 4'-chalcone carboxylic acid, also suppressed the level of secreted type IV collagenolytic enzymes. Continuous treatment of the adenocarcinoma cells with 1 or 10 µM all-trans-RA inhibited their ability to invade through a reconstituted basement membrane (Matrigel-coated filters). These results demonstrate the ability of retinoids to suppress the production of type IV collagenolytic metalloproteinases in mammary adenocarcinoma cells and inhibit their invasiveness in vitro. Such an effect in vivo may decrease the invasiveness of malignant tumors.

¹ Supported in part by USPHS National Cancer Institute Grants R01 CA41524 to M. N., R01 CA42877 to R. L., and R01 CA42475 to M. J. C. H. and Fellowship Grant 85-501 to W. R. W.

² To whom requests for reprints should be addressed, at the Department of Tumor Biology (Box 108), The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.

Received 8/29/88. Revised 12/ 9/88. Accepted 1/ 5/89.

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