Inhibition of Tumor Cell Invasiveness by Chemically Modified Tetracyclines

Y.      Gu; M.   H.   Lee; J.   E.   Roemer; L.      Musacchia; M.   L.   Golub; R.   S.   Simon

Abstract

COLO 205 is a cell line derived from a human colon carcinoma with high degradative activity towards extracellular matrix (ECM). It has been shown that COLO 205 cells produce matrix metalloproteinases (MMPs). MMPs are a family of enzymes known to degrade components of the ECM and have been implicated in tumor invasion. In the present study, we have analyzed the multiple effects of chemically modified tetracyclines (CMTs) on the expression and activity of MMPs secreted by COLO 205 cells in vitro with the aim of evaluating these compounds for potential use in management of invasive tumors. Because COLO 205 cells can degrade an interstitial ECM in serum-free medium in vitro, we have been able to compare the effects of the tetracyclines on this measure of invasive activity with their effects on proteinase expression and activity. We demonstrate here that one of the chemically modified tetracyclines, 6-deoxy-6-demethyl- 4-de(dimethylamino)tetracycline (CMT-3) can effectively inhibit ECM degradation mediated by COLO 205 cells or their conditioned medium. Gelatin zymography and immunoblots show that CMT-3 has the ability to inhibit release of MMP-2 into conditioned medium as well as to inhibit MMP-2 gelatinolytic activity, which correlates with the results from ECM degradation assays. On the basis of our findings with COLO 205 cells we have expanded our evaluation of the tetracyclines to include effects on a genetically engineered line of MDA-MB-231 breast tumor cells overexpressing MMP-9 at levels over tenfold those of the parent cell line, and on three human prostate tumor cell lines, LNCaP, DU-145, and PC-3. We show here that CMT-3 displays multiple modes of action inhibiting MMP activity, reducing levels of MMP expression, and exhibiting selective cytotoxicity towards some of the tumor cell lines.

Keywords: Tumor invasive, modified tetracyclines, colo 205, matrix metalloprotinases, plasma proteinases, multiwell microplates, zymographic analysis, radiolabeled ECM, chromogenic coupled enzyme assay, casein impregnated gels, immunoreactive MMP 1, Densitometric scans