Inhibition of breast cancer cell extracellular matrix degradative activity by chemically modified tetracyclines

BACKGROUND. Inhibition of tumour cell proliferation, invasion and metastasis by chemically modified tetracyclines has been ascribed to inhibition of matrix metalloproteinase (MMP) activity. METHODS. Exposure of the human breast carcinoma cell line MDA-MB-231 or its MMP-9-overproducing transfected clone (E-10) to 6-demethyl, 6-deoxy, 4-de [dimethylamino]-tetracycline (CMT-3), a chemically modified non-antimicrobial tetracycline followed by analysis using gelatinase activity assay, zymography, degradation of radiolabelled extracellular matrix (ECM), Western blotting, TNF-alpha ELISA and cell viability assays. RESULTS. CMT-3 treatment results in diminution in extracellular MMP-9 protein levels as well as inhibition of gelatinase activity. This prevents cell-mediated ECM degradation without inducing general cytostasis or cytotoxicity. Culturing E-10 cells in 10 or 20 mu M CMT-3 diminished secreted MMP-9 levels by 45% or 60%, respectively, but did not affect levels of most other secreted proteins, including tissue inhibitor of Metalloproteinases (TIMP-1). ECM degradation by E-10 cells or their conditioned medium was inhibited by similar to 20%-30% in the presence of 20 mu M CMT-3, reflecting inhibition of MMP-9 activity in addition to diminution of released MMP-9 levels. TNF-a levels were also diminished in E-10 conditioned medium in the presence of CMT-3, but cell viability, measured by MTS reduction and cytosolic LDH retention, was unaffected. CONCLUSIONS. It is proposed that the reduction in ECM-degradative activity reflects diminished levels of expression as well as inhibition of enzymatic activity of MMPs released by cells in the presence of CMT-3. These multiple effects of CMT-3 may offer promise for use in suppressing turnout invasion, and if used in conjunction with other chemotherapy agents, may lead to more successful treatment of cancer.