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Abstract
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‐α 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 µM 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 ∼ 20%–30% in the presence of 20 µM CMT‐3, reflecting inhibition of MMP‐9 activity in addition to diminution of released MMP‐9 levels. TNF‐α 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 tumour invasion, and if used in conjunction with other chemotherapy agents, may lead to more successful treatment of cancer.
| Abbreviations | ||
| DMEM | = | Dulbecco's modified eagle medium |
| MEM | = | minimum essential medium |
| ECM | = | extracellular matrix |
| MMP(s) | = | matrix metalloproteinase(s) |
| TIMP | = | tissue inhibitor of matrix metalloproteinase |
| SFM | = | serum free medium |
| TC | = | tetracycline |
| CMT | = | chemically modified tetracycline |
| FBS | = | foetal bovine serum |
| PBS | = | phosphate‐buffered saline |
| PMSF | = | phenylmethanesulfonyl fluoride |
| APMA | = | p‐aminophenylmercuric acetate |
| mAb | = | monoclonal antibody |
| MTS | = | 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt |
| LDH | = | lactate dehydrogenase |
| HRP | = | horse radish peroxide |
| TMB | = | tetramethylbenzidine |
| NADH | = | nicotinamide adenine dinucleotide‐hydrogen |
| VEGF | = | vascular endothelial growth factor. |
| Abbreviations | ||
| DMEM | = | Dulbecco's modified eagle medium |
| MEM | = | minimum essential medium |
| ECM | = | extracellular matrix |
| MMP(s) | = | matrix metalloproteinase(s) |
| TIMP | = | tissue inhibitor of matrix metalloproteinase |
| SFM | = | serum free medium |
| TC | = | tetracycline |
| CMT | = | chemically modified tetracycline |
| FBS | = | foetal bovine serum |
| PBS | = | phosphate‐buffered saline |
| PMSF | = | phenylmethanesulfonyl fluoride |
| APMA | = | p‐aminophenylmercuric acetate |
| mAb | = | monoclonal antibody |
| MTS | = | 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt |
| LDH | = | lactate dehydrogenase |
| HRP | = | horse radish peroxide |
| TMB | = | tetramethylbenzidine |
| NADH | = | nicotinamide adenine dinucleotide‐hydrogen |
| VEGF | = | vascular endothelial growth factor. |
Acknowledgements
The research in our laboratories has been supported by grants from the National Institute of Dental and Craniofacial Research (R01‐DE‐10985), the New York State Office of Science and Technology (SUNY Stony Brook Center for Biotechnology), the Center of Excellence Program of the Academy of Finland and TEKES, the Helsinki University Research Funds, the HUCH‐EVO (TYH 3306, TYH 2267, TI 020 Y 0002 and TYH 5306) grants, the Wilhelm and Else Stockmann Foundation, the Academy of Finland (T. Sorsa), the Invalid Foundation, the Finska Läkaresällskapet and the CollaGenex Pharmaceuticals, Inc. Y. Gu is a Predoctoral Dental Scientist Fellow of the NIDCR (F30‐DE05741).