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 microM 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 approximately 20%-30% in the presence of 20 microM CMT-3, reflecting inhibition of MMP-9 activity in addition to diminution of released MMP-9 levels. TNF-alpha 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.
Gu
Ying
Y
Department of Biochemistry, School of Dental Medicine, State University of New York at Stony Brook, NY 11794-8702, USA.
Lee
Hsi-Ming
HM
Golub
Lorne M
LM
Sorsa
Timo
T
Konttinen
Yrjö T
YT
Simon
Sanford R
SR
D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000187:drug effects*; D018093:Gelatinases / Q000037:antagonists & inhibitors*; D006801:Humans; D015151:Immunoblotting; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis*; D013754:Tetracyclines / Q000494:pharmacology*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis
D013754:Tetracyclines; D019715:Tissue Inhibitor of Metalloproteinase-1; C117155:tetracycline CMT-3; D018093:Gelatinases; D020780:Matrix Metalloproteinase 9