Inhibition of human collagenase activity by antisense oligonucleoside methylphosphonates

Oligodeoxyribonucleoside methylphosphonates (d-OMP) were synthesized whose sequences are complementary to sequences found in the mRNA coding for the 72-kDa (MMP-2) or 92-kDa (MMP-9) forms of human collagenase IV, matrix metalloproteinases (MMP) whose excessive secretion correlates with the metastatic potential of tumor cells. The effects of these oligomers on MMP-2 and MMP-9 activities secreted by HT1080 cells, a human fibrosarcoma cell line, were studied using a gelatin zymography assay. A d-OMP, M2.3, complementary to nucleotides 14 to 28 of the initiation codon region of MMP-2 mRNA selectively inhibited MMP-2 activity, whereas a d-OMP, M9.1, which was targeted to nucleotides -19 to -5 of the 5'-untranslated region of MMP-9 mRNA selectively inhibited MMP-9 activity over the concentration range 5-50 μM. At 100 μM concentration, both M2.3 and M9.1 inhibited the activities of both MMP-2 and MMP-9. These oligomers were completely stable under cell culture conditions and did not appear to adversely affect cell growth after 48 hours at concentrations up to 100 μM, although 100 μM M9.1 did reduce cell growth 30% after prolonged, 120-hours exposure. Other d-OMP tested either had no effect on collagenase activity or inhibited both MMP-2 and MMP-9 activities. The latter oligomer was complementary to MMP2 mRNA and partially complementary to MMP-9 mRNA. Oligomer M2.3 was also tested for its effects on the morphology of malignant human lung cells, BZR-T33, growing on the surface of reconstituted base membrane, Matrigel, in culture. In the absence of oligomer, the BZR-T33 cells formed extensive networks indicative of the ability of the cells to invade the Matrigel substrate. In the presence of 100 μM M2.3, BZR-T33 formed colonies of rounded cells, a morphology typical of noninvasive cells. Other non-complementary d-OMP had no effect on the morphology of BZR-T33 under these conditions. These results suggest that antisense d-OMP may be useful for inhibiting expression of collagenase in human tumor cells and for studying the role of collagenase expression in tumor cell metastasis.