Mixed backbone antisense AS-3m or VEGFR antibody inhibits tumor cell proliferation in vitro.

Cells were seeded at 1 × 10⁴ cells per well in 24 plates and treated with AS-3m (1, 5, 10 μM) on days 1 and 3 (A). Cell viability was performed on day 5 by MTT assay. Results represent the mean ± SD of quadruplicate samples. Specificity of the AS-3m ODN is shown by the lack of significant cytotoxicity in any cell line of the scrambled ODN (right panel). (B) The rhVEGF abrogates the effect of VEGF antisense. Cell lines M21 and Hey were seeded as above and were treated with 1, 5, and 10 μM AS-3m alone or with rhVEGF (10 ng/mL) on day 1 and day 2. Cell viability was measured after 72 hours. AS-3m inhibition of cell proliferation in both cell lines (black columns) could be reversed by the presence of VEGF (white columns), which did not have any appreciable effect on the growth of cells (hatched columns). The data represent the mean ± SD of 2 experiments performed in quadruplicate. (C) Cell viability studies were repeated with VEGFR-2 neutralizing antibody or unrelated (perforin polyclonal) antibody. VEGFR-2 inhibited the viability of the cell lines shown to express VEGFRs. No significant effect was seen on cell lines not expressing VEGFRs or with unrelated antibody. (D) VEGFR-1 antibody reduces proliferation in cell lines that express VEGFR-1 and VEGFR-2. Cultures of M21 or Hey were treated as in panel C with VEGFR-1 antibody, VEGFR-2 antibody, or both. VEGFR-1 reduced cell proliferation but to a lesser extent than VEGFR-2. Both antibodies result in a more pronounced decrease than with VEGFR-2 alone.