Mixed backbone antisense AS-3m inhibits VEGF mRNA and protein production.

(A) Total RNA was isolated from KS Y-1 cells treated with various concentrations of AS-3m as indicated (NT indicates not treated). Total RNA was reverse-transcribed to generate cDNA. Aliquots of the reaction mixture were removed at 5-cycle intervals to provide semiquantitative analysis for amplification of VEGF relative to β-actin as described in “Materials and methods.” Integrity of RNA in the samples was verified by β-actin amplification. (B) Band intensities shown in panel A were quantitated using the Quantity One program (Bio-Rad). Shown are estimated PCR products as a function of cycle; the units are arbitrary for each RT-PCR. Bands were undetectable below 25 cycles for VEGF. (C) Effect of AS-3m on VEGF protein production in 2 tumorigenic cell lines: Human melanoma cell line M21 (left panel) and human ovarian carcinoma cell line Hey (right panel) were treated with VEGF antisense AS-3m and the scrambled MBO at concentrations ranging from 1 to 10 μM. Supernatants were collected at 24 hours, and VEGF protein was quantitated by ELISA. The results represent the means of duplicate determinations. (D) Western blot of M21 cell lysates. Cells were treated for 8 hours with the ODNs indicated and harvested 36 hours later. Crude protein extracts were prepared from the cells, and 20 μg each was fractionated by 4% to 20% Tris-glycine polyacrylamide gel electrophoresis. Proteins were electrotransferred to nylon membranes and immunoblotted with antibodies to VEGF or IL-8 (1 μg/mL) as described in “Materials and methods.”