Figure 4

Effect of AM9D on the rate of tumor growth, final mean tumor volume, and metalloproteinase (MMP)-9 expression. (A) AM9D-treated tumors (■) grew at a slower rate than either untreated tumors (no injections) (▼), or tumors treated with control DNAzyme (▲); at the study endpoint, age 12 weeks, P <0.05. (B) Weekly intratumoral treatment of transgenic mice with 10 µg (n = 9 tumors) or 25 µg AM9D (n = 21 tumors) per tumor reduced mean tumor burden by 39.5% or 50.1%, respectively, when compared to tumors treated with control DNAzyme (n = 24 tumors) (P <0.01 ANOVA). (C) Mammary tumors treated with either 25 µg AM9D or control DNAzyme were stained with an MMP-9 antibody. (a) Mammary tumors treated with 25 µg control DNAzyme for 4 weeks showed increased MMP-9 staining (arrows) compared to (b) mammary tumors treated with AM9D for 4 weeks. Images are shown at 200× magnification; scale bar is equivalent to 100 µm. (D) Mmp9 mRNA expression levels in control DNAzyme- or AM9D-treated tumors. Total RNA was isolated and pooled from optical cutting temperature (OCT) compound-embedded tumor sections scraped from glass slides of individual control DNAzyme- or AM9D-treated tumors. Mmp9 and Bact (ß-actin) mRNA were amplified by RT-PCR and the PCR products were subjected to agarose gel and visualized by ethidium bromide staining. Lane 1, AM9D; lane 2, control DNAzyme.