Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell Apoptosis In Vivo Primarily by Inhibiting VEGF Expression and Angiogenesis
Figure 4
Ectopic VEGF expression rescues tumor cells from the antitumor effects of temsirolimus in vivo. (a) NOD/SCID mice (6 mice/group) were challenged subcutaneously with 1 × 106 HS Sultan cells stably transfected with exogenous VEGF expressing p27IRES-VEGF transfected cells (filled symbols) on the right flank and 1 × 106 control (Rev)p27IRES-VEGF transfected cells (open symbols) on the left flank. When tumor size reached approximately 500 mm3, mice were randomly assigned to receive vehicle alone, 2 mg/kg or 20 mg/kg temsirolimus IP for 10 days, as described in “Materials and Methods.” Results are presented as tumor volume (mean ± SEM). Solid bars on -axis denote days of IP treatment. Asterisk denotes significant difference () between the growth curves for p27IRES-VEGF transfected cells (filled squares) and (Rev)p27IRES-VEGF transfected cells (open squares) in the group of mice treated with 20 mg/kg temsirolimus. (b) Cleaved caspase-3 staining of isogenic HS Sultan xenografts harvested from mice at day 13 was used to identify apoptosis. Results are presented as number of cleaved caspase-3 stained cells/microscopic field (original magnification 20X), mean ± SD, fields for each tumor, and 4 tumors/group. Asterisk denotes significant difference () between control and temsirolimus-treated mice. (c) Effect of temsirolimus treatment on the MVD (#microvessel/area of microscopic field (original magnification, X20)) stained positive for CD34 and assessed as described in “Materials and Methods.” Data are mean ± SD, fields/tumor, and 4 tumors/group. Asterisk denotes significant difference () between control and temsirolimus (20 mg/kg) treated mice. (d) Representative slides of CD34-stained isogenic HS Sultan xenograft sections from mice treated with vehicle control (top panels) or 20 mg/kg temsirolimus (bottom panels) harvested on day 13. Original magnification, ×40. Arrow shows microvessel.
