Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell Apoptosis In Vivo Primarily by Inhibiting VEGF Expression and Angiogenesis
Figure 1
Antitumor effects on HS Sultan cells in vivo and in vitro. (a) Cell cycle and apoptosis analysis of HS Sultan cells. G1/S arrest induced by 48-hour incubation with rapamycin was measured by propidium iodide to determine the percentage of cells in each phase (G1, S, or sub-G1), and anticleaved caspase-3 antibody was used to determine the percentage of cells undergoing apoptosis. Results shown are mean ± SEM of 4 independent experiments. (b) NOD/SCID mice (6 mice/group) were challenged subcutaneously with HS Sultan cells. When tumor size reached approximately 500 mm3, mice were randomly assigned to receive vehicle alone or varying doses of 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. Asterisks denote significant difference () between control and temsirolimus-treated mice. (c) Cleaved caspase-3 staining of 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. (d) VEGF-specific ELISA assay for expression of VEGF collected from HS Sultan xenografts lysate harvested from mice treated with vehicle control or temsirolimus (tumors collected on day 13). Lysate was pooled ( tumor/group) and are presented as the mean ± STD. Asterisk denotes significant difference () between control and temsirolimus-treated mice. (e) Left panel. Representative slides of CD34-stained HS Sultan xenograft sections from mice treated with vehicle control (top panel) or 20 mg/kg temsirolimus (bottom panel) harvested on day 13. Original magnification, ×40. Arrow shows microvessel. Right panel. Results represent number of microvessel/area of microscopic field (original magnification, ×20) 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-treated mice.
