Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell Apoptosis In Vivo Primarily by Inhibiting VEGF Expression and Angiogenesis
Figure 2
mTOR inhibition induces apoptosis and hypoxia in HS Sultan xenografts. (a) Analysis of pimonidazole staining (a marker of hypoxia) of HS Sultan xenografts harvested from mice at day 13. The area of pimonidazole staining/microscopic field (original magnification 20X) was measured by morphometric analysis as described in the “Materials and Methods.” Data are presented as mean ± SD, fields for each tumor, and 4 tumors/group. Asterisk denotes significant difference () between control and temsirolimus-treated mice. (b) Representative photomicrographs of immunohistochemistry of serial tumor sections of control or temsirolimus-treated tumors stained for hypoxia or cleaved caspase-3. *indicates same geographical location of tumor section pairs. Arrows indicate regions of apoptosis (in panels (ii) and (iv)) that colocalize to regions of hypoxia in corresponding serial section. Panels (i) (hypoxia) and (iii) (cleaved caspase-3) show serial sections from HS Sultan xenograft harvested on day 13 from vehicle control treated mouse. Panel (ii) (hypoxia) and panel (iv) (cleaved caspase-3) show serial sections from HS Sultan xenograft harvested on day 13 from 20 mg/kg temsirolimus-treated mouse. Additional serial tumor sections are shown in panel (v) (stained for hypoxia), panel (vi) (stained for apoptosis), panel (vii) (stained for hypoxia), and panel (viii) (stained for apoptosis) from HS Sultan xenografts harvested on day 13 from 20 mg/kg temsirolimus-treated mice. Arrows and (*) in paired sections (panels (v) and (vi), panels (vii) and (viii)) indicate corresponding geographical regions. (c) Cleaved caspase-3 staining of HS Sultan xenografts harvested from mice at day 13 was used to identify apoptosis in normoxic and hypoxic regions. Hypoxic and normoxic regions were identified in pimonidazole-stained sections, and then the apoptotic index was determined in corresponding serial sections of cleaved caspase-3 stained slides. Results are number of cleaved caspase-3 stained cells/microscopic field (original magnification 20X) in either normoxic or hypoxic regions, mean ± SD, fields for each tumor, and 4 tumors/group. Asterisk denotes significant difference () between control and temsirolimus-treated mice.
