Figure 8

Chloroquine increases the pro-apoptotic effects of 3PO against LLC cells and tumors in vitro and in vivo . LLC cells were treated with 25 μM 3PO for 24 hours and LC3-II levels were measured using immunoblotting (A) and quantitative densitometry (B). Levels expressed as mean fold change LC3-II/β-actin relative to control ± SD (B). LLC cells were then treated with either vehicle or 10 or 25 μM 3PO ± either 15 or 30 μM CQ. After 24 hours of treatment, cells were stained with annexin-V and PI and measured using flow cytometry (C). Cells staining positive for both annexin-V and PI were quantitated as the percentage of the total relative to control and data is presented as the mean ± SD from three experiments (E). C57/BL6 mice were inoculated with 1x10⁶ LLC cells by subcutaneous flank injection. Mice were randomized into four treatment groups when tumors reached 150–200 mm³ and were treated by i.p. injections with either vehicle, 50 mg/kg CQ, 0.07 mg/g 3PO, or a combination of the two drugs. Tumor measurements taken over the course of treatment were used to calculate tumor mass. Data is presented as mean tumor mass ± SD (D). Tumors were fixed, paraffin embedded, and stained with an antibody directed against cleaved caspase-3 (CC3) (F). The number of cells staining positive for CC3 in five 200X fields were counted and the data is expressed as the mean ± SD from three counts (G) (*P <0.05).