Autophagy inhibition by chloroquine sensitizes HT-29 colorectal cancer cells to concurrent chemoradiation

Figure 1 Radiation-induced autophagy. A: HT-29 cells were irradiated (8 Gy) and autophagy induction was assessed by the formation of double membrane autophagosomes on transmission electron microscopy 24 h post-treatment; B: Western blot analysis of LC3 expression demonstrates increased LC3-II:I ratio and autophagy induction at 4 and 6 h post-irradiation for HT-29 and HCT116 cells, respectively; C: Increased LC3II:I ratios are seen 24 h post-irradiation at 2-8 Gy for HT-29 cells; D: GFP-labeled LC3 puncta develop within 6 h of treatment with Rapamycin (200 nmol/L) or radiation (2-8 Gy) in both HCT116 and HT-29 cell lines. Bar graphs represent quantification of percentage of cells with perinuclear punctate pattern 6 h following radiation.

Figure 2 Chemoradiation-induced autophagy. A: Western blot of LC3 in HT-29 cells harvested 24 h after treatment with RT (4 Gy), 5-FU (15 μmol/L) or 5-FU and RT; B: Western blot of LC3 in HT-29 cells harvested 24 h after treatment with RT (8 Gy), 5-FU (15 μmol/L) or 5-FU and RT; C: GFP-labeled LC3 fluorescence in HT-29 cells fixed 6 h after treatment with Rapamycin (200 nmol/L), 5-FU (15 μmol/L) or combination RT (2-8 Gy) and 5-FU (15 μmol/L). RT: Radiation therapy; 5-FU: 5-fluorouracil.

Figure 3 Radiosensitizing effects of autophagy inhibition by chloroquine. A: MTT assays in HCT-116 and HT-29 cell lines 72 h after treatment with CQ (10 μmol/L) and RT (2-8 Gy); B: Clonogenic survival of HCT-116 and HT-29 cells following treatment with CQ (0.1 and 0.5 μmol/L, respectively) and/or RT (2-8 Gy); C: Western blot in HT-29 cells harvested 24 h post-treatment with CQ (10 μmol/L) and/or RT (8 Gy); D: FACS analysis in HT-29 cells 24 h following treatment with CQ (10 μmol/L) and/or RT (8 Gy); E: Annexin V and PI staining in HT-29 cells 24 h following treatment with CQ (10 μmol/L) and/or RT (8 Gy). CQ: Chloroquine; RT: Radiation therapy.

Figure 4 Chloroquine sensitizes colorectal cancer cells to chemoradiation. Clonogenic survival of HT-29 cells treated with RT (2-8 Gy) and CQ (0.5 μmol/L), 5-FU (0.5 μmol/L) or CQ and 5-FU. CQ: Chloroquine; 5-FU: 5-fluorouracil.