Chloroquine augments radiation-induced apoptosis in NPC cells.

<p>A) Cell cycle analysis. Radiation (6 Gy) induces a G2M arrest in all 5 NPC cell lines and the immortalized nasoepithelial cell line NP69. Radiation also induces apoptosis measured by an increase in subG1 in all NPC cell lines except of C666-1. Treatment with chloroquine (20 μM) significantly augments apoptosis in radiation-sensitive NPC cell lines compared to cells radiated only (CNE-2 and HNE-1: P<0.001; CNE-1: P<0.01; HONE-1: P<0.05; Student’s t-test). The data represent the means of three independent experiments and the corresponding standard error. (B) Combined treatment with chloroquine and radiation increases the number of cells with activated caspase-3 in radiation-sensitive NPC-cell lines. Quantitative data are reported as means ± S.E.M. (triplicate samples) (Student’s t- test; <b>*</b> = P<0.05; <b>**</b> = P<0.01; <b>***</b> = P<0.001). (C) Hoechst 33258 staining. Pretreatment with chloroquine before radiation increases the percentage of cells with morphological signs of apoptosis (condensed and fragmented nuclei) in radiation-sensitive NPC cell lines CNE-2 and HNE-1, but not in the radioresistant cell line C666-1 and the immortalized nasoepithelial cell line NP69. Morphologic changes were examined under a fluorescence microscope at 200x magnification, phase contrast images are shown for cell density comparison. Data of all experiments were shown at 72h after radiation.</p

Knock-down of ATG3, ATG5, ATG6 or ATG7 by siRNA substitutes for the enhancing effect of chloroquine on radiation-induced apoptosis in NPC cells.

<p>Silencing of ATG3, ATG5, ATG6 or ATG7 by specific siRNA enhances radiation-induced apoptosis to a similar extent as chloroquine in radiation-sensitive NPC cell lines treated with scrambled siRNA as shown by an increase in subG1-DNA-content (A) or increase of active caspase-3 (B) both measured by flow cytometry. After transfection the cells were treated as described before. Quantitative data are reported as means ± S.E.M. (triplicate samples) (Student ‘s t- test; <b>*</b> = P<0.05).</p

Chloroquine blocks radiation-induced autophagy in NPC cells.

<p>(A) Immunoblot for LC-I and LC-II. Preincubation with chloroquine before radiation increases expression of LC-II in radiation-sensitive NPC cells 8h post treatment (B) Immunofluorescence for autophagic vacuoles. Combined treatment of chloroquine and radiation increases the formation of autophagic vacuoles 8h post treatment in cell lines CNE-2 and HNE-1. Autophagic vacuoles were examined under a fluorescence microscope at 200x and 400x magnification. (C) Flow cytometric analysis of autophagic vacuoles. Combined treatment of chloroquine and radiation increases the number of cells with autophagic vacuoles 8h post treatment in radiation-sensitive NPC cell lines. (Student’s t- test; <b>*</b> = P<0.05; <b>**</b> = P<0.01; <b>***</b> = P<0.001) (D) Transmission electron microscopy. Photomicrographs show normal nuclear and mitochondrial morphologies in untreated cells and cells treated with chloroquine. Especially, in irradiated CNE-2 and HNE-1 cells the number of autophagosomes is significantly increased 8h following radiotherapy and further augmented by pretreatment with chloroquine.</p