Figure 3.

Cetuximab induces autophagy that is related to the weak induction of apoptosis by cetuximab in A431 cells. (A) Cetuximab induced a weak apoptosis in A431 cells. A431 cells were either untreated or treated with cetuximab, as indicated. Cell lysates were analyzed for the levels of histone-associated DNA fragmentation in the cytosol by an ELISA. (B) Cetuximab induced appearance of membrane-associated LC3 (punctate fluorescence). A431 cells were transiently transfected with a GFP-LC3 construct for 24 h and then treated with cetuximab or untreated as indicated. The number of cells with punctate fluorescence was counted in 10 different fields under a fluorescent microscope and is shown as a percentage of the total number of cells counted. (C) Cetuximab induced formation of autophagosomes. A431 cells were either untreated or treated with cetuximab for 48 h. Cell samples were prepared for transmission electron microscopy analysis, as described in Materials and Methods. A magnified view of the electron photomicrograph shows a characteristic autophagosome. N = nucleus. (D) Inhibition of the autophagic flux led to accumulation of cetuximab-induced LC3-II. A431 cells were either untreated or treated with 10 nM cetuximab for 24 h, in the absence or presence of bafilomycin A1 (BFA, 200 nM), chloroquine (CQ, 100 µM) or NH₄Cl (50 mM). Cell lysates were analyzed by western blot. β-actin was used as protein-loading control. (E) Inhibition of caspase abolished cetuximab-induced autophagy. A431 cells were first treated with 100 µM Z-VAD-fmk or vehicle control (DMSO) for 6 h and then treated for another 24 h in the presence or absence of 10 nM cetuximab. Cell lysates were analyzed by western blot. (F–H) Lysosomal inhibition enhanced cetuximab-induced apoptosis. (F and G), A431 cells were either untreated or treated with cetuximab in presence or absence of chloroquine or NH₄Cl for 24 h, as indicated. The cell lysates were analyzed by (F) western blot and (G) an apoptosis ELISA. (H), A431 cells were either untreated or treated with cetuximab in the presence or absence of chloroquine, for 72 h, as indicated. The relative number of surviving cells was determined with an MTT assay. The optical density (O.D.) values of the treated cells at wavelength 570 nM were normalized to the O.D. value of the untreated cells (control) and expressed as a percentage of the O.D. value of the control. Values in (A, B, G and H) are means ± SD. p values for the comparisons were determined by Student's t-test.