Figure 1
mCD40L initiates a caspase-dependent mitochondrial apoptotic pathway in carcinoma cells but not normal cells. (a) mCD40L induces apoptosis (expressed as fold change versus controls) in EJ and HCT116 carcinoma cells and death is caspase-mediated as demonstrated by functional blockade of apoptosis using the pan-caspase inhibitor z-VAD (50 μM). In normal (NHU) epithelial cells, mCD40L did not cause apoptosis and was cytoprotective. Bars represent mean fold change ±s.d. (n=6). Of note, a 3-4-fold change in these apoptosis detection assays corresponded to approximately ~80–85% dead cells in Annexin V-FITC/PI flow cytometry assays.8 (b) mCD40L-induced death in EJ and HCT116, but not normal NHU, cells and involves caspase-3/7 activation. Bars represent mean fold caspase activity ±s.d. (n=5). (c) Unlike mCD40L that causes extensive apoptosis in EJ and HCT116 carcinoma cells, soluble CD40 agonist (G28-5 agonistic anti-CD40 antibody) is only weakly pro-apoptotic. Bars represent mean fold change ±s.d. (n=3). (d) CD40 ligation induces Bak and Bax expression (24 h) in EJ carcinoma cells as shown by immunoblotting. By contrast, in NHU cells, a small reduction in expression of Bax and particularly Bak was observed. CK18 detection was used for the comparison of controls (‘–’) versus mCD40L-treated cells (‘+’) to confirm equal epithelial lysate loading (as explained in the Methods). Of note, although equal amounts of protein were loaded for EJ and NHU cell lysates, normal cells naturally expressed higher levels of CK18. Results are representative of three independent experiments. (e) Immunoblotting analysis of cytoplasmic (Cyto) and mitochondrial (Mito) sub-cellular fractions following mCD40L-mediated CD40 ligation (24 h) in EJ cells. Western blot confirms induction of the mitochondrial apoptotic pathway by demonstrating mCD40L-mediated cytochrome c (Cyt c) release from the mitochondria to the cytoplasm. Detection of Bcl-2 and GAPDH was used to confirm successful sub-cellular fractionation. Results are representative of at least two independent experiments.
