Figure 6
Combination of pharmacological Trx-1 inhibitor and soluble CD40 agonist is pro-apoptotic, tumour-specific and functionally equivalent to mCD40L. (a) Normal epithelial cells (NHU) were treated with either Trx-1 inhibitor PX-12 (4μM) or soluble CD40 agonist G28-5 mAb (10μg/mL) alone, or with combination of the two (‘PX-12+G28-5’) and apoptosis was assessed (see Methods). Neither the inhibitor nor the agonist alone induced any apoptosis and nor did their combination, which was slightly cytoprotective. Bars represent mean fold change ±s.d. (n=3). (b) Combinatorial treatment of EJ and HCT116 carcinoma cells with Trx-1 inhibitor (4μM) and soluble CD40 agonist (10μg/mL) (‘PX-12+G28-5’) synergistically mediated extensive apoptosis, whereas treatment with either the inhibitor (PX-12) or agonist (G28-5) alone caused little (EJ) or relatively low (HCT116) amounts of apoptosis. Bars represent mean fold change ±s.d. (n=5). (c) Using immunoblotting, the levels of TRAF3 protein were determined in malignant epithelial cells (EJ) treated with Trx-1 inhibitor PX-12 (2μM) or soluble CD40 agonist G28-5 mAb (10μg/mL) alone, or with combination of the two (‘PX-12+G28-5’) (6 h). Solvent alone (DMSO) treated EJ cells (Control) were used as a control for experiments involving soluble agonists, whereas controls (‘–’) versus mCD40L-treated cells (‘+’) representing 3T3Neo/EJ and 3T3CD40L co-cultures (6 h), respectively, were used to detect TRAF3 protein expression following mCD40L treatment. The results demonstrate that CD40 agonist G28-5 mAb alone caused little TRAF3 induction, however its combination with PX-12 induced TRAF3 expression as strongly as did mCD40L. (d) Apoptosis triggered by the combination of Trx-1 inhibitor and soluble CD40 agonist (as above) was inhibited by the antioxidant NAC (30 mM) as well as by knockdown of ASK-1, as shown by the lack of apoptosis in ASK-1 shRNA expressing (EJ-ASK1-KD) cells in comparison to their isogenic controls (EJ-Con). Bars represent mean fold change ±s.d. (n=6).
