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. 2012 Apr 23;23(9):992–1002. doi: 10.1089/hum.2011.159

FIG. 2.

FIG. 2.

Activation and inhibition of caspase-8 affect cell viability with the expression of TRAIL and Smac transgenes. (A) Bel-7404 cells were treated with Ad-TRAIL-IETD-Smac or ZD55-TRAIL-IETD-Smac (10 MOI) and it was found that expression of TRAIL-IETD-Smac and its cleavage by caspase-8 to produce TRAIL-IETD and Smac in the Ad-TRAIL-IETD group were less than in the ZD55-TRAIL-IETD-Smac group. (B1) Bel-7404 cells were treated with ZD55-TRAIL-IETD-Smac (10 MOI) for 72 hr, either alone or in combination with caspase-8 inhibitor (Z-IETD-FMK, 20 μM). The cells were collected and cell lysates were analyzed by Western blotting for detection of expression of TRAIL and Smac, and activation of caspase-8. In addition, Ad-TRAIL-IETD-Smac was used as a control to prove that the fusion protein TRAIL-IETD-Smac is cleaved by caspase-8 activated by oncolytic virus infection. (B2) Two cancer cell lines were treated with Z-IETD-FMK (20 μM), ZD55-TRAIL (5 MOI) plus ZD55-Smac (5 MOI), and ZD55-TRAIL-IETD-Smac (10 MOI) with or without Z-IETD-FMK for the indicated times. Cell viability was determined by MTT assay. Data are presented as means±SD of three independent experiments. **p<0.01 versus ZD55-TRAIL-IETD-Smac. (C1) Expression of both TRAIL and Smac, and caspase-8 cleavage, were detected by Western blot analysis when Bel-7404 cells were treated with ZD55-TRAIL-IETD-Smac and/or 5-fluorouracil (5-FU) (0.8 μg/ml) for 72 hr. (C2) The MTT assay was used to determine the viability of Bel-7404 and Huh-7 cells treated with ZD55-TRAIL-IETD-Smac, 5-FU, or ZD55-TRAIL-IETD-Smac plus 5-FU at the indicated doses for 48 hr. Data are presented as means±SD of three independent experiments. The Student t test was used to determine significant differences, **p<0.01 versus ZD55-TRAIL-IETD-Smac.