Table 2.

Cathepsin B plays an important role in GST-MDA-7 toxicity in transformed cells

Cell type	Treatment
	Vehicle	z-VAD	LEHD	CMV vehicle	Dominant-negative caspase-9 vehicle	CMV cathepsin B inhibitor	Dominant-negative caspase-9 cathepsin B inhibitor
WT MEF + GST	2.1 ± 0.2	2.5 ± 0.2	2.0 ± 0.3
WT MEF + GST-MDA-7	17.4 ± 0.3	6.3 ± 0.6*	6.9 ± 0.4*
Cathepsin B−/− MEF + GST	1.6 ± 0.4	2.9 ± 0.7	2.7 ± 0.5
Cathepsin B−/− MEF + GST-MDA-7	6.8 ± 0.5*	2.7 ± 0.3*	3.1 ± 0.6*
GBM6 + GST				3.4 ± 0.3	5.0 ± 0.8	5.7 ± 0.4	4.8 ± 0.8
GBM6 + GST-MDA-7				24.2 ± 1.2	7.5 ± 0.2*	9.5 ± 1.1*	6.6 ± 0.2*
GBM12 + GST				2.0 ± 0.1	2.5 ± 0.2	3.6 ± 0.5	5.1 ± 0.6
GBM12 + GST-MDA-7				16.0 ± 0.6	5.3 ± 0.4*	5.4 ± 0.7*	4.5 ± 0.3*

NOTE: MEFs (WT; cathepsin B−/−) were plated and 36 h after plating were pretreated with vehicle (DMSO), pan-caspase inhibitor (z-VAD, 50 μmol/L), or caspase-9 inhibitor (LEHD, 50 μmol/L). Thirty minutes after inhibitor addition, cells were treated with GST or GST-MDA-7 (60 nmol/L). Inhibitors were resupplemented every 24 h. Cells were incubated for 72 h after which time cell viability was assessed in triplicate by trypan blue assay (±SE; n = 3). In parallel, primary human glioma cells (GBM6 and GBM12) were plated and 12 h after plating were infected with adenoviruses at a final multiplicity of infection of 50 to express as per Fig. 1: CMV or dominant-negative caspase-9. Cells were cultured for 24 h after infection and then treated with the cathepsin B inhibitor (1 μmol/L, cathepsin B inhibitor) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were incubated for 72 h after which time cell viability was assessed in triplicate by trypan blue assay (±SE; n = 3; #, P < 0.05, lower than CMV-infected vehicle-treated cells with GST-MDA-7 exposure; ##, P > 0.05, no difference between GST- and GST-MDA-7-treated cells).

^*P < 0.05, lower than vehicle-treated cells with GST-MDA-7 exposure.