Figure 2

Modulation of XIAP expression by cadmium occurs via NF-kappaB–independent, proteasome-mediated mechanism. (A) The effect of NF-κB inhibitor BAY 11-7085 and proteasome inhibitor MG132 on the expression of XIAP, cIAP1 and cIAP2 in PC-3 prostate cancer cells. Cells were treated with either BAY 11-7085 (5 μM) or MG132 (5 μM) for 16 hours. Levels of XIAP, cIAP1 and c IAP2 proteins were detected in cell lysates by immunoblotting using specific antibodies. Expression of α-actin was used to control equal protein loading. (B) Luciferase reporter assay of NF-κB activity in PC-3 cells. Cells were pre-incubated with cadmium (30 μM) or NF-κB inhibitor BAY 11-7085 (5 μM) for 3 hours followed by incubation with or w/o TNF-α (20 ng/ml) for an additional 4 hours. Columns, means of three different samples; bars, SEM. (C) Analysis of proteasome activity in PC-3 cells. Cells were incubated with cadmium (30 μM) or the proteasome inhibitor MG132 (5 μM) for 3 hours. Chymotrypsin- and trypsin-like activities were examined as described in Materials and Methods. Columns, means of three different samples; bars, SEM. (D) PC-3 cells were transfected with the N-terminally HA-tagged XIAP construct under the control of the NF-κB independent SV40 promoter. Four hours after transfection cell culture medium was replaced with medium containing either cadmium (30 μM) or MG132 (5 μM) or BAY 11-7085 (5 μM) and cells were cultured for additional 16 hours. Expression of XIAP and α-actin was detected by immunoblotting with anti-HA or anti-actin antibodies respectively. Representative data from one of three experiments is shown.