Cellular inhibitor of apoptosis 1 (cIAP-1) degradation by caspase 8 during TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

Abstract

TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as co-treatment with a second mitochondrial activator of caspases (SMAC) mimetic (which results in rapid depletion of cIAP-1), sensitizes the cells to TRAIL. TRAIL-induced loss of cIAP-1 and XIAP requires caspase activity. In particular, caspase 8 knockdown stabilizes both cIAP-1 and XIAP, while caspase 9 knockdown prevents XIAP, but not cIAP-1 degradation. Cell-free experiments confirmed cIAP-1 is a substrate for caspase 8, with likely multiple cleavage sites. These results suggest that TRAIL-mediated apoptosis proceeds through caspase 8-dependent degradation of cIAP-1. Targeted depletion of cIAP-1 by SMAC mimetics in conjunction with TRAIL may be beneficial for the treatment of human hepatobiliary malignancies.

Figure 1. Degradation of cIAP-1 and XIAP is associated with TRAIL-mediated apoptosis

(A) Hepatocellular carcinoma cells HuH-7 were treated with increasing concentrations of TRAIL (0–20 ng/ml). At the indicated time points, cell lysates were obtained and analyzed by immunoblotting for cIAP-1, cIAP-2, and XIAP. Actin was used as protein loading control. After 6 hours of treatment, apoptosis was assessed (B) by fluorescence microscopy after DAPI staining and (C) by measuring caspase 3/7 activation (DEVDase activity; relative fluorescence units - RFLU) with a fluorogenic assay, as described in Experimental Procedures. (D) Cholangiocarcinoma cells Mz-ChA-1 were treated with TRAIL (20 ng/ml). At the indicated time points, cell lysates were obtained and analyzed by immunoblotting for cIAP-1, cIAP-2, and XIAP. Actin was used as protein loading control. (E,F) Mz-ChA-1 were treated with increasing concentrations of TRAIL (0–20 ng/ml) and apoptosis was assessed by (E) fluorescence microscopy and (F) caspase 3/7 activation. Values of RFLU are expressed as fold increase over control value (untreated).

Figure 2. Knock-down of cIAP-1, but not XIAP or cIAP-2, sensitizes to TRAIL-mediated apoptosis

(A) Clones of HuH-7 cells stably transfected with shRNA against cIAP-1, cIAP-2 or XIAP were assessed by immunoblot analysis to verify efficiency of protein knock-down. Actin was included as a loading control. (B) Selected cIAP-1, cIAP-2 or XIAP shRNA clones were treated with TRAIL (5 ng/ml) for 6 hr. Apoptosis was assessed (B) morphologically and (C) by measuring caspase 3/7 activation. Data are expressed as mean ± SEM compared to TRAIL-treated HuH-7 cells. The asterisk (*) indicates statistical significance of p < 0.05.

Figure 3. SMAC mimetic induces loss of cIAP-1 and enhances sensitivity to TRAIL-mediated apoptosis

(A) HuH-7, Mz-ChA-1, and Hep3B cells were treated with the SMAC mimetic JP1584 (500 nM). At the indicated time points, cell lysates were obtained and expression of cIAP-1 and XIAP was analyzed by immunoblotting. Actin was used as loading control. (B,C) Cells were treated with medium alone (vehicle - Veh), or human recombinant TRAIL (HuH-7: 5 ng/ml; Mz-ChA-1: 2.5 ng/ml; Hep3B: 20 ng/ml), or JP1584 (500 nM), or a combination (TR+JP) for 6 hr. Apoptosis was quantitated (B) by morphological criteria and (C) by measuring caspase 3/7 activation. The asterisk (*) indicates statistical significance of p < 0.05.

Figure 4. TRAIL-mediated degradation of cIAP-1 and XIAP requires caspase activity

(A) HuH-7 cells were treated with TRAIL (20 ng/ml). At the indicated time points, cIAP-1, cIAP-2 and XIAP mRNA were quantified by real time PCR. Fold increase was determined by normalization to 18S. (B) HuH-7 cells were treated with TRAIL (20 ng/ml) in the presence or absence of the cathepsin B inhibitor CRA 025850 (10 μM; 1 hr pre-incubation), or the proteasome inhibitor MG132 (10 μM; 1 hr pre-incubation), followed by immunoblot analysis for cIAP-1 and XIAP. (C) HuH-7 cells were treated with TRAIL (20 ng/ml) in the presence or absence of the vacuolar type H⁺-ATPase inhibitor bafilomycin A1 (0.1 μM). Expression of cIAP1 and XIAP was assessed by immunoblot analysis. (D) HuH-7 cells were transiently transfected with a plasmid encoding HA-tagged cIAP-1 with the H588→A mutation in the RING domain, and treated with either TRAIL (20 ng/ml; left panel) or JP1582 (500 nM; right panel); cell lysates obtained at the indicated time points were analyzed by immunoblot for expression of HA-cIAP-1 using an antibody against the HA tag. Actin was used as loading control. (E) HuH-7 cells were treated with TRAIL (20 ng/ml) in the presence or absence of the pan-caspase inhibitor Q-VD-OPH (5 μM; 30 min pre-incubation). Expression of cIAP1 and XIAP was assessed by immunoblot analysis at the indicated time points. Actin was used as loading control. (F) Immunoblots for cIAP-1 from four independent experiments as described in (E) were quantified by densitometry. cIAP-1/Actin ratios were calculated and normalized to time zero, which was arbitrarily set at 100%. Data are expressed as mean ± SEM.

Figure 5. TRAIL-mediated degradation of cIAP-1 is caspase-8 dependent

(A) Parental HuH-7 cells or clones of HuH-7 cells stably transfected with shRNA against caspase 9 or caspase 8 were treated with TRAIL (20 ng/ml). At the indicated time points, cell lysates were obtained and analyzed by immunoblotting for cIAP-1 and XIAP; effectiveness of knockdown was verified by caspase 8 and caspase 9 immunoblot. (B) Immunoblots for cIAP-1 from independent experiments (n=3 for wild-type and 22-1; n=4 for 22-5; n=2 for 74-4 and 74-5) as described in (A) were quantified by densitometry. cIAP-1/Actin ratios were calculated at 6 hr and normalized to time zero, which was arbitrarily set at 100%. Data are expressed as mean ± SEM. (C) HuH-7 cells were treated with TRAIL (20 ng/ml) and co-immunoprecipitation of RIP1 and caspase 8 was performed at the indicated time points as described in Experimental Procedures. (D) HuH-7 cells were treated with TRAIL (20 ng/ml); total cell lysates were obtained at the indicated time points and analyzed by immunoblotting for caspase 8, RIP1, cIAP-1 and TRAF2. Actin was used as loading control. (E) Caspase 8 shRNA HuH-7 cells (clone 22-5) were treated with TRAIL (20 ng/ml); total cell lysates were obtained at the indicated time points and analyzed by immunoblotting for RIP1. Actin was used as loading control.

Figure 6. cIAP-1 is cleaved by caspase-8 in cell-free system and in vivo

(A) Recombinant human cIAP-1 was incubated with human recombinant active caspase 8 (1 U) or human recombinant active caspase 3 (0.5 unit), in the presence or absence of Q-VD-OPH (100 μM) for 30 minutes at 37°C and analyzed by immunoblot. Newly generated fragments are indicated by the arrows (left panel). As controls, recombinant human Bid or PARP were incubated with human recombinant active caspase 8 (0.25-1 U) or human recombinant active caspase 3 (0.5 unit), respectively, for 30 minutes at 37°C and analyzed by immunoblot (right panel). (B) HuH-7 cells were incubated with TRAIL (20 ng/ml) and MG132 (10 μM; 1 hr pre-incubation), in the presence or absence of Q-VD-OPH (5 μM) or z-IETD-fmk (10 μM). Cell lysates were analyzed by immunoblot for cIAP-1 and actin (loading control). The asterisk (*) indicates a fragment of approximately 32 kDa generated both in vivo and in the cell-free system.