X chromosome-linked inhibitor of apoptosis regulates cell death induction by proapoptotic receptor agonists

Abstract

Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-alpha signaling. By contrast, blockade of tumor necrosis factor-alpha does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.

FIGURE 1.

IAP antagonist BV6 synergizes with DR5 agonist antibody (αDR5) and FasL to stimulate apoptosis in cancer cells. HT1080 and UACC62 cells were treated with increasing concentrations of BV6 alone or in combination with 50 ng/ml αDR5 (upper panels) or 400 ng/ml FasL (lower panels) for 24 h. Cell viability was determined as described under “Experimental Procedures.” Results represent mean ± standard deviation from three independent experiments. Bars with numbers designate statistical significance.

FIGURE 2.

IAP antagonist BV6 potentiates DR5 agonist antibody (αDR5)- or FasL-induced caspase activation. HT1080 cells were treated for the indicated periods of time with 1 μm BV6 and 200 ng/ml αDR5 (A) or 1 μg/ml FasL (B) alone or in combination. The protein levels of c-IAP1, XIAP, caspase-8, caspase-9, caspase-3, and tubulin were analyzed by Western blotting. The arrows indicate positions of full-length and processed forms of caspases. Protein levels of c-IAP1 are shown to demonstrate BV6 activity under the experimental conditions. Tubulin was used as loading control. Caspase-8 (C), caspase-3/7 (D), or caspase-9 (E) activity was assessed using caspase-specific fluorogenic substrates.

FIGURE 3.

DR5 agonist antibody (αDR5) and IAP antagonist BV6 synergistically inhibit tumor growth. A, effect of IAP antagonist BV6 and αDR5 treatment on the viability of MDA-MB-231-X1.1 cells. Cells were treated with BV6 (100 nm) or αDR5 (200 ng/ml) alone or in combination for 24 h. Cell viability was determined as described under “Experimental Procedures.” Results represent mean ± standard deviation from three independent experiments. B, antitumor activity of BV6 and αDR5 in vivo. SCID.bg mice were injected subcutaneously with MDA-MB-231-X1.1 cells, and tumors were allowed to grow to a mean volume of ∼ 150 mm³. Tumor-bearing mice were randomly assigned into groups (five animals/group) and treated with vehicle, BV6 (q4d × 6), αDR5 (once on day 0) or combination of BV6 and αDR5 as indicated. Tumor volume measurements and calculations were done as described under “Experimental Procedures.”

FIGURE 4.

Proapoptotic synergistic activity of IAP antagonist BV6 and DR5 agonist antibody (αDR5) or FasL does not require TNFα-mediated signaling or de novo protein synthesis. A, HT1080 and UACC62 cells were treated with BV6 (20 nm), αDR5 (50 ng/ml), or FasL (50 ng/ml) in the absence or presence of TNFR1-Fc (5 μg/ml) as indicated. B, T1080 and UACC62 cells were treated with BV6 (250 nm), αDR5 (200 ng/ml), or FasL (400 ng/ml) in the absence or presence of cycloheximide (CHX, 4 μg/ml) as indicated. Cell viability was determined as described under “Experimental Procedures.” Results represent mean ± standard deviation from three independent experiments.

FIGURE 5.

Down-regulation of caspase inhibitor XIAP potentiates activity of DR5 agonist antibody (αDR5) and FasL. A and B, IAP antagonist BV6 blocks XIAP-mediated inhibition of caspases-3 and -7. BV6 or BVE6 was added to the mixture of XIAP BIR2-BIR3 (10 nm) and caspase-3 or caspase-7 (300 pm). The activity of caspase-3 (A) and capsase-7 (B) was determined as described under “Experimental Procedures.” C, down-regulation of XIAP expression potentiates proapoptotic activity of αDR5 and FasL. Cells were transfected with control scramble (Contr.), XIAP-specific siRNA oligonucleotides. 48 h later, cells were treated as indicated for 24 h. Cell viability was determined as described under “Experimental Procedures.” Results represent mean ± standard deviation from three independent experiments. The protein levels of XIAP were analyzed by Western blotting.

FIGURE 6.

Down-regulation of Bid expression reduces DR5 agonist antibody (αDR5) and FasL proapoptotic activity. Cells were transfected with control scramble (Contr.) or Bid-specific siRNA oligonucleotides. 48 h later, cells were treated as indicated for 24 h. Cell viability was determined as described under “Experimental Procedures.” Results represent mean ± standard deviation from three independent experiments. The protein levels of Bid were analyzed by Western blotting.

FIGURE 7.

Neutralization of XIAP bypasses the requirement for amplification of proapoptotic receptor agonist-induced apoptotic signaling in type II cells. Cells were transfected with control scramble (Contr.), XIAP-specific, or Bid-specific siRNA oligonucleotides alone or in combination. 48 h later, cells were treated with DR5 agonist antibody (αDR5) (200 ng/ml), FasL (400 ng/ml), or BV6 (500 nm) alone or in combination for 24 h. Cell viability was determined as described under “Experimental Procedures.” Results represent mean ± standard deviation from three independent experiments. The protein levels of XIAP and Bid were analyzed by Western blotting.