SM-164: a novel, bivalent Smac mimetic that induces apoptosis and tumor regression by concurrent removal of the blockade of cIAP-1/2 and XIAP

Abstract

Small-molecule Smac mimetics are being developed as a novel class of anticancer drugs. Recent studies have shown that Smac mimetics target cellular inhibitor of apoptosis protein (cIAP)-1/2 for degradation and induce tumor necrosis factor-alpha (TNFalpha)-dependent apoptosis in tumor cells. In this study, we have investigated the mechanism of action and therapeutic potential of two different types of novel Smac mimetics, monovalent SM-122 and bivalent SM-164. Our data showed that removal of cIAP-1/2 by Smac mimetics or small interfering RNA is not sufficient for robust TNFalpha-dependent apoptosis induction, and X-linked inhibitor of apoptosis protein (XIAP) plays a critical role in inhibiting apoptosis induction. Although SM-164 is modestly more effective than SM-122 in induction of cIAP-1/2 degradation, SM-164 is 1,000 times more potent than SM-122 as an inducer of apoptosis in tumor cells, which is attributed to its much higher potency in binding to and antagonizing XIAP. SM-164 induces rapid cIAP-1 degradation and strong apoptosis in the MDA-MB-231 xenograft tumor tissues and achieves tumor regression, but has no toxicity in normal mouse tissues. Our study provides further insights into the mechanism of action for Smac mimetics and regulation of apoptosis by inhibitor of apoptosis proteins. Furthermore, our data provide evidence that SM-164 is a promising new anticancer drug for further evaluation and development.

Figure 1

Nonpeptidic, monovalent, and bivalent Smac mimetics bind to cIAP-1/2 and XIAP proteins with high affinities and antagonize XIAP. A, chemical structures of monovalent SM-122 and bivalent SM-164. B, competitive binding of Smac mimetics and their control compounds to recombinant XIAP and cIAP-1 proteins containing both BIR2 and BIR3 domains and to cIAP-2 protein containing only the BIR3 domain, as determined using fluorescence polarization assays. C, dose-dependent inhibition of caspase-3/caspase-7 activity by recombinant XIAP containing both BIR2 and BIR3 domains and antagonism of Smac mimetics against XIAP to restore the activity of caspase-3/caspase-7.

Figure 2

Monovalent and bivalent Smac mimetics induce apoptosis in the MDA-MB-231 cancer cell line in a caspase-3– and caspase-8–dependent manner. MDA-MB-231 cells were treated as indicated. Apoptosis was analyzed by propidium iodide (PI)/Annexin V double staining using flow cytometry (A); cell viability was determined by trypan blue dye exclusion assay (B); and cleavage of caspases and PARP was analyzed by Western blotting (C). D, cells were transfected with siRNA against caspase-3, caspase-8, and caspase-9 for 48 h, followed by treatment with SM-164 (middle) or SM-122 (right) for 48 h. Knockdown efficacy was examined by Western blotting and cell growth inhibitory activity by a WST assay. E, cells were treated with Z-DEVD-FMK (25 μmol/L) or Z-IETD-FMK (25 μmol/L) for 1 h, followed by treatment with a Smac mimetic for 48 h. Cell growth inhibitory activity was examined by a WST assay. Points, mean of triplicates; bars, SD.

Figure 3

Smac mimetics induce TNFα-dependent apoptosis and cIAP-1 degradation. A, MDA-MB-231 cells were pretreated with or without TNFα-or TRAIL- blocking antibody, followed by treatment with Smac mimetics. Cell viability was determined with trypan blue assay. B, HCT116 colon cancer cells were treated with SM-164 alone, TNFα alone, or the combination for 48 h. Cell growth inhibition was determined by a WST assay. C and D, MDA-MB-231 cells were treated with Smac mimetics as indicated and the levels of cIAP-1 and XIAP were examined by Western blotting. E, MDA-MB-231 or HCT116 cells treated with or without MG-132, followed by the treatment with 100 nmol/L of SM-122 or SM-164. The levels of cIAP-1 were examined by Western blotting.

Figure 4

Removal of cIAP-1/2 alone is not sufficient to induce robust TNFα-dependent apoptosis. MDA-MB-231 (A) and SK-OV-3 (B) cell lines were treated with SM-122 alone, TNFα alone, or the combination for 24 h. The levels of cIAP-1 and cIAP-2 were examined by Western blotting and cell viability was determined by trypan blue assay. MDA-MB-231 (C) and SK-OV-3 (D) cell lines were transfected with siRNA against cIAP-1, cIAP-2, both cIAP-1 and cIAP-2, or control siRNA for 48 h, followed by treatment with TNFα for 24 h. The levels of cIAP-1 and cIAP-2 were examined by Western blotting and cell viability was determined by trypan blue assay.

Figure 5

XIAP plays an important role in mediating TNFα-dependent apoptosis induced by Smac mimetics. A, MDA-MB-231 cell line was transfected with XIAP siRNA for 48 h, followed by the treatment of SM-122 or SM-164 in combination with TNFα (10 ng/mL) for 48 h. Cell viability was determined by trypan blue dye exclusion. B, HCT 116 XIAP^+/+ and XIAP^−/− cell lines were treated with SM-122 or SM-164 alone or in combination with TNFα (0.1 ng/mL) as indicated. PARP cleavage and activation of caspase-3 were examined by Western blotting and cell growth inhibition by a WST assay. C, Jurkat cell lines were treated with SM-122, SM-164, TNFα (300 ng/mL) alone, or the combination of SM-122 or SM-164 with TNFα for 24 h. Apoptosis was analyzed by propidium iodide staining/Annexin V double staining using flow cytometry.

Figure 6

SM-164 induces rapid cIAP-1 degradation and robust apoptosis in tumor tissues and achieves tumor regression, but causes minimal toxicity to mouse tissues. A, SCID mice (two mice per group) bearing established MDA-MB-231 xenograft tumors were treated with a single i.v. dose of SM-164, Taxotere (TXT), or vehicle (VEH). Tumor tissues were harvested at the indicated time points. Degradation of cIAP-1 and XIAP, activation of caspases, and PARP cleavage in tumor tissues were analyzed by Western blotting. B, SCID mice (two mice per group) bearing established MDA-MB-231 xenograft tumors were treated with a single i.v. dose of SM-122 or vehicle. Tumor tissues were harvested at the indicated time points. cIAP-1 degradation and PARP cleavage in tumor tissues were analyzed by Western blotting. Tumor tissues harvested from mice treated with SM-164 were included as controls. C, apoptosis by TUNEL staining. To score apoptosis, at least 1,000 cells were counted under a microscope. D, SCID mice (two to three mice per group) bearing established MDA-MB-231 xenograft tumors were treated with a single dose of SM-164 (5 mg/kg, i.v.) or vehicle. Tumor and mouse tissues were harvested at the 24-h time point and examined by H&E staining. Xenograft tumor tissues treated with SM-164 were shown with cell shrinkage, nuclear pyknosis, and chromatin condensation. E, SCID mice (8–10 per group) bearing established MDA-MB-231 xenograft tumors were treated i.v. with SM-164, Taxotere, or vehicle. Points, mean tumor volume; bars, SE.