Peptides Mimicking the Unique ARTS-XIAP Binding Site Promote Apoptotic Cell Death in Cultured Cancer Cells

Abstract

Purpose: XIAP [X-linked inhibitor of apoptosis (IAP) protein] is the best characterized mammalian caspase inhibitor. XIAP is frequently overexpressed in a variety of human tumors, and genetic inactivation of XIAP in mice protects against lymphoma. Therefore, XIAP is an attractive target for anticancer therapy. IAP antagonists based on a conserved IAP-binding motif (IBM), often referred to as "Smac-mimetics," are currently being evaluated for cancer therapy in the clinic. ARTS (Sept4_i2) is a mitochondrial proapoptotic protein which promotes apoptosis by directly binding and inhibiting XIAP via a mechanism that is distinct from all other known IAP antagonists. Here, we investigated the ability of peptides derived from ARTS to antagonize XIAP and promote apoptosis in cancer cell lines.Experimental Design: The ability of synthetic peptides, derived from the C-terminus of ARTS, to bind to XIAP, stimulate XIAP degradation, and induce apoptosis was examined. We compared the response of several cancer cell lines to different ARTS-derived peptides. Pull-down assays were used to examine binding to XIAP, and apoptosis was evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, caspase activation, and Western blot analyses of caspase substrates.Results: The C-terminus of ARTS contains a unique sequence, termed ARTS-IBM (AIBM), which is important for binding to XIAP and cell killing. AIBM peptides can bind to XIAP-BIR3, penetrate cancer cells, reduce XIAP levels, and promote apoptosis.Conclusions: Short synthetic peptides derived from the C-terminus of ARTS are sufficient for binding to XIAP and can induce apoptosis in cancer cells. These results provide proof-of-concept for the feasibility of developing ARTS-based anticancer therapeutics.

Electro-hyperthermia up-regulates tumour suppressor Septin 4 to induce apoptotic cell death in hepatocellular carcinoma

“…This observation is in agreement with recent findings that SEPT4, which encodes the IAP (inhibitor of apoptosis protein) antagonist ARTS, is significantly down-regulated in human HCC cells and regulates susceptibility to apoptosis, suggesting a tumour-suppressor role of SEPT4 in HCC [12]. Sept4-null mice had increased numbers of stem and progenitor cells, elevated X-linked IAP (XIAP) protein, increased resistance to cell death, and accelerated tumour development in a Myc-dependent manner [18,19], and synthetic peptides mimicking the unique ARTS-XIAP binding site promoted apoptosis in cultured cancer cells [20]. Notably, mEHT treatment commonly induced p21 levels in both HepG2 (with wt p53) and Huh7 cells (with Y220C mt p53), suggesting that the mEHT may be applied to HCC patients regardless of their p53 genetic status.…”

Degradation of Bcl-2 by XIAP and ARTS Promotes Apoptosis

“…Media were supplemented with 10% heat inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate and 2 mM glutamine (Biological Industries). Knocked-down (KD) ARTS HeLa stable cell line and Bcl-2-HeLa KD lines were established by using short hairpin RNAs (shRNAs) as described in (Edison et al, 2012a). Cells were grown in the presence of 0.5 mg/ml G418 (Sigma).…”

From antimicrobial to anticancer peptides. A review

“…Apart from LfcinB, other peptides have been recently described as apoptosis inducers in leukemia cells (Edison et al, 2012; Labelle et al, 2012). Pep2 and Pep3 are short synthetic peptides derived from the C-terminus of the proapoptotic mitochondrial protein ARTS and were shown to efficiently kill cells from human leukemia (Edison et al, 2012).…”