Caspase-3 cleaves XIAP in a positive feedback loop to sensitize melanoma cells to TRAIL-induced apoptosis

Abstract

Successful treatment of melanoma is still challenging, because metastasis remain chemoresistant and radioresistant. Accordingly, combinational treatments involving death ligands are mandatory. In a recent study from our lab, the majority out of 18 melanoma cell lines remained resistant against treatment with the death ligand TRAIL (tumor necrosis factor related apoptosis inducing ligand). Resistance was shown to be mainly due to incomplete processing of caspase-3 into catalytically inactive p21 by binding of the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP). Coirradiation with sub-lethal ultraviolet (UV) B caused depletion of XIAP resulting in synergistic sensitization of all but two melanoma cell lines to TRAIL. We show here the XIAP depletion to essentially require initial caspasemediated cleavage, which promotes proteasomal degradation of XIAP. Utilizing specific caspase inhibitors and small interfering RNA-mediated knockdown, we further identified caspase-3 to be responsible for performing the initial cleavage of XIAP after UVB treatment. Additional evidence suggests an accelerated mitochondrial outer membrane permeabilization in response to co-treatment with TRAIL and UVB, which directs the release of XIAP antagonizing factors including Smac. Distraction of XIAP consequently liberates caspase-3 to autocatalytically process into active p17. Activated caspase-3 cleaves XIAP and further enhances its activation in a positive regulatory feedback loop. The molecular mechanism discovered here appears to have broader implications, because cleavage of XIAP was also shown to accompany cisplatin-induced sensitization of melanoma cells to TRAIL.

Embelin prevents LMP1-induced TRAIL resistance via inhibition of XIAP in nasopharyngeal carcinoma cells

“…P0018A; Beyotime Institute of Biotechnology). Subsequent to incubation with the indicated TRAIL treatment doses (20,40,60, 80 and 100 ng/ml) for various lengths of time (4,8,12,18 and 24 h), cells were harvested, and the cell pellets were suspended in 500 µl binding buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 140 mM NaCl, 1 mM MgCl 2 , 5 mM KCl and 2.5 mM CaCl 2 ] at a density of 1x10 6 cells⁄ml. Samples were incubated with 1 µl annexin V-fluorescein isothiocyanate (FITC) and 5 µl PI for 5 min at room temperature in the dark and measured on a FACSort flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).…”

“…Friboulet et al (19) showed that EBV-positive NPC cell lines expressed increased levels of inhibitor of apoptosis proteins (IAPs), which have anti-apoptotic functions. Additionally, X-linked inhibitor of apoptosis protein (XIAP) is a member of the IAP family that inhibits caspases and induces TRAIL resistance (20).…”

Cellular Mechanisms Controlling Caspase Activation and Function

“…As a direct inhibitor of these caspases, XIAP has been detected on the apoptosome (Bratton et al 2001(Bratton et al , 2002. In a recent study of melanoma cell lines, XIAP was also subject to caspase cleavage in a positive feedback loop that reduces caspase inhibition and XIAP levels through proteasomal degradation (Hornle et al 2011). Although XIAP is the best-characterized vertebrate member of the IAP family with regard to direct binding and inhibition of caspases, one recent study showed that cIAP1 can specifically block apoptosis downstream of cytochrome c release by binding to and inhibiting active caspase-9 within the apoptosome, precluding downstream activation of procaspase-3 (Burke et al 2010).…”

Oxidative Stress Induced by Curcumin Promotes the Death of Cutaneous T-cell Lymphoma (HuT-78) by Disrupting the Function of Several Molecular Targets

“…Activation of NF-kB enhances the expression of antiapoptotic proteins such as inhibitor of apoptosis proteins (IAP), X-linked IAP (XIAP), Bcl-2, Bcl-xL, and c-FLIP (4). Human IAP proteins, including cIAP-1, cIAP-2, XIAP, are known to inhibit apoptosis by inhibiting effector caspases, caspase-7 and -3 (5).…”