15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) sensitizes human leukemic HL-60 cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through Akt downregulation

Abstract

While tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising new agent for the treatment of cancer, resistance to TRAIL remains a therapeutic challenge. Identifying agents to use in combination with TRAIL to enhance apoptosis in leukemia cells would increase the potential utility of this agent as a therapy for leukemia. Here, we show that 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), a natural ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), can sensitize TRAIL-resistant leukemic HL-60 cells to TRAIL-induced apoptosis. The sensitization to TRAIL-induced apoptosis by 15d-PGJ2 was not blocked by a PPARgamma inhibitor (GW9662), suggesting a PPARgamma-independent mechanism. This process was accompanied by activation of caspase-8, caspase-9, and caspase-3 and was concomitant with Bid and PARP cleavage. We observed significant decreases in XIAP, Bcl-2, and c-FLIP after cotreatment with 15d-PGJ2 and TRAIL. We also observed the inhibition of Akt expression and phosphorylation by cotreatment with 15d-PGJ2 and TRAIL. Furthermore, inactivation of Akt by Akt inhibitor IV sensitized human leukemic HL-60 cells to TRAIL, indicating a key role for Akt inhibition in these events. Taken together, these findings indicate that 15d-PGJ2 may augment TRAIL-induced apoptosis in human leukemia cells by down-regulating the expression and phosphorylation of Akt.

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“…Cytosolic and mitochondrial proteins were separated as previously described (20,22). In brief, cells treated with 0.1% DMSO or with STC for indicated times were collected and resuspended in mitochondrial isolation buffer and protease inhibitor cocktail (Boehringer Mannheim) supplemented with 10 mmol/L digitonin.…”

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“…Cytosolic and mitochondrial proteins were separated as previously described [18]. Briefly, cells treated with or without fucoidan for 48 h were collected and resuspended in mitochondrial isolation buffer (20 mM Hepes-KOH, pH 7.5, 210 mM sucrose, 70 mM mannitol, 1 mM EDTA, 1 mM dithiothreitol, 1.5 mM MgCl 2 , 10 mM KCl) and protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany) supplemented with 10 mM digitonin.…”

15d-PGJ2 Induces Apoptosis by Reactive Oxygen Species–mediated Inactivation of Akt in Leukemia and Colorectal Cancer Cells and Shows In vivo Antitumor Activity

“…Total RNA was extracted from cultured cells with 0.1% DMSO and 5, 10, or 20 μmol/L 15d-PGJ 2 for 24 h or 20 μmol/L 15d-PGJ 2 for 12 h in the presence of Z-VAD according to previously published methods (31). An aliquot of 1 μg of total RNA from each sample was reverse transcribed to cDNA using First-Strand cDNA Synthesis Kit (Pharmacia LKB Biotechnology) according to manufacturer's instructions with oligo (dT) primer.…”

“…An aliquot of 1 μg of total RNA from each sample was reverse transcribed to cDNA using First-Strand cDNA Synthesis Kit (Pharmacia LKB Biotechnology) according to manufacturer's instructions with oligo (dT) primer. For detection of the human Akt1 and Akt2 mRNA, PCR was done as described previously (31).…”