Purpose: Recent studies have shown that 15-deoxy-Δ 12, 14 -prostaglandin J 2 (15d-PGJ 2 ), a natural ligand for peroxisome proliferator-activated receptor-γ (PPARγ), inhibits cell proliferation and induces apoptosis. The specific molecular mechanisms underlying this effect remain to be elucidated. We examined whether 15d-PGJ 2 has antitumor activity in vitro and in vivo, and investigated the underlying mechanism. Experimental Design: We examined 15d-PGJ 2 -induced apoptosis in human leukemia cells in the context of mitochondrial injury, oxidative damage, and signaling pathway disturbances. In addition, we investigated the antitumor effect of 15d-PGJ 2 in a mouse CT-26 s.c. tumor model and HL-60 leukemia xenograft model. Results: 15d-PGJ 2 induced apoptosis in leukemia and colorectal cancer cells in a dosedependent manner and led to generation of reactive oxygen species (ROS) through mitochondria and NADPH oxidase activation, activation of JNK, and inactivation of Akt, a serine/threonine-specific protein kinase. Constitutive activation of Akt for an engineered myristoylated protein prevented 15d-PGJ 2 -mediated apoptosis but not ROS generation. Collectively, these findings suggest a hierarchical model of apoptosis induced by 15d-PGJ 2 in human leukemia cells: oxidative injury represents a primary event resulting in Akt inactivation, which in turn leads to mitochondrial injury and apoptosis. Moreover The peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor belonging to the nuclear receptor superfamily (1, 2) and is expressed in some myeloid leukemic cell lines (3). PPARγ agonists include the natural ligands 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ; refs. 4, 5) and lysophosphatidic acid (6), and the synthetic thiazolidinedione class of antidiabetic drugs such as ciglitazone (7). The cyclopentenone PG 15d-PGJ 2 is a product of the cyclooxygenase pathway and is the final metabolite of PGD 2 (8). 15d-PGJ 2 has highly reactive structures that contain an α, β-unsaturated ketone susceptible to nucleophilic addition reactions. For example, the cyclopentenone ring of 15d-PGJ 2 covalently modifies cellular proteins such as the p65 and p50 subunits of NF-κB (9, 10). The potent antiproliferative and antiviral effects of cyclopentenone PGs are attributed to this reactivity (11).15d-PGJ 2 has recently received attention because it functions as a potential regulator of diverse processes, including cell growth, differentiation, and inflammation. It also exerts antitumorigenic activity; for example, 15d-PGJ 2 activation of PPARγ significantly inhibits cell growth and induces apoptosis in several types of cancer cells (12)(13)(14)(15). Some effects of 15d-PGJ 2 are exerted through its interaction with PPARγ (4, 5); however, more recent evidence indicates that 15d-PGJ 2 can also act independently of PPARγ (16). Until now, the molecular mechanism of the antineoplastic activity of 15d-PGJ 2 has not been fully elucidated. In addition, controversial data on the role of PPARγ in colon cancer sugg...

No published data are available about the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and the role of PPARgamma in retinoblastoma protein (RB)-deficient human colorectal cancer (CRC) cells (SNU-C4 and SNU-C2A). Our aim was to investigate whether PPARgamma is expressed in SNU-C4 and SNU-C2A cells and to elucidate possible molecular mechanisms underlying the effect of pioglitazone, a synthetic ligand for PPARgamma, on cell growth in these cell lines. RT-PCR and Western blot analysis showed that both human CRC cell lines expressed PPARgamma mRNA and protein. Pioglitazone inhibited the cell growth of both cell lines through G2/M phase block and apoptosis. In addition, pioglitazone caused a down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bcl-2, and cyclooxygenase-2 (COX-2) under conditions leading to PPARgamma down-regulation. These results suggest that pioglitazone may have therapeutic relevance or significance in the treatment of human CRC, and the down-regulation of XIAP, Bcl-2, and COX-2 may contribute to pioglitazone-induced apoptosis in these and other RB-deficient cell lines and tumors.

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.

Supplementary Data from 15d-PGJ₂ Induces Apoptosis by Reactive Oxygen Species–mediated Inactivation of Akt in Leukemia and Colorectal Cancer Cells and Shows <i>In vivo</i> Antitumor Activity

<div>Abstract<p><b>Purpose:</b> Recent studies have shown that 15-deoxy-Δ^{12, 14}-prostaglandin J₂ (15d-PGJ₂), a natural ligand for peroxisome proliferator–activated receptor-γ (PPARγ), inhibits cell proliferation and induces apoptosis. The specific molecular mechanisms underlying this effect remain to be elucidated. We examined whether 15d-PGJ₂ has antitumor activity <i>in vitro</i> and <i>in vivo</i>, and investigated the underlying mechanism.</p><p><b>Experimental Design:</b> We examined 15d-PGJ₂–induced apoptosis in human leukemia cells in the context of mitochondrial injury, oxidative damage, and signaling pathway disturbances. In addition, we investigated the antitumor effect of 15d-PGJ₂ in a mouse CT-26 s.c. tumor model and HL-60 leukemia xenograft model.</p><p><b>Results:</b> 15d-PGJ₂ induced apoptosis in leukemia and colorectal cancer cells in a dose-dependent manner and led to generation of reactive oxygen species (ROS) through mitochondria and NADPH oxidase activation, activation of JNK, and inactivation of Akt, a serine/threonine-specific protein kinase. Constitutive activation of Akt for an engineered myristoylated protein prevented 15d-PGJ₂–mediated apoptosis but not ROS generation. Collectively, these findings suggest a hierarchical model of apoptosis induced by 15d-PGJ₂ in human leukemia cells: oxidative injury represents a primary event resulting in Akt inactivation, which in turn leads to mitochondrial injury and apoptosis. Moreover, 15d-PGJ₂ markedly reduced growth of mouse CT-26 s.c. tumors and HL-60 xenograft tumors and down-regulated p-Akt and Akt expression <i>in vivo</i>.</p><p><b>Conclusions:</b> These results suggest that Akt inactivation through ROS production may contribute to 15d-PGJ₂–induced apoptosis in leukemia and colorectal cancer cell lines and that 15d-PGJ₂ may have therapeutic relevance in the treatment of human leukemia and colorectal cancer. (Clin Cancer Res 2009;15(17):5414–25)</p></div>