Sung-Won Shin

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

Purpose: Recent studies have shown that 15-deoxy-Δ12, 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 in vitro and in vivo, and investigated the underlying mechanism. Experimental Design: 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. Results: 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 in vivo. Conclusions: 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)

Differential effects of triterpene glycosides, frondoside A and cucumarioside A2-2 isolated from sea cucumbers on caspase activation and apoptosis of human leukemia cells

Frondoside A is a pentaoside having an acetyl moiety at the aglycon ring and xylose as a third monosaccharide residue. Cucumarioside A2‐2 is a pentaoside having glucose as a third monosaccahride unit. We compared the effects of frondoside A and A2‐2 for cell death‐inducing capability with close attention paid to structure–activity relationships. Both frondoside A and A2‐2 strongly induced apoptosis of leukemic cells. Frondoside A‐induced apoptosis was more potent and rapid than A2‐2‐induced apoptosis. A2‐2‐induced but not frondoside A‐induced apoptosis was caspase‐dependent. This suggests that holothurians may induce apoptosis of leukemic cells caspase‐dependently or ‐independently, depending on the holothurian structure.

Stichoposide C Induces Apoptosis through the Generation of Ceramide in Leukemia and Colorectal Cancer Cells and Shows In Vivo Antitumor Activity

Purpose: Marine triterpene glycosides that are physiologically active natural compounds isolated from sea cucumbers (holothurians) and sponges have antifungal, cytotoxic, and antitumor activities, whose specific molecular mechanisms remain to be elucidated. In this study, we examined if and through which mechanisms stichoposide C (STC) from Thelenota anax (family Stichopodidae) induces apoptosis in leukemia and colorectal cancer cells. Experimental Design: We examined STC-induced apoptosis in human leukemia and colorectal cancer cells in the context of mitochondrial injury and signaling pathway disturbances, and investigated the antitumor effect of STC in mouse CT-26 subcutaneous tumor and HL-60 leukemia xenograft models. Results: We found that STC induces apoptosis in these cells in a dose-dependent manner and leads to the activation of Fas and caspase-8, cleavage of Bid, mitochondrial damage, and activation of caspase-3. STC activates acid sphingomyelinase (SMase) and neutral SMase, which resulted in the generation of ceramide. Specific inhibition of acid SMase or neutral SMase and siRNA knockdown experiments partially blocked STC-induced apoptosis. Moreover, STC markedly reduced tumor growth of HL-60 xenograft and CT-26 subcutaneous tumors and increased ceramide generation in vivo. Conclusions: Ceramide generation by STC, through activation of acid and neutral SMase, may in part contribute to STC-induced apoptosis and antitumor activity. Thus, STC may have therapeutic relevance for human leukemia and colorectal cancer. Clin Cancer Res; 18(21); 5934–48. ©2012 AACR.

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

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-Δ12,14-prostaglandin J2 (15d-PGJ2), a natural ligand for peroxisome proliferator-activated receptor γ (PPARγ), 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 PPARγ inhibitor (GW9662), suggesting a PPARγ-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.

Overexpression of PGC‑1α enhances cell proliferation and tumorigenesis of HEK293 cells through the upregulation of Sp1 and Acyl-CoA binding protein

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC‑1α), a coactivator interacting with multiple transcription factors, regulates several metabolic processes. Although recent studies have focused on the role of PGC‑1α in cancer, the underlying molecular mechanism has not been clarified. Therefore, we evaluated the role of PGC‑1α in cell proliferation and tumorigenesis using human embryonic kidney (HEK)293 cells and colorectal cancer cells. We established stable HEK293 cell lines expressing PGC‑1α and examined cell proliferation, anchorage-independent growth, and oncogenic potential compared to parental HEK293 cells. To identify the molecular PGC‑1α targets for increased cell proliferation and tumorigenesis, the GeneFishing™ DEG (differentially expressed genes) screening system was used. Western blot analysis and immunofluorescence staining were performed for a regulated gene product to confirm the results. Forced expression of PGC‑1α in HEK293 cells promoted cell proliferation and anchorage-independent growth in soft agar. In addition, HEK293 cells that highly expressed PGC‑1α showed enhanced tumor formation when subcutaneously injected into the bilateral flanks of immunodeficient mice. The results of the GeneFishing DEG screening system identified one upregulated gene (Acyl-CoA binding protein; ACBP). Real-time RT-PCR, western blot analysis, and immunofluorescence staining showed that ACBP was markedly increased in HEK293 cells stably overexpressing PGC‑1α (PGC‑1α-HEK293 cells) compared to those expressing an empty vector. In PGC‑1α, ACBP, and specificity protein 1 (Sp1) siRNA knockdown experiments in PGC‑1α-HEK293 and SNU-C4 cells, we also observed inhibition of cell proliferation, reduced expression of antioxidant enzymes, and increased H2O2-induced reactive oxygen species production and apoptosis. These findings suggest that PGC‑1α may promote cell proliferation and tumorigenesis through upregulation of ACBP. We provide evidence that increased Sp1 expression might contribute to enhanced ACBP expression by PGC‑1α. The current results also suggest that PGC‑1α, whose expression is related to enhanced cell proliferation and tumorigenesis, may be a good candidate molecular target for cancer therapy.

Induction of Apoptosis and Antitumor Activity by Stichoposide D through the Generation of Ceramide in Human Leukemia Cells

Marine triterpene glycosides are physiologically active natural compounds isolated from sea cucumbers(holothurians). It was demonstrated that they have a wide range of biological activities, including antifungal, cytotoxic, and antitumor effects. A previous study showed that stichoposide C (STC) isolated from Thelenota anax induces apoptosis through generation of ceramide by activation of acid sphingomyelinase (SMase) and neutral SMase in human leukemia cells. In this study, we investigated whether STD, a structural analog of STC, can induce apoptosis and examined the molecular mechanisms for its activity. It was found that STC and STD induce apoptosis in a dose- and time-dependent manner and lead to the activation of caspase-8, mitochondrial damage, activation of caspase-9, and activation of caspase-3 in K562 and HL-60 cells. STC activates acid SMase and neutral SMase, which results in the generation of ceramide. Specific inhibition of acid SMase or neutral SMase partially blocked STC-induced apoptosis, but not STD-induced apoptosis. In contrast, STD generates ceramide through the activation of ceramide synthase. Specific inhibition of ceramide synthase partially blocked STD-induced apoptosis, but not STC-induced apoptosis. Moreover, STC and STD markedly reduced tumor growth of HL-60 xenograft tumors and increased ceramide generation in vivo. These results indicate that STC and STD can induce apoptosis and have antitumor activity through the different molecular mechanisms, because they have a different sugar residue attached to aglycones. Thus, these results suggest that their actions are affected by a sugar residue attached to aglycones and they can be used as anticancer agents in the treatment of leukemia.

Abstract 4441: Stichoposides induce apoptosis through the generation of ceramide in human leukemia cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Marine triterpene glycosides are physiologically active natural compounds, isolated from sea cucumbers (holothurians) and demonstrate a wide spectrum of biological effects. Stichoposides are extracted from Thelenota anax (family Stichopodidae). Previous studies demonstrated that stichoposides had antifungal, cytotoxic, and antitumor activities. However, the specific molecular mechanisms underlying this effect remain to be elucidated. Stichoposide-induced apoptosis was examined in human leukemia cells in relation to mitochondrial injury and perturbations in signaling pathways. Stichoposide C (STC) induces apoptosis in these cells in a dose-dependent manner and leads to the activation of FAS, recruitment of FADD, activation of caspase-8, cleavage of Bid, mitochondrial damage and activation of caspase-3. In contrast, stichoposide D (STD) induces apoptosis in these cells in a dose-dependent manner and causes to activation of extrinsic pathway, not to activation of intrinsic pathway. In addition, STC- and STD-induced apoptosis are mediated by ER stress pathway, showing that Salubrinal, ER pathway inhibitor, inhibits apoptosis by STC and STD. STD activated acid sphingomyelinase (SMase) and neutral SMase, resulting in the generation of ceramide, but STC slightly activated them. By using specific inhibitor for acid SMase or neutral SMase and siRNA knockdown experiments, stichoposides-induced apoptosis was blocked. Thus, these results indicate that ceramide generation by stichoposides may contribute to stichoposides-induced apoptosis. Thus, these data suggest that stichoposides may have therapeutic relevance in the treatment of human leukemia. This study was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) through the Medical Research Center for Cancer Molecular Therapy at Dong-A University (R13-2002-044-05002-0). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4441.