行政院國家科學委員會補助專題研究計畫成果報告
※※※※※※※※※※※※※※※※※※※※※※※※※※
※ ※
※ PPAR 配位体之抗癌化及抗發炎作用機制 ※
※ ※
※※※※※※※※※※※※※※※※※※※※※※※※※※
計畫類別：;個別型計畫 □整合型計畫 計畫編號：NSC 90－2320－B－038－050－
執行期間：90 年 8 月 1 日至 91 年 7 月 31 日
計畫主持人：梁 有 志
共同主持人：林 仁 混
計畫參與人員：劉正典, 潘憲, 林時宜, 何元順, 洪苓方, 蔡淑慧.
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
執行單位：台北醫學大學醫學系
中 華 民 國 91 年 10 月 18 日
行政院國家科學委員會專題研究計畫成果報告
PPAR 配位体之抗癌化及抗發炎作用機制
Study on the molecular mechanisms of anti-carcinogenic and anti-inflammatory effects of peroxisome proliferator-activated receptor (PPAR) ligands
計畫編號：NSC 90-2320-B-038-050
執行期限：90 年 8 月 1 日至 91 年 7 月 31 日
主持人：梁 有 志 執行機構及單位名稱: 台北醫學大學醫學系 共同主持人：林 仁 混 執行機構及單位名稱: 台大醫學院生化所 計畫參與人員：劉正典, 潘憲, 林時宜, 何元順, 洪苓方, 蔡淑慧.
執行機構及單位名稱: 台北醫學大學醫學系
一、中文摘要
花生四烯酸的多種前列腺素衍生物已知可經由活化 peroxisome proliferator-activated receptor (PPAR)或其他路徑引起細胞凋亡。本研究計畫試驗了多種 eicosanoids 對細胞增殖的 抑制能力。結果發現 prostaglandin J2 (PGJ2)的代謝衍生物 15-deoxy-U12, 14-PGJ2 (15d-PGJ2)及
PGA1，可有效抑制胃腫瘤細胞株 AGS 的生長，且造成此細胞凋亡。過渡表現 PPARα 或 PPARγ
的細胞，並不會增加 15d-PGJ2及 PGA1所造成的細胞凋亡，顯示 15d-PGJ2及 PGA1引起細胞
凋亡非經由活化 PPAR 的路徑。然而 15d-PGJ2及 PGA1會依濃度及時間的增加，提昇 JNK 及
Caspase-3 的活化；若細胞先轉殖 dominant-negative c-Jun N-terminal kinase (DN-JNK)則可減少
JNK 及 Caspase-3 的活性且降低細胞凋亡的數目，這些結果顯示 15d-PGJ2及 PGA1可活化
JNK，且活化 JNK 與造成細胞凋亡有相關性。
關鍵詞：前列腺素、細胞凋亡、JNK
ABSTRACT
Cyclopentenone prostaglandins (CyPGs) derivatives of arachidonic acid have been suggested to exert growth-inhibitory activity through peroxisome proliferator-activated receptor
(PPAR)-dependent and -independent mechanisms. Here we examined that various eicosanoids on the inhibition of cell proliferation, and found that the terminal derivative of PGJ2 metabolism, 15-deoxy-U12, 14-PGJ2 (15d-PGJ2), and PGA1 markedly inhibited growth and induced apoptosis in AGS gastric carcinoma cells. There were no significant increase in DNA-fragmentation in the cells with overexpression of PPARα or PPARγ plasmid, indicating that 15d-PGJ2 and PGA1 induced apoptosis through PPAR-independent pathway, Moreover, 15d-PGJ2 and PGA1 activated the c-Jun N-terminal kinase (JNK), and the caspase-3 activity in dose- and time-dependent manners. To further examine the role of JNK signaling cascades in apoptosis induced by 15d-PGJ2 and PGA1, we transfected dominant-negative (DN) mutants of JNK into the cells to analyze the apoptotic
characteristics of cells expressing DN-JNK plasmid following exposure to 15d-PGJ2 and PGA1. Expression of DN-JNK proteins repressed both of endogenous JNK and caspase-3 activity, and subsequently inhibited apoptosis induced by 15d-PGJ2 and PGA1. These results suggest that CyPGs, such as 15d-PGJ2 and PGA1, activates the JNK signaling pathway, and that JNK activation may be involved in 15d-PGJ2 and PGA1 induced cell death.
Key words: prostaglandins; apoptosis; JNK
INTRODUCTION
The c-Jun N-terminal kinases (JNKs) members of mitogen-activated protein kinase (MAPKs), are classic stress-activated protein kinase (1), can be activated by proinflammatory cytokines (2, 3) and environmental stress such as UV light (4), γ-irradiation (5), heat shock, osmotic stress (6), shear stress (7), growth factor withdrawal (8), ceramide (9), protein synthesis inhibitor (2), and treatment with chemotherapy drugs including paclitaxel (10), adriamycin, vinblastine (11), and etoposide (12).
Recently, JNK had play an important mediator of apoptosis signaling (13). For example, JNK activation was associated with apoptosis in PC-12 cells deprived of NGF (14). In addition,
nonneuronal embryonic fibroblasts with targeted disruptions of the jnk1 and jnk2 genes, were able to inhibit apoptosis (15). These reports suggested that activation of JNK can be associated with induction of apoptosis, however, the mechanism(s) and functional role for JNK in the regulation of apoptosis are not clear (4).
Prostaglandins are oxygenated metabolites of arachidonic acid (AA), and divided into two groups, conventional prostaglandins and cyclopentenone prostaglandins (CyPGs) according to their mechanisms of action (16). The conventional eicosanoids, such as PGE2 and PGD2, bind to cell surface receptor and transduces signal into cells. The CyPGs, such as the A and J series of PGs, lack cell surface receptors but directly enter the cells and exert their actions in the nuclei, including induction of cell growth arrest and cell differentiation (16). Several proteins have been identified to be induced by the CyPGs, such as heat shock proteins (17), γ-glutamylcysteine synthetase (18), collagen (19), gadd 45 (20), and heme oxygenase (21). Arachidonic acid is first converted to PGH2 by cyclooxygenase and subsequently converted to one of several related products, including PGD2, PGE2, PGF2α, PGI2 and thromboxane A2, through the action of specific PG synthases. 15d-PGJ2 is derived from PGD2 and involves sequential conversion of PGD2, PGI2, U12-PGJ2 and 15d-PGJ2
(16). 15d-PGJ2 has been shown to bind to and activate peroxisome proliferator-activated receptor γ (PPARγ) and is therefore an intracellular target of this cyclopentenone prostaglandin (22).
The CyPGs have been demonstrated that possess potent antiproliferative and antitumor activities, but their mechanisms of action are complex and not well understood. Recently it was shown that PGD2 metabolites, such as PGJ2, U12-PGJ2, and 15d-PGJ2, were potential inducers of intracellular oxidative stress that mediated the cytotoxic effects in human neuroblastoma cells (23).
Uchida and colleagues showed that the 15d-PGJ2-induced accumulation of p53 resulted in the activation of a death-inducing caspase cascade mediated by Fas and the Fas ligand in neurons (24).
Another studies showed that de novo gene transcription was necessary for 15d-PGJ2-induced apoptosis in breast cancer cells (25). The current investigation was undertaken to study the effects of arachidonic acid and its metabolites on human gastric cells death. The results demonstrated that 15d-PGJ2 and PGA1 induced apoptosis, activated JNK and caspase-3 activity in AGS cells.
Moreover, the cells were decrease in apoptosis with overexpression of DN-JNK plasmid, indicating that induction of apoptosis by 15d-PGJ2 and PGA1may be mediated the activation of JNK.
MATERIALS AND METHODS
Materials
PGA1, PGJ2, and LTB4 were purchased from Caymen Chemical (Ann Arbor, MI), and arachidonic acid, PGD2, PGE2, PGF2α, PGH2, TBX2 and 15d-PGJ2 were purchased from Biomol (Plymouth Meeting, PA).
Cell Culture
The human AGS gastric carcinoma cell lines (CCRC 60102) were obtained from Food Industry Research and Development Institute, Hsinchu, Taiwan, and cultured in DMEM/F12
containing 10% heat-inactivated fetal bovine serum (Gibco BRL, Grand Island, NY). For all assays, cells were plated in 6-cm dishes at 5×106 cells per dish and allowed to grow for 24 h.
Western Blot
Equal amounts of total cellular protein (50 µg) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto Immobilon-P membrane (Millipore, Bedford, MA) as described previously (26). The membrane was then incubated with an anti-PARP or JNK antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) and subsequently probed with anti-mouse antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence kits (Amersham, Arlington, IL).
Kinase Assay
Equal amounts of total cellular protein (200 µg) were immunoprecipitated with JNK1 or ERK1 specific antibody (Santa Cruz Biotechnology) and protein A/G-PLUS agarose for 12 h at 4℃.
Kinase assay was performed in kinase buffer with Gst-c-Jun fusion protein (for JNK, Santa Cruz Biotechnology) or myelin basic protein (for ERK, Sigma) as substrates as previously described (27).
Plasmids and Transition Transfection
The DN-JNK plasmid was constructed from human JNK1 with double point mutant of Thr183 to Ala and Tyr185 to Phe (28). The PPARγ expression plasmid and PPARα expression plasmid were generously provided by Professor Christopher K. Glass (California University) and Professor Bart Staels (Université de Lille), respectively (29, 30). AGS cells were seeded in 6-cm dishes, and transfected with the mock, DN-JNK, PPARγ, or PPARα expression plasmid using
LipofectAMINETM 2000 (Gibco) for 48 h (31). After transfection, the cells were treated with 15d-PGJ2 or PGA1, and collected cells for DNA fragmentation, caspase activity, or kinase assay.
DNA Fragmentation
After treatment with tested drugs, the cells were washed with PBS and detected the DNA fragmentation by Cell Death Detection ELISA kits according to the manufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany).
Caspase-3 Activity Assay
Briefly, cells were washed with cold PBS twice, and extracted the cytosolic fraction with extraction buffer (12.5 mM Tris, pH7.4, 2 mM DTT, 0.125 mM EDTA, 5% glycerol, and 1 µg/ml leupeptin, and 1 µg/ml aprotinin) by repeated cycles of freezing and thawing. Cell lysate (150 µg) were incubated with 10 µM fluorescence substrate, Ac-DEVD-AMC (Calbiochem, La Jolla, CA) in assay buffer (50 mM Tris, pH7.4, 1 mM EDTA and 10 mM EGTA) at 37°C for 30 min in dark. The fluorescence intensity of the cleaved substrate was measured using a fluorescence
spectrophotometer (Hitachi F-3000) by Ex 380 nm/Em 460 nm (32).
Flow Cytometry
SubG1 population of the cell cycle was analyzed by flow cytometry as described previously (26). Briefly, cells were trypsinized, washed once with PBS, and fixed in 75% ethanol for 1 h at -20°C. Fixed cells were then washed with PBS, incubated with 0.5 ml PBS containing 0.05%
RNase and 0.5% Triton X-100 for 30 min at 37°C, and stained with propidium iodide. The stained cells were analyzed using a FACscan laser flow cytometer (Becton Dickinson, San Jose, CA).
RESULTS
15d-PGJ2 and PGA1 Inhibited the Growth of Human AGS Gastric Carcinoma Cells To examine whether CyPGs inhibit growth of AGS cells, we screened a large number of arachidonic acid and its metabolites, and found that PGA1, the PGD2 itself and its metabolites, such as PGJ2 and 15d-PGJ2 significantly inhibited the growth of AGS cells (Fig. 1A). Among the classes of PGs examined, 15d-PGJ2 and PGA1 were most effective. 10 µM 15d-PGJ2 and 20 µM PGA1
reduced the cell number (Fig. 1) by 80% and 50%, respectively. The terminal metabolites of PGJ2,
15d-PGJ2, represented the most potent growth inhibitor. PGD2 and PGJ2, the precursors of
15d-PGJ2, were less effective than 15d-PGJ2. The inhibitory order was 15d-PGJ2 > PGA1 > PGJ2 >
PGD2. Arachidonic acid, PGE2, PGF2α, PGH2, LTB4, and TXB2, were ineffective to inhibit the
growth at the concentration of 50 µM. Thus, a reactive α, β-unsaturated carbonyl group in the cyclopentane ring, such as 15d-PGJ2, PGA1, and PGJ2 (Fig. 1B), which renders this portion of the molecule able to form Michael assucts with cellular nucleophilics and covalently modify specific protein (33, 34), is essential for apoptosis.
We next examined the effects of different concentrations of 15d-PGJ2 and PGA1 on the induction of AGS cells apoptosis. As shown in Fig. 2A, 15d-PGJ2 and PGA1 induced DNA fragmentation in dose- and time- (Data not shown) dependent manners. DNA fragmentation was approximately 75% and 68% after 12 h incubation with 10 µM 15d-PGJ2 and 20 µM PGA1,
respectively. Moreover, we measured the numbers of cells with a subG1 DNA content as a measure of apoptosis and observed an increase in the apoptosis cells (Fig. 2B). Previous studies have
indicated that 15d-PGJ2 activates caspase-3 in a variety of cancer cell types (35-37), so this parameter was monitored as a measure of the rate and extent of apoptosis. As shown in Fig. 2C, caspase-3 activity was detected in AGS cells with 10 µM 15d-PGJ2 and 20 µM PGA1 at 8 h. The substrate of caspase-3, poly(ADP ribose) polymerase (PARP) also cleaved in 15d-PGJ2 (Fig. 2D) and PGA1 (Data not shown). These results indicated that 15d-PGJ2 and PGA1 inhibited the growth through induction of apoptotic cell death and the caspase-3 pathway.
15d-PGJ2 and PGA1 Induced Apoptosis Through PPAR-Independent Pathway
Since 15d-PGJ2 or PGA1 was a potent agonist of PPARα and PPARγ. Activation of PPARα or PPARγ can promotes cells apoptosis in other cells (38). To examine this possibility, AGS cells were transfected with mock, PPARα, or PPARγ expression plasmid, and treated with 15d-PGJ2 or PGA1. The DNA fragmentation were measured and shown the results in Fig. 3. 15d-PGJ2 and PGA1 induced DNA fragmentation in mock-expression AGS cells, however, it was equally effective in PPARα or PPARγ-overexpression AGS cells. Treatment of mock-expression AGS cells with 10 µM 15d-PGJ2 and 10 µM PGA1 resulted in induction of DNA fragmentation by 75% and 28%,
respectively. However, similar effects of 15d-PGJ2 and PGA1 were observed in PPARα or
PPARγ-overexperssion AGS cells. These results indicated that overexpression of PPARα or PPARγ
did not enhance the apoptosis, and induction of apoptosis may be mediated PPAR-independent pathway in the cells with 15d-PGJ2 or PGA1.
15d-PGJ2 and PGA1 Activated the JNK and Caspase-3 Activity
Since JNK is a classic stress-activated protein kinase and play an important role in drugs inducing apoptosis. To examine whether activity of JNK participated in the 15d-PGJ2 and
PGA1-induced apoptosis, we determined the kinase activity by an immunocomplex kinase assay as described in “Materials and Methods.” As shown in Fig. 4, the activation of JNK was observed at the earliest measured time point of 1.5 h at a concentration of 10 µM 15d-PGJ2 and 20 µM PGA1. The activation of JNK by 15d-PGJ2 or PGA1 was a time- and dose-dependent manner (Fig. 4).
Dominant-Negative Mutants of JNK Blocked Apoptosis Induced by 15d-PGJ2 or PGA1 To assess directly the involvement of JNK in apoptosis by 15d-PGJ2 or PGA1,
dominant-negative (DN) mutants of JNK plasmid were used. As shown in Fig. 5, overexpression of DN-JNK significantly inhibited 15d-PGJ2 and PGA1-induced DNA fragmentation and caspase-3 activity. To verify that overexpression of DN-JNK efficiently decreased endogenous JNK activity, we determined the total JNK activity in both mock and DN-JNK expression cells by
immunocomplex kinase assay. As shown in Fig. 5C, transfection of DN mutant of JNK exactly resulted in the reduction of JNK activity about ＞50﹪induced by 15d-PGJ2 and PGA1. These results indicated that the JNK-mediated induction of apoptosis is important for the treatment of 15d-PGJ2 and PGA1 in AGS cells.
DISCUSSION
We here show that 15d-PGJ2 and PGA1-induced apoptosis in human AGS gastric carcinoma cells requires the activation of JNK. Activation of PPARγ has also been reported to induce apoptosis in various cell types, including choriocarcinoma cells, breast cancer cells, endothelial cells and
macrophages (39-41). Recently, Harris and Phipps showed that PGD2 and 15d-PGJ2 have potent antiproliferative effects through activation of PPARγ (42). However, here we have described a different, PPAR-independent mechanism that explains the ability of 15d-PGJ2 and PGA1 to induce apoptosis in AGS cells.
The specified J2-derivatives are potential metabolites of PGD2 that are sequentially formed from this prostaglandin. A comparison of the PG biosynthetic pathway and the induction of apoptosis profile revealed that induction of apoptosis was mediated mainly by the metabolites of PGD2, the most active of which is the terminal metabolite of 15d-PGJ2. The induction potency was 15d-PGJ2＞PGJ2＞PGD2, indicating a gain in biological potency as the catabolism of PGD2
proceeds. CyPGs are reactive compounds that possess a α, β-unsaturated carbonyl group in the cyclopentenone ring. These groups are very susceptible to nucleophilic addition reactions with sulfhydryl groups of cysteine residues of proteins or of the tripeptide glutathione (43). Since it has been demonstrated that PGA1 formed a bisconjugate with thiol-containing proteins (44), and 15d-PGJ2 formed a covalent adduct with the p50 subunit of NFκB (45), which resulted in
modulation of protein functions. Based on functional evidence, a modification of IKK (46) and of the p65 subunit of NFκB (47) by 15d-PGJ2 has also been proposed. In this study, we showed that 15d-PGJ2 and PGA1 activated the JNK pathway. Thus, whether 15d-PGJ2 and PGA1 activate JNK via interaction with JNK itself or upstream proteins of JNK deserves further study.
Activation of the JNK signaling pathway has been mechanistically implicated in regulation of apoptosis. In contrast, several reports also indicated that JNK play a role in promoting or preventing apoptosis, depending on both cell type and apoptotic-triggering stimuli (48-50). On the other hand, JNK activation may be a stress response secondary to apoptosis itself, rather than a primary
mediator in apoptotic pathway (50). However, we showed that activation of JNK reached a peak at early 2 h with 15d-PGJ2 and PGA1, in which the apoptotic cells are still minimal (Data not shown).
These results suggested that JNK activation was not a secondary response to 15d-PGJ2 and
PGA1-induced apoptosis. In addition, 15d-PGJ2 and PGA1 caused a more sustained pattern of JNK activation (Fig. 4), suggesting that activation of JNK was directly linked to cell death in 15d-PGJ2
and PGA1 treatment cells. Since several reports have demonstrated that the duration of JNK
activation is a determining factor for cell proliferation or death (4). Transient JNK activation leaded cell proliferation not death, such as activation of JNK by PMA plus ionomycin.
In summary, we have found that 15d-PGJ2 and PGA1 represent the most potent inducers of apoptosis in AGS cells, and that JNK is directly involved in 15d-PGJ2 and PGA1-induced program cell death. The present study may therefore represent a first step in establishing a link between the activation of JNK and apoptosis induced by 15d-PGJ2 and PGA1.
REFERENCES
1. Hibi M, Lin A, Smeal T, Minden A, Karin M. Identification of an oncoprotein- and
UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Develop 1993;7:2135-48.
2. Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J, Woodgett JR.
The stress-activated protein kinase subfamily of c-Jun kinases. Nature 1994;369:156-60.
3. Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, Davis RJ. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 1995;270:7420-26.
4. Chen YR, Meyer CF, Tan TH. Persistent activation of c-Jun N-terminal kinase 1 (JNK1) in gamma radiation-induced apoptosis. J Biol Chem 1996;271:631-4.
5. Zanke BW, Boudreau K, Rubie E, Winnett E, Tibbles LA, Zon L, Kyriakis J, Liu FF, Woodgett JR. The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat. Curr Biol 1996;6:606-13.
6. Galcheva-Gargova Z, Derijard B, Wu IH, Davis RJ. An osmosensing signal transduction
pathway in mammalian cells. Science 1994;265:806-8.
7. Li YS, Shyy JY, Li S, Lee J, Su B, Karin M, Chien S. The Ras-JNK pathway is involved in shear-induced gene expression. Mol Cell Biol 1996;16:5947-54.
8. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science1995;270:1326-31.
9. Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer MJ, Szabo E, Zon LI, Kyriakis JM, Haimovitz-Friedman A, Fuks Z, Kolesnick RN. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 1996;380:75-9.
10. Srivastava RK, Mi QS, Hardwick JM, Longo DL. Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis. Pro Nat Acad Sci USA 1999;96:3775-80.
11. Fan M, Goodwin ME, Birrer MJ, Chambers TC. The c-Jun NH(2)-terminal protein kinase/AP-1 pathway is required for efficient apoptosis induced by vinblastine. Cancer Res 2001;61:4450-8.
12. Osborn MT, Chambers TC. Role of the stress-activated/c-Jun NH2-terminal protein kinase pathway in the cellular response to adriamycin and other chemotherapeutic drugs. J Biol Chem 1996;271:30950-5.
13. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell 200;103:239-52.
14. Le-Niculescu H, Bonfoco E, Kasuya Y, Claret FX, Green DR, Karin M. Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death. Mol Cell Biol 1999;19:751-63.
15. Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, Bar-Sagi D, Jones SN, Flavell RA, Davis RJ. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 2000;288:870-4.
16. Fukushima M. Biological activities and mechanisms of action of PGJ2 and related compounds:
an update. Prostagland Leukotri Essent Fatty Acids 1992;47:1-12.
17. Santoro MG, Garaci E, Amici C. Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells. Proc Nat Acad Sci USA 1989;86:8407-11.
18. Ohno K, Higaki J, Takechi S, Hirata M. Specific role of an alpha,beta-unsaturated carbonyl group in gamma-glutamylcysteine synthetase induction by prostaglandin A2. Chem Biol Interact 1990;76:77-87.
19. Tasaki Y, Takamori R, Koshihara Y. Prostaglandin D2 metabolite stimulates collagen synthesis by human osteoblasts during calcification. Prostaglandins 1991;41:303-13.
20. Ohtani-Fujita N, Minami S, Mimaki S, Dao S, Sakai T. p53-Independent activation of the gadd45 promoter by Delta12-prostaglandin J2. Biochem Biophy Res Commun
1998;251:648-52.
21. Koizumi T, Odani N, Okuyama T, Ichikawa A, Negishi M. Identification of a cis-regulatory element for delta 12-prostaglandin J2-induced expression of the rat heme oxygenase gene. J Biol Chem 1995;270:21779-84.
22. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell 1995;83:813-9.
23. Kondo M, Oya-Ito T, Kumagai T, Osawa T, Uchida K. Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress. J Biol Chem 2001;276:12076-83.
24. Kondo M, Shibata T, Kumagai T, Osawa T, Shibata N, Kobayashi M, Sasaki S, Iwata M, Noguchi N, Uchida K. 15-Deoxy-∆12,14-prostaglandin J2: The endogenous electrophile that induces neuronal apoptosis. Proc Nat Acad Sci USA 2002;99:7367-72.
25. Clay CE, Atsumi GI, High KP, Chilton FH. Early de novo gene expression is required for 15-deoxy-Delta 12,14-prostaglandin J2-induced apoptosis in breast cancer cells. J Biol Chem 2001;276:47131-5.
26. Liang YC, Lin-Shiau SY, Chen CF, Lin JK. Inhibition of cyclin-dependent kinases 2 and 4 activities as well as induction of Cdk inhibitors p21 and p27 during growth arrest of human breast carcinoma cells by (-)-epigallocatechin-3-gallate. J Cell Biochem 1999;75:1-12.
27. Liang YC, Huang YT, Tsai SH, Lin-Shiau SY, Chen CF, Lin JK. Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages. Carcinogenesis 1999;20:1945-52.
28. Lin SY, Liang YC, Ho YS, Tsai SH, Pan S, Lee WS. Involvement of both extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways in the
12-o-tetradecanoylphorbol-13-acetate-induced upregulation of p21Cip1 in colon cancer cells.
Mol Carcinogenesis 2002; In press.
29. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature 1998;391:79-82.
30. Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Torra IP, Teissier E, Minnich A, Jaye M, Duverger N, Brewer HB, Fruchart JC, Clavey V, Staels B. PPAR-alpha and PPAR-gamma
activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nature Med. 2001;7:53-8.
31. Liang YC, Tsai DC, Lin-Shiau SY, Chen CF, Lin JK. Suppression of inducible cyclooxygenase and inducible nitric oxide synthase through peroxisome proliferator-activated receptor-γ by apigenin and related-flavonoids in macrophages. FEBS Lett 2001;496:12-8.
32. Shiah SG, Chuang SE, Kuo ML. Involvement of Asp-Glu-Val-Asp-directed, caspase-mediated mitogen-activated protein kinase kinase 1 cleavage, c-Jun N-terminal kinase activation, and subsequent bcl-2 phosphorylation for paclitaxel-induced apoptosis in HL-60 cells. Mol Pharmacol 2001;59:254-62.
33. Fukushima M. Prostaglandin J2--anti-tumour and anti-viral activities and the mechanisms involved. Eicosanoids 1990;3:189-99.
34. Rossi A, Elia G, Santoro MG. 2-Cyclopenten-1-one, a new inducer of heat shock protein 70 with antiviral activity. J Biol Chem 1996;271:32192-6.
35. Rohn TT, Wong SM, Cotman CW, Cribbs DH. 15-deoxy-delta12,14-prostaglandin J2, a specific ligand for peroxisome proliferator-activated receptor-gamma, induces neuronal apoptosis. Neuro Report 2001;12:839-43.
36. Chattopadhyay N, Singh DP, Heese O, Godbole MM, Sinohara T, Black PM, Brown EM.
Expression of peroxisome proliferator-activated receptors (PPARS) in human astrocytic cells:
PPARgamma agonists as inducers of apoptosis. J Biol Chem 2000;61:67-74.
37. Keelan JA, Sato TA, Marvin KW, Lander J, Gilmour RS, Mitchell MD. 15-Deoxy-Delta (12,14)-prostaglandin J(2), a ligand for peroxisome proliferator-activated receptor-gamma, induces apoptosis in JEG3 choriocarcinoma cells. Biochem Biophy Res Commun
1999;262:579-85.
38. Chinetti G, GriglioS, Antonucci M, Torra IP, Delerive P, Majd Z, Fruchart JC, Chapman J, Najib J, Staels B. Activation of proliferator-activated receptors alpha and gamma induces apoptosis of human monocyte-derived macrophages. J Biol Chem 1998;273:25573-80.
39. Elstner E, Müller C, Koshizuka K, Williamson EA, Park D, Asou H, Shintaku P, Said JW, Heber D, Koeffler DP. Ligands for peroxisome proliferator-activated receptorγ and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc Nat Acad Sci USA 2001;95:8806-11.
40. Bishop-Bailey D, Hla T. Endothelial cell apoptosis induced by the peroxisome
proliferator-activated receptor (PPAR) ligand 15-deoxy-Delta12, 14-prostaglandin J2. J Biol Chem 1999;274:17042-8.
41. Chinetti G, Griglio S, Antonucci M, Torra IP, Delerive P, Majd Z, Fruchart JC, Chapman J, Najib J, Staels B. Activation of proliferator-activated receptors alpha and gamma induces apoptosis of human monocyte-derived macrophages. J Biol Chem 1998;273:25573-80.
42. Harris SG, Phipps RP. Prostaglandin D(2), its metabolite 15-d-PGJ(2), and peroxisome
proliferator activated receptor-gamma agonists induce apoptosis in transformed, but not normal, human T lineage cells. Immunology 2002;105: 23-34.
43. Atsmon J, Sweetman BJ, Baertschi SW, Harris TM, Roberts LJ 2nd. Formation of thiol
conjugates of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2.
Biochemistry 1990;29:3760-5.
44. Parker J, Ankel H. Formation of a prostaglandin A2-glutathione conjugate in L1210 mouse leukemia cells. Biochem Pharmacol 1992;43:1053-60.
45. Cernuda-Morollon E, Pineda-Molina E, Canada FJ. and Perez-Sala D. 15-Deoxy-Delta
12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit. J Biol Chem 2001;276:35530-6.
46. Rossi A, Kapahi P, Natoli G, Takahashi T, Chen Y, Karin M, Santoro MG. Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase. Nature 2000;403:103-8.
47. Straus DS, Pascual G, Li M, Welch JS, Ricote M, Hsiang CH, Sengchanthalangsy LL, Ghosh G, Glass CK. 15-deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway. Proc Nat Acad Sci USA 2000;97:4844-9.
48. Zanke BW, Lee C, Arab S, Tannock IF. Death of tumor cells after intracellular acidification is dependent on stress-activated protein kinases (SAPK/JNK) pathway activation and cannot be inhibited by Bcl-2 expression or interleukin 1beta-converting enzyme inhibition. Cancer Res 1998;58:2801-8.
49. Nishina H, Fischer KD, Radvanyi L, Shahinian A, Hakem R, Rubie EA, Bernstein A, Mak TW, Woodgett JR, Penninger JM. Stress-signalling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3. Nature 1997;385:350-3.
50. Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK)--from inflammation to development. Curr Opin Cell Biol 1998;10:205-19.
LEGENDS TO FIGURES
Fig. 1. Effect of arachidonic acid (AA) and its metabolites on the growth of AGS cells. (A). Cells were treated with 50 µM AA, LTB4, PGD2, PGE2, PGF2α, PGH2 or TXB2, or 20 µM PGA1, PGJ2, or 10 µM 15d-PGJ2 for 24 h. Cell numbers were measured by counting the number of trypan blue-excluding cells. The values were expressed as mean ± S.E. of triplicate test. *, p < 0.05 versus control. (B). Structures of arachidonic acid and its metabolites.
Fig. 2. Effect of 15d-PGJ2 and PGA1 on the DNA fragmentation, caspase-3 activity, and PARP cleavage in AGS cells. Cells were treated with different concentration of PGA1 or 15d-PGJ2 for 15 h. (A). DNA fragmentation was determined by the histone-associated DNA-fragments as described in “Materials and Methods.” The values were expressed as mean ± S.E. of triplicate test. *, p < 0.05 versus control. (B). Cells were treated with 5 µM 15d-PGJ2 or 10 µM PGA1, and subjected to DNA content analysis by flow cytometer. The percentages of total cells with subG1 apoptotic DNA are indicated. (C). Cells were treated with 10 µM 15d-PGJ2 or 20 µM PGA1 for 4 h and 8 h as indicated, and cell extracts were subjected to caspase-3 assay as described in “Materials and Methods”. The values were expressed as mean ± S.E. of triplicate test. *, p < 0.05 versus control. (D). Cells were treated with different concentrations of 15d-PGJ2 for 15 h. Total cell lysates (50 µg) were subjected to Western blot by using anti-PARP antibody. The intact (112 kD) and cleaved (86 kD) species of PARP were indicated in right of the panel.
Fig. 3. Effect of overexpression of PPARα or PPARγ plasmid on the induction of DNA
fragmentation by 15d-PGJ2 or PGA1. Cells were transfected with PPARα or PPARγ expression plasmid for 48 h, and treated with different concentrations of 15d-PGJ2 or PGA1 for 15 h. DNA fragmentation was determined by the histone-associated DNA-fragments as described in “Materials and Methods.” The values were expressed as mean ± S.E. of triplicate test. *, p < 0.05 versus mock control.
Fig. 4. Effect of 15d-PGJ2 and PGA1 on the activation of JNK in AGS cells. (A). Cells were treated with 10 µM 15d-PGJ2 or 20 µM PGA1 for various times as indicated. (B). Cells were treated with different concentrations of 15d-PGJ2 or PGA1 for 3 h. JNK1 was immunoprecipitated from cell lysates and incubated with purified GST-c-Jun fusion protein as substrate in an in vitro kinase assay
as described in “Materials and Methods”.
Fig. 5. Effect of dominant-negative JNK on the blocking of endogenous JNK activity, DNA fragmentation, and caspase-3 activity induced by 15d-PGJ2 or PGA1. Cells were transfected with mock expression plasmid or DN-JNK expression plasmid for 48 h. (A). Transfected cells were treated with 10 µM 15d-PGJ2 and 20 µM PGA1 for 15 h. DNA fragmentation was determined by the histone-associated DNA-fragments as described in “Materials and Methods.” The values were expressed as mean ± S.E. of triplicate test. *, p < 0.05 versus control. (B). Transfected cells were treated with 10 µM 15d-PGJ2 or 20 µM PGA1 for 3 h or 8 h, and determined the caspase-3 activity (8 h), and JNK activity (3 h) as described in “Methods and Methods.” The values were expressed as mean ± S.E. of triplicate test. *, p < 0.05 versus control.
