Thiazolidenediones Mediate Apoptosis in Prostate Cancer Cells in Part Through Inhibition of Bcl-Xl/Bcl-2 Functions Independently of PPAR;
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Research Article Thiazolidenediones Mediate Apoptosis in Prostate Cancer Cells in Part through Inhibition of Bcl-xL/Bcl-2 Functions Independently of PPAR; Chung-Wai Shiau, Chih-Cheng Yang, Samuel K. Kulp, Kuen-Feng Chen, Chang-Shi Chen, Jui-Wen Huang, and Ching-Shih Chen Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, Ohio Abstract proliferator-activated receptor g (PPARg; ref. 1). This family of g Certain members of the thiazolidenedione family of the PPAR agonists improves insulin sensitivity by increasing tran- scription of certain insulin-sensitive genes involved in the peroxisome proliferator-activated receptor ; (PPAR;) ago- metabolism and transport of lipids, thus representing a new class nists, such as troglitazone and ciglitazone, exhibit anti- of p.o. antidiabetic agents. More recently, certain thiazolidene- tumor effects; however, the underlying mechanism remains diones, especially troglitazone and ciglitazone, have also been inconclusive. This study shows that the effect of these shown to inhibit the proliferation of many cancer cell lines that thiazolidenedione members on apoptosis in prostate cancer express high levels of PPARg, including but not limited to those of cells is independent of PPAR; activation. First, close colon, prostate, breast, and liposarcoma (2). As PPARg-mediated structural analogues of thiazolidenediones, whereas devoid effects of thiazolidenediones promote the differentiation of of PPAR; activity, retain the ability to induce apoptosis preadipocytes, one school of thought attributes the same with equal potency. Second, both PC-3 (PPAR;-expressing) mechanism to the terminal differentiation and cell cycle arrest of and LNCaP (PPAR;-deficient) cells are sensitive to apopto- tumor cells (3). However, the PPARg-activated target genes that sis induction by troglitazone and its PPAR;-inactive mediate the antiproliferative effects remain elusive as genomic analogue irrespective of their PPAR; expression status. responses to PPARg activation in cancer cells are highly Third, rosiglitazone and pioglitazone, potent PPAR; ago- complicated (4). Reported causal mechanisms include attenuated nists, show marginal effects on apoptosis even at high expression of protein phosphatase 2A (5), cyclins D1 and E, concentrations. Evidence indicates that the apoptotic effect inflammatory cytokines and transcription factors (2), and of troglitazone, ciglitazone, and their PPAR;-inactive increased expression of an array of gene products linked to growth analogues 5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2- regulation and cell maturation (4). On the other hand, several lines ylmethoxy)-benzylidene]-2,4-thiazolidine-dione (D2-TG) and of evidence indicate that the inhibitory effect of thiazolidenediones 5-[4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine- on tumor cell proliferation was independent of PPARg activation. 2,4-dione, respectively, is in part attributable to their ability For example, the antitumor effects seem to be structure specific, to inhibit the anti-apoptotic functions of Bcl-xL and Bcl-2. irrespective of potency in PPARg activation (i.e., troglitazone and Treatment of PC-3 cells with troglitazone or D2-TG led to ciglitazone are active, whereas rosiglitazone and pioglitazone are reduced association of Bcl-2 and Bcl-xL with Bak, leading to not). Also, there exists a 3-order-of-magnitude discrepancy between caspase-dependent apoptosis. Bcl-xL overexpression protects the concentration required to produce antitumor effects and that LNCaP cells from apoptosis induction by troglitazone and to mediate PPARg activation. To date, an array of non-PPARg D2-TG in an expression level–dependent manner. Consider- targets have been implicated in the antitumor activities of ing the pivotal role of Bcl-xL/Bcl-2 in regulating mitochon- troglitazone and/or ciglitazone in different cell systems, which drial integrity, this new mode of mechanism provides include intracellular Ca2+ stores (6); phosphorylating activation of a framework to account for the PPAR;-independent action extracellular signal-regulated kinases (7, 8), c-Jun-NH -kinase, and of thiazolidenediones in inducing apoptosis in cancer cells. 2 p38 (9); up-regulation of early growth response-1 (10), p27kip1 (11), Moreover, dissociation of these two pharmacologic activities p21WAF/CIP1 (12), p53, and Gadd45 (13); and altered expression of provides a molecular basis to develop novel Bcl-xL/Bcl-2 Bcl-2 family members (9). However, some of these targets seem to inhibitors, of which the proof of principle is illustrated by be cell type–specific owing to differences in signaling pathways a D2-TG analogue with potent in vivo antitumor activities. regulating cell growth and survival in different cell systems. (Cancer Res 2005; 65(4): 1561-9) In light of the potential use of thiazolidenediones in prostate cancer prevention and treatment (14, 15), signaling mechanisms Introduction whereby these PPARg agonists inhibit the proliferation of prostate cancer cells represent the focus of this investigation. We report Thiazolidenediones, including troglitazone, rosiglitazone, piogli- here the development of novel thiazolidenedione derivatives that tazone, and ciglitazone, are synthetic ligands of the peroxisome lack activity in PPARg activation but retain the ability to induce apoptosis in two prostate cancer cell lines with distinct PPARg expression status, suggesting that these two pharmacologic Requests for reprints: Ching-Shih Chen, Division of Medicinal Chemistry and activities are unrelated. More importantly, we show that Pharmacognosy, College of Pharmacy, The Ohio State University, Room 336, Parks Hall, thiazolidenedione-mediated apoptosis was attributable in part to 500 West 12th Avenue, Columbus, OH 43210. Phone: 614-688-4008; Fax: 614-688-8556; E-mail: [email protected]. the inhibition of the anti-apoptotic functions of Bcl-xL and Bcl-2 I2005 American Association for Cancer Research. by disrupting the BH3 domain-mediated interactions with www.aacrjournals.org 1561 Cancer Res 2005; 65: (4). February 15, 2005 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research proapoptotic Bcl-2 members. From a translational perspective, death. In brief, 1 Â 106 cells were cultured in a T-25 flask in 10% fetal bovine dissociation of these two pharmacologic activities (i.e., PPARg serum–containing medium for 24 hours, and were treated with the test agents at various concentrations in serum-free medium for 24 hours. Both activation and Bcl-xL/Bcl-2 inhibition) provides a molecular basis 5 to use 5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)- floating and adherent cells were collected; cell lysates equivalent to 5 Â 10 cells were used in the ELISA. benzylidene]-2,4-thiazolidine-dione (D2-TG) as a scaffold to Western Blot Analysis of Cytochrome c Release into the Cytoplasm. generate a novel class of Bcl-xL/Bcl-2 inhibitors. Accordingly, we Cytosolic-specific, mitochondria-free lysates were prepared according to an developed a structurally optimized D2-TG derivative (TG-88) with established procedure (16). In brief, after individual treatments for 24 hours, high in vivo potency in inhibiting PC-3 tumor growth. both the incubation medium and adherent cells in T-75 flasks were collected and centrifuged at 200 Â g for 5 minutes. The pellet fraction was recovered, placed on ice, and triturated with 300 AL of a chilled hypotonic Materials and Methods lysis solution [50 mmol/L PIPES-KOH (pH 7.4) containing 220 mmol/L Reagents. Troglitazone and ciglitazone were purchased from Sigma mannitol, 68 mmol/L sucrose, 50 mmol/L KCl, 5 mmol/L EDTA, 2 mmol/L (St. Louis, MO) and Cayman Chemical (Ann Arbor, MI), respectively. MgCl2, 1 mmol/L DTT, and a mixture of protease inhibitors including 100 Rosiglitazone and pioglitazone were prepared from the respective com- Amol/L 4-(2-aminoethyl)benzenesulfonyl fluoride, 80 nmol/L aprotinin, 5 mercial capsules by solvent extraction followed by recrystallization or Amol/L bestatin, 1.5 Amol/L E-64 protease inhibitor, 2 Amol/L leupeptin, chromatographic purification. D2-TG, 5-[4-(1-methyl-cyclohexylmethoxy)- and 1 Amol/L pepstatin A]. After a 45-minute incubation on ice, the mixture benzylidene]-thiazolidine-2,4-dione (D2-CG), 5-{4-[2-(methyl-pyridin-2-yl- was centrifuged at 200 Â g for 10 minutes. The supernatant was collected in amino)-ethoxy]-benzylidene}-thiazolidine-2,4-dione (D2-RG), 5-{4-[2-(5-eth- a microcentrifuge tube, and centrifuged at 14,000 rpm for 30 minutes. An yl-pyridin-2-yl)-ethoxy]-benzylidene}-thiazolidine-2,4-dione (D2-PG; Fig. 1A), equivalent amount of protein (50 Ag) from each supernatant was resolved and TG-88 are thiazolidenedione derivatives with attenuated or unappreci- in 10% SDS-polyacrylamide gel. Bands were transferred to nitrocellulose able activity in PPAR activation, of which the synthesis will be published membranes and analyzed by immunoblotting with anti–cytochrome c elsewhere. The identity and purity (z99%) of these synthetic derivatives were antibodies as described below. verified by proton nuclear magnetic resonance, high-resolution mass Immunoblotting. Cells in T-75 flasks were collected by scraping and spectrometry, and elemental analysis. For in vitro experiments, these agents suspended in 60 AL of PBS. Two microliters of the suspension was taken for at various concentrations were dissolved in DMSO, and were added to cells in protein analysis using the Bradford assay kit (Bio-Rad, Hercules, CA). The medium with a final DMSO concentration of 0.1%. For the in vivo study, TG-88 same volume of 2Â SDS-PAGE sample loading buffer [100 mmol/L Tris-HCl was prepared as a suspension by sonication in a vehicle consisting of 0.5% (pH 6.8), 4% SDS, 5% h-mercaptoethanol, 20% glycerol, and 0.1% methylcellulose and polysorbate 80 in sterile water. The pan-caspase bromophenol blue] was added to the remaining solution. The mixture inhibitor Z-VAD-FMK was purchased from BD Biosciences (Bedford, MA). was sonicated briefly and then boiled for 5 minutes. Equal amounts of The Cell Death Detection ELISA kit was purchased from Roche Diagnostics proteins were loaded onto 10% SDS-PAGE gels.