Molecular Cancer Therapeutics 43 Up-regulation of c-Jun-NH2-kinase pathway contributes to the induction of mitochondria-mediated apoptosis by A-tocopheryl succinate in human prostate cancer cells Ke Zu,1 Lesleyann Hawthorn,2 and Clement Ip1 Introduction Departments of 1Cancer Chemoprevention and 2Cancer Genetics, Vitamin E is a generic term for two distinct, but Roswell Park Cancer Institute, Buffalo, New York structurally related, groups of compounds: tocopherols and tocotrienols. Numerous studies have convincingly shown the growth-inhibitory activities of vitamin E Abstract compounds in a variety of cancer cell lines (1–5). Among Previously, A-tocopheryl succinate (A-TOS) has been the various naturally occurring vitamin E compounds and reported to induce caspase-mediated apoptosis in PC-3 their synthetic derivatives, a-tocopheryl succinate (a-TOS), human prostate cancer cells. Caspase-9 was among a redox-silent analogue of a-tocopherol, is the most several initiator caspases activated by A-TOS, suggest- commonly used form in in vitro studies of cancer research ing a potential contribution of the intrinsic apoptotic (6, 7). The chemotherapeutic effects of a-TOS include pathway in mediating the response to A-TOS. Gene repressed cell proliferation, cell cycle block, reduced expression microarray was carried out as a screen to DNA synthesis, and induction of apoptosis (3, 8–15). identify novel signaling molecules modulated by A-TOS, Of these different mechanisms, the last one has been with a special focus on those known to play a role in studied most extensively. mitochondria-mediated apoptosis. We discovered that It is generally accepted that there are two principal Ask1, GADD45B, and Sek1, three key components of apoptotic cascades: the receptor-mediated extrinsic path- the stress-activated mitogen-activated protein kinase way and the mitochondria-mediated intrinsic pathway. In pathway, are novel targets of A-TOS. Western blot the presence of external stimuli such as inflammatory analysis showed increased levels of phospho-Sek1 and cytokines or withdrawal of growth factors, specific ligands phospho-c-Jun-NH2-kinase (JNK) in addition to total bind to death receptors such as Fas, leading to the assembly Ask1, GADD45B, and Sek1. A-TOS also altered JNK- of a death-inducing signal complex, and the recruitment specific phosphorylation of Bcl-2 and Bim in a manner and activation of initiator caspase-8. Independent of the consistent with enhanced mitochondrial translocation of above mechanism, the mitochondria-mediated pathway is Bax and Bim. Because the expression level of most Bcl-2 activated by internal death stimuli such as reactive oxygen family members remained unchanged, the posttransla- species, culminating in the release of cytochrome c from the tional modification of Bcl-2 and Bim by JNK is likely to mitochondria into the cytoplasm. Following the binding of be a driving force in A-TOS activation of the intrinsic cytochrome c to Apaf-1, procaspase-9 is recruited to the apoptotic pathway. Based on our findings, we propose complex and activated by the cofactor Apaf-1 through the a working model to capture the salient features of the caspase recruitment domain. Once activated, initiator A apoptotic signaling circuitry of -TOS. [Mol Cancer Ther caspase-8 and caspase-9 cleave and activate downstream 2005;4(1):43–50] effector caspase-3, -6, and -7 to complete the escalation of the caspase cascade. The whole process is geared ultimately to the execution of apoptotic cell death (16, 17). The Bcl-2 family proteins are critical regulators of the mitochondrial pathway of apoptosis (16–19). They are believed to modulate the permeability of the mitochondrial membrane directly and thereby control the release of Received 9/9/04; revised 10/29/04; accepted 11/8/04. cytochrome c. Based on the Bcl-2 homology domain Grant support: Supported by grant CA 91990 from the National Cancer Institute, and partially supported by core resources of the Roswell Park structures and protein functions, the Bcl-2 family can be Cancer Institute Cancer Center support grant P30 CA 16056 from the classified into three groups: the antiapoptotic members National Cancer Institute. (containing all four BH domains) such as Bcl-2 and Bcl-xL, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked the proapoptotic members (containing only BH1, BH2, and advertisement in accordance with 18 U.S.C. Section 1734 solely to BH3 domains) such as Bax and Bak, and the BH3 domain– indicate this fact. only members such as Bad, Bim, and Bid. Apoptotic Requests for reprints: Clement Ip, Department of Cancer Chemoprevention, signaling triggers Bax translocation from the cytosol to Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-8875; Fax: 716-845-8100. the outer mitochondrial membrane. In contrast to Bax, Bak E-mail: [email protected] is localized primarily in the outer mitochondrial mem- Copyright C 2005 American Association for Cancer Research. brane. When cued by apoptotic signals, Bax and Bak Mol Cancer Ther 2005;4(1). January 2005 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. 44 Apoptosis signaling induced by a-tocopheryl succinate oligomerize and form heterotetrameric channels for the exit Quantitation of Apoptosis by Terminal Deoxynucleo- of cytochrome c. Bcl-2 and Bcl-xL, on the other hand, tidyl Transferase-Mediated Nick End Labeling prevent apoptosis by inhibiting the formation of the Bax/ PC-3 cells were plated at a density of 4,000 cells/cm2 Bak heterotetrameric channels. Furthermore, the phosphor- in T175 culture flasks. At 72 hours after seeding, cells ylation of the Bcl-2 protein is involved in the regulation of were exposed to 20 or 40 Amol/L a-TOS for 24 hours. its function. The BH3 domain–only proteins are usually Adherent cells harvested by mild trypsinization were localized in the cytosol and function as death ligands to the pooled together with detached cells. The cell pellets were multidomain Bcl-2 family members. Upon activation by then fixed in 1% (w/v) paraformaldehyde in PBS apoptotic stimuli, the BH3 domain–only proteins translo- (pH 7.4), washed in PBS, and stored in 70% ethanol at cate to the mitochondria; the above process is regulated by 4jC overnight. The ethanol solution was subsequently different mechanisms. For example, Bim is sequestered in removed following centrifugation, and cells were treated the cytosol by binding to the dynein motor complexes. with the enzyme terminal deoxynucleotidyl transferase- Phosphorylation by c-Jun-NH2-kinase (JNK) disrupts the mediated nick end labeling, labeled with bromodeoxyur- binding motif of Bim and facilitates the release of Bim, thus idine, and stained with fluorescein fPRB-1 antibody and making it available to antagonize Bcl-2 in the mitochondria. propidium iodide using the apo-bromodeoxyuridine kit Previously, we found that caspase-9, the key initi- from Phoenix Flow Systems (San Diego, CA) as per the ator caspase of mitochondria-mediated apoptosis, is manufacturer’s protocol. Apoptotic cells were counted by activated in PC-3 human prostate cancer cells treated with flow cytometry, and the data were analyzed with the a-TOS (15). The significance of the mitochondrial pathway WinList software. in a-TOS-induced apoptosis has similarly been impli- Oligonucleotide Array Analysis cated by Kline et al. and Neuzil et al. in different cell PC-3 cells were plated at a density of 4,000 cells/cm2 models (20–22). In the present study, we used gene expres- in 15-cm culture dishes. Synchronization of cells was sion microarray as a first step to identify novel signaling achieved by starving in serum-free medium for 48 hours. molecules modulated by a-TOS, with a special focus on After returning to regular growth medium for 6 hours, those that are known to play a role in mitochondria- cells were exposed to regular growth medium or mediated apoptosis. Based on further research aimed at 20 Amol/L a-TOS for 24 hours. Total RNA and protein validating the discovery, a working model is proposed to were then isolated using TRIzol (Life Technologies, Inc., delineate the molecular mechanism that may underlie the Gaithersburg, MD). The experiment was repeated twice proapoptotic activity of a-TOS. more, and total RNA collected from the three replicates were pooled and submitted to microarray analysis using Materials and Methods the U95A chip from Affymetrix (Santa Clara, CA). Cell Culture Condition Biotinylated cRNA probe generation, array hybridization, The PC-3 human prostate cancer cell line was purchased and other procedures were carried out according to the from American Type Culture Collection (Manassas, VA). standard Affymetrix GeneChip protocol. Fluorescence Cells were cultured in RPMI 1640 medium supplemented intensity for each chip was captured with a Hewlett- with 10% fetal bovine serum, 100 units/mL penicillin, Packard laser confocal scanner. Three data analysis 100 Ag/mL streptomycin, and 2 mmol glutamine, and parameters from the Affymetrix Microarray Suite soft- j maintained in an atmosphere of 5% CO2 in a 37 C ware were used to determine a change in gene humidified incubator. Ethanol was used to dissolve a- expression between the control and treatment samples: TOS (Sigma, St. Louis, MO); the final concentration of detection (a qualitative measure of the presence or ethanol in the culture medium was kept at 0.2% (v/v). absence of a particular transcript), change (a qualitative MTT Cell Growth Assay measure of the increase or decrease of a particular This assay is based on the conversion of the yellow transcript), and signal log ratio (a quantitative measure tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- of the increase or decrease of a particular transcript). A trazolium bromide (MTT) to purple formazan crystals detailed description of the analysis of the microarray data by metabolically active cells.