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

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

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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. It provides a quanti- was published previously (23). For a single comparison tative determination of cell viability. Cells were seeded in between two groups, a log2-transformed treatment/control 24-well plates at a density of 8,000 cells/mL. At 72 hours signal ratio of z1orVÀ1 was chosen as the criterion for after seeding, cells were treated with a-TOS at different induction or repression, respectively. These values are concentrations as specified in Results. The experiment was recommended by guidelines described in the Affymetrix done in triplicate. After 24, 48, or 72 hours of treatment, Data Analysis Fundamentals Manual. 200 AL of MTT was added to each well of cells, and the Cell Lysis and Fractionation plate was incubated for 4 hours at 37jC. The MTT crystals PC-3 cells were plated at a density of 4,000 cells/cm2 in from both attached and floating cells were solubilized in T175 culture flasks. At 72 hours after seeding, cells were isopropanol, and the solution was centrifuged to pellet the exposed to 40 Amol/L a-TOS for 12 or 24 hours. Whole cellular debris. Spectrophotometric absorbance of each cell lysate was prepared using 1Â cell lysis buffer (Cell sample was measured at 570 nm using a Spectra Microplate Signaling Technology, Beverly, MA), and protein concen- Reader (SLT, Australia). tration determined by using the bicinchoninic acid protein

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assay kit (Pierce Biotechnology, Rockford, IL). The cytosol Results and mitochondria-enriched fractions were collected using Sensitivity of PC-3 Cells to a-TOS the cytochrome c release apoptosis assay kit from The effect of a-TOS on the viability of PC-3 cells was Oncogene Research Products (San Diego, CA) as per the assessed by the MTT assay (Fig. 1). Treatment with a-TOS manufacturer’s protocol. Briefly, adherent cells harvested at a concentration of 10 Amol/L did not produce any by mild trypsinization were pooled together with de- change in cell number even after 72 hours. Raising the 7 tached cells. Approximately 5 Â 10 cells were collected concentration to 20 Amol/L inhibited cell viability by for each sample. After washing with ice-cold PBS, cell f10% and f40% at 48 hours and 72 hours, respectively. pellets were resuspended in 1 mL of 1Â cytosol extraction Further increases of a-TOS in the culture medium resulted buffer containing 1 mol/L DTT and protease inhibitors. in a greater magnitude of inhibition with a shorter exposure The cell suspension was then incubated on ice for 10 time. Thus, at a concentration of 40 Amol/L, a-TOS was minutes, and the cells were homogenized in a tissue able to reduce cell number by f50% only after 24 hours. grinder on ice for 30 to 50 passes. The homogenate was Based on the above data, it is evident that a-TOS inhibited j centrifuged at 700 Â g for 10 minutes at 4 C, and the the accumulation of PC-3 cells in a time-dependent and supernatant was centrifuged at 10,000 Â g for 30 minutes dose-dependent manner. j at 4 C. The supernatant from the high-speed centrifuge Induction of Apoptosis by a-TOS was collected as the cytosol fraction. The cell pellets from In an experiment aimed at determining the proapoptotic the high-speed centrifuge were resuspended in 0.1 mL of effect of a-TOS, exponentially growing PC-3 cells were mitochondrial extraction buffer containing 1 mol/L DTT incubated for 24 hours in the presence of 20 or 40 Amol/L and protease inhibitors, and saved as the mitochondrial a-TOS. Apoptosis was measured by flow cytometric analysis fraction. Western blot was used to analyze for cytochrome of cells labeled with bromodeoxyuridine. DNA fragmenta- c release in the cytosol fraction, and for Bcl-2 family tion by cellular nucleases is a signature event of apoptosis. protein translocation in the mitochondrial fraction. This process results in a substantial appearance of free Western Blot Analysis 3V-hydroxyl ends in genomic DNA, which can be earmarked f Briefly, 50 Ag of protein was resolved over 10% to 15% by labeling with bromodeoxyuridine in the presence of SDS-PAGE and transferred to polyvinylidene difluoride terminal deoxynucleotidyl transferase. As shown in Fig. 2A, membrane. The blot was blocked in blocking buffer cells in the right region of each cytogram are bromodeox- [5% nonfat dry milk, 10 mmol Tris (pH 7.5), 10 mmol yuridine-positive. The percentage of apoptotic cells was NaCl and 0.1% Tween 20] at 37jC for 1 hour, incubated quantitated from three independent experiments and with the primary antibody overnight at 4jC, followed by shown in Fig. 2B. A concentration of 20 mol/L a-TOS incubation with an anti-mouse or anti-rabbit horseradish caused only a marginal increase in apoptosis. Raising the peroxidase-conjugated secondary antibody (Bio-Rad, Her- concentration to 40 mol/L led to a 5-fold increase cules, CA) at room temperature for 30 minutes. Individual compared with the control. Thus in agreement with the proteins were visualized by an enhanced chemilumines- cell number data, the apoptotic response to a-TOS was cence kit obtained from Amersham Pharmacia Biotech rather steep in the dose range between 20 and 40 mol/L. (Piscataway, NJ). Immunoreactive bands were quantitated by volume densitometry using the ImageQuant software (Molecular Dynamics, Sunnyvale, CA), and normalized to loading controls. The following polyclonal antibodies (source) were used in this study: anti-PARP, phospho-Bcl-2 (Ser70), Bcl-xL, Bax, Bak, Ask1, Sek1, phospho-Sek1, JNK, and phospho- JNK (Cell Signaling Technology), anti-Bim (BD PharMin- gen, San Diego, CA), and anti-phospho-Bim EL (Upstate Cell Signaling Solutions, Charlottesville, VA). The following monoclonal antibodies (source) were used in this study: anti-cytochrome c (Oncogene Re- search Products), anti-Bcl-2 (BD PharMingen), anti- GADD45 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-h-actin (Sigma), anti-glyceraldehyde-3-phosphate dehydrogenase (Chemicon, Temecula, CA), and anti-heat shock protein 70 (StressGen Biotechnologies Corp., Victoria, BC, Canada). Statistical Analysis Figure 1. Sensitivity of PC-3 cells to a-TOS. Cells were exposed to 10, The Student’s two-tailed t test was used to determine 20, 30, or 40 Amol/L a-TOS for 24, 48, or 72 h. Results are expressed as percentages of the control. Values of < 100% represent growth statistical significance between treatment and control inhibition. Columns, mean (n = 5); bars, SE. *, P < 0.05, statistically values, and P < 0.05 was considered significant. different compared with the untreated control.

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Identification of Novel a-TOS Responsive Apoptosis- Table 1. Changes of a-TOS-responsive apoptosis-related genes Related Genes In order to avoid the confounding effect of cell death Gene Gene Name Change Signal but still in a position to capture the early response genes Symbol log2 ratio which are exquisitely sensitive to a-TOS, we exposed PC-3 AKT1 v-akt murine thymoma Decrease À2.7 cells to 10 Amol/L a-TOS for only 24 hours for the viral oncogene homologue 1 microarray experiment. As noted in Methods, genes with APP Amyloid h (A4) Increase 1.9 z V alog2-transformed treatment/control ratio of 1or À1 precursor protein are considered to be up-regulated or down-regulated by ASK1 MAPK kinase kinase 5 Increase 2.8 a-TOS, respectively. Based on the above analysis, we CGB Chorionic gonadotropin, h Decrease À2.1 identified more than 600 genes responsive to a-TOS. With polypeptide the resources provided by the NetAffx Analysis Center on CLDN3 Claudin 3 Decrease À1.6 the Affymetrix web site, we narrowed the roster to those C-MYC v-myc myelocytomatosis viral Decrease À1.8 genes that are implicated in the signal transduction of oncogene homologue apoptosis. The information is summarized in Table 1. One FXR1 Fragile X mental retardation, Increase 2.2 thing caught our attention immediately from this list of 21 autosomal homologue 1 GADD45B Growth arrest and DNA- Increase 2.0 genes. Three upstream components of the stress-activated damage inducible, beta h boldface kinase cascade, ASK1, GADD45 , and SEK1 ( in HDAC1 Histone deacetylase 1 Decrease À2.7 Table 1), were all induced by a-TOS with a respective HSPB2 Heat shock 27 kDa protein 2 Decrease À2.5 signal log ratio of 2.8, 2.0, and 1.4. Because the JNK NFKB3 v-rel reticuloendotheliosis viral Decrease À1.5 pathway is known to mediate the apoptotic response of oncogene homologue A cancer cells, we thought it would be reasonable to do PAR1 Coagulation factor II Decrease À1.8 additional experiments with these three genes. (thrombin) receptor To confirm the array data, we used Western blot PAR4 PRKC, apoptosis, WT1, Increase 1.4 analysis to measure the changes in protein level of Ask1, regulator PRKCZ Protein kinase C, Decrease À2.1 PSEN1 Presenilin 1 Decrease À3.3 RAF1 v-raf-1 murine leukemia viral Decrease À3.4 oncogene homologue 1 SEK1 MAPK kinase 4 Increase 1.4 SOCS2 Suppressor of cytokine Increase 2.0 signaling 2 STK3 Serine/threonine kinase 3 Increase 2.4 SURVIVIN Baculoviral IAP repeat- Decrease À21.6 containing 5 (survivin) TNFRSF25 Tumor necrosis factor receptor Decrease À1.7 super family, member 25

GADD45h, and Sek1. The results are shown in Fig. 3. We quantified the intensity of the protein bands by densitometry and expressed the results as fold of change induced by a-TOS (Table 2). The protein levels of all three molecules were markedly increased. In the other half of Table 2, we presented the array data, but this time, calculated as fold of change also in order to match the Western data. The conversion was done by taking the value of 2 and raised it to the power of the signal log ratio. The exercise is meant to illustrate that there is a reasonably close correlation between the changes in transcript and protein levels. Activation of JNK Pathway by a-TOS To further investigate the effect of a-TOS on the activity of the JNK pathway components, PC-3 cells were exposed Figure 2. Quantitation of apoptosis by terminal deoxynucleotidyl to 40 Amol/L a-TOS for 12 or 24 hours and whole cell transferase-mediated nick end labeling in PC-3 cells treated with 20 or lysates were analyzed by Western blot. The higher dose 40 Amol/L a-TOS for 24 h. A, cytograms from flow cytometric analysis. and shorter time protocol were designed to see how quickly Right of each cytogram, apoptotic cells. B, percentages of apoptotic cells. Columns, mean (n = 3); bars, SE. *, P < 0.05, statistically cells respond to a-TOS induction of the stress-activated different compared with the untreated control. signaling pathway. Because this is a kinase cascade,

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includingBax,Bak,andBim.Wholecelllysateand mitochondria-enriched fraction were used to analyze for protein level and translocation changes, respectively. Because a-TOS did not alter the protein level of Bcl-xL, we used this mitochondria-resident protein as a loading control for mitochondrial fraction Western blot. As shown in Fig. 6, a-TOS significantly increased the amount of Bax protein in whole cell lysate at 24 hours. Interestingly, an increase of Bax protein in the mitochondria was observed as early as 12 hours, suggesting that the induction of Bax translocation to the mitochondria by a-TOS was independent of the increase in expression level. In contrast to Bax, the protein level of Bak in either whole cell lysate or mitochondria was Figure 3. Western blot analysis of TRIzol-isolated protein samples not affected by a-TOS. pooled from PC-3 cells treated with 20 Amol/L a-TOS for 24 h for The protein level of Bim EL, a generally expressed confirmation of the array data. The protein levels of Ask1, GADD45h, and isoform of Bim, was not changed in whole cell lysate of Sek1 were determined with actin as the loading control. Results shown are a-TOS-treated cells. However, the amount of Bim EL representative of two independent experiments. protein was significantly increased in the mitochondria. The phosphorylation status of Bim EL at Ser65, a JNK- we were naturally interested in the phosphorylation status specific site (25), was also evaluated. Phospho-Bim EL of the proteins, especially when microarray data do not was not cleanly resolved in whole cell lysate due to the furnish information on posttranslational changes. As presence of too many nonspecific signals. However, an shown in Fig. 4, a-TOS markedly increased the expression increase in phospho-Bim EL was observed in the mito- level of Ask1, GADD45, Sek1, and phospho-Sek1 as early as chondria of a-TOS treated cells. 12 hours; the magnitude of the increase seemed to increase Cytochrome c Release and PARP Cleavage with longer exposure. Consistent with the above findings, To confirm the biological significance of a-TOS activation phospho-JNK was also noticeably increased, although the of the intrinsic apoptotic pathway, Western blot analysis expression level of total JNK was not affected. Given the was done to study cytochrome c release and PARP above observations, the following question came to mind: cleavage. Cytochrome c release is a known outcome of an is JNK activation by a-TOS associated with phosphoryla- activated mitochondrial pathway, whereas PARP cleavage tion of some members of the Bcl-2 family? is a sensitive marker for caspase-mediated apoptosis. Effect of a-TOS on Bcl-2 Family Members As shown in Fig. 7, a-TOS induced cytochrome c release The effect of a-TOS on the antiapoptotic proteins Bcl-2 and Bcl-xL was examined first by Western blot. As shown in Fig. 5, the expression level of both proteins remained unchanged with a-TOS treatment. However, phosphorylation of Bcl-2 at Ser70, a phosphorylation site specific to JNK (24), was found to increase more than 100-fold by a-TOS at 12 hours based on densitometric analysis. This robust enhancement was sustained for at least up to 24 hours. No change was detected with Bcl-xL. The next experiment was designed to determine the effect of a-TOS on the proapoptotic Bcl-2 family proteins,

Table 2. Comparison between array and Western analysis

Gene Array analysis Western analysis

Difference Fold of Difference Fold of c call change* call change-

ASK1 Increase 7.0 Increase 7.4 GADD45h Increase 4.0 Increase 2.6 SEK1 Increase 2.6 Increase 1.9 Figure 4. Western blot analysis of the JNK pathway in PC-3 cells treated with 40 Amol/L a-TOS for 12 or 24 h. Ask1, GADD45h, Sek1, *The value is obtained by raising 2 to the power of signal log ratio (Table 1). phospho-Sek1, total JNK, and phospho-JNK were determined in whole cell -cThe value is the treatment/control signal ratio normalized by actin from lysate with actin as the loading control. Results shown are representative the densitometric analysis. of two independent experiments.

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and PARP cleavage in a time-dependent manner. Further- more, the fact that cytochrome c release happened before PARP cleavage supports the role of the mitochondrial pathway in apoptosis signaling by a-TOS.

Discussion a-TOS is considered an anticancer agent with chemotherapeutic and chemopreventive potential because of its high proapoptotic activity in malignant cells. Several studies have shown that the mitochondria pathway is central to a-TOS-induced apoptosis, but the underlying mechanism has not been fully delineated (15, 20–22). In this study, we successfully used microarray analysis to identify potential molecular triggers that might be critical to a-TOS mitochondria-mediated apoptosis signaling. We discovered that Ask1, GADD45h, and Sek1, three key Figure 6. Western blot analysis of the proapoptotic Bcl-2 family members in PC-3 cells treated with 40 Amol/L a-TOS for 12 or 24 h. components of the stress-activated mitogen-activated pro- Whole cell lysate and the mitochondrial fraction were used to detect tein kinase (MAPK) pathway, are novel targets of a-TOS. changes in the expression level or mitochondrial translocation, respec- Additional Western blot analysis showed that an increased tively. Glyceraldehyde-3-phosphate dehydrogenase and Bcl-xL were used level of phospho-Sek1 could be responsible for the as the loading controls for whole cell lysate and the mitochondrial fraction, respectively. Results shown are representative of two independent activation of JNK. We also found that a-TOS altered JNK- experiments. specific phosphorylation of Bcl-2 and Bim in such a way as to hasten the mitochondrial translocation of Bax and Bim. Because the expression level of most of the Bcl-2 family proteins can bind to and activate MTK1/MEKK4, leading proteins remain unchanged, the posttranslational modifi- to the activation of downstream MAPK cascade mole- cation of Bcl-2 and Bim by activated JNK is likely to play a cules, including Sek1 and JNK. Sek1 is a dual-specificity key role in mediating the apoptotic response to a-TOS. protein kinase that can directly activate JNK by phos- Ask1 is a MAPK kinase kinase (27–29). In the presence phorylation on Thr183 and Tyr185 (32). The present of stress-related stimuli, Ask1 activates Sek1, which in study is the first to report that a-TOS activates the JNK turn activates JNK. Overexpression of Ask1 has been pathway through enhancing the gene expression of a shown to induce mitochondria-dependent caspase cleav- string of upstream molecules. The implication of stress age in various cell models, and the activation of Ask1 is signaling by a-TOS opens up a new dimension in required for stress-induced and cytokine-induced apo- elucidating the anticancer mechanism of a-TOS. ptosis. Taken together, Ask1 seems to function as a JNK activation has been reported to be essential for proapoptotic signaling molecule when cells are subjected stress-activated apoptosis (32). There are two possible to various kinds of stress. GADD45h is a member of a mechanisms to support the proapoptotic action of JNK. group of genes whose transcripts are induced rapidly First, JNK can activate the transcription factor c-Jun by under the conditions of growth arrest and DNA damage. phosphorylation, leading to c-Jun/AP-1-regulated gene Saito et al. (30, 31) have reported that GADD45-like expression of death receptor ligands, such as FasL (33).

Figure 7. Western blot analysis of PARP cleavage and cytochrome c Figure 5. Western blot analysis of the antiapoptotic Bcl-2 family release in PC-3 cells treated with 40 Amol/L a-TOS for 12 or 24 h. PARP members in PC-3 cells treated with 40 Amol/L a-TOS for 12 or 24 h. cleavage was determined in whole cell lysate with heat shock protein 70 Phospho-Bcl-2 at Ser70, Bcl-2, and Bcl-xL were determined in whole cell (HSP70) as the loading control. Cytochrome c release was determined in lysate with glyceraldehyde-3-phosphate dehydrogenase as the loading the cytosolic fraction with glyceraldehyde-3-phosphate dehydrogenase as control. Results shown are representative of two independent the loading control. Results shown are representative of two independent experiments. experiments.

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Second, JNK may contribute to the initiation of mitochon- Although the model is built largely on the strength of dria-mediated apoptosis via phosphorylation of the Bcl-2 descriptive data, it is congruent with the idea that JNK is a family proteins (24, 25, 34, 35). Our present study focuses key player in orchestrating a favorable climate to facilitate mainly on the latter. Recent evidence indicates that JNK cytochrome c release from the mitochondria and apoptosis phosphorylates Bcl-2 at Ser70 to dampen its antiapoptotic induction by a-TOS. To confirm our working model, activity (36). Because the antiapoptotic activity of Bcl-2 additional studies will be required to establish the primarily involves an inhibition of Bax mitochondrial functional significance of activating the JNK pathway. translocation (37, 38), our findings that JNK activation by Overexpression and knockout studies of ASK1, GADD45h, a-TOS is associated with increased phospho-Bcl-2 and Bax and SEK1 will be appropriate in order to gain a better protein in the mitochondria lend credence to a cause and understanding of the biological role of up-regulation of effect relationship. these three genes in a-TOS apoptosis signaling. Moreover, JNK may also affect the proapoptotic Bcl-2 family PC-3 is an aggressive androgen-independent prostate members (25, 34, 39, 40). Kline et al. (20) recently showed cancer cell line. Demonstration of similar mechanisms in that in human breast cancer cells, a-TOS induces JNK other prostate cancer cell lines and/or in vivo models activation and Bax mitochondrial translocation. Functional would greatly increase the relevance of proposed mecha- knockout of Bax significantly reverses a-TOS-induced nisms to the human disease. apoptosis, suggesting a key role of Bax in this process. The presence of a chemical JNK inhibitor prevents Bax from undergoing conformational changes and interferes with its Acknowledgments proapoptotic activity. However, the mechanism by which We thank Dr. Yan Dong for her advice, and Cassandra Hayes and Earl JNK regulates Bax mitochondrial translocation remains Timm for their technical assistance. unclear. Structural studies of Bim and Bax implicate a direct interaction of these two proteins that leads to spontaneous References activation of Bax (41). Our observation that JNK activation is 1. Rama BN, Prasad KN. Study on the specificity of a-tocopheryl (vitamin accompanied by phosphorylation of Bim EL and its release E) acid succinate effects on melanoma, glioma and neuroblastoma cells in from cytosolic sequestration might provide a missing link culture. Proc Soc Exp Biol Med 1983;174:302 – 7. between JNK and Bax mitochondrial translocation in a-TOS 2. Prasad KN, Edwards-Prasad J. Effects of tocopherol (vitamin E) acid succinate on morphological alterations and growth inhibition in melanoma treated cells. cells in culture. Cancer Res 1982;42:550 – 5. On the basis of our findings, we propose a working model 3. Yu W, Simmons-Menchaca M, Gapor A, Sanders BG, Kline K. Induction to integrate the apoptotic signaling circuitry of a-TOS (Fig. 8). of apoptosis in human breast cancer cells by tocopherols and tocotrienols. Some notable vignette captured in the diagram includes (a) Nutr Cancer 1999;33:26 – 32. 4. Weber T, Lu M, Andera L, et al. Vitamin E succinate is a potent novel the activation of the Ask1/Sek1/JNK pathway, (b) phos- antineoplastic agent with high selectivity and cooperativity with tumor pho-Bim as a possible tether between JNK and Bax necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) in vivo. translocation, and (c) the convergence of multiple JNK Clin Cancer Res 2002;8:863 – 9. targets on the mitochondrial pathway of apoptosis. 5. Kline K, Lawson KA, Yu W, Sanders BG. Vitamin E and breast cancer prevention: current status and future potential. J Mammary Gland Biol Neoplasia 2003;8:91 – 102. 6. Neuzil J. Vitamin E succinate and cancer treatment: a vitamin E prototype for selective antitumour activity. Br J Cancer 2003;89: 1822 – 6. 7. Prasad KN, Kumar B, Yan XD, Hanson AJ, Cole WC. a-Tocopheryl succinate, the most effective form of vitamin E for adjuvant cancer treatment: a review. J Am Coll Nutr 2003;22:108 – 17. 8. Israel K, Sanders BG, Kline K. RRR-a-tocopheryl succinate inhibits the proliferation of human prostatic tumor cells with defective cell cycle/ differentiation pathways. Nutr Cancer 1995;24:161 – 9. 9. Israel K, Yu W, Sanders BG, Kline K. Vitamin E succinate induces apoptosis in human prostate cancer cells: role for Fas in vitamin E succinate-triggered apoptosis. Nutr Cancer 2000;36:90 – 100. 10. Ni J, Chen M, Zhang Y, Li R, Huang J, Yeh S. Vitamin E succinate inhibits human prostate cancer cell growth via modulating cell cycle regulatory machinery. Biochem Biophys Res Commun 2003;300:357 – 63. 11. Simmons-Menchaca M, Qian M, Yu W, Sanders BG, Kline K. RRR-a- tocopheryl succinate inhibits DNA synthesis and enhances the production and secretion of biologically active transforming growth factor-h by avian retrovirus-transformed lymphoid cells. Nutr Cancer 1995;24:171 – 85. 12. You H, Yu W, Sanders BG, Kline K. RRR-a-tocopheryl succinate induces MDA-MB-435 and MCF-7 human breast cancer cells to undergo differentiation. 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Downloaded from mct.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Up-regulation of c-Jun-NH2-kinase pathway contributes to the induction of mitochondria-mediated apoptosis by α -tocopheryl succinate in human prostate cancer cells

Ke Zu, Lesleyann Hawthorn and Clement Ip

Mol Cancer Ther 2005;4:43-50.

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