Tanshinone IIA Facilitates TRAIL Sensitization by Up-regulating DR5

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Chemical Research in Toxicology

Tanshinone IIA facilitates TRAIL sensitization by up-regulating DR5 through the ROS-JNK-CHOP signaling axis in human ovarian carcinoma cell lines

Chia-Che Chang,‡§║┴1 Cheng-Ping Kuan,#1 Jyun-Yi Lin,† Jui-Sheng Lai,# Tsing-Fen Ho†*

†

Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan;

‡

Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan;

§

Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan;

║

Ph D program in Translational Medicine, National Chung Hsing University, Taichung, Taiwan;

┴

Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, Taiwan;

#

Division of Biotechnology, Taiwan Agricultural Research Institute, Wufeng, Taiwan;

*

Corresponding author. Tel.: +886 4 22391647; Fax: +886 4 22396761. E-mail address: [email protected] (T.-F. Ho)

1

These two authors contributed equally to this work.

ABSTRACT 1

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Tanshinone IIA (TIIA) extracted from Salvia miltiorrhiza has been shown to possess antitumor and TRAIL-sensitizing activity. The involvement of DR5 in the mechanism whereby TIIA exerts its effects is unknown. This study aimed to explore the mechanism underlying TIIA augmentation of TRAIL-induced cell death in ovarian carcinoma cells. Cell viability was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of relating signaling proteins. Transcriptional activation was explored by dual-luciferase reporter assay. We found that TIIA sensitized human ovarian carcinoma cells to TRAIL-induced extrinsic apoptosis. Combined treatment with subtoxic concentrations of TIIA and TRAIL was more effective than single treatments with respect to cytotoxicity, clonogenic inhibition, and the induction of caspase-8 and PARP activity in ovarian carcinoma cell lines TOV-21G and SKOV3. TIIA induced DR5 protein and mRNA expression in a concentration-dependent manner. DR5/Fc treatment markedly suppressed the TRAIL cytotoxicity enhanced by TIIA. These results indicate that DR5 plays an essential role in TIIA-induced TRAIL sensitization and that induction of DR5 by TIIA is mediated through up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP). Knockdown of CHOP gene expression by shRNA attenuated DR5 up-regulation and rescued cell viability under the treatment of TIIA-TRAIL combination. TIIA promoted JNK-mediated signaling to up-regulated CHOP and thereby inducing DR5 expression as shown by the ability of a JNK inhibitor potently suppressed the TIIA-mediated activation of 2


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CHOP and DR5. In addition, the quenching of ROS using NAC prevented induction of JNK phosphorylation and CHOP induction. Furthermore, inhibition of ROS by NAC significantly attenuated TRAIL sensitization by TIIA. Taken together, these data suggest that TIIA enhances TRAIL-induced apoptosis by upregulating DR5 receptors through the ROS-JNK-CHOP signaling axis in human ovarian carcinoma cells.

Keywords: Tanshinone IIA; TRAIL; DR5; ROS; JNK; CHOP

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INTRODUCTION Epithelial ovarian carcinoma (EOC) is the leading cause of death among gynecological malignancies in women worldwide. Most patients are diagnosed at an advanced stage and have a poor prognosis. Currently, the standard treatment for advanced ovarian cancer is surgical tumor resection followed by chemotherapy.1 However, most patients relapse with tumors that acquire resistance to chemotherapy. Therefore, novel therapeutic agents and strategies are needed for treating this devastating disease. We identified a novel anti-ovarian cancer agent, tanshinone IIA (TIIA), a natural product derived from the roots of Salvia miltiorrhiza.2 We revealed that TIIA induces proapoptotic and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitizing effects, implicating this active compound as a potential therapeutic agent or TRAIL-based therapy for cancer chemoprevention or chemotherapy. TIIA is reported to possess anti-inflammatory, anti-oxidative, and anti-tumor activities.3-8 The ability of TIIA to induce apoptosis in cancer cells has been established, and several molecular targets have been identified, including activation of JNK,9 p38 MAPK10 and p53,11,12 induction of reactive oxygen species (ROS),13 as well as inhibition of PI3K/AKT,14 STAT3,15 CHOP,16 HIF-1α and VEGF.7 Whereas apoptosis is generally initiated by either the intrinsic or extrinsic stimuli. The intrinsic pathway is trigger by cellular damage leads to the cleavage and consequent activation of caspase-9, which in turn sets of the caspase cascade 4


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that stimulates apoptosis. The extrinsic pathway is triggered by binding of tumor necrosis factor-α (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), or Fas ligands to their receptors, leads to cleavage and activation of caspase-8, which is recruited to the “death-inducing signal complex” (DISC) to initiate apoptosis. Cellular FLICE-inhibitory protein (c-FLIP) is structurally homologous to caspase-8, therefore it competes with caspase-8 for binding to the DISC complex but is devoid of caspase activity, thus precluding caspase-8 activation in a dominant-negative manner. However, the mechanism whereby TIIA activates the extrinsic pathway has not been fully elucidated. TRAIL is a potential candidate for anti-cancer therapy based on its selective suppression of tumor growth in vivo and in vitro with little or no effect on normal cells.17,18 TRAIL belongs to the TNF family which includes cytokines such as TNF-α and Fas ligands. The soluble TRAIL is homo-trimer which initiates extrinsic apoptosis through binding to death receptors (DRs) 4 and/ 5 expressed on the cell surface in a variety of tumor cells.19,20 Although TRAIL effectively induces the apoptosis of cancer cells, repeated application leads to resistance through multiple mechanisms.21,22 Suggested mechanisms include dysregulation of death receptors and the defective production of DISC.23-25 Thus, agents that can up-regulate death receptors have the potential to sensitize the apoptotic effects of TRAIL.26,27 In this study, we explored the role of DR5 in TRAIL sensitization by TIIA in EOC cells. Recently, numerous signaling molecules are known to trigger DR5 induction, including 5



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activation of mitogen-activated protein kinases (MAPKs) and the binding of CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) transcription factor to DR5 promoter.28 The signaling event involved in survival, growth arrest, or programmed cell death includes activation of the MAPK pathway. The MAPK pathways consist of JNK, p38 MAPK and ERK. The JNK/p38-MAPK pathway plays a central role in apoptosis,29 especially chemical-induced apoptosis, whereas ERK is involved in cell survival, proliferation, differentiation and migration. In this study, we present evidence supporting that TIIA up-regulates DR5 expression through activating the ROS-JNK-CHOP signaling axis, leading to sensitization of EOC cells to TRAIL-induced apoptosis.

MATERIALS AND METHODS Reagents Tanshinone IIA (TIIA, Lot number 00020043-010, purity > 96.1%) was purchased from ChromaDex Inc. (Irvine, CA, USA). Purified TIIA was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM and was stored in the dark at −20 °C until use. The final concentration of DMSO used in all experiments was 0.1% (v/v). Recombinant human TRAIL (Gibco; Grand Island, NY, USA) was prepared as a 100 µg/mL stock solution and stored in aliquots at −20 °C before use. 2’, 7’-dichlorodihydrofluororescein diacetate (H2DCFDA) was purchased from Sigma. Puromycin·2HCl, NAC (N-acetylcysteine), 6


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SB203580 (p38 MAPK-specific inhibitor) and SP600125 (JNK-specific inhibitor) were purchased from TOCRIS (Ellisville, Missouri, USA). Thapsigargin (Calbiochem; Darmstadt, Germany) was prepared as a 1 mM stock solution in DMSO. Human recombinant DR5/Fc chimeric protein was purchased from R&D Systems (Minneapolis, MN, USA). Cell culture Three human ovarian cancer cell lines CaOV3 (ATCC HTB-75), TOV-21G (BCRC 60407) and SKOV3 (ATCC HTB-77) were used in this study. CaOV3 cells were grown in DMEM medium with 10% heat-inactivated fetal bovine serum (FBS) (Gibco; Grand Island, NY, USA). TOV-21G cells were grown in a 1:1 mixture of MCDB 105 and medium 199 with 15% FBS. SKOV3 cells were grown in McCoy’s 5α medium with 10% FBS supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin. The media and supplements were purchased from Invitrogen (Carlsbad, CA, USA). All cell lines were cultured at 37 °C in a 5% CO2 atmosphere. Cell viability assays The effect of TRAIL or TIIA on cell viability was determined by MTS assay as previously described.30 In brief, cultures were established in 96-well flatbottom microtiter plates in growth medium containing 10% FBS. Cell suspensions (100 µL; 5–15×103 cells) were added to each well and allowed to attach overnight. Medium was then changed and cells were maintained in media alone or in the presence of the indicated amount of drug in a final 7



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volume of 150 µL 2%-FBS culture medium. After 48 h of incubation, the medium in each well was replaced with 100 µL of fresh phenol-red–free culture medium, followed by addition of 20 µL of One Solution Reagent MTS (Promega; Madison, WI, USA). The plate was then incubated for 2 h at 37 °C in a humidified, 5% CO2 environment before measuring the absorbance (A) at 490 nm using a Sunrise absorbance reader (Tecan; Grodig, Austria). Cell viability was calculated as (Asample − Ablank) / (Acontrol − Ablank) × 100%, while Cytotoxicity = 100% − cell viability (%). All experiments were repeated at least three times with triplicated samples in each experiment. Determination of combination index value (CI) The effect of drug combination was evaluated with Calcusyn software v2.1 (Biosoft) using the Chou-Talalay method.31 In this model, CI value < 1.0 indicate synergism of drug combination, whereas CI value > 1.0 indicate antagonistic effect. Synergistic effects can be further graded based on the calculated CI value: < 0.1: very strong, 0.1−0.3: strong synergism, 0.3−0.7: synergism, 0.7−0.85: moderate synergism, 0.85−0.9: slight synergism, and 0.9−1.1: additive effect. Clonogenic assay The effect of TRAIL or TIIA on colony formation was determined by clonogenic assay as previously described.30 In brief, cells were plated onto 60 mm dishes at a density of 5×105 cells/dish, incubated overnight, and treated with the indicated doses of drugs for 24 h. At the 8


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end of drug treatments, cells were washed twice with phosphate-buffered saline (PBS) and then trypsinized to determine cell numbers. Drug-treated cells were then seeded at a density of 200 cells per 60 mm-dish in triplicate for each treatment. TOV-21G cells were then allowed to form colonies by incubation in drug-free medium for 10 days. To count the numbers of colonies, the cell monolayer was rinsed twice with PBS, followed by staining with 1% crystal violet solution in 30% ethanol. Colonies comprising 50 or more cells were counted under a microscope. The plating efficiency was calculated as the ratio of the number of colonies counted to the number of cells seeded. A plating efficiency of 50–60% was routinely achieved. The same procedure was repeated for at least 3 times. Western blot analysis Immunoblotting was performed as previously described.32 Briefly, whole cell lysates were boiled in sample buffer (100 mM Tris–HCl, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 10% dithiothreitol). Equal amounts of protein (20–50 µg) were separated by SDS-PAGE using a 10–15% polyacrylamide gel. After electrophoresis, the proteins were transferred onto a FluoroTrans W membrane (Pall Life Sciences, USA), blocked with 5% skim milk, and then probed with antibodies. Antibodies against caspase-8 (NEB 9746), DR5 (NEB 3696), cleaved PARP (NEB 9546), JNK (NEB 9258), p-JNK (NEB 9251), p38 (NEB 9212), and p-p38 (NEB 9211) were purchased from Cell Signal (Beverly, MA, USA). The antibody against CHOP/GADD153 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 9



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The β-tubulin antibody was purchased from Sigma. The signals were detected with using enhanced superSignal West Pico chemiluminescence (Pierce/Life Technologies; South San Francisco, CA, USA). Analysis of cell surface expression of DR5 To analyze the cell surface expression of DR5, cells were treated with 0, 1, 3, 10 µM TIIA or 100 ng/mL TRAIL for 24 h, stained with either PE mouse IgG1, κ isotype control or PE mouse anti-human DR5 antibody (BD Pharmingen TM) for 30 min at 4 °C according to the manufacturer’s instructions, resuspended in phosphate-buffered saline, and finally analyzed by flow cytometry. Quantitative real-time reverse transcription polymerase chain reaction The levels of DR5 and CHOP mRNA were determined by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA extracted from mock- or drug-treated cells was isolated using the guanidinium thiocyanate method (Trizol; Invitrogen) following the manufacturer's protocol. Total RNA (10 µg) was reverse-transcribed into 1st strand cDNA using ImProm-II Reverse Transcription reagents (Promega; Madison, WI, USA) using random hexamers as the primer. PCR reactions were set up in a 20 µL reaction volume using SYBR Green PCR Master Mix (Applied Biosystems, USA) with the following primer pairs: DR5 forward, 5’-AgTCAgAgCATCTgCTggAAC-3’; DR5 reverse, 5’-AgCACTgTCTCAgAgTCTCAg-3’; CHOP forward, 10


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5’-ACAgAgCCAAAATCAgAgCTg-3’; and CHOP reverse, 5’-AAgCACATCTgCTTTCAggTg-3’. Gene expression levels were normalized to that of TATA-binding protein (TBP). Final results were expressed as the ratio of copy numbers of CHOP mRNA to TBP mRNA and are presented as mean ± SEM of 3 independent experiments. Dual-luciferase reporter assays TOV-21G cells were transiently transfected with the CHOP promoter luciferase reporter plasmid (pCHOP-Luc) in combination with plasmid-expressing β-galactosidase using the jet PEI transfection reagent (Polyplus, New York, NY, USA) and allowed to grow for an additional 24 h. Subsequently, cells were treated with various doses of TIIA (0, 10, and 20 µM) for 24 h, and the drug-treated cell lysates were prepared and subjected to the dual luciferase assay according to the manufacturer's protocol. The internal control plasmid pGL4.18 was included in all transfections. To normalize transfection efficiency, we used the Tropix Dual-light reporter gene assay system according to the manufacturer's instructions (Applied Biosystems/Life Technologies, South San Francisco, CA, USA). Luciferase activity was normalized to β-galactosidase activity, and final data were presented as the fold-change in luciferase activity between treated and untreated cells. Evaluation of intracellular ROS levels The levels of ROS in TOV-21G cells were examined and determined by flow cytometry (BD 11



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FACSCaliburTM). 2’, 7’-dichlorofluorescin diacetate (H2DCFDA) is a cell-permeable non-fluorescent probe. 2’, 7’-dichlorofluorescin diacetate is de-esterified intracellularly and turns to highly fluorescent 2’, 7’-dichlorofluorescein upon oxidation.33 Cells were treated with or without TIIA for 0.5 h to determine the influence of ROS. Cells were harvested and resuspended in 500 µL of H2DCFDA solution (10 µM), incubated at 37 °C for 30 min, washed twice and analyzed by flow cytometry. shRNA interference for knockdown of CHOP on TOV-21G cell line For retroviral infection, 293T cells were plated on 10-cm dishes at a density of 1 × 106 cells/dish. After 24 h, the cells were transfected with the retroviral constructs pMKO.1-puro or pMKO.1-puro-CHOP-shRNA,34 and viral particles were harvested at 24 and 48 h post-transfection. TOV-21G cells (0.5–1×105/well) were seeded in 6-cm–well plates to yield 50–60% confluence. After 24 h, cells were transfected using jetPEI transfection reagent (Polyplus) with 2 µg/mL puromycin and either empty pMKO.1 vector or the CHOP expression plasmid (pMKO.1-shCHOP). At 48 h after transfection, cells from each well were passed to 96-well plates and selected in medium supplemented with puromycin (2 µg/mL) for 14 days. Downregulated CHOP expression in these transient transfectants was verified by immunoblotting. Statistical analysis All assays were repeated in at least three independent experiments, and the results are 12


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expressed as the mean ± standard error of mean (SEM). Differences between groups were examined for statistical significance using the unpaired two-tailed Student's t-test. Statistical significance was regarded at p < 0.05.

RESULTS TIIA sensitizes human ovarian cancer cells to TRAIL-induced extrinsic apoptosis To explore the effect of TIIA on TRAIL-induced cell death, TRAIL sensitivity was first examined in an array of human ovarian carcinoma cell lines. To this end, the viability of CaOV3, SKOV3 and TOV-21G cells after 48 h-treatment with TRAIL (0–200 ng/mL) was determined. We found that CaOV3 cells were highly sensitive to TRAIL-induced cytotoxicity, with an IC50 of 19.22 ng/mL (Figure 1A). In contrast, both SKOV3 and TOV-21G cells were resistant to the cytotoxic effects of TRAIL. Notably, functional blockade of TRAIL binding to DR5 using the recombinant human DR5/Fc chimeric protein was found to completely rescue the viability of TRAIL-treated CaOV3 cells (Figure 1A). This result illustrates that DR5-mediated apoptotic signaling is required for TRAIL-induced killing of CaOV3 cells. Given their resistance to TRAIL-induced cytotoxicity, SKOV3 and TOV-21G cells were used to investigate TIIA–induced TRAIL sensitization. SKOV3 and TOV-21G cells were also treated for 48 h with increased dosage (0−30 µM) of TIIA for follow-up evaluation of cell viability (Figure 1B). We found that TIIA suppressed the viability in a concentration-dependent manner, with TOV-21G more sensitive to TIIA treatment (IC50: 4.9 ± 0.6 µM) than SKOV3 (IC50 > 30 µM). Importantly, subtoxic doses of TIIA (1–3 µM) effectively sensitized TOV-21G cells to TRAIL-induced killing (Figure 1C). Likewise, TIIA increased TRAIL sensitivity of SKOV3 cells, though requiring higher drug doses. As shown in Figure 1C, 5 ng/mL of TRAIL started to significantly reduce viability in TOV-21G cells 13



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treated with TIIA alone at doses of 1 µM, 2 µM and 3µM. In SKOV3 cells, 50 ng/mL of TRAIL began to induce significant viability reduction when cells were treated with TIIA alone at doses of 1 µM, 2 µM and 3 µM. Furthermore, isobologram analyses were performed to characterize the effect of TIIA-TRAIL combination. It is clear to note that treatment with indicated doses of TRAIL in combination with various concentrations of TIIA all yield combination indexes lower than 1.0, thus confirming that TIIA synergistically enhances TRAIL-induced cell death (Figure 1D). Particularly, a marked synergism was observed in TIIA (2 µM)-TRAIL (100 ng/mL) in TOV-21G cells and in TIIA (5 µM) -TRAIL (100 ng/mL) in SKOV3 cells, which yield combination index (CI) values of 0.36 and 0.56, respectively. At the biochemical level, co-treatment with TIIA markedly enhanced the levels of cleavage and thus activation of caspase-8 and PARP elicited by TRAIL alone in both cell lines tested (Figure 1E), confirming the capability of TIIA to up-regulate TRAIL-induced extrinsic death receptor apoptosis. Lastly, the effect of TIIA on the clonogenic assay of TRAIL-treated TOV-21G cells was examined. As shown in Figure 1F, treatment with either drug alone led to a mild decrease in colony formation (71.3 ± 5.1% and 74.2 ± 2.4% of solvent control after treatment with TRAIL or TIIA alone, respectively). By contrast, TRAIL combined with TIIA showed a marked decrease in the number of colony formation (40.1 ± 2.3% of untreated cells; p < 0.005). TIIA up-regulates DR5 to facilitate TRAIL sensitization Accumulating evidence has highlighted the increased expression level of DR5 as one of the critical mechanisms responsible for overcoming TRAIL resistance.22,23,35 Thus, we asked whether TIIA induces TRAIL sensitization by up-regulating DR5 expression. Our results show that TIIA concentration-dependently increases the levels of DR5 protein in TOV-21G and SKOV3 cells (Figure 2A). It is known that the death receptor-induced pathway leads to 14


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the recruitment of caspase-8 or -10 to the DISC for activation. DISC signaling can be inhibited by expression of c-FLIP, a physiologic dominant-negative mutant of caspase-8 that leads to the formation of a signaling inactive DISC.36 Intriguingly, the level of c-FLIP remained relatively constant in TOV-21G cells and was even elevated in SKOV3 cells on TIIA stimulation (Figure 2A). Additionally, we found that TIIA (0, 10, and 20 µM) also elicited a concentration-dependent increase in DR5 mRNA levels (Figure 2B), with the increase in DR5 mRNA expression reaching its maximal level as early as 3 h after TIIA treatment (Figure 2C). We further performed flow cytometry analysis to validate that the increase in DR5 levels induced by TIIA corresponds to the increased DR5 expression at the cell surface. As shown in Figure 2D, TIIA significantly elevated DR5 levels at the cell surface as low as 1 µM, whereas stimulation of TRAIL (100 ng/mL) failed to induce cell-surface DR5 expression. Lastly, we elucidated the role of DR5 up-regulation in TIIA-induced TRAIL sensitization. The proapoptotic action of DR5 in TOV-21G and SKOV3 cells was functionally blocked using the DR5/Fc chimeric protein, followed by TRAIL-TIIA co-treatment and subsequent evaluation of cell viability. In both TOV-21G and SKOV3 cells, DR5/Fc treatment markedly suppressed the level of TRAIL-induced cytotoxicity enhanced by TIIA (Figure 2E). In particular, the cytotoxicity induced by TRAIL-TIIA co-treatment decreased from 80.2 ± 9.4% to 43.9 ± 13.0% (p < 0.005) and from 70.4 ± 19.2% to 38.3 ± 15.0% (p < 0.01) in TOV-21G and SKOV3 cells, respectively. 15



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TIIA transcriptionally induces CHOP for DR5 up-regulation We next sought to elucidate the role of CHOP in TIIA-induced DR5 up-regulation. TIIA treatment led to a time-dependent increase in CHOP and DR5 protein expression (Figure 3A). Real-time RT-PCR analysis revealed that the level of CHOP mRNA was increased by TIIA treatment in both a concentration- and time-dependent manner (Figure 3B). To further explore whether TIIA up-regulates CHOP at the transcriptional level, we generated a luciferase reporter construct for probing the activity of the CHOP promoter. TOV-21G cells were treated for 24 h with various doses of TIIA (0, 10, 20 µM) or 100 nM of thapsigargin (TG), a well-known ER stress inducer, followed by dual luciferase reporter assay to monitor the CHOP promoter activity. As shown in Figure 3C, TIIA treatment induced dose-dependent activation of the CHOP promoter. To determine the role of TIIA-mediated CHOP promoter induction in DR5 up-regulation and TRAIL sensitization, we examined the effect of TIIA in TOV-21G cells expressing CHOP shRNA. TIIA-TRAIL co-treatment failed to up-regulate DR5 and elicit PARP cleavage in shCHOP cells (Figure 3D). Thus, TIIA mediates up-regulation of DR5 for TRAIL sensitization through the CHOP promoter. TIIA-induced up-regulation of CHOP and DR5 requires JNK Given the involvement of JNK and p38 MAPK in apoptosis induction and the role of JNK in the regulation of CHOP and DR5 expression, we tested the effect of TIIA on JNK and p38 MAPK phosphorylation. As shown in Figure 4A, TIIA treatment led to a 16


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concentration-dependent increase in the levels of phosphorylated JNK (Thr183/Tyr185) and phosphorylated p38 MAPK (Thr180/Tyr182), indicating that TIIA induced the activation of both JNK and p38 MAPK. The addition of a JNK inhibitor potently suppressed TIIA-mediated induction of CHOP and DR5, whereas inhibition of p38 MAPK exerted no effect (Figure 4B). Thus, TIIA likely promotes JNK-mediated signaling to up-regulate CHOP, thereby inducing DR5 expression. ROS induced by TIIA is required for JNK activation and CHOP induction We observed a clear increase in ROS production in TOV-21G cells following TIIA treatment (Figure 5A). Cells pre-treated with the ROS scavenger NAC potently suppressed the TIIA-evoked JNK phosphorylation and CHOP induction, thereby abrogating PARP to cleavage (Figure 5B). Inhibition of ROS by NAC significantly attenuated TRAIL-induced cell death potentiated by TIIA, as the viability of TOV-21G cells co-treated with TIIA and TRAIL was rescued by NAC from 19.3 ± 1 % to 31.6 ± 4.9 % of drug-untreated cells (p < 0.005) (Figure 5C).

DISCUSSION This study investigated the mechanisms whereby TIIA induces TRAIL sensitization. We observed that TIIA treatment markedly sensitized the TRAIL-resistant human EOC cell

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lines TOV-21G and SKOV3 to TRAIL-induced cell death and increased caspase-8 and PARP processing (Figure 1). Furthermore, we provide evidence that DR5 up-regulation underlies TIIA-mediated TRAIL sensitization (Figure 2). Additionally, CHOP was found to be transcriptionally induced by TIIA and was proved to be essential for TIIA-elicited DR5 up-regulation (Figure 3). We confirmed that TIIA-mediated up-regulation of CHOP and DR5 requires JNK, whose activation is induced by ROS evoked by TIIA (Figure 4). To the best of our knowledge, this study is the first to report that TIIA engages the ROS-JNK-CHOP signaling axis to up-regulate DR5 for sensitizing cells to TRAIL-induced extrinsic apoptosis (Figure 5).

Effective cancer therapeutics must induce cell death selectively, affecting malignant tumor cells but not normal cells. TRAIL is considered a promising candidate for medical applications because it triggers apoptosis in a variety of tumor cells but shows little toxicity in normal cells.37 One mechanism that may contribute to the cancer-specific cytotoxicity of TRAIL is the differential expression of its receptors. To date, 5 TRAIL-specific receptors have been identified. DR5 and DR4 are proapoptotic TRAIL receptors, while decoy receptors (DcR1, DcR2), and osteoprotegerin (OPG) contain mutations in the death domain and are unable to transmit apoptotic signals.38,39 DR4 and/or DR5 are expressed in most cancer cell lines, whereas DcR expression is less frequent and does not appear to correlate with resistance to TRAIL.40 According to the numerous clinical trials of TRAIL, one major 18


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obstacle facing the use of TRAIL for treatment is the resistance exhibited by many cancer cells.38 Such resistance occurs in cells with low levels of DR4 and DR5 expression on the cell surface. To our knowledge, this is the first report demonstrating that DR5 receptors play an essential role in the sensitization effect of TIIA on TRAIL’s proapoptotic action in TRAIL-resistant human ovarian cancer cells. Several chemicals induce re-sensitization to TRAIL-mediated apoptosis through upregulation of DR5, a TRAIL receptor.27,41-46 Our results show that TIIA strongly affects the expression of DR5, and blockade of DR5 expression leads to the inhibition of TIIA-mediated TRAIL sensitization (Figure 2). An alternative way to circumvent TRAIL resistance is through down-regulation of c-FLIP, an endogenous inhibitor of caspase-8 activation.22,23,47 Resistance to TRAIL has also been correlated with high levels of c-FLIP which shows a structurally similar to caspase-8 but has no protease activity. Along this line, we noticed an elevated expression of c-FLIP in SKOV3 cells after TIIA treatment whereas the levels of c-FLIP remained relatively constant in TIIA-treated TOV-21G cells (Figure 2A). These results therefore excludes the possible involvement of c-FLIP in TIIA-induced TRAIL sensitization, and also might be a reason why TIIA failed to cause profound cell death in TRAIL-treated SKOV3 cells as that in TOV-21G. Thus, we propose that TIIA might serve as an effective adjunctive reagent in re-sensitizing TRAIL-resistant cells to TRAIL-induced apoptosis through up-regulation of DR5. These findings provide a foundation for developing TIIA as a TRAIL sensitizer that can overcome 19



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TRAIL resistance.

In light of numerous reports that induction of DR5 is transcriptionally regulated by CHOP,28,48 we examined the role of CHOP in DR5 induction. We found that CHOP plays a critical role in the expression of DR5 induced by TIIA. Results of Western blot analysis, real time RT-PCR or luciferase assay, and gene-silencing suggest that TIIA-mediated DR5 induction in TOV-21G cells occurs at the transcriptional level via increased CHOP gene transcription. We observed that TIIA treatment led to a time-dependent increase in the level of CHOP protein, along with the induction of DR5 expression (Figure 3A). In addition, TIIA induced up-regulation of CHOP mRNA (Figure 3B) and activation of the CHOP promoter (Figure 3C). Furthermore, silencing of CHOP abolished the effect of TIIA-mediated TRAIL sensitization on induction of DR5 and apoptosis (Figure 3D). Taken together, this evidence indicates that CHOP plays an essential role in DR5 regulation and the sensitization of TRAIL on TIIA treatment.

We examined the involvement of the signaling molecules JNK and p38 MAPK in this process. Figure 4 shows that JNK and p38 were activated by TIIA; pretreatment with a JNK inhibitor, but not a p38 inhibitor, decreased TIIA-induced upregulation of CHOP and DR5, suggesting that JNK activation may be responsible for TIIA-induced upregulation of CHOP and DR5. This finding is consistent with previous studies showing that JNK-dependent

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CHOP induction regulates apoptosis.49 ROS triggers a variety of signal transduction pathways that lead to cell growth, differentiation, or death. Important downstream mediators of ROS-induced signaling include MAPKs,50 such as JNK and p38 MAPK. We next investigated whether scavenging of ROS could abolish the TIIA-induced sensitization of cells to TRAIL. Figure 5 shows that cell treatment with TIIA alone induced the upregulation of CHOP, the phosphorylation of JNK, and PARP cleavage; these activities were all dramatically inhibited by addition of the ROS scavenger NAC. We also found that induction of ROS is critical for the sensitization of cells to TRAIL by TIIA. Quenching of ROS also abrogated the effect of TIIA on TRAIL-induced apoptosis. These results suggest that ROS generated by TIIA play an essential role in CHOP-dependent DR5 up-regulation and in the increase in apoptosis induced by TIIA plus TRAIL. It is worth noting that NAC treatment or CHOP silencing induces significant but not complete rescue of cell viability under the treatment of TRAIL-TIIA combination. This finding suggested that the NAC-JNK-CHOP-DR5 signaling axis identified in this study represents one of the mechanisms underlying TIIA-mediated TRAIL sensitization. In fact, previous studies have demonstrated a number of mechanisms involved in the sensitization of cancer cell lines to TRAIL-induced apoptosis. These include up-regulation of death receptors, increased DISC formation, upregulation of proapoptotic proteins and suppression of anti-apoptotic proteins. Our recent study identified that down-regulation of anti-apoptotic survivin also plays an 21



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important role in TIIA-induced TRAIL sensitization (Lin et al., Phytomedicine, accepted).51 In conclusion, as summarized in Figure 6, the present study provides the first mechanistic evidence that pretreatment of cells with TIIA effectively recovers or enhances the sensitivity of TRAIL in human EOC cells. TIIA markedly sensitized TRAIL-resistant human EOC cell lines TOV-21G and SKOV3 to TRAIL-induced cell death, likely due to the increased level of processing/activation of caspase-8 and PARP. We identified DR5 up-regulation as a key mechanism leading to TIIA-mediated TRAIL sensitization. Additionally, CHOP was found to be transcriptionally induced by TIIA and was later proved responsible for TIIA-induced DR5 up-regulation. Moreover, we confirmed that TIIA-elicited up-regulation of CHOP and DR5 requires p-JNK, whose activation is evoked by ROS initiated by TIIA. Collectively, these lines of evidence are the first to support the notion that TIIA engages the ROS-JNK-CHOP signaling axis to up-regulate DR5, thereby sensitizing cells to TRAIL-induced extrinsic apoptosis.

AUTHOR INFORMATION Corresponding Author Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, 666 Buzih Road, Taichung 40601, TAIWAN; Tel.: +886 4 22391647; Fax: +886 4 22396761. E-mail address: [email protected] (T.-F. Ho) 22


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Funding Source This work was supported by grants from the following donors: Central Taiwan University of Science and Technology, Taichung, Taiwan (CTU102-P-17 and CTU103-P-18); National Chung Hsing University and Agricultural Research Institute, Council of Agriculture, Executive of Yuan, R.O.C. (NCHU-TARI 9904 and NCHU-TARI 10104); Taichung Veterans General Hospital and National Chung Hsing University, Taichung, Taiwan (TCVGH-NCHU997606); and The Ministry of Education, Taiwan, R.O.C. under the ATU plan.

ABBREVIATIONS TIIA, Tanshinone IIA; DR5, death receptor 5; c-FLIP, Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein; FADD, Fas-associated death domain; ROS, reactive oxygen species; CHOP, C/EBP homologous protein; JNK, c-Jun N-terminal kinase; TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand; z-VAD.fmk, N-benzyloxycarbonyl Val-Ala-Asp (O-methyl)-fluoromethylketone; PARP, poly(ADP-ribose)polymerase; NAC, (N-acetylcysteine); EOC, epithelial ovarian carcinoma.

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Figure 1

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Figure 1. TIIA enhances the TRAIL-induced extrinsic apoptotic pathway in human EOC cell lines. (A) Effect of TRAIL on the viability in EOC cells. Cell viability was determined by MTS assay. Cells were exposed to TRAIL (0–200 ng/mL) for 48 h. CaOV3 cells were pre-incubated with or without DR5/Fc chimeric protein blocking DR5 receptor (1 µg/mL) for 1 h and stimulated for 48 h by TRAIL. (B) Effect of TIIA on the viability in SKOV3 and TOV-21G cells. Cells were treated without (DMSO control) or with increasing doses of TIIA (1 µM–30 µM) for 48 h, followed by determination of cell viability using MTS assay. (C) Cells were treated for 1 h with TIIA (0, 1, 2, or 3 µM for TOV-21G; 0, 1, 3, or 5 µM for SKOV3), followed by the indicated doses of TRAIL for a total 48 h, and cell viability was determined. The values are expressed as the means ± SEM. * p < 0.05, ** p < 0.01, ***p < 0.005, significant difference compared to TIIA only treatment. (D) The dose effect curves were generated by Calcusyn software to fit the experimental points. × symbol designates the combination effect. Cells were exposed to the combination in a non-constant ratio. CI analysis to determine synergy was carried out using Calcusyn software as described in Method. (E) Western blotting of extracts from TOV-21G and SKOV3 cells treated with vehicle control, TRAIL (100 ng/mL) alone, TIIA (2 µM for TOV-21G; 5 µM for SKOV3) alone, or a combination of both drugs were assayed for caspase-8, cleaved PARP and

β-tubulin. (F) Clonogenic assay was performed to determine the survival effect of TIIA-TRAIL co-treatment in TOV-21G cells. ***p < 0.005. 33



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Figure 2

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Figure 2. TIIA increases expression of DR5 protein that regulates sensitivity to TRAIL. (A) TOV-21G and SKOV3 cells were treated with 0, 10 or 20 µM of TIIA for 24 h, and cell extracts were prepared for Western blotting of DR5 and c-FLIP. (B) The expression of DR5 mRNA was determined by real-time RT-PCR. TOV-21G and SKOV3 cells were treated with 0, 10, or 20 µM of TIIA for 12 h (TOV-21G) or 3 h (SKOV3). (C) Cells were treated with 10

µM of TIIA for 0, 3, 6, or 12 h; DR5 mRNA expression was then measured. (D) Effect of TIIA or TRAIL on the surface expression of DR5 in TOV-21G and SKOV3 cells with flow cytometry. Black histograms, isotype control; gray histrograms, solvent control; black line, treated cells with TIIA or TRAIL. (E) After treatment with TIIA for 2 h (2 µM for TOV-21G; 5 µM for SKOV3), cells were pre-incubated without or with 1 µg/mL DR5/Fc for 2 h. Cells were then treated with 100 ng/mL TRAIL for 24 h and the cytotoxicity determined by MTS assay. *p < 0.05; **p < 0.01; ***p < 0.005.

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Figure 3

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Figure 3. TIIA induces upregulation of CHOP to increase DR5 expression in TOV-21G cells. (A) Cells were treated with 10 µM TIIA for 0, 3, 6, 12, or 24 h followed by Western blot analysis of CHOP and DR5. (B) TIIA increases the levels of CHOP mRNA concentrationand time-dependently. Cells were treated with 0, 10, or 20 µM TIIA for 12 h (left) and with 20 µM TIIA for 0, 3, 6, 12 or 18 h (right); the expression of CHOP mRNA was then measured. (C) Effect of TIIA on CHOP promoter activity. TOV-21G cells were treated with TIIA or thapsigargin (TG) for 24 h after transfection with a reporter comprising the CHOP promoter (pGL4-CHOP-Luc) in a β-galactosidase expression vector. TG, an ER stress inducer. (D) TOV-21G cells were transfected with shRNA directed against CHOP mRNA. Transfected cells were treated with or without 2 µM TIIA plus 50 ng/mL TRAIL for 24 h, and cell viability was assessed using MTS assay. Western blotting was used to confirm CHOP knockdown and examine its effect on TIIA-induced DR5 up-regulation. *p < 0.05; ***p < 0.005.

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Figure 4

Figure 4. JNK-mediated signaling up-regulates CHOP, leading to DR5 induction. (A) Immunoblot represents the phosphorylation of JNK and p38 upon TIIA treatment in TOV-21G cells. The sample blots were stripped and reported with antibodies for non-phosphorylated proteins to verify equal protein loading. (B) TOV-21G cells were pretreated with p38 MAPK inhibitor (SB203580) or JNK inhibitor (SP600125) for 1 h and then treated with 10 µM TIIA for 24 h. Western blotting was used to analyze the extracts for CHOP and DR5 expression.

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Figure 5

Fig. 5. JNK phosphorylation and CHOP induction by TIIA are ROS-dependent. (A) TOV-21G cells were treated with TIIA (gray line, vehicle control; dotted line, 10 µM; solid line, 20 µM), and labeled with H2DCFDA to examine ROS production. (B) Inhibition of p-JNK, decrease in CHOP and PARP cleavage by NAC in TOV-21G cells. Cells were pretreated with NAC for 1 h and then treated with 10 µM TIIA for 24 h; whole-cell lysates were analyzed by immunoblot. (C) Pretreatment with antioxidants blocks the cell death induced by TIIA plus TRAIL. TOV-21G cells were pretreated with 10 mM NAC for 1 h and further treated with 2 µM TIIA plus 100 ng/mL TRAIL for 24 h. Cell viability was assessed using MTS assay (**p < 0.01).

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Figure 6

Fig. 6. Diagram showing the mechanism by which tanshinone IIA (TIIA) sensitizes TRAIL-resistant epithelial ovarian carcinoma (EOC) to TRAIL. TIIA enhances TRAIL-induced apoptosis by up-regulating the expression of DR5 via ROS-JNK-CHOP signal cascade.

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209x106mm (300 x 300 DPI)


Tanshinone IIA Facilitates TRAIL Sensitization by Up-regulating DR5

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