Sensitization of Tumor Necrosis Factor-Related Apoptosis-Inducing

Oct 16, 2014 - Ligand (TRAIL)-Resistant Primary Prostate Cancer Cells by. Isoegomaketone from ... bioactive behavior of primary tumors.7 Therefore, fu...
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Sensitization of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Resistant Primary Prostate Cancer Cells by Isoegomaketone from Perilla f rutescens Ju-Hye Lee,†,‡ Hyun-Dong Cho,† Il-Yun Jeong,§ Mi-Kyung Lee,† and Kwon-Il Seo*,† †

Department of Food and Nutrition and ‡Research Institute of Basic Science, Sunchon National University, Suncheon 540-742, Republic of Korea § Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongup 580-185, Republic of Korea S Supporting Information *

ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently in clinical trials as a cancer treatment due to its ability to induce apoptosis selectively in cancer cells. Nevertheless, the risk of developing resistance warrants the development of sensitizers that can overcome resistance to TRAIL. In this study, isoegomaketone (1) acted as a synergistic TRAIL sensitizer by mediating up-regulation of DR5 expression in primary prostate cancer RC-58T/h/SA#4 cells. Combined with 1, TRAIL exhibited enhanced apoptotic activity in a human prostate cancer cell line designated RC-58T/h/SA#4, as indicated by increases in annexin V-positive and sub-G1 cell populations as well as condensation of chromatin or apoptotic bodies. Combined treatment also activated caspases-8, -9, and -3; increased the protein levels of Bax, AIF, and cytosolic cytochrome c; and induced PARP cleavage while reducing Bcl-2 protein expression. Human recombinant DR5 Fc chimera efficiently attenuated 1-induced apoptosis, thereby demonstrating the critical role of DR5 in 1-mediated apoptotic cell death. Furthermore, DR5 expression induced by 1 was mediated via a ROS-independent pathway that required CHOP and p53. Overall, these findings provide evidence that 1 potentiates TRAIL-mediated apoptosis through up-regulation of DR5 via a ROSindependent pathway. This suggests that 1 has potential for increasing the effectiveness of prostate cancer therapy with TRAIL.

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recruit and activate Fas-associated death domain (FADD), initiator caspases such as caspases-8, -9, and -10, and effector caspases such as caspase-3, which is ultimately responsible for apoptosis.12−14 However, some cancer cells are resistant to TRAIL-mediated apoptosis.15−17 Several previous studies have demonstrated that the prostate cancer cell lines ALVA-31, PC-3, and DU-145 are highly sensitive to apoptosis induced by TRAIL, whereas the TSU-Pr1 and JCA-1 cell lines are moderately sensitive and the LNCaP cell line is resistant.18 Naturally occurring compounds sensitize TRAIL-resistant cancer cells as well as augment the apoptotic activity of TRAIL. Failure to undergo apoptosis has been implicated in the resistance of cancer cells to TRAIL and is thus related to tumor development. Several previous studies have reported that TRAIL-resistant prostate cancer cells can be sensitized by natural compounds, including curcumin, ursolic acid, flavonoid epigallocatechin gallate, and resveratrol.15−1719−23

rostate cancer is one of the most commonly diagnosed cancers in men.1,2 Most prostate cancers are slow-growing, and symptoms begin to emerge only when the tumor mass grows large enough to constrict the urethra.3 If tumors are detected early and confined to the prostate gland, treatment can be successful. There is much interest in the early prevention of prostate cancer since treatment becomes far more difficult when the cancer spreads to other areas of the body. The human prostate cancer cell lines designated LNCaP, PC3, and DU-145, which are derived from bone, brain, and lymph node metastases, respectively, have been widely used in the study of prostate cancer.4−6 However, it remains particularly difficult to duplicate the genetic makeup or stimulate the bioactive behavior of primary tumors.7 Therefore, further studies using primary prostate cells are urgently needed to inform preclinical or early clinical research. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent due to its capacity to selectively kill cancer cells without toxic effects on normal cells.8−10 TRAIL induces apoptosis by binding to TRAIL receptors death receptor 4 (DR4) and death receptor 5 (DR5).11 It has been shown that activation of these receptors leads to the formation of homo- or heterocomplexes that can © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 17, 2014

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Perilla frutescens (L.) Britt., commonly referred to as perilla, is an annual plant native to eastern Asia, and it is a member of the mint family Lamiaceae. Perilla plants have a long history of use in traditional Chinese medicine. The compounds isolated from P. f rutescens include rosmarinic acid, luteolin, apigenin, ferulic acid, (+)-catechin, and caffeic acid.24 Today, perilla seed oil and its dried leaf extracts are used for a variety of health-promoting purposes. Oil of P. f rutescens was reported to contain perillaketone and isoegomaketone (1) as main constituents.25 Compound 1 has been reported to possess anti-inflammatory and anticancer activities.26,27 However, there is a lack of data to support 1 for the sensitization of TRAIL-resistant prostate cancer cells, including primary prostate cancer cells. The aim of the present study was to investigate the potential sensitizing effects of isoegomaketone (1) on TRAIL-mediated apoptosis in human primary prostate cancer cells.

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RESULTS AND DISCUSSION Isoegomaketone (1) Sensitizes TRAIL-Resistant Primary Human Prostate Cancer RC-58T/h/SA#4 Cells. Human prostate cancer cell lines (LNCaP, PC-3, and RC58T/h/SA#4) were treated with different concentrations (10, 25, 50, 100, 150, and 200 ng/mL) of 1. When PC-3 cells were treated with 0−200 ng/mL of TRAIL for 24 h, cellular viability decreased as determined by the sulforhodamine B (SRB) assay. In contrast, LNCaP and RC-58T/h/SA#4 cells were relatively resistant to TRAIL treatment (Figure 1A). These results show that the primary prostate cancer cell line RC-58T/h/SA#4 is resistant to TRAIL. In parallel with our results, previous studies have shown that PC-3 cells are sensitive to TRAIL-induced apoptosis, whereas LNCap cells are resistant.23 To investigate the potential effects of combined 1 and TRAIL treatment on primary prostate cancer cell growth, RC58T/h/SA#4 cells were treated with various doses of 1 with or without TRAIL (Figure 1B). Co-treatment with 1 and TRAIL significantly increased RC-58T/h/SA#4 cell death, suggesting that 1 sensitized RC-58T/h/SA#4 cells to TRAIL. Next, the cytotoxic effect of 1 and/or TRAIL was also evaluated on human normal prostate epithelial cells (RWPE-1). As shown in Figure 1C, RC-58T/h/SA#4 cells were shown to be more sensitive to 1 and TRAIL treatment than normal cells. These results suggest that 1 was less cytotoxic against normal cells compared to RC-58T/h/SA#4 cells. Isoegomaketone (1) Sensitizes TRAIL-Mediated Apoptosis in RC-58T/h/SA#4 Cells. Apoptosis, known as programmed cell death, is a morphological and biochemical change characterized by chromatin condensation, formation of apoptotic bodies, accumulation of the sub-G1 cell population, and DNA fragmentation.28 To examine whether or not 1 could sensitize RC-58T/h/SA#4 cells to TRAIL-mediated apoptosis, the cells were treated with 1 alone or in combination with TRAIL for 24 h. As shown in Figure 2A, condensation of chromatin or apoptotic bodies were greatly enhanced upon combined 1 and TRAIL treatment. The percentage of annexin V-positive and PI-negative RC-58T/h/SA#4 cells treated with 1

Figure 1. Effect of IK (1) and TRAIL treatment, alone or in combination, on growth of prostate cancer cells. (A) LNCap, RC58T/h/SA#4, and PC-3 cells were treated with the indicated concentrations of TRAIL (10−200 ng/mL) for 24 h, and their cell viability was analyzed by SRB assay. (B) RC-58T/h/SA#4 cells were treated with 1 and/or TRAIL at the indicated concentrations for 24 h. Cell viability was measured by SRB assay. (C) RC-58T/h/SA#4 cells and normal human prostate (RWPE-1) cells were treated with 20 μM 1 and 100 ng/mL TRAIL, alone or in combination, for 24 h, after which their cell viability was determined by SRB assay. Data are presented as means ± SD of three independent experiments. **p < 0.01 and ***p < 0.001 by Student’s t-test.

alone or in combination with TRAIL is shown in Figure 2B. The percentage of apoptotic cells was significantly higher (p < 0.05) upon cotreatment compared to control. To determine whether or not 1 enhances the effect of TRAIL on the sub-G1 cell population, the rate of apoptosis in RC-58T/h/SA#4 cells was measured by flow cytometry. As shown in Figure 2C, the sub-G1 cell population was negligible for untreated control cells, whereas cotreatment with 1 and TRAIL for 24 h increased accumulation of RC-58T/h/SA#4 cells in the sub-G1 phase. These results indicate that cotreatment with 1 and TRAIL significantly elevated the rate of apoptosis in RC-58T/h/SA#4 cells compared with control. Sensitization to TRAIL-Mediated Apoptosis by Isoegomaketone (1) Is Dependent on Caspase. Caspases play a key role in TRAIL-mediated signaling events such as apoptosis and gene induction.29,30 The pan-caspase inhibitor zVAD-fmk significantly suppressed synergistic cell death as well as accumulation of sub-G1 phase cells upon cotreatment with TRAIL and 1, suggesting that combined 1 and TRAIL treatment induced caspase-dependent apoptotic cell death (Figure 3A and B). Additionally, immunoblotting showed that activation of caspases-8, -9, and -3 was enhanced in cells B

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Isoeomaketone (1) Sensitizes TRAIL-Mediated Apoptosis through Up-regulation of DR5. Binding of TRAIL to death receptors results in activation of caspase-8 or -10, which in turn activates downstream effector caspases such as caspases3 and -7 for induction of apoptosis.36,37 Up-regulation of death receptors on the cell surface may enhance TRAIL signaling in sensitive cells or sensitize TRAIL-resistant cells. To explore the role of DR5 in the apoptotic mechanism induced by 1 and TRAIL cotreatment, expression of DR5 was measured by immunoblot analysis. Expression of DR5, but not DR4, in 1treated cells was elevated in a dose- and time-dependent manner (Figure 4A). To determine whether or not this increase in DR5 expression could be responsible for 1 and TRAILinduced cell death, a DR5 Fc chimera was used. For this, RC58T/h/SA#4 cells were treated with 5 mM DR5 Fc chimera prior to combined treatment with 1 and TRAIL. Treatment with DR5 Fc chimera significantly reduced the 1 and TRAILinduced sub-G1 cell population (Figure 4B), abolished 1 and TRAIL-induced activation of caspases-8, -9, and -3, and inhibited cleavage of the caspase-3 substrate PARP (Figure 4C). These results indicate that up-regulation of DR5 may be an initiating event in the mechanism of 1 and TRAIL-induced apoptosis. Isoeomaketone (1) Sensitizes TRAIL-Mediated Apoptosis through Induction of p53 and CHOP. p53 and CCAAT/enhancer binding protein homologous protein (CHOP) have been reported to induce TRAIL receptors.38,39 To investigate whether p53 or CHOP is required for induction of DR5 by 1, expression of p53 or CHOP was determined by immunoblot analysis. Compound 1 was found to induce both p53 and CHOP in RC-58T/h/SA#4 cells in a time- and dosedependent manner (Figure 5A and B). These results suggest that p53 and CHOP are involved in induction of DR5 by 1. These findings are supported by previous studies showing that CHOP up-regulates the promoter activity of DR5 through the CHOP-binding site. Further, CHOP has been demonstrated as a p53 target gene in response to hypoxia and DNA damage.40 Therefore, activation of p53 and CHOP may amplify death receptor-mediated apoptotic signaling in primary prostate cells exposed to both 1 and TRAIL. Sensitization to TRAIL-Mediated Apoptosis by Isoegomaketone (1) Is Independent of ROS. It has been shown that induction of DR5 is mediated through ROS generation.15,41 To investigate whether or not ROS production is implicated in 1 and TRAIL-induced apoptosis, RC-58T/h/ SA#4 cells were treated with N-acetyl-L-cysteine (NAC) before cotreatment with 1 and TRAIL, after which the viability of the cells was examined by the SRB assay. As shown in Figure 6A, NAC treatment did not affect 1 and TRAIL-reduced cell growth. In contrast, previous studies have shown that ROS generation acts as a key initiator of TRAIL-induced apoptosis as well as CHOP and DR5 expression.42,43 Most likely, ROSinduced up-regulation of death receptors depends on cell type. Furthermore, compound 1 had no effect on ROS generation compared to untreated control cells (Figure 6B). These results indicate that ROS generation is not implicated in 1 and TRAILinduced apoptosis. In conclusion, this study is the first to demonstrate that isoegomaketone (1) sensitizes primary prostate cancer cells to TRAIL by triggering up-regulation of DR5, leading to caspasedependent and -independent TRAIL-mediated apoptosis. The present findings suggest that isoegomaketone (1) may be combined with death ligands to treat TRAIL resistance in

Figure 2. Effect of IK (1) and TRAIL treatment, alone or in combination, on apoptosis in RC-58T/h/SA#4 cells. Cells were treated with 1 (20 μM) and/or TRAIL (100 ng/mL) for 24 h. (A) Nuclear fragmentation was assessed by fluorescent microscopy using bisbenzimide (×400). (B) Quantitative analysis of apoptosis was determined using an annexin V-FITC/PI kit. (C) The distribution of cells in sub-G1 phases was analyzed by flow cytometry. Three independent experiments were performed, and means ± SD are presented. **p < 0.01 and ***p < 0.001 by Student’s t-test.

cotreated with 1 and TRAIL, indicating that both extrinsic and intrinsic apoptosis pathways were enhanced by 1 and TRAIL cotreatment. Isoegomaketone (1) or TRAIL alone had no effect on the cleavage of pro-caspases-8, -9, and -3 compared to untreated control (Figure 3C). Isoegomaketone (1) Sensitizes TRAIL-Mediated Apoptosis Signaling by Triggering the Mitochondrial Pathway. To determine whether or not changes in apoptotic regulatory proteins are associated with 1-induced TRAIL sensitization, expression levels of pro-apoptotic Bax and antiapoptotic Bcl-2, which regulate the mitochondrial pathway,31 were determined by immunoblot analysis. Expression of Bax was up-regulated while Bcl-2 expression was downregulated in 1 and TRAIL cotreated cells, suggesting activation of apoptosis in RC-58T/h/SA#4 cells in response to combined 1 and TRAIL treatment (Figure 3C). Bax/Bcl-2 regulates the release of cytochrome c and AIF from mitochondria into the cytosol.32,33 It is worthwhile to define the caspase-independent cell death machinery involved in the alternate pathway activated by 1. AIF released from mitochondria and translocated into the nucleus mediates cell death through a caspase-independent pathway.34,35 Isoegomaketone (1) and TRAIL cotreated cells showed significantly reduced levels of cytochrome c and AIF in mitochondria, whereas levels were simultaneously elevated within the cytosol (Figure 3D). These findings clearly indicate that combined treatment with 1 and TRAIL led to the activation of caspase-dependent and -independent apoptotic pathways. C

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Figure 3. Effect of isoegomaketone (1) and TRAIL treatment, alone or in combination, on caspase activation and expression of Bcl-2 family proteins as well as release of AIF and cytochrome c from mitochondria into the cytosol in RC-58T/h/SA#4 cells. Cells were pretreated with 10 μM z-VADfmk for 2 h prior to 24 h of 20 μM 1 and/or 100 ng/mL TRAIL treatment. (A) Cell viability analyzed by SRB assay and (B) the distribution of cells in sub-G1 phases were analyzed by flow cytometry. Cells were treated with 1 (20 μM) and/or TRAIL (100 ng/mL) for 24 h and then harvested. (C) Total cell lysates were analyzed by immunoblot using specific antibodies. (D) Mitochondrial and cytosolic fractions were prepared as described in the Experimental Section. The fractions were analyzed by immunoblot using specific antibodies. Cell Culture. RC-58T/h/SA#4 cells were obtained from the Center for Prostate Disease Research (Washington, DC, USA). RWPE-1 cells (human prostate epithelial cells) were purchased from ATCC (Manassas, VA, USA). Cells were cultured in DMEM medium supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin (100 μg/mL) in an incubator containing a humidified atmosphere of 5% CO2 at 37 °C. Cell Viability Assay. Cell viability was measured by SRB (SigmaAldrich) assay. Cancer cells were seeded at a concentration of 5 × 104 cells/mL and incubated with 1 for 24 h with or without TRAIL (Sigma-Aldrich) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were then fixed with 12% trichloroacetic acid. After 1 h of incubation at 4 °C, cells were washed three times with water. Cells were then stained with 0.4% SRB at room temperature for 1 h and then washed three times with 1% acetic acid. Bound SRB was dissolved with 10 mM Tris buffer, after which absorbance of the 96-well plate was measured using a microplate reader (Molecular Devices Inc., Sunnyvale, CA, USA). Hoechst 33258 Staining. Characteristic apoptotic morphological changes were assessed by fluorescent microscopy using bisbenzimide (Hoechst 33258) (Sigma Chemical, St Louis, MO, USA) staining. Briefly, cells were seeded onto six-well plates at a density of 1 × 106 cells per well, followed by treatment with 1 for 24 h with or without TRAIL. Cells were then washed and fixed with 1% glutaraldehyde and stained with 5 μg/mL of bisbenzimide for 10 min at room temperature. Cells were examined using a fluorescence microscope (Olympus Optical Co. Ltd., Tokyo, Japan) to determine nuclei fragmentation and chromatin condensation. Annexin V/Propidium Iodide (PI) Staining. RC-58T/h/SA#4 cells (1 × 105 cells/mL) were seeded onto six-well plates and treated

primary prostate cancer cells without impairing its tumor selectivity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Isogeomaketone (1) was isolated from P. f rutescens (L.) Britt. cv. Chookyoupjaso as previously described.26 In brief, the above-ground portion of P. f ructesces (L.) Britt. cv. Chookyoupjaso was extracted with MeOH at room temperature over 3 d. After filtration, the MeOH extract was evaporated and partitioned using ethyl acetate, butanol, and water. The soluble ethyl acetate fraction was separated by column chromatography on silica gel using a hexane−ethyl acetate gradient. The fraction obtained was further separated to yield 1. The purity (>97%) was determined using NMR spectroscopy and HPLC (Figures S1−S3, Supporting Information). For the present study, the compound was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) and stored at −20 °C. The final concentration of DMSO was 0.1% (v/v) in cell culture system. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA, penicillin, streptomycin, and an antimycotic were purchased from Gibco BRL (Rockville, MD, USA). The general caspase inhibitor z-VAD-fmk was obtained from R&D Systems (Minneapolis, MN, USA). Anti-Bax, antiBcl-2, anticaspase-3, anticaspase-8, anticaspase-9, anti-p53, anticytochrome c, anti-AIF, antipoly(ADP-ribose) polymerase (PARP), and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-CHOP was purchased from Cell Signaling Technology (Beverly, MA, USA). An enhanced chemiluminesence (ECL) kit was purchased from GE Healthcare (Buckinghamshire, UK). A mitochondria isolation kit was purchased from Thermo Scientific Pierce (Rockford, IL, USA). D

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Figure 4. Role of DR5 in cell death induced by isoeomaketone (1) and TRAIL treatments. (A) Cells were treated with 1 (20 μM) for the indicated time. Whole cell extracts were analyzed by immunoblotting using DR4 and DR5 antibodies. Cells were pretreated with 100 ng/mL DR5 Fc chimera for 2 h prior to 24 h of 1 (20 μM) and/or TRAIL (100 ng/mL) treatment. (B) The distribution of cells in the sub-G1 phase was analyzed by flow cytometry. Three independent experiments were performed, and means ± SD are presented. **p < 0.01 and ***p < 0.001 by Student’s t-test. (C) Total cell lysates were analyzed by immunoblot using specific antibodies.

Figure 5. Effect of isoegomaketone (1) treatment on the expression of p53 and CHOP proteins in RC-58T/h/SA#4 cells. Protein levels of p53, CHOP, and β-actin in RC-58T/h/SA#4 cells were analyzed by immunoblot using specific antibodies following treatment with 1 for the indicated (A) concentrations or (B) times.

Figure 6. Effect of isoegomaketone (1) treatment on ROS generation in RC-58T/h/SA#4 cells. (A) Cells were pretreated with 5 mM NAC for 2 h prior to 24 h of 1 (20 μM) and/or TRAIL (100 ng/mL) treatment. Cell viability was measured by SRB assay. Data are presented as means ± SD of three independent experiments. **p < 0.01 and ***p < 0.001 by Student’s t-test. (B) Cells were treated with indicated concentrations of 1 for 30 min. Intracellular ROS levels were investigated by flow cytometry using DCFH-DA.

with 1 with or without TRAIL for 24 h. Apoptotic cells were detected using an annexin V Alexa Fluor 488/PI apoptosis kit (Santa Cruz Biotechnology), according to the manufacturer’s instructions. Cells were washed twice with ice-cold phosphate-buffered saline (PBS), resuspended in PBS (100 μL), and incubated with annexin V labeling solution (5 μL) for 30 min at 4 °C in the dark. Cells were then incubated in 1× buffer solution (200 μL) and labeled with PI. The percentage of apoptotic cells was determined by a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA). Cell Cycle Analysis. Cells were seeded at a density of 1 × 105 cells per well in six-well plates and cultured for 24 h in DMEM. After culture, cells were treated with 1 for 24 h with or without TRAIL. Cells were then collected and fixed in ice-cold 70% ethanol in media and stored at 4 °C overnight. After resuspension, cells were washed and incubated with 1 μL of RNase (1 mg/mL) (Sigma-Aldrich), 20 μL of propidium iodide (1 mg/mL) (Sigma-Aldrich), and 500 mL of PBS at 37 °C for 30 min. After staining, flow cytometry was used to analyze sub-G1 DNA content.

Immunoblot Analysis. RC-58T/h/SA#4 cells were seeded at a density of 5 × 104 cells/mL in a 100 mm dish and cultured for 24 h in DMEM medium in a humidified atmosphere of 5% CO2 at 37 °C. After culture, cells were treated with 1 for 24 h with or without TRAIL and then collected by centrifugation. Pelleted cells were lysed in lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 1 mM PMSF, 2 μg/mL of aprotinin) for 30 min on ice. To examine the subcellular localization of cytochrome c and AIF, cytosolic extracts were prepared according to the manual provided in the mitochondria isolation. Protein content of the supernatant was measured by BCA protein assay (Thermo Scientific Pierce). Protein samples (10 μg of protein/lane) were separated by 12% SDS-PAGE at 100 V of constant voltage/slab for 1.5 h. Proteins were then transferred onto nitrocellulose membranes. After blocking with 2.5% and 5% bovine serum albumin for 1 h at 37 °C, membranes were E

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incubated with anticaspase-3, anticaspase-8, anticaspase-9, anti-Bax, anti-Bcl 2, anticytochrome c, anti-AIF, anti-PARP, anti-p53, and antiCHOP primary antibodies overnight at 4 °C. Next, membranes were washed with T-TBS, incubated with horseradish peroxidase-coupled secondary antibodies for 1 h at 4 °C, and then washed again with TTBS. Antibody binding was detected using an ECL kit as previously described.44 Measurement of ROS Generation. Production of intracellular ROS was detected by flow cytometry using dichlorofluoresceindiacetate (DCFH-DA) (Sigma-Aldrich). Briefly, RC-58T/h/SA#4 cells were plated at a density 5 × 105 cells/well, allowed to attach overnight, and exposed to 1 with or without TRAIL for 30 min. Then, the wells were stained with DCFH-DA (10 μM) for 30 min at 37 °C, and the fluorescence intensity in the cells was determined using flow cytometry. Statistical Analysis. Data were analyzed by Student’s t-test to evaluate significant differences. Levels of *p < 0.05, **p < 0.01, and ***p < 0.001 were regarded as statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

Spectroscopic data of isoeomaketone (1). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82 61 750 3655. Fax: +82 61 750 3650. E-mail: seoki@ sunchon.ac.kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors wish to thank Dr. J. S. Rhim of the Center for Prostate Disease Research for providing the RC-58T/h/SA#4 primary human prostate cancer cells. This work was supported by a National Research Foundation of Korean Grant funded by the Korean Government (Ministry of Education, Science and Technology, NRF-2011-355-F00049).

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