Steroidal Constituents from the Edible Sea Urchin

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ABSTRACT Bioassay-directed fractionation and purification were used to isolate 12 steroids (1–12) from a CH2Cl2 extract of the edible Vietnamese sea urchin ...
JOURNAL OF MEDICINAL FOOD J Med Food 18 (1) 2015, 45–53 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.3105

Steroidal Constituents from the Edible Sea Urchin Diadema savignyi Michelin Induce Apoptosis in Human Cancer Cells Nguyen Phuong Thao,1,2 Bui Thi Thuy Luyen,1,2 Eun Ji Kim,3 Jung Il Kang,3 Hee Kyoung Kang,3 Nguyen Xuan Cuong,2 Nguyen Hoai Nam,2 Phan Van Kiem,2 Chau Van Minh,2 and Young Ho Kim1 1

College of Pharmacy, Chungnam National University, Daejeon, Korea. Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), Caugiay, Hanoi, Vietnam. 3 Department of Pharmacology, School of Medicine, Institute of Medical Sciences, Jeju National University, Jeju, Korea.

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ABSTRACT Bioassay-directed fractionation and purification were used to isolate 12 steroids (1–12) from a CH2Cl2 extract of the edible Vietnamese sea urchin Diadema savignyi Michelin. The cytotoxic activity of the CH2Cl2 extract and 12 steroids was evaluated in three human cancer cell lines (HL-60, PC-3, and SNU-C5). Relative to the effects of the positive control, mitoxantrone, the CH2Cl2 extract (with an inhibitory concentration of 50% [IC50] values ranging from 1.37 – 0.15 to 3.11 – 0.15 lg/mL) and compounds 2 (with IC50 values ranging from 5.29 – 0.11 to 6.80 – 0.67 lM) and 11 (with IC50 values ranging from 4.95 – 0.07 to 6.99 – 0.28 lM) exhibited potent cytotoxic effects against all three tested human cancer cell lines. In addition, the CH2Cl2 extract and compounds 2 and 11 were found to induce apoptosis. The induction of apoptosis was accompanied by alterations of the apoptosis-related protein expression, inactivation of ERK1/2 mitogen-activated protein kinase signaling, and decreased c-Myc expression. These data suggest that compounds 2 and 11 from the edible sea urchin D. savignyi may have potential for the treatment of colon cancer, leukemia, and prostate cancer as complementary cancer remedies.

KEY WORDS:  apoptosis  cytotoxic  Diadema savignyi  echinoderm  HEL-299  HL-60  PC-3  SNU-C5

cations, much attention has been focused on natural compounds in plants, marine organisms, and microorganisms.5 Natural products have many pharmacological applications, especially with regard to their potential for use in cancer chemoprevention. Natural marine products have recently become the focus of increased research interest due to their potential pharmacological activities and lower toxicity.6,7 Oxysterols, or oxygenated derivatives of cholesterol, are produced through autooxidation or in vivo enzymatic processes and have been identified in blood, mammalian tissues and cells, and processed foods. Oxysterols have emerged as intriguing substances with diverse biological activities.8,9 For example, they suppress the expression of genes that are involved in the positive balance of cellular cholesterol. Furthermore, oxysterols are cytotoxic and inhibit cell growth.10,11 Recently, it was shown that oxysterol-induced cell death shares many common features with apoptotic cell death,12 which plays an important role in the balance between cell proliferation and cell death. A wide range of stimuli can trigger cell death, which is an irreversible process.13,14 The sea urchin Diadema savignyi Michelin is an invertebrate in the family Diadematidae, order Diadematoida, class Echinoidea, and phylum Echinodermata. They are abundant in the Vietnamese sea and have been used as a

INTRODUCTION

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omatic cells are generated by mitosis, and the vast majority of them die by apoptosis, the physiological process of cellular suicide.1 Cancer can occur as the result of a disruption of this balance due to an increase in cell proliferation, decreased cell death, or both.2 Cancer is often characterized by the uncontrolled proliferation of cells with a loss of cell cycle regulation and apoptosis. Programmed cell death, which is also known as apoptosis, is a tightly controlled process by mechanisms that play an important role in many normal processes.3 More than half of currently available drugs are natural compounds or related to them, and in the case of cancer, this proportion surpasses 60%.4 Most of the anticancer drugs currently used in chemotherapy are cytotoxic to normal cells and cause immunotoxicity not only affecting tumor development but also impairing the patient’s recovery. To find new mediManuscript received 27 November 2013. Revision accepted 23 July 2014. Address correspondence to: Hee Kyoung Kang, PhD, Department of Pharmacology, School of Medicine, Institute of Medical Sciences, Jeju National University, Jeju 690756, Republic of Korea, E-mail: [email protected] or Young Ho Kim, PhD, College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea, E-mail: [email protected]

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health food. However, no reports on its chemical constituents and/or biological activities have been published. In a continuation of our recent investigation of Vietnamese echinoderms,15–18 a CH2Cl2 extract of D. savignyi was found to exhibit significant cytotoxic effects. In this study, we demonstrate the promotive effects of this CH2Cl2 extract and of 12 steroids (1–12, Fig. 1) from the edible sea urchin D. savignyi on the induction of apoptosis through the regulation of mitogen-activated protein kinase (MAPK) signaling in promyelocytic leukemia (HL-60), prostate cancer (PC-3), and colorectal cancer (SNU-C5) cells.

Cell culture and reagents HL-60 (Human promyelocytic leukemia cells), PC-3 (Human prostate cancer cells), SNU-C5 (Human prostate cancer cells), and HEL-299 (Human embryonic lung cells) cell lines were obtained from the Korea Cell Line Bank (KCLB) and were grown in RPMI 1640 (Hyclone, Logan, UT, USA) medium that was supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL and 100 mg/mL, respectively) at 37C in a humidified 5% CO2 atmosphere (Gibco, Inc., Grand Island, NY, USA). The exponentially growing cells were used throughout the experiments.

MATERIALS AND METHODS Western blot analysis

Marine material The samples of D. savignyi Michelin were collected in Nha Trang, Khanhhoa, Vietnam, in December 2011 and identified by MSc. Nguyen Thi My Ngan (Institute of Oceanography). Voucher specimens (No. DS-11-2011_01) were deposited at the Institute of Marine Biochemistry and Institute of Oceanography, VAST, Vietnam. Compounds From the CH2Cl2 extract of the edible Vietnamese sea urchin D. savignyi, 12 steroids 1–12 were isolated and their structures were elucidated (see Supplementary Data; Supplementary Data are available online at www.liebertpub .com/jmf). Stock solutions of tested compounds in dimethyl sulfoxide (DMSO) were prepared, kept at - 20C, and diluted to the final concentration in fresh media before each experiment. The final DMSO concentration did not exceed 0.5% in any experiment to avoid affecting cell growth.

HL-60 (3 · 105 cells/mL), PC-3 (5 · 104 cells/mL), and SNU-C5 (1 · 105 cells/mL) cells were treated with the IC50 values of the CH2Cl2 extract and compounds 2 and 11 for 12, 24, and 48 h. After treatment, the cells were harvested and washed twice with cold phosphate-buffered saline. The cells were lysed with lysis buffer (50.0 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2.0 mM EDTA, 1.0 mM EGTA, 1.0 mM NaVO3, 10.0 mM NaF, 1.0 mM dithiothreitol, 1.0 mM phenylmethylsulfonylfluoride, 25.0 lg/mL aprotinin, 25.0 lg/mL leupeptin, and 1% Nonidet P-40) and kept on ice for 30 min at 4C. The lysates were centrifuged at 18,461 g at 4C for 15 min. The supernatants were stored at - 20C until use. Protein content was determined by the Bradford assay.19 Equal amounts of lysates were separated on 8–15% SDSPAGE gels and then transferred onto a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) by glycine transfer buffer (192 mM glycine, 25.0 mM Tris-HCl [pH 8.8], and 20% MeOH, v/v) at 200 mA for 2 h. After blocking with 5% nonfat dried milk, the membrane was

FIG. 1. The chemical structures of steroids 1–12 from the edible sea urchin D. savignyi.

STEROIDAL CONSTITUENTS FROM DIADEMA SAVIGNYI

incubated with a primary antibody against Bcl-2 (1:500), Bax (1:1000), cleaved PARP (1:1000), cleaved caspase-9 (1:1000), cleaved caspase-3 (1:1000), ERK1/2 (1:1000), phospho-ERK1/2 (1:1000), c-Myc (1:1000), and b-actin (1:5000) antibodies and incubated with a secondary HRP antibody (1:5000; Vector Laboratories, Burlingame, VT, USA) at room temperature. The membrane was exposed on X-ray films (AGFA, Mortsel, Belgium), and protein bands were detected using a WEST-ZOL plus Western Blot Detection System (iNtRON, Gyeonggi-do, Korea). Statistical analysis The results are expressed as the mean – standard deviation. All assays were performed in at least three independent experiments. Statistical significance was assessed by the

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analysis of variance using the paired Student’s t-test (*P < .05, **P < .01, and ***P < .001). RESULTS Chemistry analysis Using various chromatographic experiments, 12 steroids (1–12) were isolated from the CH2Cl2 extract of the Vietnamese sea urchin D. savignyi (Figs. 1 and 2). Their structures were identified as cholest-8-ene-3b,5a,6b,7a-tetraol (1), cholest-8(14)-ene-3b,5a,6b,7a-tetraol (2), cholest-7ene-3b,5a,6b-triol (3), cholest-7-ene-3b,5a,6a,9a-tetraol (4), cholest-7-ene-6-one-3b,5a,9a-triol (5), cholest-5-ene-3b,7adiol (6), cholest-5-ene-3b,7b-diol (7), cholest-5-ene-7bmethoxy-3b-ol (8), campesterol (9), cholest-5-ene-3b-sulfat

FIG. 2. Extraction and isolation process of steroids 1–12 from D. savignyi.

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sodium (10), cholest-6-ene-5a,8a-epidioxy-3b-ol (11), and cholest-5-ene-3b-ol (12), on the basis of spectral and chemical evidence in agreement with literature reports (see Supplementary Data). Of these, compounds 1 and 2 were isolated for the first time from a natural source. Biological activities The cytotoxic effect of the CH2Cl2 extract of the sea urchin D. savignyi was examined against three human cancer cell lines (HL-60, PC-3, and SNU-C5) using the MTT assays. Treatment of these cell lines with the CH2Cl2 extract for 72 h yielded an inhibitory concentration of 50% (IC50) values of 1.37 – 0.15, 2.87 – 0.19, and 3.11 – 0.15 lg/mL, respectively. Subsequently, all of the isolated steroids (1– 12) from D. savignyi were evaluated for their cytotoxic effects on the same cell lines after 72 h of exposure using the MTT assays (Table 1). Compounds 2 and 11 exhibited potent cytotoxic activity in all three tested cell lines with IC50 values of 5.29 – 0.11, 5.49 – 0.22, and 6.80 – 0.67 lM, and 4.95 – 0.07, 6.99 – 0.28, and 6.14 – 0.22 lM, respectively. In comparison, the positive control, mitoxantrone, had IC50 values of 6.80 – 0.90, 5.17 – 0.34, and 19.00 – 1.40 lM, respectively (Table 1). Compound 8 exhibited potent cytotoxicity against HL-60 and PC-3 cells. Compounds 6 and 9 also significantly inhibited the proliferation of the HL-60 cell line with IC50 values of 7.89 – 0.18 and 5.01 – 0.29 lM, respectively. Moderate cytotoxicity was observed with compounds 1, 4, 7, and 10, whereas a weak effect of compound 3 was evident in all three cancer cell lines. Compound 12 showed no cyTable 1. The Cytotoxic Effects of the Ch2Cl2 Extract and Isolated Compounds 1–12 From Diadema savignyi IC50 values (lM) Compounds 1 2 3 4 5 6 7 8 9 10 11 12 CH2Cl2 extract (lg/mL) Mitoxantrone

HL-60 (leukemia)

PC-3 (prostate)

SNU-C5 (colorectal)

25.62 – 1.20 5.29 – 0.11 48.17 – 1.29 28.59 – 0.46 19.35 – 0.29 7.89 – 0.18 22.36 – 0.84 5.33 – 0.03 5.01 – 0.29 26.41 – 0.81 4.95 – 0.07 NI 1.37 – 0.15

40.43 – 1.45 5.49 – 0.22 74.06 – 3.46 27.41 – 0.50 24.40 – 0.46 29.22 – 0.17 27.94 – 0.63 9.22 – 0.67 22.26 – 0.59 68.87 – 6.08 6.99 – 0.28 NI 2.87 – 0.19

36.96 – 2.35 6.80 – 0.67 NI 31.36 – 1.29 29.19 – 0.30 30.70 – 0.27 31.20 – 1.04 23.73 – 0.75 27.08 – 1.17 NI 6.14 – 0.22 NI 3.11 – 0.15

6.80 – 0.90

5.17 – 0.34

19.00 – 1.40

Results are the means – SD of three independent experiments in triplicate. Mitoxantrone (an anticancer agent) was used as a positive control. Compounds/extract were tested at a maximum concentration of 100 lM and 100 lg/mL, respectively. IC50, concentration that inhibits 50% of cell growth; NI, no inhibition (values < 100 lM are considered active); SD, standard deviation.

Table 2. The Effects of the Ch2Cl2 Extract And Compounds 2 and 11 on Hel-299 Cells Compounds/extract 2 11 CH2Cl2 extract (lg/mL) Mitoxantrone

IC50 values (lM) HEL-299 (embryonic lung) NI NI 9.62 – 0.66 4.40 – 0.13

Results are the means – SD of three independent experiments in triplicate. Mitoxantrone was used as a positive control. NI, no inhibition.

totoxic effects compared with the positive control (Table 1). Based on the results, compounds 8 and 9 exhibited selective cytotoxicity toward HL-60 cells. This observation suggests that the methoxy group at the C-7 and/or the methyl group at C-24 might play an important role in the cytotoxicity of the isolated steroids. This notion was supported by previous studies.15,20,21 When HEL-299 cells (a normal cell line) were treated with the CH2Cl2 extract, cytotoxicity was observed, with an IC50 value of 9.62 – 0.66 lM (Table 2). However, when the extract was applied at a concentration equal to its IC50 for each human cancer cell line, HEL-299 cells showed greater viability than the three cancer cell lines (Table 3). Compounds 2 and 11 barely inhibited the growth of HEL-299 cells ( < 10%) at concentrations of approximately 50.0 lM (Table 2). Effect of CH2Cl2 extract and compounds 2 and 11 on the induction of apoptosis The isolated steroids might induce cell death by inducing apoptosis. To determine whether the CH2Cl2 extract and compounds 2 and 11 were able to induce apoptosis in the three human cancer cell lines, we screened for apoptotic characteristics, including a cell cycle arrest and nuclear morphological changes, in extract-treated cells. When the cell cycle distribution was analyzed after 48 h of treatment with the CH2Cl2 extract or compounds 2 and 11, an increase in sub-G1 hypodiploid cells was observed at concentrations equal to the IC50 (Fig. 3). Since nuclear morphological changes are critical markers of cell apoptosis, we performed Hoechst staining to confirm the induction of nuclear morphological changes in the tested samples. The CH2Cl2 extract, as well as compounds 2 and 11, induced the production of apoptotic bodies in HL-60, PC-3, and SNU-C5 cells (Fig. 4). Table 3. The Effects of the Ch2Cl2 Extract on the Cell Viability in Hel-299 Cels IC50 values (lg/mL) 1.37 – 0.15 (HL-60 cells) 2.87 – 0.19 (PC-3 cells) 3.11 – 0.15 (SNU-C5 cells)

Cell viability (%) HEL-299 (embryonic lung) 84.3 75.6 78.0

Results are the means – SD of three independent experiments in triplicate.

FIG. 3. The degree of apoptosis represented as the DNA content measured by flow cytometric analysis in HL-60 (A), PC-3 (B), and SNU-C5 (C) cells for 48 h.

FIG. 4. The degree of apoptosis represented as the fluorescent image of nuclei in HL-60 (A), PC-3 (B), and SNU-C5 (C) cells by Hoechst 33342 staining. HL-60, PC-3, and SNU-C5 cells were treated with the IC50 values of the CH2Cl2 extract and compounds 2 and 11 for 48 h. DNA-specific fluorescent dye, Hoechst 33342 (culture medium at a final concentration of 10.0 lg/mL) was directly added to media and apoptotic bodies were observed with an inverted fluorescent microscope that was equipped with an IX-71 Olympus camera and photographed (magnification · 200). Color images available online at www.liebertpub.com/jmf

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FIG. 5. The effects of CH2Cl2 extract on the levels of Bcl-2, Bax, cleaved caspase-9, caspase-3, cleaved PARP, and on the activation of ERK1/2 mitogen-activated protein kinase (MAPK) and c-Myc in HL-60, PC-3, and SNU-C5 cells for 12, 24, and 48 h. Color images available online at www.liebertpub.com/jmf

Effect of CH2Cl2 extract and compounds 2 and 11 on the regulation of apoptosis-related proteins To investigate the possible mechanism underlying the induction of apoptosis by the CH2Cl2 extract and compounds 2 and 11 in human cancer cells, we examined the expression of anti-apoptotic proteins and pro-apoptotic proteins by Western blot analysis. The B-cell CLL/lymphoma 2 (Bcl-2) family is separated into two subfamilies that either inhibit (anti-apoptotic proteins such as Bcl-2 and Bcl-xL) or promote (pro-apoptotic proteins such as Bax, Bid, and Bak)

apoptosis. It has been reported that the ratio of Bcl-2 to Bax is another reliable marker for apoptosis.22 Treatment with the CH2Cl2 extract and compounds 2 and 11 decreased the level of Bcl-2, while the level of Bax decreased in a time-dependent manner. When caspase cascade activation was examined, the CH2Cl2 extract, as well as compounds 2 and 11, increased the cleavage of procaspase-9 and procaspase-3 in a time-dependent manner. Activation of caspase-3 was demonstrated by the proteolytic cleavage of PARP (116 kDa) to 89-kDa cleavage products (Figs. 5–7). These data indicate that the CH2Cl2 extract and compounds 2 and 11 induced

FIG. 6. The effects of compound 2 on the levels of Bcl-2, Bax, cleaved caspase-9, caspase-3, cleaved PARP, and on the activation of ERK1/2 MAPK and c-Myc in HL-60, PC-3, and SNU-C5 cells for 12, 24, and 48 h. Color images available online at www.liebertpub.com/jmf

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FIG. 7. The effects of compound 11 on the levels of Bcl-2, Bax, cleaved caspase-9, caspase-3, cleaved PARP, and on the activation of ERK1/2 MAPK and c-Myc in HL-60, PC-3, and SNU-C5 cells for 12, 24, and 48 h. Color images available online at www.liebertpub.com/jmf

apoptosis in HL-60, PC-3, and SNU-C5 cells through the regulation of apoptosis-related proteins. Effect of CH2Cl2 extract and compounds 2 and 11 on the regulation of ERK1/2 MAPK and c-Myc The members of the MAPK kinase family mediate a wide variety of cellular behaviors in response to extracellular stimuli. Three of the four main subgroups, ERK, JNK, and p38, serve as a nexus for signal transduction and play a vital role in cellular apoptosis.23 Among MAPK proteins, ERK1/2 contributes to the stabilization of c-Myc, an oncoprotein. ERK1/2 is targeted to mitochondria, where it prevents the release of apoptogenic proteins, is involved in the response to oxidative insults, regulates cholesterol transport, and takes part in the disposal of damaged organelles. It also plays a major role in complex survival responses, leading to carcinogenesis by orchestrating transient signals and transcription modulation in different subcellular locations.24 We further examined the effect of the CH2Cl2 extract and compounds 2 and 11 on intracellular signaling via the activation of ERK1/2 MAPK and the expression of c-Myc in HL60, PC-3, and SNU-C5 cells. The CH2Cl2 extract and compounds 2 and 11 decreased the phosphorylation of ERK1/2. In addition, the downregulation of phospho-ERK1/2 was accompanied by a decrease in c-Myc (Figs. 5–7). DISCUSSION During apoptosis, cells undergo morphological changes such as membrane blebbing, chromatin condensation, nuclear fragmentation, and the formation of apoptotic bodies, as well as biochemical changes, including the alteration of apoptosis-related protein levels. It has been demonstrated

that the phosphorylation/dephosphorylation states of some regulatory proteins are crucial events along the pathways controlling cell growth and apoptosis. A well-established apoptotic signaling cascade is regulated by MAPK.24,25 Two key molecular signaling pathways are implicated in the induction of apoptotic cell death. The first is the extrinsic pathway, which is activated by a death receptor from outside the cell; the second is the intrinsic pathway, which is activated by the Bcl-2 protein family and downstream mitochondrial signals from inside the cell.26 Cancer chemotherapy has improved gradually with the development of novel antitumor drugs.27,28 While the treatment of certain malignancies with chemotherapy has been both successful and encouraging, the efficacy of these approaches has frequently been limited by drug resistance in the tumors themselves and by side effects on normal tissues and cells.29,30 Many reports have indicated that natural products and compounds have potential antitumor activity via the induction of apoptosis.31,32 One of the more attractive strategies considered in current cancer chemotherapy is dietary or pharmaceutical manipulation to induce the death of malignant cells via apoptosis.33 An accumulating body of evidence suggests that naturally occurring compounds and many chemotherapeutic agents with anticancer effects can trigger apoptosis in cancer cells.2 Oxysterols are polyfunctional biological effectors that can prevent the growth of various cell types,34 exerting differential cytotoxic effects on normal versus malignant cells.35,36 The mechanism of oxysterol action involves effects on membrane formation and homeostasis by inhibiting endogenous cholesterol synthesis and insertion into phospholipid bilayers.37 It is interesting to note that membraneaffecting chemicals influence the cell cycle. To elucidate the

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cytotoxic mechanism, we investigated whether the inhibitory effects of the CH2Cl2 extract and compounds 2 and 11 on the proliferation of HL-60, PC-3, and SNU-C5 cells might arise from the induction of apoptosis (see Supplementary Data). Apoptotic cells exhibit typical characteristics such as membrane blebbing, cell shrinkage, chromatin condensation, and an increased population of sub-G1 hypodiploid cells.38 Therefore, we examined the apoptotic characteristics of cancer cells after treatment with CH2Cl2 extract and compounds 2 and 11 at IC50 levels for 48 h. Flow cytometry revealed that the percentage of sub-G1 hypodiploid cells exposed to the CH2Cl2 extract, compounds 2 and 11, was significantly increased after 48 h compared with the control (Fig. 3). These results suggests the induction of apoptosis, and were further supported by an increase in the number of apoptotic bodies in treated cells stained with the cell-permeable DNA dye, Hoechst 33342, and visualized using fluorescence microscopy (Fig. 4). Bcl-2 proteins play important roles in regulating mitochondrial permeabilization and caspase activation. The apoptosis-inducing effect is more dependent on the balance of Bcl-2 and Bax than on the quantity of Bcl-2 alone. Typically, the ratio of Bcl-2 to Bax protein expression is used as an index of apoptosis.39,40 Moreover, the pro-apoptotic members of the Bcl-2 family proteins induce apoptosis by stimulating the release of cytochrome c from mitochondria, resulting in the cleavage and activation of caspase-9. On activation, caspase-9 initiates a protease cascade leading to the rapid activation of caspase-3, an effector caspase in cells undergoing apoptosis.41–43 Therefore, to determine the possible mechanism underlying the induction of apoptosis, we monitored the expression of apoptosis-related proteins such as Bcl-2, Bax, cleaved-caspase-9, caspase-3, and PARP in HL-60, PC-3, and SNU-C5 cells. When treated with the IC50 values of the CH2Cl2 extract and compounds 2 and 11, the levels of these apoptosis-related proteins were altered. Specifically, Bcl-2 was decreased, while the levels of Bax and cleavage of caspase-9, caspase-3, and PARP were increased in a time-dependent manner (Figs. 5–7). This suggests that the CH2Cl2 extract and compounds 2 and 11 induced apoptosis by modulating the expression of apoptosis-related proteins in HL-60, PC-3, and SNU-C5 cells. The MAPK signaling pathways induce either cell proliferation or cell death depending on the cell type and stimulus.25 To establish the MAPK-dependent mechanism of apoptosis induced by the CH2Cl2 extract and compounds 2 and 11, we examined the activation of ERK1/2 MAPK in HL-60, PC-3, and SNU-C5 cells. Treatment with either the CH2Cl2 extract or compounds 2 and 11 significantly decreased the levels of phospho-ERK1/2 levels (Figs. 5–7). c-Myc plays an important role in cell progression through the cell cycle, and its regulation has been correlated with the occurrence of apoptosis in various systems. Several studies have reported that activation of the ERK1/2 MAPK pathway contributes to the stabilization of c-Myc.24 Interestingly, decreased c-Myc and phospho-ERK1/2 levels were observed in HL-60, PC-3, and SNU-C5 cells treated with CH2Cl2 extract or compounds 2 and 11 (Figs. 5–7). These

results suggest that these the extract or compounds inhibited ERK1/2 MAPK signaling and downregulated c-Myc in HL60, PC-3, and SNU-C5 cells. In summary, a CH2Cl2 extract and steroids isolated from the sea urchin D. savignyi exhibited potent in vitro cytotoxic activity against HL-60, PC-3, and SNU-C5 human cancer cells. Moreover, the extract and compounds 2 and 11 induced apoptosis in the three cancer cell lines via inactivation of the ERK1/2 MAPK pathway and the downregulation of c-Myc. Sea urchin is a common ingredient in Vietnamese foods. This study is the first to demonstrate the cytotoxic potential of chemical constituents of D. savignyi. Oxysterol derivatives are strong regulators of gene expression and are known to regulate cholesterol synthesis by transcriptionally and post-transcriptionally inhibiting a key enzyme.34 Additional investigations will be necessary to develop the CH2Cl2 extract and isolated steroids from this species as a complementary cancer remedy and/or an ingredient in functional foods or nutraceuticals.

ACKNOWLEDGMENTS This study was supported by the Vietnam Ministry of Science and Technology (MOST), VAST (code: VAST.TÐ.ÐAB.03/13–15), and the Priority Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2009-0093815), Republic of Korea. The authors are grateful to the Institute of Chemistry, VAST, and KBSI for the provision of the spectroscopic instrument. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist with this work for any of the authors who are involved in this study. REFERENCES 1. Gerl R, Vaux DL: Apoptosis in the development and treatment of cancer. Carcinogenesis 2005;25:263–270. 2. Bold RJ, Termuhlen PM, McConkey DJ: Apoptosis, cancer and cancer therapy. Surg Oncol 1997;6:133–142. 3. Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456–1462. 4. Newman DJ, Cragg GM: Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70:461–467. 5. Cragg GM, Newman DJ: Plants as a source of anticancer agents. J Ethnopharmacol 2005;100:72–79. 6. Jeune MAL, Diaka JK, Brown J: Anticancer activities of pomegranate extracts and genistein in human breast cancer cells. J Med Food 2005;8:469–475. 7. Wona HJ, Han CH, Kim YH, et al.: Induction of apoptosis in human acute leukemia Jurkat T cells by Albizzia julibrissin extract is mediated via mitochondria-dependent caspase-3 activation. J Ethnopharmacol 2006;106:383–389. 8. Lund E, Bjorkhem I: Selective decrease of the viability and the sterol content of proliferating versus quiescent glioma cells exposed to 25-hydroxycholesterol. Acc Chem Res 1995;28:241–249. 9. Smith LL, Johnson BH: Biological activities of oxysterols. Free Radic Biol Med 1989;7:285–332.

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