Steroidal Constituents from the Edible Sea Urchin Diadema savignyi Michelin Induce Apoptosis in Human Cancer Cells

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 μg/mL) and compounds 2 (with IC₅₀ values ranging from 5.29±0.11 to 6.80±0.67 μM) and 11 (with IC₅₀ values ranging from 4.95±0.07 to 6.99±0.28 μM) 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.

Introduction

Somatic 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 medications, 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 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.

FIG. 1.

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

Materials and Methods

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 −20°C, 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.

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 37°C in a humidified 5% CO₂ atmosphere (Gibco, Inc., Grand Island, NY, USA). The exponentially growing cells were used throughout the experiments.

Western blot analysis

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 μg/mL aprotinin, 25.0 μg/mL leupeptin, and 1% Nonidet P-40) and kept on ice for 30 min at 4°C. The lysates were centrifuged at 18,461 g at 4°C for 15 min. The supernatants were stored at −20°C until use. Protein content was determined by the Bradford assay.¹⁹ Equal amounts of lysates were separated on 8–15% SDS-PAGE 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 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 β-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 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-3β,5α,6β,7α-tetraol (1), cholest-8(14)-ene-3β,5α,6β,7α-tetraol (2), cholest-7-ene-3β,5α,6β-triol (3), cholest-7-ene-3β,5α,6α,9α-tetraol (4), cholest-7-ene-6-one-3β,5α,9α-triol (5), cholest-5-ene-3β,7α-diol (6), cholest-5-ene-3β,7β-diol (7), cholest-5-ene-7β-methoxy-3β-ol (8), campesterol (9), cholest-5-ene-3β-sulfat sodium (10), cholest-6-ene-5α,8α-epidioxy-3β-ol (11), and cholest-5-ene-3β-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.

FIG. 2.

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

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 μg/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).

Table 1.

The Cytotoxic Effects of the Ch₂Cl₂ Extract and Isolated Compounds 1–12 From Diadema savignyi

	IC₅₀ values (μM)
Compounds	HL-60 (leukemia)	PC-3 (prostate)	SNU-C5 (colorectal)
1	25.62±1.20	40.43±1.45	36.96±2.35
2	5.29±0.11	5.49±0.22	6.80±0.67
3	48.17±1.29	74.06±3.46	NI
4	28.59±0.46	27.41±0.50	31.36±1.29
5	19.35±0.29	24.40±0.46	29.19±0.30
6	7.89±0.18	29.22±0.17	30.70±0.27
7	22.36±0.84	27.94±0.63	31.20±1.04
8	5.33±0.03	9.22±0.67	23.73±0.75
9	5.01±0.29	22.26±0.59	27.08±1.17
10	26.41±0.81	68.87±6.08	NI
11	4.95±0.07	6.99±0.28	6.14±0.22
12	NI	NI	NI
CH₂Cl₂ extract (μg/mL)	1.37±0.15	2.87±0.19	3.11±0.15
Mitoxantrone	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 μM and 100 μg/mL, respectively.

IC₅₀, concentration that inhibits 50% of cell growth; NI, no inhibition (values<100 μM are considered active); SD, standard deviation.

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 μM, and 4.95±0.07, 6.99±0.28, and 6.14±0.22 μM, respectively. In comparison, the positive control, mitoxantrone, had IC₅₀ values of 6.80±0.90, 5.17±0.34, and 19.00±1.40 μM, 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 μM, 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 cytotoxic 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 μM (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 μM (Table 2).

Table 2.

The Effects of the Ch₂Cl₂ Extract And Compounds 2 and 11 on Hel-299 Cells

	IC₅₀ values (μM)
Compounds/extract	HEL-299 (embryonic lung)
2	NI
11	NI
CH₂Cl₂ extract (μg/mL)	9.62±0.66
Mitoxantrone	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.

Table 3.

The Effects of the Ch₂Cl₂ Extract on the Cell Viability in Hel-299 Cels

	Cell viability (%)
IC₅₀ values (μg/mL)	HEL-299 (embryonic lung)
1.37±0.15 (HL-60 cells)	84.3
2.87±0.19 (PC-3 cells)	75.6
3.11±0.15 (SNU-C5 cells)	78.0

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

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).

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 μg/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

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 apoptosis in HL-60, PC-3, and SNU-C5 cells through the regulation of apoptosis-related proteins.

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

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

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

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 HL-60, 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 membrane-affecting chemicals influence the cell cycle. To elucidate the 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 HL-60, 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.

Footnotes

Acknowledgments

This study was supported by the Vietnam Ministry of Science and Technology (MOST), VAST (code: VAST.TĐ.ĐAB.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.

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Supplementary Material

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Steroidal Constituents from the Edible Sea Urchin Diadema savignyi Michelin Induce Apoptosis in Human Cancer Cells

Abstract

Introduction

Materials and Methods

Marine material

Compounds

Cell culture and reagents

Western blot analysis

Statistical analysis

Results

Chemistry analysis

Biological activities

Effect of CH2Cl2 extract and compounds 2 and 11 on the induction of apoptosis

Effect of CH2Cl2 extract and compounds 2 and 11 on 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

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Supplementary Material

Effect of CH₂Cl₂ extract and compounds 2 and 11 on the induction of apoptosis

Effect of CH₂Cl₂ extract and compounds 2 and 11 on 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