Advanced
Steroids from the Cold Water Starfish Ctenodiscus crispatus with Cytotoxic and Apoptotic Effects on Human Hepatocellular Carcinoma and Glioblastoma Cells
Steroids from the Cold Water Starfish Ctenodiscus crispatus with Cytotoxic and Apoptotic Effects on Human Hepatocellular Carcinoma and Glioblastoma Cells
Bulletin of the Korean Chemical Society. 2014. Aug, 35(8): 2335-2341
Copyright © 2014, Korea Chemical Society
  • Received : March 03, 2014
  • Accepted : April 14, 2014
  • Published : August 20, 2014
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Tran Hong Quang
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
Dong-Sung Lee
Hanbang Body-Fluid Research Center, Wonkwang University, Iksan 570-749, Korea
Se Jong Han
Korea Polar Research Institute, KORDI, Incheon 406-840, Korea
Il Chan Kim
Korea Polar Research Institute, KORDI, Incheon 406-840, Korea
Joung Han Yim
Korea Polar Research Institute, KORDI, Incheon 406-840, Korea
Youn-Chul Kim
Hanbang Body-Fluid Research Center, Wonkwang University, Iksan 570-749, Korea
Hyuncheol Oh
Hanbang Body-Fluid Research Center, Wonkwang University, Iksan 570-749, Korea

Abstract
Chemical investigation on the methanol extract of the starfish Ctenodiscus crispatus resulted in the isolation of five steroids, (22 E ,24ξ)-26,27-bisnor-24-methyl-5α-cholest-22-en-3β,5,6β,15α,25-pentol 25- O -sulfate ( 1 ), (22 E ,24 R ,25 R )-24-methyl-5α-cholest-22-en-3β,5,6β,15α,25,26-hexol 26- O -sulfate ( 2 ), (28 R )-24-ethyl-5α-cholesta-3β,5,6β,8,15α,28,29-heptaol-24-sulfate ( 3 ), (25 S )-5α-cholestane-3β,5,6β,15α,16β,26-hexaol ( 4 ), and Δ7-sitosterol ( 5 ). Their structures were identified by extensive spectroscopic analyses, including 1D, 2D NMR and MS and chemical methods. Compound 4 showed cytotoxicity against human hepatoma HepG2 and glioblastoma U87MG cells via inhibition of cell growth and induction of apoptosis. Induction of apoptosis by 4 was demonstrated by cell death, DNA fragmentation, increased Bax/Bcl-2 protein ratio and the activation of caspase-3, caspase-9 and poly (ADP-ribose) polymerase (PARP).
Keywords
Introduction
Marine natural products are considered as a rich source of bioactive substances with high structural diversity. Since the first researches on marine natural product reported in 1960s, over 20,000 natural products have been discovered from marine organisms, 1 and a remarkable number of marine natural products are currently in different phases of clinical trials. 2,3 Among marine organisms, the research on starfish has expanded rapidly over the past few years, which has been prompted by the discovery of a variety of unique structures and the interest in their pharmacological properties. 4 Various secondary metabolites including steroids, steroidal glycosides, anthraquinones, alkaloids, phospholipids, peptides, and fatty acids were reported from starfish, in which polyhydroxysteroids are known as the predominant secondary metabolites. 4 In this study, the starfish Ctenodiscus crispatus (Ctenodiscidae family) was collected at the Sea of Okhotsk. Previous investigations on chemical composition of C. crispatus have shown the presence of some polyhydroxylated steroids. 5,6 As a part of our on-going search for bioactive secondary metabolites from marine organisms, this study describes the isolation and identification of five steroids ( 1-5 ) from the cold water starfish C. crispatus .
In this study, cytotoxic effects of five isolated steroids ( 1-5 ) were evaluated toward two human carcinoma cell lines, human hepatocellular carcinoma (HepG2) and human glioblastoma (U87MG). HepG2 is one of the most frequent malignancies worldwide and is associated with a high rate of metastasis, 7 and U87MG (or grade IV astrocytoma) is the most common primary malignant brain tumor that only weakly responsive to the current therapeutic strategies. 8,9 Based on the result of this primary screening step, compound 4 was selected for further studies on molecular action mechanism of induction of apoptosis in HepG2 and U87MG cells.
Experimental
Extraction and Isolation. The starfish C. crispatus (09CST3- DR7), identified by Dr. Joung Han Yim, was collected at the Sea of Okhotsk (N 53°22.626'E 144°24.200') on January 28, 2009, and was stored in a freezer until being extracted. A voucher specimen is preserved in KOPRI. The frozen sample (122.0 g) was extracted with MeOH in room temperature. The MeOH extract was concentrated in vacuo to provide a residue (DR7, 2441.8 mg). The residue (DR7) was chromatographed over reversed phase (RP) YMC C 18 silica gel column, stepwise eluting with MeOH in H 2 O (20%; 40%; 60%; 80%; and 100% v/v) to give 6 fractions, DR71-6. Fraction DR74 (162.9 mg) was subjected to C 18 silica gel column chromatography (CC), using MeOH-H 2 O (13:10) to provide three subfractions DR741-3. Subfraction DR742 was then subjected to silica gel CC, eluted with EtOAc-MeOH-H 2 O (35:10:1) to provide compound 3 (5.0 mg) and three subfractions, DR7421, DR7423, and DR7424. Subfraction DR7424 was further purified by silica gel CC, using CHCl 3 -MeOH-H 2 O (20:10:1) as eluents to give compounds 1 (2.0 mg) and 2 (5.0 mg). Fraction DR75 (514.4 mg) was subjected to RP C 18 silica gel CC, eluting with MeOH-H 2 O (4:1) to yield 2 subfractions, DR751 and DR752. Subfraction DR752 was subjected to RP C 18 CC, eluting with MeOH-H 2 O (4:1) and further purified by silica gel CC, using CH 2 Cl 2 - MeOH-H 2 O (40:10:1) as eluents to obtain compound 4 (5.0 mg). Fraction DR76 (203.4 mg) was separated by silica gel CC, eluting with n-hexane-EtOAc (4:1) and CH 2 Cl 2 -MeOH (7:1) to give 6 subfractions, DR761-6. From subfraction DR762, compound 5 (4.0 mg) was isolated by Sephadex LH-20 CC, using CH 2 Cl 2 -MeOH (10:1) as eluents.
(22E,24ξ)-26,27-Bisnor-24-methyl-5α-cholest-22-en-3β, 5,6β,15α,25-pentol 25-O-sulfate (1): White, amorphous powder;
PPT Slide
Lager Image
+4.8° ( c 0.1, MeOH). HR-ESITOFMS m / z 515.2679 [M]− (calcd. for C 26 H 43 O 8 S, 515.2679). 1 H NMR (400 MHz, CD 3 OD) and 13 C NMR (100 MHz, CD 3 OD): see Table 1 .
1H and13C NMR data for compounds1,1a, and3
PPT Slide
Lager Image
aSpectra were recorded in CD3OD. b400 MHz. c100 MHz. dSignals are interchangeable.
(22E,24R,25R)-24-Methyl-5α-cholest-22-en-3β,5,6β,15α,25,26-hexol 26-O-sulfate (2): White, amorphous powder;
PPT Slide
Lager Image
+10.5° ( c 0.1, MeOH). HR-ESITOFMS m / z 559.2926 [M] (calcd. for C 28 H 47 O 9 S, 559.2941). 1 H NMR (400 MHz, CD 3 OD) δ 4.00 (m, H-3), 3.45 (br s, H-6), 1.12 (d, J = 10.4 Hz, H-14), 3.93 (m, H-15), 1.44 (m, H-17), 0.74 (s, H 3 -18), 1.16 (s, H 3 -19), 1.01 (d, J = 6.4 Hz, H 3 -21), 5.29 (m, H-22), 5.42 (dd, J = 8.4, 15.6 Hz, H-23), 2.27 (m, H-24), 3.84 (d, J = 10.0 Hz, H-26a), 3.94 (dd, J = 10.0 Hz, H-26b), 1.14 (s, H 3 -27), 0.99 (d, J = 6.4 Hz, H 3 -28). 13 C NMR (100 MHz, CD 3 OD) δ 31.6 (C-1), 33.5 (C-2), 68.3 (C-3), 41.4 (C-4), 76.6 (C-5), 76.4 (C-6), 35.2 (C-7), 31.2 (C-8), 46.6 (C-9), 39.4 (C-10), 22.1 (C-11), 41.5 (C-12), 44.8 (C-13), 63.6 (C- 14), 74.2 (C-15), 42.3 (C-16), 54.9 (C-17), 14.0 (C-18), 17.4 (C-19), 41.1 (C-20), 21.1 (C-21), 138.8 (C-22), 130.4 (C- 23), 45.3 (C-24), 74.3 (C-25), 74.6 (C-26), 22.3 (C-27), 15.4 (C-28).
(28R)-24-Ethyl-5α-cholesta-3β,5,6β,8,15α,28,29-heptaol-24-sulfate (3): White, amorphous powder;
PPT Slide
Lager Image
+32.0° ( c 0.5, MeOH). HR-ESITOFMS m / z 591.3207 [M] (calcd. for C 29 H 51 O 10 S, 591.3203). 1 H NMR (400 MHz, CD 3 OD) and 13 C NMR (100 MHz, CD 3 OD): see Table 1 . 1 H NMR (400 MHz, pyridine- d 5 ) δ 4.88 (m, H-3), 4.29 (br s, H-6), 1.71 (H-14), 4.87 (m, H-15), 1.49 (m, H-17), 1.28 (s, H 3 -18), 1.82 (s, H 3 -19), 1.65 (m, H-20), 0.93 (d, J = 6.8 Hz, H 3 -21), 1.65 (m, H-22), 1.35 (m, H-23), 1.61 (m, H-24), 1.96 (m, H-25), 1.02 (d, J = 6.4 Hz, H 3 -26), 0.92 (d, J = 6.4 Hz, H 3 -27), 4.45 (m, H-28), 4.75 (dd, J = 8.4, 10.4 Hz, H-29a), 4.58 (dd, J = 3.6, 10.4 Hz, H-29b). 13 C NMR (100 MHz, pyridine- d 5 ) δ 31.8 (C-1), 34.4 (C-2), 67.3 (C-3), 42.5 (C-4), 75.9 (C-5), 78.0 (C-6), 42.0 (C-7), 76.7 (C-8), 48.8 (C-9), 39.1 (C-10), 19.5 (C-11), 42.5 (C-12), 44.9 (C-13), 66.4 (C-14), 69.3 (C-15), 40.9 (C-16), 55.0 (C-17), 15.6 (C-18), 18.2 (C-19), 35.9 (C-20), 19.0 (C-21), 35.9 (C-22), 23.3 (C-23), 47.5 (C- 24), 29.4 (C-25), 20.7 (C-26), 20.6 (C-27), 71.3 (C-28), 72.5 (C-29).
(25S)-5α-Cholestane-3β,5,6β,15α,16β,26-hexaol (4): Colorless gum;
PPT Slide
Lager Image
+10.1° ( c 0.9, MeOH). HR-ESITOFMS m / z 513.3426 [M + COOH] (calcd. for C 28 H 49 O 8 , 513.3427). ESIMS m / z 433 [M + H − 2H 2 O] + . 1 H NMR (400 MHz,CD 3 OD) δ 3.33 (m, H-3), 3.47 (br s, H-6), 1.26 (m, H-14), 3.74 (dd, J = 2.0, 10.0 Hz, H-15), 3.97 (dd, J = 2.0, 8.0 Hz, H-16), 0.99 (m, H-17), 0.90 (s, H 3 -18), 1.17 (s, H 3 -19), 0.96 (d, J = 6.8 Hz, H 3 -21), 1.57 (H-25), 4.02 (dd, J = 6.4, 10.8 Hz, H-26a), 3.43 (dd, J = 6.4, 10.8 Hz, H-26b), 0.91 (d, J = 6.8 Hz, H 3 -27). 13 C NMR (100 MHz, CD 3 OD) δ 31.6 (C-1), 33.5 (C-2), 68.3 (C-3), 41.4 (C-4), 76.5 (C-5), 76.3 (C-6), 35.1 (C-7), 31.1 (C-8), 46.5 (C-9), 39.3 (C-10), 21.9 (C-11), 41.9 (C-12), 44.7 (C-13), 60.9 (C-14), 85.0 (C-15), 82.9 (C- 16), 59.9 (C-17), 15.0 (C-18), 17.4 (C-19), 30.9 (C-20), 18.6 (C-21), 37.4 (C-22), 24.8 (C-23), 34.9 (C-24), 36.9 (C-25), 68.4 (C-26), 17.2 (C-27).
Δ7-Sitosterol (5): White, amorphous powder;
PPT Slide
Lager Image
+5.5° ( c 0.7, CHCl 3 ). ESIMS m / z 415 [M + H] + . 1 H NMR (400 MHz, CDCl 3 ) δ 3.50 (m, H-3), 5.09 (br s, H-7), 0.47 (s, H 3 -18), 0.72 (s, H 3 -19), 0.86 (d, J = 6.0 Hz, H 3 -21), 0.81 (d, J = 6.4 Hz, H 3 -26), 0.80 (d, J = 6.4 Hz, H 3 -27), 0.86 (d, J = 6.8 Hz, H3-29). 13 C NMR (100 MHz, CDCl 3 ) δ 37.1 (C-1), 31.1 (C-2), 70.7 (C-3), 37.6 (C-4), 40.2 (C-5), 29.6 (C-6), 117.4 (C-7), 139.6 (C-8), 49.5 (C-9), 34.2 (C-10), 21.5 (C-11), 39.5 (C-12), 43.4 (C-13), 55.0 (C-14), 23.0 (C-15), 27.9 (C- 16), 56.1 (C-17), 11.8 (C-18), 13.0 (C-19), 36.2 (C-20), 18.8 (C-21), 33.9 (C-22), 26.2 (C-23), 45.8 (C-24), 29.1 (C-25), 19.8 (C-26), 19.0 (C-27), 22.9 (C-28), 11.9 (C-29).
Solvolysis of Compound 1. Compound 1 (3.0 mg) was dissolved in a 2 mL of dioxane-pyridine (1:1 v/v). The solution was stirred and heated at 100 ℃ for 5 h. The reaction mixture was concentrated in vacuo , and the dry residue was chromatographed over preparative TLC using CH 2 Cl 2 -MeOH (4:1) to give 1a (1.0 mg).
(22E,24ξ)-24-Methyl-26,27-bisnor-5α-cholest-22-en-3β,5,6β,15α,25-pentol (1a): white, amorphous powder,
PPT Slide
Lager Image
+12.6° ( c 0.06, MeOH). HR-ESITOFMS m / z 481.3181 [M + COOH] (calcd. for C 27 H 45 O 7 , 481.3165). 1 H NMR (400 MHz, CD 3 OD) and 13 C NMR (100 MHz, CD 3 OD): see Table 1 .
Preparation of the (S)- and (R)-MTPA Esters. Compound 3 (2.0 mg) was treated with ( R )-(−)- or ( S )-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (10 μL) in dry pyridine (400 μL) for 24 h at room temperature. The reaction was monitored by silica gel TLC and stopped when the original spot had disappeared. The reaction product was purified by preparative silica gel TLC using CH 2 Cl 2 -MeOH (5:1) as eluent to give ( S )- ( 3a ) and ( R )-MTPA esters ( 3b ), respectively. The 1H NMR spectra of the esters were recorded in pyridine- d 5 and the assignments were done by 1 H- 1 H COSY spectra. Selected 1 H NMR (pyridine- d 5 , 400 MHz) of ( 3a ): 6.18 (H-3), 6.13 (H-28), 5.94 (H-15), 4.76 (H-29), 4.26 (H-6), 3.71 (OCH 3 ), 3.64 (OCH 3 ), 3.59 (OCH 3 ), 1.87 (H-25), 1.67 (H-24), 1.42 (H-20), 1.36 (H-23), 0.96 (d, J = 6.8 Hz, H 3 -21 and H 3 -26), 0.92 (d, J = 6.8 Hz, H 3 -27). Selected 1 H NMR (pyridine- d 5 , 400 MHz) of ( 3b ): 6.13 (H-3), 5.99 (H-28), 5.86 (H-15), 4.78 (H-29), 3.91 (H-6), 3.89 (OCH 3 ), 3.75 (OCH 3 ), 3.65 (OCH 3 ), 1.72 (H-25), 1.58 (H-24), 1.27 (H-20), 1.27 (H-23), 0.90 (d, 6.8 Hz, H 3 -21), 0.85 (d, J = 6.8 Hz, H 3 -26), 0.81 (d, J = 6.8 Hz, H 3 -27).
Results and Discussion
Compound 1 was isolated as white amorphous powder. The 1 H and 13 C NMR spectra contained signals characteristic of a common 3β,5α,6β,15α-tetrahydroxy-substituted cholestane nucleus ( Table 1 ). The 1 H and 13 C spectra further showed the presence of one oxymethylene group (C-25) and one double bond at C-22 of the side chain of 1 . The downfield chemical shift of the oxymethylene group (δ 73.5) suggested location of a sulfate group at C-25. By analyzing the HMQC, HMBC, and 1 H− 1 H COSY spectra, the positions of the functionalities and side chain of 1 were fully assigned. The molecular formula of 1 was established as C 26 H 43 O 8 S based on the NMR data along with a molecular anion peak at m / z 515.2679 [515.2679 required, Δ 0.0 mmu] in the HR-ESITOFMS (negative ion mode). Based on these analyses, in combination with the comparison of the NMR data of 1 with those of the reported steroid, 6 the structure of 1 was established as (22 E ,24ξ)-26,27-bisnor-24-methyl-5α-cholest-22-en-3β,5,6β,15α,25-pentol 25- O -sulfate ( Figure 1 ).
PPT Slide
Lager Image
Structures of compounds 1-5, 1a, 3a, and 3b.
Upon solvolysis in a dioxane-pyridine mixture followed by preparative TLC, compound 1 afforded a desulfated derivative 1a . The 1 H and 13 C NMR spectra of 1a were identical to those of 1 , except for the upfield shifted chemical shifts of the oxymethylene (δ 3.27 and 3.40/68.4, C-25), showing that the sulfate group at C-25 has been removed ( Table 1 ). Furthermore, the molecular formula of 1a was established as C 26 H 44 O 5 by a pseudomolecular ion m / z 481.3181 [M + COOH] (481.3165 required, Δ −1.6 mmu) in the HR-ESITOFMS. Thus, the structure of 1a was identified as (22 E ,24ξ)-24-methyl-26,27-bisnor-5α-cholest-22-en-3β,5,6β,15α,25-pentol, representing a new steroidal derivative ( Figure 1 ).
Compound 3 was isolated as white, amorphous powder. The molecular formula of 3 was identified as C 29 H 51 O 10 S by analysis of 1D and 2D NMR data and a molecular anion m / z 591.3207 [591.3203 required, Δ −0.4 mmu] in the negative ion mode of HR-ESITOFMS. Comparative analysis indicated that the NMR data of 3 was identical with those of the known steroid, 24-ethyl-5α-cholesta-3β,5,6β,8,15α,28,29-heptaol-24-sulfate. 5 Since the absolute configuration at C-28 has not been determined, 5 the Mosher’s method has been applied to determine the stereochemistry at this position. The ( S )- and ( R )-MTPA esters of 3 ( 3a and 3b , respectively) were prepared from the corresponding ( R )- and ( S )-MTPA chlorides, respectively. Analysis of 1 H NMR and COSY spectra allowed the assignment of the proton chemical shifts for the two diastereomeric esters 3a and 3b in proximity of the esterified carbon (C-28). Calculation of Δδ values (δ S − δ R ) for protons neighboring C-28 led to the assignment of the 28 R configuration ( Table 2 ). Finally, the structure of 3 was established as (28 R )-24-ethyl-5α-cholesta-3β,5,6β,8,15, 28,29-heptaol-24-sulfate ( Figure 1 ).
1H NMR chemical shift differences (Δδ) for the MTPA esters of3
PPT Slide
Lager Image
aΔδ (ppm) = δS − δR
Three remaining sterols were determined to be (22 E ,24 R ,25 R )-24-methyl-5α-cholest-22-en-3β,5,6β,15α,25,26-hexol 26- O -sulfate ( 2 ), 6 (25 S )-5α-cholestane-3β,5,6β,15α,16β,26-hexaol ( 4 ), 10 and Δ7-sitosterol ( 5 ) 11 by comparing their NMR and MS data with those reported in the literature ( Figure 1 ).
The toxic potentials of five steroids ( 1-5 ) were evaluated toward the viability of HepG2 and U87MG cells using MTT assay (see Supporting information). As shown in Figure 2(a) and 2(b) , cell viability was decreased in a dose-dependent manner after treatment with varying concentration of 4 for 24 h, whereas other steroids had no effect (data not shown). From this primary result, compound 4 was selected for further studies to elucidate the underlying mechanism of cell death induction in HepG2 and U87MG cells.
PPT Slide
Lager Image
Effects of compound 4 on cell viability. HepG2 and U87MG cells were incubated for 24 h with various concentrations of compound 4 (10−200 μM). Cell viability was determined by MTT assay. Bar represents the mean ± S.D. of three independent experiments. *P < 0.05 vs. control.
Apoptosis, a process of programmed cell death, is a physiological mechanism, characterized by specific morphological and biochemical changes such as chromatin condensation, DNA fragmentation and protein cleavage. 12 To evaluate whether the cytotoxic effect of 4 against HepG2 and U87MG cells was mediated via apoptosis, we initially performed DNA fragmentation assay using ELISA (see Supporting information). After the treatment of 4 (10-200 μM) with HepG2 and U87MG cells for 24 h, the amount of fragmented DNA increased by 1.1, 1.8, 3.9, and 6.6-fold and 1.3, 2.6, 3.1, and 5.1-fold compared with the control, respectively ( Figure 3 ). This result revealed that the cytotoxic effect of 4 might be derived from the induction of apoptosis.
PPT Slide
Lager Image
Effects of compound 4 on DNA fragmentation in HepG2 and U87MG cells. Cells were incubated for 24 h with various concentrations of compound 4 (10−200 μM). DNA fragmentation was evaluated using the Cellular DNA fragmentation ELISA kit. Bar represents the mean ± S.D. of three independent experiments. *P < 0.05 vs. control.
Apoptosis is tightly regulated by the complex interplay of individual members of the Bcl-2 family. 13 To further determine the molecular mechanisms of apoptotic induction of 4 , we next examined the expression of Bcl-2 family proteins in HepG2 and U87MG cells (see Supporting information). The Bcl-2 family proteins, consisting of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins, play crucial roles in the regulation of apoptosis. After the treatment with 4 , the expression of Bcl-2 proteins was decreased dose-dependently, whereas the expression of Bax protein accumulated with increasing concentrations ( Figure 4 ). Densitometric tracing showed that the ratio of Bax/Bcl-2 exhibited a dose-dependent increase in both HepG2 and U87MG cells. This data indicated that 4 induced apoptosis by interfering with the expression of Bcl-2 family proteins resulting in up-regulation of Bax:Bcl-2 ratio ( Figure 4 ).
PPT Slide
Lager Image
Effects of compound 4 on the expression of Bcl-2 family proteins in HepG2 and U87MG cells. Cells were incubated for 24 h with various concentrations of compound 4 (10–200 μM). Western blot analyses for proteins expression were performed as described in Experimental. Representative blots of three independent experiments are shown. Relative changes of Bax/Bcl-2 ratio were quantitated using an image program. *P < 0.05 vs. control.
An increase in the ratio of Bax:Bcl-2 can stimulate the release of cytochrome c from mitochondria into the cytosol, resulting in the activation of caspases and PARP. 14,15 Activated caspase-3 is the key executioner of apoptosis that leads to the cleavage of key cellular proteins including PARP, which is serves as a marker of apoptosis when cleaved. 16 Therefore, we further examined the effects of 4 on the cleavage of caspases and the proteolytic cleavage of PARP in HepG2 and U87MG cells (see Supporting information). The treatment with 4 (10-200 μM) for 24 h significantly increased the cleavage of caspase-3, caspase-9 and PARP in a dose-dependent manner ( Figure 5 ). A colorimetric assay was further confirmed the increased caspase-3 and caspase-9 activity in HepG2 and U87MG cells treated with 4 as shown in Figure 6 . These results strongly indicate that the observed antitumoric effects of 4 were mediated through the apoptotic process.
PPT Slide
Lager Image
Effects of compound 4 on caspase-3, caspase-9, and PARP activation in HepG2 and U87MG cells. Cells were incubated for 24 h with various concentrations of compound 4 (10–200 μM). Western blot analyses for proteins expression were performed as described in Experimental. Representative blots of three independent experiments are shown.
PPT Slide
Lager Image
Effects of compound 4 on caspase-3 and caspase-9 activity in HepG2 and U87MG cells. Cells were incubated for 24 h with various concentrations of compound 4 (10−200 μM). Caspase-3 and caspase-9 activities were measured by a colorimetric assay. Bar represents the mean ± S.D. of three independent experiments. *P < 0.05 vs. control.
Conclusion
In the present study, five known steroids, including three sulfated steroids, were isolated from the cold water starfish C. crispatus . In addition, a new polyhydroxylated steroidal derivative, (22 E ,24ξ)-24-methyl-26,27-bisnor-5α-cholest-22-en-3β,5,6β,15α,25-pentol ( 1a ) was prepared from desulfation of compound 1 . The modified Mosher’s method was applied to assign the absolute configuration at C-28 of the unusual stigmastane side chain of compound 3 . The cytotoxic effects of five isolated steroids ( 1-5 ) were evaluated against two human carcinoma cell lines (HepG2 and U87MG). Among the isolated compounds, only compound 4 induced apoptosis of HepG2 and U87MG cells through the enhanced expression of Bax and the activation of caspase pathways.
Acknowledgements
This research was supported by a grant from the Ministry of Oceans and Fisheries’ R&D project (PM13030).
References
Hu G. P. , Yuan J. , Sun L. , She Z. G. , Wu J. H. , Lan X. J. , Zhu X. , Lin Y. C. , Chen S. P. 2011 Mar. Drugs 9 514 -    DOI : 10.3390/md9040514
Molinski T. F. , Dalisay D. S. , Lievens S. L. , Saludes J. P. 2009 Nat. Rev. Drug Discov. 8 69 -    DOI : 10.1038/nrd2487
Mayer A. M. , Glaser K. B. , Cuevas C. , Jacobs R. S. , Kem W. , Little R. D. , McIntosh J. M. , Newman D. J. , Potts B. C. , Shuster D. E. 2010 Trends Pharmacol. Sci. 31 255 -    DOI : 10.1016/j.tips.2010.02.005
Dong G. , Xu T. , Yang B. , Lin X. , Zhou X. , Yang X. , Liu Y. 2011 Chem. Biodivers. 8 740 -    DOI : 10.1002/cbdv.200900344
Kicha A. A. , Kalinovskii A. I. , Ivanchina N. V. , El'kin Y. N. , Stonik V. A. 1994 Russ. Chem. Bull. 43 1726 -    DOI : 10.1007/BF00703498
Kicha A. A. , Ivanchina N. V. , Kalinovsky A. I. , Dmitrenok P. S. , Stonik V. A. 2005 Russ. Chem. Bull. 54 1266 -    DOI : 10.1007/s11172-005-0392-3
Parkin D. M. , Bray F. , Devesa S. 2001 Eur. J. Cancer 8 S4 -
Sanchez-Martin M. 2008 Curr. Stem Cell Res. Ther. 3 1997 -
Hösli P. , Sappino A. P. , de Tribolet N. , Dietrich P. Y. 1998 Ann. Oncol. 9 589 -    DOI : 10.1023/A:1008267312782
Iorizzi M. , Bryan P. , McClintock J. , Minale L. , Palagiano E. , Maurelli S. , Riccio R. , Zollo F. 1995 J. Nat. Prod. 58 653 -    DOI : 10.1021/np50119a003
Zhang X. , Cambrai A. , Miesch M. , Roussi S. , Raul F. , Aoude- Werner D. , Marchioni E. 2006 J. Agric. Food. Chem. 54 1196 -    DOI : 10.1021/jf052761x
Saraste A. , Pulkki K. 2000 Cardiovasc. Res. 45 528 -    DOI : 10.1016/S0008-6363(99)00384-3
Manion M. K. , Hockenbery D. M. 2003 Cancer Biol. Ther. 2 S105 -
Yang J. , Liu X. , Bhalla K. , Kim C. N. , Ibrado A. M. , Cai J. , Peng T. I. , Jones D. P. , Wang X. 1997 Science 275 1129 -    DOI : 10.1126/science.275.5303.1129
Kluck R. M. , Bossy-Wetzel E. , Green D. R. , Newmeyer D. D. 1997 Science 275 1132 -    DOI : 10.1126/science.275.5303.1132
Mi Y. , Thomas S. D. , Xu X. , Casson L. K. , Miller D. M. , Bates P. J. 2003 J. Biol. Chem. 278 8572 -    DOI : 10.1074/jbc.M207637200