New Rare Triterpene Glycosides from Pacific Sun Star, Solaster pacificus, and Their Anticancer Activity

Six previously unknown triterpene glycosides, pacificusosides L–Q (1–6), and two previously known triterpene glycosides, cucumariosides B1 (7) and A5 (8), were isolated from an alcoholic extract of Pacific sun star, Solaster pacificus. The structures of 1–6 were determined using 1D and 2D NMR, ESIMS, and chemical modifications. Compound 1 is a rare type of triterpene glycoside with non-holostane aglycon, having a linear trisaccharide carbohydrate chain. Pacificusosides M–P (2–5) have new structures containing a Δ8(9)-3,16,18-trihydroxy tetracyclic triterpene moiety. This tetracyclic fragment in sea star or sea cucumber triterpene glycosides was described for the first time. All the compounds under study exhibit low or moderate cytotoxic activity against colorectal carcinoma HCT 116 cells, and breast cancer MDA-MB-231 cells were assessed by MTS assay. Compound 2 effectively suppresses the colony formation of cancer cells at a non-toxic concentration, using the soft-agar assay. A scratch assay has shown a significant anti-invasive potential of compound 2 against HCT 116 cells, but not against MDA-MB-231 cells.


Introduction
Triterpene glycosides are typical secondary metabolites of sea cucumbers (class Holothuroidea, phylum Echinodermata).These glycosides most commonly have the so-called holostane type of aglycon containing lanostan-3β-ol with (18,20)-lactone in the E-ring of the pentacyclic triterpene core and a carbohydrate chain consisting of five or six monosaccharide residues [1,2].The terminal monosaccharide residue (D-glucose or D-xylose) in most cases has an additional methoxyl group at C-3, and the carbohydrate chain may contain from one to four sulfate groups.However, rarer triterpene glycosides with a non-holostane type of aglycon [3] or with shorter carbohydrate chains were also found in sea cucumbers.Basically, these secondary metabolites, often exhibiting marked cytotoxic properties, are produced by holothurians to protect themselves against predators [4].
Triterpene glycosides were identified in several starfish species (class Asteroidea, phylum Echinodermata) but their presence in starfish is a rare occurrence.Recently, we have reported about 11 new-to-science triterpene glycosides, pacificusosides A-K, and four previously known triterpene glycosides, cucumariosides A 10 , C 1 , C 2 , and D, isolated from the Pacific sun star Solaster pacificus [5,6].It is likely that some starfish species, being active predators, use sea cucumbers as food and accumulate triterpene glycosides in their bodies.
Since each sea cucumber species has its own set of triterpene glycosides, these compounds can be considered as food markers.We previously assumed that starfish can modify sea cucumber triterpene glycosides by their own enzyme systems [5,6].Thus, we found new triterpene glycosides in S. pacificus (Kuril population), presumably specialized in preying on sea cucumbers of the genus Eupentacta, which had not previously been reported for this sea cucumber genus [5,6].This suggests that starfish can modify the most toxic triterpene glycosides by oxidizing and removing part of the aglycon side chain or by reducing the number of monosaccharide residues in the carbohydrate chain of their molecules.This mode of metabolism of triterpene glycosides in S. pacificus can be considered as an adaptive mechanism developed for such a specialized diet.
The interest in sea cucumber triterpene glycosides is explained not only by their unusual chemical structure, but also by the diverse biological activities that these compounds exhibit.Thus, antifungal [7,8], bactericidal, hemolytic, antiviral, antiparasitic [9], and immunomodulatory properties [10] of these glycosides were described.Many authors indicate that sea cucumber triterpene glycosides have a significant antitumor potential [11].For example, frondoside A from Cucumaria frondosa inhibits proliferation of AsPC-1 human pancreatic cancer cells by induction of apoptosis of these cells via the mitochondrial pathway and activation of the caspase cascade [12].Frondoside A decreases the viability of MDA-MB-231 human breast cancer cells in a concentration-and time-dependent manner by activation of p53, followed by emergence of caspases 9 and 3/7 cell death pathways in MDA-MB-231 cells [13].Furthermore, frondoside A has a potent antimetastatic effect on a syngeneic murine model of metastatic breast cancer [14].We also showed that pacificusoside C and cucumariosides C 1 and C 2 from S. pacificus almost completely suppress the colony formation of HT-29, RPMI-7951, and MDA-MB-231 cells at a non-toxic concentration [5].Moreover, pacificusoside D and cucumarioside D from S. pacificus at non-toxic concentrations have the highest inhibiting effect on colony formation of SK-MEL-2 cancer cells, and significantly inhibit neoplastic transformation of JB6 Cl41 cells induced by chemical carcinogens (EGF and TPA) or ionizing radiation (X-rays and UVB) [6].
As a continuation of our research on biologically active low-molecular-weight compounds from the Pacific sun star S. pacificus (order Valvatida, family Solasteridae) we herein report the results of studies of six new triterpene glycosides, pacificusosides L-Q (1-6), and two previously known triterpene glycosides, cucumarioside B 1 (7) and cucumarioside A 5 (8).In this paper, we describe their isolation, structure elucidation, and their effects on the viability, colony formation, and invasion of cancer cells.

Isolation and Structure Elucidation of Compounds 1-8 from S. pacificus
An ethanol extract from a sun star, S. pacificus, was sequentially separated by chromatography on columns with Polychrome 1, Si gel, and Florisil followed by high-performance liquid chromatography (HPLC) on reverse phase Diasfer-110-C18, Discovery C18, and YMC-Pack Pro C18 columns.As a result, six previously unknown triterpene glycosides, referred to as pacificusosides L-Q (1-6), and two previously known triterpene glycosides (7 and 8) were obtained (Figure 1).Compounds 7 and 8 were identified by comparing their 1 H, 13 C NMR, and MS spectra to those reported for cucumariosides B 1 and A 5 from the sea cucumber Eupentacta fraudatrix [1].
The chemical shifts of protons and carbons of four CH 3 groups (δ H 1.03 s, 1.18 s, 1.32 s, 1.33 s; δ C 23.9, 17.3, 28.6, 33.9), the 7(8)-double bond [δ H 5.62 brd (J = 7.0); δ C 122.7, 147.3], and the lactone carbonyl (δ C 180.7) were present in the 1 H and 13 C NMR spectra of the pentacyclic core of compound 1 (Tables 1 and 2, Figures S3-S8).The resonances of an acetate group were not observed in the 1 H and 13 C NMR spectra of 1.The respective sequences of protons of polycyclic moiety of 1 shown in Figure 2 were determined by the 1 H-1 H COSY and HSQC experiments (Figures S5 and S6).The major HMBC correlations depicted in Figure 2 confirmed the overall structure of the triterpene nucleus of glycoside 1 (Figure S7).The common 5α/9β/10β/13β/14α stereochemistry of the polycyclic moiety and 3β-configuration of oxygenated substituent in 1 were defined from the ROESY crosspeaks (Figures 3 and S8).A CH 3 group (δ H 1.74 s; δ C 23.0) and a 20,22-double bond (δ H 5.06 s, 4.99 s; δ C 139.9, 113.9) were observed in the 1 H and 13 C NMR spectra of aglycon side chain of compound 1 (Tables 1 and 2, Figures S3 and S4).The overall structure of the side chain was supported by major HMBC: The chemical shifts of protons and carbons of four CH3 groups (δH 1.03 s, 1.18 s, 1.32 s, 1.33 s; δC 23.9, 17.3, 28.6, 33.9), the 7(8)-double bond [δH 5.62 brd (J = 7.0); δC 122.7, 147.3], and the lactone carbonyl (δC 180.7) were present in the 1 H and 13 C NMR spectra of the pentacyclic core of compound 1 (Tables 1 and 2, Figures S3-S8).The resonances of an acetate group were not observed in the 1 H and 13 C NMR spectra of 1.The respective sequences of protons of polycyclic moiety of 1 shown in Figure 2 were determined by the 1 H-1 H COSY and HSQC experiments (Figures S5 and S6).The major HMBC correlations depicted in Figure 2 confirmed the overall structure of the triterpene nucleus of glycoside 1 (Figure S7).The common 5α/9β/10β/13β/14α stereochemistry of the polycyclic moiety and 3β-configuration of oxygenated substituent in 1 were defined from the ROESY crosspeaks (Figures 3 and S8).A CH3 group (δH 1.74 s; δC 23.0) and a 20,22-double bond (δH 5.06 s, 4.99 s; δC 139.9, 113.9) were observed in the 1 H and 13 C NMR spectra of aglycon side chain of compound 1 (Tables 1 and 2, Figures S3 and S4 16)-lactone-3β-ol aglycon [5,6].3, Figures S3-S8).Along with NMR spectrum information, ESIMS/MS data confirmed the presen three monosaccharide residues in the carbohydrate chain of glycoside 1.The 1 H spectrum of 1 showed a resonance of CH3 group of 6-deoxy-sugar unit at δH 1.64.β figurations of all the glycosidic bonds were determined by the coupling constants of meric protons (7.1-7.5 Hz).The chemical shifts of protons and carbons and coupling stants of protons of monosaccharide units in the oligosaccharide moiety of glycos were identified by the 1D TOCSY, 1 H-1 H COSY, HSQC, HMBC, and ROESY experim (Table 3, Figures S3−S8).The NMR spectroscopic data of the carbohydrate chain ex coincided with those of the terminal β-D-xylopyranosyl residue and the internal 2-O stituted β-D-quinovopyranosyl and 2-O-substituted β-D-xylopyranosyl residues i earlier reported 1 H and 13 C NMR spectra of the known cucumariosides B1 and B2 [1].С peaks between H-1 of Xylp-I and C-3 (H-3) of aglycon; H-1 of Quip and C-2 (H-2) of X and H-1 of Xylp-II and C-2 (H-2) of Quip in the HMBC and ROESY spectra allowed determine the attachment of the oligosaccharide moiety to aglycon and the positio interglycosidic linkages (Table 3, Figures S7 and S8).The D-configuration for all the osaccharide units that constitute the carbohydrate chain of 1 was identified in the fo ing way.At the first stage, glycoside 1 was hydrolyzed by 2 M TFA.Next, the resu mixture of monosaccharides was processed by (R)-(−)-octanol, followed by acetyl and GC analysis.S16).The IR spectrum of compound 2 showed that hydroxy (3426 and olefinic (1632 cm -1 ) groups were present (Figure S17).The absorption band of tone was absent in the IR spectrum of compound 2, but there was an absorption ba acetate carbonyl (1716 cm -1 ).The chemical shifts of protons and carbons of four groups (δH 1.05 s, 1.11 s, 1.32 s, 1.02 s; δC 19.2, 16.3, 27.7, 26.4), the 8(9)-double bon 132.7, 136.5), an OAc group (δH 2.17 s, δC 21.4,170.1), and two oxygenated groups [δH 3.27 dd (J = 12.0, 4.3), δC 88.7] and CH2-18 (δH 4.17 m, 3.93 m; δC 62.2) were observ the 1 H and 13 C NMR spectra of the tetracyclic core of compound 2 (Tables 1 and 2 Along with NMR spectrum information, ESIMS/MS data confirmed the presence of three monosaccharide residues in the carbohydrate chain of glycoside 1.The 1 H NMR spectrum of 1 showed a resonance of CH 3 group of 6-deoxy-sugar unit at δ H 1.64.β Configurations of all the glycosidic bonds were determined by the coupling constants of anomeric protons (7.1-7.5 Hz).The chemical shifts of protons and carbons and coupling constants of protons of monosaccharide units in the oligosaccharide moiety of glycoside 1 were identified by the 1D TOCSY, 1 H-1 H COSY, HSQC, HMBC, and ROESY experiments (Table 3, Figures S3-S8).The NMR spectroscopic data of the carbohydrate chain exactly coincided with those of the terminal β-D-xylopyranosyl residue and the internal 2-O-substituted β-Dquinovopyranosyl and 2-O-substituted β-D-xylopyranosyl residues in the earlier reported 1 H and 13 C NMR spectra of the known cucumariosides B 1 and B 2 [1].Cross-peaks between H-1 of Xyl p -I and C-3 (H-3) of aglycon; H-1 of Qui p and C-2 (H-2) of Xyl p -I; and H-1 of Xyl p -II and C-2 (H-2) of Qui p in the HMBC and ROESY spectra allowed us to determine the attachment of the oligosaccharide moiety to aglycon and the positions of interglycosidic linkages (Table 3, Figures S7 and S8).The D-configuration for all the monosaccharide units that constitute the carbohydrate chain of 1 was identified in the following way.At the first stage, glycoside 1 was hydrolyzed by 2 M TFA.Next, the resulting mixture of monosaccharides was processed by (R)-(−)-octanol, followed by acetylation and GC analysis.Finally, comparison of retention times of the resulting octyl glycoside acetates with the respective derivatives of standard monosaccharides (D-xylose and D-quinovose) showed their almost complete identity (Figures S11-S15).On the basis of these data, the structure of pacificusoside L (1) was elucidated as  S16).The IR spectrum of compound 2 showed that hydroxy (3426 cm −1 ) and olefinic (1632 cm −1 ) groups were present (Figure S17).The absorption band of γ-lactone was absent in the IR spectrum of compound 2, but there was an absorption band of acetate carbonyl (1716 cm −1   1 H and 13 C NMR spectra of the tetracyclic core of compound 2 (Tables 1  and 2, Figures S18-S23).The respective sequences of protons in the triterpene nucleus from C-1 to C-3, C-5 to C-7, C-11 to C-12, and C-15 to C-17 were determined by the 1    S22).The common 5α/10β/13β/14α stereochemistry of the triterpene nucleus and a 3β,16β-configurations of the oxygenated substituents in 2 were defined from the ROESY cross-peaks (Figures 3 and S23).
Moreover, in the 1 H NMR spectrum of 2, four resonances of the anomeric protons of monosaccharide units at δ H 4.79, 5.16, 4.97, and 5.20 were observed, which, in the HSQC experiment, correlated with carbon signals at δ C 105.5, 105.5, 104.9, and 106.0, respectively, together with the signal of O-CH 3 at δ H 3.85, which, in the HSQC experiment, was correlated with a carbon signal at δ C 60.5 (Table 3, Figures S18-S23 S25).
Along with NMR spectrum information, ESIMS/MS data confirmed the presence of four monosaccharide residues in the carbohydrate chain of glycoside 2. In the 1 H NMR spectrum of 2, a resonance of CH 3 group of 6-deoxy-sugar unit at δ H 1.76 was observed.β-Configurations of all the glycosidic bonds were determined by the coupling constants of anomeric protons (7.5-8.0Hz).The chemical shifts of protons and carbons and coupling constants of protons of monosaccharide units in the oligosaccharide moiety of glycoside 2 were identified by the 1D TOCSY, 1   Along with NMR spectrum information, ESIMS/MS data confirmed the presen four monosaccharide residues in the carbohydrate chain of glycoside 2. In the 1 H N spectrum of 2, a resonance of CH3 group of 6-deoxy-sugar unit at δH 1.76 was observe Configurations of all the glycosidic bonds were determined by the coupling constan The NMR spectroscopic data of the carbohydrate chain exactly coincided with those of the terminal 3-O-Me-β-xylopyranosyl residue and the internal 3-O-substituted β-glucopyranosyl, 4-O-substituted β-quinovopyranosyl, and 2-O-substituted β-xylopyranosyl residues in the earlier reported 1 H and 13 C NMR spectra of the known cucumarioside A 5 [1].Cross-peaks between H-1 of Xyl p and C-3 (H-3) of aglycon; H-1 of Qui p and C-2 (H-2) of Xyl p ; H-1 of Glc p and C-4 (H-4) of Qui p ; H-1 of 3-O-Me-Xyl p and C-3 (H-3) of Glc p in the HMBC and ROESY spectra allowed us to determine the attachment of the oligosaccharide moiety to aglycon and the positions of interglycosidic linkages (Table 3, Figures S22 and S23).The D-series of monosaccharide units was expected to be similar to that in co-occurring glycoside 1.
A comparison of the 1 H, 13 C NMR and MS spectra and an application of extensive 2D NMR analysis of compounds 2-6 showed that the oligosaccharide moiety of 2 is identical to that in compounds 3-6 (Figures S33, S34, S43, S44, S52 and S53), while compounds 2-6 differ from each other in triterpene aglycons only (Tables 1-3).
The molecular formula of  S26).A comparison of the molecular weights (MWs) of 3 and 2 showed that the difference between 3 and 2 was 42 atomic mass units (amu's).Most of the signals in the NMR spectra of 3 attributable to triterpene nucleus were similar to those of 2, except some resonances belonging to D-ring.The signals of H-15 (m), H-16 (m), and H-17 (m) in 3 were upfield-shifted from δ H 2.26 to 2.07, from δ H 5.85 to 5.02, and from δ H 2.32 to 2.15, respectively, compared to those of 2. Also, in the 1 H NMR spectrum of 3, there was no resonance of acetate group at δ H 2.17 s (CH 3 CO).In the 13   S35).Thus, the MWs of 4 and 5 differed by 2 amu's.The IR spectrum of the mixture of 4 and 5 demonstrated the presence of only hydroxy (3439 cm −1 ) and olefinic (1632 cm −1 ) groups (Figure S36).It was found that compounds 4, 5, and 3 differed from each other only in signals of their side chains, on the basis of a thorough comparison of their 1 H and 13 C NMR data (Tables 1-3, Figures S37 and S38).
We previously suggested that the unusual triterpene glycosides in S. pacificus could be produced by the biosynthetic enzyme systems from related dietary glycosides [5,6].Such modifications can occur either through oxidation followed by degradation of the aglycon side chain [5] or through reduction in the number of monosaccharide units in the oligosaccharide chain of triterpene glycosides [6].It is confirmed, in part, by a significant decrease in the toxicity of the modified compounds, and can be considered as an adaptation mechanism of starfish specialized in preying on sea cucumbers.The unusual pacificusosides M-Q (2-6) have much in common with the A-series cucumariosides from E. fraudatrix [1].Thus, pacificusoside M is almost identical to cucumarioside A 8 , and pacificusoside O is similar to cucumarioside A 9 , except for the position of the double bond in the triterpene aglycon: ∆ 8 (9) in pacificusosides M and O and ∆ 7 in cucumariosides A 8 and A 9 .This fact allows the assumption that pacificusosides M-Q (2-6) are also products of modification of the starfish biosynthetic enzyme systems and are derived by isomerization of the double bond 7(8) to 8 (9).Thus, the isomerization of the double bond in the triterpene aglycon may be another way to utilize toxic triterpene glycosides taken up with food by the sun star, S. pacificus.

Investigation of Biological Activities 2.2.1. Cytotoxicity of Compounds 1-3 and 6-8 against Normal and Cancer Cells
Cancer is known to be a complex process characterized by mutation and selection for cells with progressively increasing capacity for proliferation, survival, invasion, and metastasis [18][19][20].In the present study, the effect of compounds 1-3 and 6-8on important hallmarks of cancer such as viability, proliferation, and invasion of cancer cells was investigated.
We determined the cytotoxicity of compounds 1-3 and 6-8 against human embryonic kidney HEK 293, colorectal carcinoma HCT 116, and breast cancer MDA-MB-231 cells by the MTS assay, after 24 h of treatment with the compounds.We calculated the inhibiting concentration that cause death of 50% of cells (IC 50 ) and the selectivity index (SI) of the compounds tested (Table 4).Compounds 1, 6, 7, and 8 proved to be non-selective against cancer cells and exhibited cytotoxic activity against HEK 293, HCT 116, and MDA-MB-231 cells, to a varying degree (Table 4).Moderate cytotoxic effect of compounds 2 and 3 was observed against normal HEK 293 cells and cancer HCT 116 or MDA-MB-231 cells.IC 50 of compound 2 was estimated at 18.6 µM against HCT 116 cells (SI = 1.0) and 15.5 µM against MDA-MB-231 cells (SI = 1.2).Compound 3 possessed less cytotoxic activity than compound 2, with its IC 50 value of 42.2 µM against HCT 116 cells (SI = 1.25) and 36.7 µM against MDA-MB-231 cells (SI = 1.4) (Table 4).Since compound 2 exhibited high cytotoxic activities against the tested cell lines, it was chosen for the further study of its colony-inhibiting and anti-invasive effects at low non-toxic concentrations of 0.6, 1.25, 2.5, and 5 µM.The chemotherapeutic drug, cisplatin, was used as positive control.IC 50 of cisplatin against HEK 293, HCT 116, and MDA-MB-231 cells was determined to be 64.6 µM, 40.2.9 µM, and 34.5 µM, respectively, after 48 h of cell incubation (Table 4).

Colony-Inhibiting Activity of Compound 2 in Cancer Cells HCT 116 and MDA-MB-231
The soft-agar clonogenic assay was applied in order to assess the effect of compound 2 on the colony formation of human colorectal carcinoma HCT 116 and breast cancer MDA-MB-231 cells.As a result, compound 2 at concentrations of 0.6, 1.25, 2.5, and 5 µM decreased the number of colonies of HCT 116 cells by 11, 24, 52, and 97%, respectively (Figure 6a,b) and MDA-MB-231 cells by 20, 39, 61, and 80%, respectively (Figure 6c,d).

Anti-Invasive Activity of Compound 2 in Cancer Cells HCT 116 and MDA-MB-231
Metastasis is the leading cause of cancer mortality [21].The metastatic cascade is a multistep process in which cancer cells are destroyed, from the primary tumor to distant parts and tissues [22].Migration and plasticity of cancer cells, as well as the environment such as stromal and endothelial cells, are essential in metastasis [23].In this study, the migration ability (invasive potential) of colorectal carcinoma HCT 116 cells and breast cancer MDA-MB-231 cells and the anti-invasive effect of compound 2 were determined by the "wound-healing", or scratch, method.HCT 116 cells were found to migrate by 49, 71, and 100% slower after 24, 48, and 72 h of cell incubation, respectively, compared to control at the time point of 0 h (Figure 7a).MDA-MB-231 cells showed a higher migration speed than HCT 116 cells, and were able to completely heal the "experimental wound" after 24 h of incubation, compared to control at 0 h (Figure 7c).

Anti-Invasive Activity of Compound 2 in Cancer Cells HCT 116 and MDA-MB-231
Metastasis is the leading cause of cancer mortality [21].The metastatic cascade is a multistep process in which cancer cells are destroyed, from the primary tumor to distant parts and tissues [22].Migration and plasticity of cancer cells, as well as the environment such as stromal and endothelial cells, are essential in metastasis [23].In this study, the migration ability (invasive potential) of colorectal carcinoma HCT 116 cells and breast cancer MDA-MB-231 cells and the anti-invasive effect of compound 2 were determined by the "wound-healing", or scratch, method.HCT 116 cells were found to migrate by 49, 71, and 100% slower after 24, 48, and 72 h of cell incubation, respectively, compared to control at the time point of 0 h (Figure 7a).MDA-MB-231 cells showed a higher migration speed than HCT 116 cells, and were able to completely heal the "experimental wound" after 24 h of incubation, compared to control at 0 h (Figure 7c).We found that compound 2 at concentrations of 0.6, 1.25, 2.5, and 5 µM inhibited the migration of HCT 116 cells by 5, 17, 30, and 37% after 24 h of treatment, respectively, compared to non-treated cells (control, 24 h).A treatment of HCT 116 cells with compound 2 (0.6, 1.25, 2.5, and 5 µM) for 48 h led to the suppression of cells' migration by 8, 18, 42, and 41%, respectively, compared to control at 48 h.Also, compound 2 reduced the migration of HCT 116 cells by 3, 3, 23, and 41% after 72 h of treatment, respectively, compared to control at 72 h (Figure 7a,b).On the other hand, compound 2 slightly influenced the migration of MDA-MB-231 cells, and at 5 µM inhibited cells' migration only by 15% after 24 h of treatment, compared to control at 24 h (Figure 7c,d).
Thus, we assessed the cytotoxicity of compounds 1-3 and 6-8 against human embryonic kidney HEK 293, colorectal carcinoma HCT 116, and breast cancer MDA-MB-231 cells.All the compounds under study exhibited low or moderate cytotoxic activity against the cell lines tested.Unfortunately, we could not find any selectivity of the effect of these We found that compound 2 at concentrations of 0.6, 1.25, 2.5, and 5 µM inhibited the migration of HCT 116 cells by 5, 17, 30, and 37% after 24 h of treatment, respectively, compared to non-treated cells (control, 24 h).A treatment of HCT 116 cells with compound 2 (0.6, 1.25, 2.5, and 5 µM) for 48 h led to the suppression of cells' migration by 8, 18, 42, and 41%, respectively, compared to control at 48 h.Also, compound 2 reduced the migration of HCT 116 cells by 3, 3, 23, and 41% after 72 h of treatment, respectively, compared to control at 72 h (Figure 7a,b).On the other hand, compound 2 slightly influenced the migration of MDA-MB-231 cells, and at 5 µM inhibited cells' migration only by 15% after 24 h of treatment, compared to control at 24 h (Figure 7c,d).
Thus, we assessed the cytotoxicity of compounds 1-3 and 6-8 against human embryonic kidney HEK 293, colorectal carcinoma HCT 116, and breast cancer MDA-MB-231 cells.All the compounds under study exhibited low or moderate cytotoxic activity against the cell lines tested.Unfortunately, we could not find any selectivity of the effect of these triterpene glycosides towards cancer cells.Nevertheless, compound 2 effectively suppressed the colony formation activity of HCT 116 and MDA-MB-231 cells at non-toxic concentrations in a dose-dependent manner, and inhibited migration of HCT 116 cells after 24, 48, and 72 h of treatment.This suggests that even highly toxic sea cucumber (or starfish) triterpene glycosides can be considered promising antitumor agents.Further investigations into the molecular mechanism of the anticancer effect of these compounds are expected to provide sufficient scientific evidence for research and applied purposes.

Animal Material
Specimens of S. pacificus were collected as described previously [5,6], near Iturup Island in the Sea of Okhotsk, at a depth of 10-20 m during the 42nd scientific cruise of R/V Akademik Oparin in August 2012.A voucher specimen (no.042-112) was deposited at the marine specimen collection of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, Vladivostok, Russia.

Acid Hydrolysis and Determination of Absolute Configurations of Monosaccharides
Absolute configurations of monosaccharides of compounds 1 (1.5 mg) and 2 (2.0 mg) were determined by a method published earlier [5,6].

Cell Lines and Culture Conditions
The human embryonic kidney HEK 293 cells (ATCC ® CRL-1573™), breast cancer MDA-MB-231 cells (ATCC ® HTB-26™), and colorectal carcinoma HCT 116 cells (ATCC ® CCL-247™) were purchased from the American Type Culture Collection (Manassas, VA, USA).The HEK 293 and MDA-MB-231 cells were cultured in DMEM; the HCT 116 cells were cultured in the McCoy's 5A medium at 37 • C in a humidified atmosphere containing 5% CO 2 .The culture media were supplemented with 10% heat-inactivated FBS and a 1% penicillin/streptomycin solution.The number of passages was carefully controlled, and contamination by Mycoplasma was monitored on a regular basis.

Preparation of Compounds
Compounds 1-3, and 6-8 were dissolved in sterile dimethyl sulfoxide (DMSO), to prepare stock concentrations of 20 mM.Cells were treated with serially diluted 1-3, 6-8 (0.3-100 µM) (with the culture medium used as diluent) (the final concentration of DMSO was less than 0.5%).The vehicle control was the cells treated with an equivalent volume of DMSO (the final concentration was less than 0.5%) for all of the presented experiments.
The IC 50 concentration was calculated using the AAT-Bioquest ® online calculator [24].The selectivity index (SI) was calculated as described previously [25], using the following formula: SI = IC 50 of the compounds for normal cells (HEK 293) divided by IC 50 of the same compounds for the human colorectal adenocarcinoma and breast cancer (HCT 116 and MDA-MB-231) cell lines.Both IC 50 and SI values are provided in Table 4.

Scratch Assay
HCT 116 and MDA-MB-231 cells (3 × 10 5 cells/mL) were seeded in 6-well plates and grown to 80% confluence for 24 h.After removing the culture medium, the cells' monolayer was scraped with a 200 µL sterile pipette tip, to create a straight scratch.Then, the cells were treated with compound 2 at concentrations of 0.6, 1.25, 2.5, and 5 µM and incubated for 24, 48, and 72 h.All experiments were set up in triplicate for each group.For image analysis, the cell migration into the wound area was photographed at the stages of 0, 24, 48, and 72 h through a Motic AE 20 microscope and using the ImageJ v. 1.50i software, bundled with 64-bit Java 1.6.0_24(NIH, Bethesda, MD, USA).The cell migration distance was estimated by measuring the width of the wound, and was expressed as the percentage of each control (0 h) in relation to the mean of the wound-closure area.

Statistical Analysis
All the assays were performed in at least triplicate.The results were expressed as mean ± standard deviation (SD).The obtained data were statistically processed by the one-way analysis of variance (ANOVA) and the Tukey's HSD test with significance levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 7 .
Figure 7.The effect of compound 2 on migration of human colorectal carcinoma HCT 116 cells and breast cancer MDA-MB-231 cells.(a,b) HCT 116 and (c,d) MDA-MB-231 cells were treated with 2 (0.6, 1.25, 2.5, 5 µM) for 24, 48, and 72 h.The cell migration distance was estimated by measuring the width of the wound, and was expressed as the percentage of each control in relation to the mean of the wound-closure area.All experiments were set up at least in triplicate (n = 9 for control and compound; n is the number of photographs).The magnification of the representative photos is ×10.The results are expressed as mean ± standard deviation (SD).The asterisks (* p < 0.05, ** p < 0.01) indicate a significant decrease in migration of cells treated with the compound, compared to control.

Figure 7 .
Figure 7.The effect of compound 2 on migration of human colorectal carcinoma HCT 116 cells and breast cancer MDA-MB-231 cells.(a,b) HCT 116 and (c,d) MDA-MB-231 cells were treated with 2 (0.6, 1.25, 2.5, 5 µM) for 24, 48, and 72 h.The cell migration distance was estimated by measuring the width of the wound, and was expressed as the percentage of each control in relation to the mean of the wound-closure area.All experiments were set up at least in triplicate (n = 9 for control and compound; n is the number of photographs).The magnification of the representative photos is ×10.The results are expressed as mean ± standard deviation (SD).The asterisks (* p < 0.05, ** p < 0.01) indicate a significant decrease in migration of cells treated with the compound, compared to control.