Sulfated Polyhydroxysteroid Glycosides from the Sea of Okhotsk Starfish Henricia leviuscula spiculifera and Potential Mechanisms for Their Observed Anti-Cancer Activity against Several Types of Human Cancer Cells

Three new monosulfated polyhydroxysteroid glycosides, spiculiferosides A (1), B (2), and C (3), along with new related unsulfated monoglycoside, spiculiferoside D (4), were isolated from an ethanolic extract of the starfish Henricia leviuscula spiculifera collected in the Sea of Okhotsk. Compounds 1–3 contain two carbohydrate moieties, one of which is attached to C-3 of the steroid tetracyclic core, whereas another is located at C-24 of the side chain of aglycon. Two glycosides (2, 3) are biosides, and one glycoside (1), unlike them, includes three monosaccharide residues. Such type triosides are a rare group of polar steroids of sea stars. In addition, the 5-substituted 3-OSO3-α-L-Araf unit was found in steroid glycosides from starfish for the first time. Cell viability analysis showed that 1–3 (at concentrations up to 100 μM) had negligible cytotoxicity against human embryonic kidney HEK293, melanoma SK-MEL-28, breast cancer MDA-MB-231, and colorectal carcinoma HCT 116 cells. These compounds significantly inhibited proliferation and colony formation in HCT 116 cells at non-toxic concentrations, with compound 3 having the greatest effect. Compound 3 exerted anti-proliferative effects on HCT 116 cells through the induction of dose-dependent cell cycle arrest at the G2/M phase, regulation of expression of cell cycle proteins CDK2, CDK4, cyclin D1, p21, and inhibition of phosphorylation of protein kinases c-Raf, MEK1/2, ERK1/2 of the MAPK/ERK1/2 pathway.

Starfish of the genus Henricia Gray, 1840 (order Spinulosida, family Echinasteridae) inhabit mainly temperate and arctic waters.About 50 species of starfish belong to this genus.Henricia spp.are widely distributed in the North Pacific Ocean, especially in the Bering and Okhotsk Seas.Species of the genus Henricia are highly variable.Many of them are very close to each other, making their identification difficult in some cases [16,17].It is known that sea stars contain a variety of polar steroid metabolites.So far, efforts have been made to study the steroid composition of seven species of the genus, namely, H. leviuscula (earlier erroneously named as H. laeviuscola) [5,18], H. downeyae [19,20], H. sanguinolenta, H. leviuscula leviuscula, H. aspera, H tumida, and H. derjugini [5].Most of the isolated polar steroids are either sulfated or non-sulfated polyhydroxysteroids or structurally related to them monoglycosides with a monosaccharide residue at C-3 of the aglycon or so-called "two-chains" glycosides with two monosaccharide units attached at different positions of the aglycon, namely, in the steroid nucleus at C-3 and in the steroid side chain at C-24.Summarily, these studies demonstrate a high diversity of polar steroids in sea stars of the genus Henricia, which is consistent with the wide biological variability of species in this genus.However, "classical" asterosaponins, which are monosulfated steroid oligoglycosides with five to six monosaccharide residues, were not found in the species studied, with the exception of henricioside A from H. leviuscula [18].
Steroid metabolites isolated from sea stars of the genus Henricia exhibited diverse biological effects.Thus, some compounds have shown antifungal activity [18], the ability to inhibit cell division of fertilized sea urchin eggs [20], cytotoxic activity against non-smallcell lung human carcinoma [19], and hemolytic effect against mouse erythrocytes [5].In addition, leviusculoside G from H. leviuscula was shown to induce apoptosis in cancer cells and decrease the pro-carcinogenic transformation of normal cells.A possible molecular mechanism was proposed through the induction of p53-dependent apoptosis and inhibition of AP-1, NF-κB, and ERKs activities.Thereby, steroid metabolites isolated from Henricia spp.are of interest for further study of their structures and biological activity, especially as anti-cancer and cancer-preventive compounds [21].
In continuation of the study on the chemical constituents of the sea stars [5], herein we report the results of our investigation of polar steroid metabolites from an ethanolic extract of the Far Eastern starfish Henricia leviuscula spiculifera H.L. Clark, 1901 (order Spinulosida, family Echinasteridae), collected near Urup Island (Kuril Islands) in the Sea of Okhotsk.We have isolated and structurally elucidated four new polyhydroxysteroid glycosides 1-4.The anti-cancer activity of 1-3 against several types of human cancer cells has been investigated.A bioassay of compound 4 was not carried out since it was isolated in insufficient amounts.In addition, the influence of 3, the most active of the tested compounds, on the cell cycle, regulation of expression of cell cycle proteins, and inhibition of phosphorylation of protein kinases has been studied.

Structure Determination of Compounds 1-4
Three new monosulfated polyhydroxysteroid glycosides and one new unsulfated related monoside were isolated from an ethanolic extract of the sea star Henricia spiculifera by means of chromatographic techniques (column chromatography on Polychrom 1, Si gel, and Florisil followed by reverse-phase high-pressure liquid chromatography on Diasfer-110-C18 and YMC-Pack Pro C18 columns).These substances were designated as spiculiferosides A (1), B (2), C (3), and D (4) (Figure 1).+ showed the presence of a sulfate group in 1 (Figure S1).The IR spectrum of 1 revealed absorption bands due to hydroxy (3441 and 1641 cm −1 ) and sulfate (1265 cm −1 ) groups.The 13 C-NMR and DEPT spectra of 1 exhibited the presence of 45 carbon atoms in the molecule, including 5 methyls, 11 methylenes, 24 methines, two quaternary carbon atoms, one oxygenated tertiary carbon, and two methoxyl groups (Figures S2 and S3).The 1 H-and 13 C-NMR spectra of 1 (Tables 1 and 2 S1).The IR spectrum of 1 revealed absorption bands due to hydroxy (3441 and 1641 cm −1 ) and sulfate (1265 cm −1 ) groups.The 13 C-NMR and DEPT spectra of 1 exhibited the presence of 45 carbon atoms in the molecule, including 5 methyls, 11 methylenes, 24 methines, two quaternary carbon atoms, one oxygenated tertiary carbon, and two methoxyl groups (Figures S2 and S3).The 1 H-and 13 C-NMR spectra of 1 (Tables 1 and 2 and S6).The relative configurations 3β, 4β, 6β, and 15α of hydroxyl substituents in the steroid core and the 5α-cholestane skeleton in 1 were determined based on proton correlations in the ROESY spectrum from H-3 to Hα-1 and H-5; from H-5 to Hα-7 and H-9; from H-14 to H-9 and H-17; from H 3 -18 to Hβ-11, Hβ-12, and H-15; and from H 3 -19 to Hβ-1, and Hβ-2 (Figures 3 and S7).The main cross peaks of protons and carbon atoms in the HMBC spectrum confirmed the general structure of the steroid aglycon in 1 (Figures 2 and S8).The resonance value of the methyl group H 3 -21 at δ H 0.90, as well as the presence in the ROESY spectrum of correlations of protons from Hβ-12 to H 3 -21, from H-17 to H 3 -21, and from H 3 -18 to H-20, indicated a 20R configuration of the asymmetric center [23,24].
In the 1 H-NMR spectrum of 1, three chemical shifts of anomeric protons were observed at δ H 4.44, 5.00, and 4.34, associated with signals of carbon atoms at δ C 102.6, 109.2, and 104.9 in the HSQC spectrum, respectively.These data indicated the existence of three monosaccharide residues in glycoside 1.The coupling constants 7.  S1).Therefore, according to the ESIMS/MS and NMR spectra, molecule 1 contains hexose, di-O-methyl-pentose, and sulfoxypentose units.Acid hydrolysis of glycoside 1 with 2 M CF 3 COOH yielded three monosaccharides, which, after obtaining 2-octylglycoside derivatives by treatment with (R)-(-)-2-octanol and subsequent acetylation according to the procedure of Leontein et al. [25], were identified by GC as 2,4-di-O-methyl-D-xylose, L-arabinose, and D-glucose.The sequences of protons and the carbon atoms associated with corresponding protons, as well as the relative proton configurations of monosaccharide residues, were assigned using 1 H-1 H COSY, HSQC, HMBC, and ROESY experiments (Table 2, Figures 2 and 3).Irradiation of anomeric protons in 1D TOCSY experiments allowed us to refine the chemical shifts and coupling constants of the carbohydrate moiety protons.The spectral data of the two monosaccharide units were in good agreement with those for the terminal residues of 2,4-di-O-methyl-β-D-xylopyranose [22] and β-D-glucopyranose [26].The position of the terminal 2,4-di-O-methyl-β-D-xylopyranose unit at C-3 of aglycon was confirmed by the cross-peaks between H-1 ′ and H-3, C-3, the linkage of the terminal residue of β-D-glucopyranose to C-5 ′′ of the internal residue of α-L-arabinofuranose was indicated by the cross-peaks between H-1 ′′′ and H 2 -5 ′′ , C-5 ′′ , and the linkage of the α-L-arabinofuranose residue to C-24 of aglycon was fixed by the cross-peaks between H-1 ′′ and H-24, C-24 in the ROESY and HMBC spectra, respectively.A comparison of the proton and carbon signals of the internal monosaccharide residue of glycoside 1 with those of the five-substituted α-L-arabinofuranose residue of kurilensoside B from the starfish Hippasteria kurilensis [27] showed that the chemical shifts of H-3 ′′ and C-3 ′′ were deshielded from δ H 3.94 to 4.67 and from δ C 79.2 to 84.7, respectively, and the signal of C-2 ′′ was shielded from δ C 83.8 to 82.0.These facts clearly revealed the location of the sulfate group at C-3 ′′ of 5 ′′ -substituted residue of α-L-arabinofuranose in 1.In addition, the signal of C-1 ′′ at δ C 109.2 unambiguously indicated the α-configuration of the anomeric center of the arabinofuranose residue [28].The 24S configuration was proposed based on the similarity of the 13 C-NMR spectroscopic data for the side chain of glycoside 1 with those for other related (24S)-24-O-α-L-arabinofuranosides previously isolated from starfish [29][30][31].Consequently, the structure of spiculiferoside A (1) was elucidated as the (24S Glycoside 1 is a triglycoside and contains two carbohydrate moieties, one of which is attached to C-3 of the steroid core, and the other is located at C-24 of the aglycon side chain.Only five such "two-chain" triglycosides from see stars were previously known [27,32,33].In addition, the five-substituted 3-OSO 3 -α-L-Araf residue was found for the first time in steroid glycosides from starfish. Spiculiferoside B ( 2 S9).A detailed comparison of the 1 H-, 13 C-NMR, DEPT, COSY, HSQC, HMBC, and ROESY spectroscopic data of glycoside 2 (Tables 1 and 2, Figures 2, 3, and S10-S16) with the corresponding data of glycoside 1 showed that 2 had the same 3β,4β,6β,8,15α,24-hexahydroxy-5α-cholestane aglycon, glycosylated at C-3 with a 2,4-di-O-methyl-β-D-xylopyranose residue, and at C-24 with a sulfated α-L-arabinofuranose residue, and differed from 1 only in the absence of a terminal β-D-glucopyranose residue.A comparison of the signals of the protons and carbon atoms of the monosaccharide unit at C-24 in glycoside 2 with the corresponding signals of the terminal α-L-arabinofuranose residue of forbeside J [22] showed that the resonance of C-3 ′′ was deshielded from δ C 78.7 to 84.5; the resonances of C-2 ′′ and C-4 ′′ were shielded from δ C 84.0 to 82.1 and from δ C 85.0 to 84.4, respectively, and the signal of H-3 ′′ was deshielded from δ H 3.86 to 4.46 in accordance with αand β-effects of sulfation.In this way, the position of a sulfate group in the α-L-arabinofuranose in 2 was defined as C-3 ′′ .Compound 2 was subjected to mild solvolysis with a mixture of dioxane and pyridine to give desulfated derivative 2a, which was identified by comparison of the HRESIMS and 1 H-, 13 C-NMR, and HSQC data (Experimental section, Figures S17-S20) with those of forbeside J [22].As a result, the absolute configuration at C-24 in 2 was proposed as S by analogy with forbeside J (2a).On the basis of the above-mentioned data, the structure of spiculiferoside B ( 2 1 and 2).

The Effect of Compounds 1-3 on Cell Viability and Proliferation of Human Normal and Cancer Cells
In the present work, the effect of compounds 1, 2, and 3 on cell viability of human embryonic kidney HEK293, melanoma SK-MEL-28, breast cancer cells MDA-MB-231, and colorectal carcinoma HCT 116 cells was determined by MTS assay in a 24 h cell treatment (Figure 4).

The Effect of Compounds 1-3 on Cell Viability and Proliferation of Human Normal and Cancer Cells
In the present work, the effect of compounds 1, 2, and 3 on cell viability of human embryonic kidney HEK293, melanoma SK-MEL-28, breast cancer cells MDA-MB-231, and colorectal carcinoma HCT 116 cells was determined by MTS assay in a 24 h cell treatment (Figure 4).The tested compounds were less cytotoxic against normal cells, HEK293, and two types of cancer cells, SK-MEL-28 and MDA-MB-231 (Figure 4A-C).On the other hand, these compounds were found to suppress cell viability of colorectal carcinoma cells HCT 116 more effectively, with great impact of compound 3 (Figure 4D).Compound 1 at concentrations of 1, 10, 50, and 100 μM inhibited cell viability of HCT 116 cells by 0%, 2%, 6%, and 35%, respectively; compound 2 inhibited cell viability at 1, 10, 50, and 100 μM-0%, 0%, 6%, and 30%, respectively, while 3 at the same experimental conditions suppressed the cell viability by 0%, 0%, 37%, and 54%, respectively (Figure 4D).IC50 was reached only for compound 3, which was 87.6 μM with a selective index (SI) of 1.5 after 24 h of HCT 116 cells' treatment (Figure 4D).

The Effect of Compounds 1-3 on the Colony Formation of Human Colorectal Carcinoma Cells
More promising data were obtained in the results of the studies on the effects of 1-3 on microcolony formation by tumor cells.In the present study, the colony-inhibiting activity was investigated in HCT 116 cells using the soft agar assay.Non-toxic concentrations of 10, 20, and 40 μM of the investigated compounds were chosen for further experiments.All the tested compounds were found to significantly decrease colonies'

The Effect of Compounds 1-3 on the Colony Formation of Human Colorectal Carcinoma Cells
More promising data were obtained in the results of the studies on the effects of 1-3 on microcolony formation by tumor cells.In the present study, the colony-inhibiting activity was investigated in HCT 116 cells using the soft agar assay.Non-toxic concentrations of 10, 20, and 40 µM of the investigated compounds were chosen for further experiments.All the tested compounds were found to significantly decrease colonies' numbers of colorectal carcinoma cells dose-dependently (Figure 6).Compound 1 at concentrations of 10, 20, and 40 µM inhibited colony formation in HCT 116 cells by 18%, 39%, and 65%, respectively (Figure 6A); 2-by 25%, 48%, and 81%, respectively (Figure 6B), and 3-by 19%, 56%, 87%, respectively (Figure 6C).Compound 3 was found to have the most significant colony-inhibiting activity against HCT 116 cells among all the compounds studied and was, therefore, selected for further investigation of the molecular mechanism of its anti-cancer action.

The Effect of Compounds 1-3 on the Colony Formation of Human Colorectal Carcinoma Cells
More promising data were obtained in the results of the studies on the effects of 1-3 on microcolony formation by tumor cells.In the present study, the colony-inhibiting activity was investigated in HCT 116 cells using the soft agar assay.Non-toxic concentrations of 10, 20, and 40 μM of the investigated compounds were chosen for further experiments.All the tested compounds were found to significantly decrease colonies' numbers of colorectal carcinoma cells dose-dependently (Figure 6).Compound 1 at concentrations of 10, 20, and 40 μM inhibited colony formation in HCT 116 cells by 18%, 39%, and 65%, respectively (Figure 6A); 2-by 25%, 48%, and 81%, respectively (Figure 6B), and 3-by 19%, 56%, 87%, respectively (Figure 6C).Compound 3 was found to have the most significant colony-inhibiting activity against HCT 116 cells among all the compounds studied and was, therefore, selected for further investigation of the molecular mechanism of its anti-cancer action.

The Effect of Compound 3 on Cell Cycle Progression and Molecular Mechanism of Anti-cancer Action in Human Colorectal Carcinoma Cells
The fundamental abnormality that leads to the development of cancer is the continuous, unregulated proliferation of cancer cells.Instead of responding appropriately to signals that control normal cell behavior, cancer cells grow and divide uncontrollably, invading normal tissues and organs and eventually spreading throughout the body [35].Since compound 3 inhibited proliferation and colony formation of colorectal cancer cells

The Effect of Compound 3 on Cell Cycle Progression and Molecular Mechanism of Anti-cancer Action in Human Colorectal Carcinoma Cells
The fundamental abnormality that leads to the development of cancer is the continuous, unregulated proliferation of cancer cells.Instead of responding appropriately to signals that control normal cell behavior, cancer cells grow and divide uncontrollably, invading normal tissues and organs and eventually spreading throughout the body [35].Since compound 3 inhibited proliferation and colony formation of colorectal cancer cells HCT 116, we checked whether compound 3 could regulate cell cycle distribution by flow cytometric analysis.Cell cycle progression was examined after treatment of HCT 116 cells with 10, 20, and 40 µM of 3 for 72 h.
It was found that the treatment of HCT 116 cells with 3 resulted in a dose-dependent increase in cells in the G2/M phase compared to the control group.Compound 3 at 10, 20, and 40 µM was shown to increase the amount of HCT 116 cells in G2/M phase by 16%, 42%, and 71%, respectively, with a corresponding reduction in the percentage of cells in the G0/G1 phase by 0%, 15%, and 25%, respectively, and S phase by 9%, 11%, and 20%, respectively, compared to the control group (Figure 7A,B).These data suggest that the inhibition of cell proliferation of HCT 116 cells is mainly associated with the induction of G2/M cell cycle arrest.and 40 μM was shown to increase the amount of HCT 116 cells in G2/M phase by 16%, 42%, and 71%, respectively, with a corresponding reduction in the percentage of cells in the G0/G1 phase by 0%, 15%, and 25%, respectively, and S phase by 9%, 11%, and 20%, respectively, compared to the control group (Figure 7A,B).These data suggest that the inhibition of cell proliferation of HCT 116 cells is mainly associated with the induction of G2/M cell cycle arrest.Next, we turned our attention to the molecular mechanism of anti-cancer action of compound 3 associated with the inhibition of cell proliferation of HCT 116 cells via the regulation of a series of important cell cycle proteins and the activation of mitogen-activated protein kinases (MAPK) by Western Blot assay.Extracellular-signal-related kinase p44/42 MAPK (Erk1/2) is known to be an important participant in the MAPK signaling pathway [36].ERK1/2 plays a well-established role in regulating cell cycle progression by activation of multiple transcription factors such as Elk1, c-Jun, c-Myc, and c-Fos, which control the expression of proteins important for cell-cycle progression, including Cyclin D1 and p21WAF1/CIP1 [37].Cyclin-dependent kinases (CDK) are major Next, we turned our attention to the molecular mechanism of anti-cancer action of compound 3 associated with the inhibition of cell proliferation of HCT 116 cells via the regulation of a series of important cell cycle proteins and the activation of mitogen-activated protein kinases (MAPK) by Western Blot assay.Extracellular-signal-related kinase p44/42 MAPK (Erk1/2) is known to be an important participant in the MAPK signaling pathway [36].ERK1/2 plays a well-established role in regulating cell cycle progression by activation of multiple transcription factors such as Elk1, c-Jun, c-Myc, and c-Fos, which control the expression of proteins important for cell-cycle progression, including Cyclin D1 and p21WAF1/CIP1 [37].Cyclin-dependent kinases (CDK) are major players in cell proliferation that regulate cell cycle checkpoints and transcription events in response to extracellular and intracellular signals.CDK dysregulation is certain to be a hallmark of cancer and an attractive target in cancer therapy.CDK activity is primarily regulated by the binding of CDK catalytic subunits to Cyclin partners and CDK inhibitors.The complex formed by CDK4 and Cyclin D1 has been strongly implicated in the control of cell proliferation and prognoses in human malignancies [38].In this regard, we examined the influence of 3 on the expression of CDK2, CDK4, Cyclin D1, and p21.The investigated compound was found to dose-dependently down-regulate the expression of CDK2 and Cyclin D1 but not CDK4.The expression of the inhibitor of CDK/Cyclin complex-p21 was significantly increased by 3 compared to non-treated HCT 116 cells (Figure 7C,D).The treatment of HCT 116 cells by 3 was demonstrated to cause the inhibition of phosphorylation of c-Raf, MEK1/2, and ERK1/2 kinases (Figure 7C,D).
Our results provided evidence that the coordinated alteration of the expression of cell cycle proteins and inhibition of the phosphorylation of the ERK1/2 MAPK signaling cascade were likely the basis of the anti-cancer effect of compound 3 on the proliferation of colorectal carcinoma cells HCT 116.
HEK293 cells were grown in MEM medium; SK-MEL-28 and MDA-MB-231 cells were cultured in DMEM medium, while HCT 116 cells were maintained in McCoys' 5A medium according to the manufacturer's instructions.Culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin solution.The cells were cultured at 37 • C in a humidified atmosphere containing 5% CO 2 .

Cell Viability Assay
The CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay kit (MTS) was used for cell viability analysis and performed according to the standard protocol.Briefly, cells (1 × 10 4 /200 µL) were seeded in 96-well plates and incubated for 24 h in a humidified atmosphere containing 5% CO 2 .Then, they were treated with DMSO (control) and compounds T1, B1, and B2 at 1, 10, 50, and 100 µM for an additional 24 h.The MTS reagent (20 µL/well) was added to the cell culture medium and incubated at a 37 • C incubator for 2 h.Cell viability was examined at 490/630 nm using a Power Wave XS microplate reader (BioTek, Winooski, VT, USA).
The concentration at which the compounds exert half of their maximal inhibitory effect on cell viability (IC 50 ) was calculated by the AAT-Bioquest ® online calculator [39].The selectivity index (SI) was calculated as described previously [40] using the following formula: SI = IC 50 of the compounds in normal cell (HEK293)/IC 50 of the same compounds in human colorectal adenocarcinoma cell line (HCT 116).

Cell Proliferation Assay
HCT 116 cells (8 × 10 3 /200 µL) were seeded in 96-well plates and incubated for 24 h in a CO 2 incubator.The cells' monolayers were washed with phosphate-buffered saline (PBS) to remove unattached cells.The attached cells were incubated with fresh medium containing DMSO (control) and B2 (0-100 µM) for 24, 48, and 72 h.Subsequently, the cells were incubated with 15 µL MTS reagent for 2 h, and the absorbance of each well was measured at 490/630 nm using a microplate reader (Power Wave XS, USA).

Anchorage-Independent Cell Growth Assay
Soft agar assay was performed as described previously [41].Briefly, the cells were counted and seeded into 6-well plates at a density of 8×10 3 /per well with 0.3% BME agar containing 10% FBS and DMSO (control) or various concentrations of compounds 1, 2, and 3 (10, 20, and 40 µM).The number of the colonies was determined using a Motic microscope AE 20 and ImageJ software bundled with 64-bit Java 1.8.0_112 (NIH, Bethesda, MD, USA) 14 days later.

Cell Cycle Assay
HCT 116 cells (3 × 10 5 ) were seeded in 60 mm dishes and incubated for 24 h in a CO 2 incubator.The attached cells were treated by DMSO (control) or 3 (10,20, and 40 µM) for 72 h.Then, cells were harvested, washed with ice-cold 1× PBS, and fixed with 70% ethanol.HCT 116 cells were incubated overnight at -20 • C, and then fixed cells were collected by centrifugation at 4000 rpm for 10 min and rinsed with 1× PBS.The cell pellet was resuspended in Muse™ Cell Cycle Reagent (MCH100106, Luminex, Austin, TX, USA), and the cells were incubated for 30 min at RT in the dark.The DNA content was assessed by measuring the fluorescence intensity by flow cytometry (Muse™ Cell Analyzer).Results were expressed as a percentage of cells in the G0/1, S, and G2/M phases of the cell cycle, associated with the DNA content profile histograms 3.13.Western Blot Assay HCT 116 cells (1.0 × 10 5 /mL) were seeded in 100 mm dishes and incubated for 24 h at 37 • C in a CO 2 incubator.The cells were treated by DMSO (control) or 3 (10,20, and 40 µM) for 72 h.Then, cells were harvested and lysed by 1× cell lysis buffer ("Cell Signaling Technology", Danvers, MA, USA) according to the manufacturer's protocol.Cells' protein content was determined by the DC protein assay (Bio-Rad, Hercules, CA, USA).Lysates of protein (20-40 µg) were exposed to 10% or 12% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (PVDF) (Millipore, Burlington, MA, USA).The membranes were blocked with 5% non-fat milk (Bio-Rad) for 1 h and then incubated with the respective specific primary antibody at 4 • C overnight.Protein bands were visualized using an enhanced chemiluminescence reagent (ECL) (Bio-Rad, Hercules, CA, USA) after hybridization with an HRP-conjugated secondary antibody.

Statistical Analysis
All of the assays were performed in at least three independent experiments.Results are expressed as the mean ±standard deviation (SD).Statistical procedures were performed using one-way ANOVA and Tukey's HSD tests with * p < 0.05, ** p < 0.01, and *** p < 0.001.

Conclusions
Polar steroid compounds from the ethanolic extract of the Sea of Okhotsk starfish Henricia leviuscula spiculifera were investigated.New monosulfated steroid glycosides, spiculiferosides A, B, and C, and a new unsulfated related monoglycoside, spiculiferoside D, were isolated, and their chemical structures were characterized.Three of them contain two carbohydrate chains, which are located at positions C-3 and C-24 of the polyhydroxylated cholestane aglycone.Spiculiferosides B and C are biosides, and spiculiferoside A, in contrast, has three monosaccharide residues.Previously, only five such "two-chains" triglycosides were known from sea stars, kurilensosides A, B, C, and I, found in the Far Eastern starfish Hippasteria kurilensis [27,32], and planciside D isolated from the tropical starfish Acanthaster planci [33], which also contain two carbohydrate patterns attached to the steroid core and aglycon side chain.The 5-substituted 3-OSO 3 -α-L-Araf residue of spiculiferoside A was discovered and described for the first time in steroid glycosides of starfish.Moreover, the 3-OSO 3 -α-L-Araf residue that comprised spiculiferosides B and C, was previously found only in one steroid glycoside from the sea star Oreaster reticulatus [34].Interestingly, we did not find "classical" oligoglycosides (asterosaponins) in the starfish H. leviuscula spiculifera as in most previously studied species of the genus Henricia.
Spiculiferosides A, B, and C exhibited moderate cytotoxic activities against human embryonic kidney HEK293, melanoma SK-MEL-28, breast cancer MDA-MB-231, and colorectal carcinoma HCT 116 cell lines but significantly inhibited proliferation and colony formation in HCT 116 cells.Spiculiferoside C demonstrated the highest anti-cancer activity among the investigated compounds.The molecular mechanism of anti-cancer action of this compound was associated with the induction of cell cycle arrest at the G2/M phase

Spiculiferoside A ( 1 )
has the molecular formula C 45 H 77 O 22 SNa determined from the peak of [M − Na] − ion at m/z 1001.4622 in the (-)HRESIMS and from the peak of the cationized molecule [M + Na] + at m/z 1047.4419 in the (+)HRESIMS.The fragment ion peak at m/z 97 [HSO 4 ] − in the (−)ESIMS/MS spectrum of the ion with m/z 1001 [M − Na] − and the fragment ion peaks at m/z 927 [(M + Na) − NaHSO 4 ] + and 143 [Na 2 HSO 4 ] + in the (+)ESIMS/MS spectrum of the ion with m/z 1047 [M + Na]

Figure 1 .
Figure 1.The chemical structures of spiculiferosides A (1), B (2), C (3), and D (4).Analysis of the 1 H-1 H COSY and HSQC correlations made it possible to establish the spin systems of protons and the corresponding sequences of carbon atoms from C-1 to C-7, from C-9 to C-12 through C-11, from C-14 to C-17, from C-20 to C-21, and from C-22 to C-27 (Figures 2, S5 and S6).The relative configurations 3β, 4β, 6β, and 15α of hydroxyl substituents in the steroid core and the 5α-cholestane skeleton in 1 were determined based on proton correlations in the ROESY spectrum from H-3 to Hα-1 and H-5; from H-5 to Hα-7 and H-9; from H-14 to H-9 and H-17; from H 3 -18 to Hβ-11, Hβ-12, and H-15; and from H 3 -19 to Hβ-1, and Hβ-2 (Figures 3 and S7).The main cross peaks of protons and carbon atoms in the HMBC spectrum confirmed the general structure of the steroid aglycon in 1 (Figures 2 and S8).The resonance value of the methyl group H 3 -21 at δ H 0.90, as well as the presence in the ROESY spectrum of correlations of protons from Hβ-12 to H 3 -21, from H-17 to H 3 -21, and from H 3 -18 to H-20, indicated a 20R configuration of the asymmetric center [23,24].In the 1 H-NMR spectrum of 1, three chemical shifts of anomeric protons were observed at δ H 4.44, 5.00, and 4.34, associated with signals of carbon atoms at δ C 102.6, 109.2, and 104.9 in the HSQC spectrum, respectively.These data indicated the existence of three monosaccharide residues in glycoside 1.The coupling constants 7.5 and 7.7 Hz of two anomeric protons exhibited the β-glycosidic bonds of the corresponding monosaccharide residues, and a wide singlet of the third anomeric proton showed the presence of an α-glycosidic bond in this monosaccharide residue.The ESIMS/MS spectrum of the [M − Na] − ion with m/z 1001 revealed fragment ion peaks corresponding to the loss in a hexose at m/z 839 [(M − Na) -C 6 H 10 O 5 ] − and the simultaneous loss in a hexose and a di-O-methyl-pentose at m/z 679 [(M − Na) − C 6 H 10 O 5 − C 7 H 12 O 4 ] − .Respectively, the ESIMS/MS spectrum of the [M + Na] + ion with m/z 1047 exhibited fragment ion peaks arising due to the loss in a hexose at m/z 885 [(M + Na) − C 6 H 10 O 5 ] + , the simultaneous loss in a hexose and a di-O-methyl-pentose at m/z 725 [(M + Na) − C 6 H 10 O 5 − C 7 H 12 O 4 ] + , the simultaneous loss in a hexose and a sulfoxypentose at m/z 651 [(M + Na) − C 6 H 10 O 5 − C 5 H 7 O 7 SNa] + ,

5 and 7 . 7
Hz of two anomeric protons exhibited the β-glycosidic bonds of the corresponding monosaccharide residues, and a wide singlet of the third anomeric proton showed the presence of an α-glycosidic bond in this monosaccharide residue.The ESIMS/MS spectrum of the [M − Na] − ion with m/z 1001 revealed fragment ion peaks corresponding to the loss in a hexose at m/z 839 [(M − Na) -C 6 H 10 O 5 ] − and the simultaneous loss in a hexose and a di-O-methyl-pentose at m/z 679 [(M − Na) − C 6 H 10 O 5 − C 7 H 12 O 4 ] − .Respectively, the ESIMS/MS spectrum of the [M + Na] + ion with m/z 1047 exhibited fragment ion peaks arising due to the loss in a hexose at m/z 885 [(M + Na) − C 6 H 10 O 5 ] + , the simultaneous loss in a hexose and a di-O-methyl-pentose at m/z 725 [(M + Na) − C 6 H 10 O 5 − C 7 H 12 O 4 ] + , the simultaneous loss in a hexose and a sulfoxypentose at m/z 651 [(M + Na) − C 6 H 10 O 5 − C 5 H 7 O 7 SNa] + , and the simultaneous loss in a hexose, a sulfoxypentose, and a di-O-methyl-pentose at m/z 491 [651 − C 7 H 12 O 4 ] + (Figure

Figure 4 .
Figure 4.The cytotoxic activity of compounds 1, 2, and 3 against human normal and cancer cells.(A) Human embryonic kidney HEK293, (B) melanoma SK-MEL-28, (C) breast cancer MDA-MB-231, and (D) colorectal carcinoma HCT 116 cells were treated with 1, 2, and 3 (1, 10, 50, and 100 μM) for 24 h.MTS assay was used to evaluate cytotoxicity of compounds.IC50-the concentration at which the compounds exert half of their maximal inhibitory effect on cell viability.The data results are presented as mean ± SD for triplicate experiments.A one-way ANOVA and Tukey's HSD test for multiple comparisons indicated the statistical significance (* p < 0.05).

Figure 4 .
Figure 4.The cytotoxic activity of compounds 1, 2, and 3 against human normal and cancer cells.(A) Human embryonic kidney HEK293, (B) melanoma SK-MEL-28, (C) breast cancer MDA-MB-231, and (D) colorectal carcinoma HCT 116 cells were treated with 1, 2, and 3 (1, 10, 50, and 100 µM) for 24 h.MTS assay was used to evaluate cytotoxicity of compounds.IC 50 -the concentration at which the compounds exert half of their maximal inhibitory effect on cell viability.The data results are presented as mean ± SD for triplicate experiments.A one-way ANOVA and Tukey's HSD test for multiple comparisons indicated the statistical significance (* p < 0.05).

Figure 7 .
Figure 7.The effect of compound 3 on cell cycle regulation and the expression of cell cycle markers and MAPK kinases in human colorectal carcinoma cells HCT 116.(A,B) HCT 116 cells were treated with 3 at 10, 20, and 40 μM for 72 h.The percentage of cells in G0/G1, S, and G2/M phases was determined using a Muse cell analyzer.Histograms from a representative experiment show the effect of 3 on cell cycle profile.(C) The regulation of expression of cell cycle markers, MAPK, and β-actin by 3 (10, 20, and 40 μM) after 72 h of treatment of HCT 116 cells.(D) Relative band density was measured using the Quantity One 1D analysis software version 4.6.7.Band density was normalized to β-actin total level.Results are presented as mean ± standard deviation (SD).A one-way ANOVA and Tukey's HSD test for multiple comparisons indicated the statistical significance (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 7 .
Figure 7.The effect of compound 3 on cell cycle regulation and the expression of cell cycle markers and MAPK kinases in human colorectal carcinoma cells HCT 116.(A,B) HCT 116 cells were treated with 3 at 10, 20, and 40 µM for 72 h.The percentage of cells in G0/G1, S, and G2/M phases was determined using a Muse cell analyzer.Histograms from a representative experiment show the effect of 3 on cell cycle profile.(C) The regulation of expression of cell cycle markers, MAPK, and β-actin by 3 (10, 20, and 40 µM) after 72 h of treatment of HCT 116 cells.(D) Relative band density was measured using the Quantity One 1D analysis software version 4.6.7.Band density was normalized to β-actin total level.Results are presented as mean ± standard deviation (SD).A one-way ANOVA and Tukey's HSD test for multiple comparisons indicated the statistical significance (* p < 0.05; ** p < 0.01; *** p < 0.001).