New Conjugates of Polyhydroxysteroids with Long-Chain Fatty Acids from the Deep-Water Far Eastern Starfish Ceramaster patagonicus and Their Anticancer Activity

Four new conjugates, esters of polyhydroxysteroids with long-chain fatty acids (1–4), were isolated from the deep-water Far Eastern starfish Ceramaster patagonicus. The structures of 1–4 were established by NMR and ESIMS techniques as well as chemical transformations. Unusual compounds 1–4 contain the same 5α-cholestane-3β,6β,15α,16β,26-pentahydroxysteroidal moiety and differ from each other in the fatty acid units: 5′Z,11′Z-octadecadienoic (1), 11′Z-octadecenoic (2), 5′Z,11′Z-eicosadienoic (3), and 7′Z-eicosenoic (4) acids. Previously, only one such steroid conjugate with a fatty acid was known from starfish. After 72 h of cell incubation, using MTS assay it was found that the concentrations of compounds 1, 2, and 3 that caused 50% inhibition of growth (IC50) of JB6 Cl41 cells were 81, 40, and 79 µM, respectively; for MDA-MB-231 cells, IC50 of compounds 1, 2, and 3 were 74, 33, and 73 µM, respectively; for HCT 116 cells, IC50 of compounds 1, 2, and 3 were 73, 31, and 71 µM, respectively. Compound 4 was non-toxic against tested cell lines even in three days of treatment. Compound 2 (20 µM) suppressed colony formation and migration of MDA-MB-231 and HCT 116 cells.

formation and migration of human breast cancer and colorectal carcinoma cells were investigated using soft agar and wound healing assays.
Fatty acid units in 2 were identified by ECL values of fatty acids in GC analysis and mass spectra of FAME-2 and DMOXs-2 derivatives in GC-MS [16] similar to compound 1. GC-MS analysis showed the existence of one major component, which was identified as methyl 11 Z-octadecenoate. Minor components and their percentage are shown in Table 3. Thereby, the structure of 2 was established to be (25S)-5α-cholestane-3β,6β,15α,16β-tetraol-26-yl 11 Z-octadecenoate.
Many natural compounds do not possess direct cytotoxic activity but are able to suppress cell viability time-dependently [17]. That is why the cytostatic activity of  It should be noted that investigated compounds did not exert a selective effect on cancer cells, because the viability of normal cells was suppressed as well. Therefore we checked the assumption whether compounds 1-4 were able to influence the process of carcinogenesis (colony formation, growth, and migration of cancer cells) at the non-toxic concentration of 20 µM. The formation of colonies is one of the most stringent characteristics for malignant transformation in cells [18]. In the present study the soft agar assay was used to investigate the effect of compounds 1-4 on capability of cancer cells to form colonies. It should be noted that investigated compounds did not exert a selective effect on cancer cells, because the viability of normal cells was suppressed as well. Therefore we checked the assumption whether compounds 1-4 were able to influence the process of carcinogenesis (colony formation, growth, and migration of cancer cells) at the non-toxic concentration of 20 µM.

The Effect of Compounds 1-4 on the Colony Formation and Growth of Human Cancer Cells
The formation of colonies is one of the most stringent characteristics for malignant transformation in cells [18]. In the present study the soft agar assay was used to investigate the effect of compounds 1-4 on capability of cancer cells to form colonies.

The Effect of Compounds 1-4 on Migration of Human Cancer Cells
The metastasis process is proven to be the leading cause of cancer-related death. Metastasis is a multistep process that includes migration and invasion of cancer cells, hallmarks of malignancy [19]. Therefore, we investigated the ability of compounds 1-4 to inhibit the migration of breast cancer MDA-MB-231 cells and colorectal carcinoma HCT 116 cells with high metastatic potential. It was demonstrated that compounds 1 and 2 (at concentration 20 µM) were able to prevent migration of MDA-MB-231 cells by 42% and 50%, respectively, compared to control after 48 h of incubation ( Figure  5). Compounds 3 and 4 possessed moderate inhibitory activity against migration of MDA-MB-231 cells. On the other hand, the migration of HCT 116 cells was almost completely inhibited by compound 2 (with the percentage of migration prevention being 73%). Compounds 1, 3, and 4 prevented HCT 116 cell migration by 36%, 30%, and 24%, respectively ( Figure 5). Compounds 1, 3, and 4 prevented HCT 116 cell migration by 36%, 30%, and 24%, respectively ( Figure 5).

The Effect of Compounds 1-4 on Migration of Human Cancer Cells
The metastasis process is proven to be the leading cause of cancer-related death. Metastasis is a multistep process that includes migration and invasion of cancer cells, hallmarks of malignancy [19]. Therefore, we investigated the ability of compounds 1-4 to inhibit the migration of breast cancer MDA-MB-231 cells and colorectal carcinoma HCT 116 cells with high metastatic potential. It was demonstrated that compounds 1 and 2 (at concentration 20 µM) were able to prevent migration of MDA-MB-231 cells by 42% and 50%, respectively, compared to control after 48 h of incubation ( Figure 5). Compounds 3 and 4 possessed moderate inhibitory activity against migration of MDA-MB-231 cells. On the other hand, the migration of HCT 116 cells was almost completely inhibited by compound 2 (with the percentage of migration prevention being 73%) . Compounds 1, 3, and 4 prevented HCT 116 cell migration by 36%, 30%, and 24%, respectively ( Figure 5). Compounds 1, 3, and 4 prevented HCT 116 cell migration by 36%, 30%, and 24%, respectively ( Figure 5).

General Procedures
Optical rotations were determined on a PerkinElmer 343 polarimeter (Waltham, MA, USA). IR spectra were determined on a Bruker OPUS Vector-22 infrared spectrophotometer in CDCl3. The 1 Hand 13 C-NMR spectra were recorded on Bruker Avance III 700 spectrometer (Bruker, Germany) at 700.13 and 176.04 MHz, respectively, and chemical shifts were referenced to the corresponding residual solvent signal (δH 3.30 / δC 49.0 for CD3OD). The HRESIMS spectra were recorded on a Bruker Impact II Q-TOF mass spectrometer (Bruker, Germany); the samples were dissolved in MeOH (c 0.001 mg/mL). HPLC separations were carried out on an Agilent 1100 Series chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a differential refractometer; Discovery C18 (5 µm, 250 × 10 mm, Supelco, Bellefonte, USA) column was used. GC and GC-MS analyses were performed on a GC 2010 chromatograph with a flame ionization detector and a gas chromatograph couple to a mass spectrometer GCMS-QP5050, both from Shimadzu (Japan). Fused silica capillary columns Supelcowax 10 and MDN-5S (both columns 30 m, 0.25 mm ID, 0.25 µm film, Supelco, USA) were used in the apparatus. Low-pressure liquid column chromatography was carried out with the Si gel KSK (50-160 µm, Sorbpolimer, Krasnodar, Russia). Sorbfil Si gel plates (4.5 × 6.0 cm, 5-17 µm, Sorbpolimer, Krasnodar, Russia) were used for thin-layer chromatography.

Animal Material
Specimens of Ceramaster patagonicus Sladen, 1889 (order Valvatida, family Goniasteridae), were collected at a depth of 150-300 m in the Sea of Okhotsk near Iturup Island during 42nd scientific cruise of the research vessel Akademik Oparin, in August 2012. Species identification was carried out The magnification of representative photos is ×10. Results are expressed as the mean ± standard deviation (SD). The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant decrease in migration of cells treated with compounds compared with the control.

General Procedures
Optical rotations were determined on a PerkinElmer 343 polarimeter (Waltham, MA, USA). IR spectra were determined on a Bruker OPUS Vector-22 infrared spectrophotometer in CDCl 3 . The 1 H-and 13 C-NMR spectra were recorded on Bruker Avance III 700 spectrometer (Bruker, Germany) at 700.13 and 176.04 MHz, respectively, and chemical shifts were referenced to the corresponding residual solvent signal (δ H 3.30/δ C 49.0 for CD 3 OD). The HRESIMS spectra were recorded on a Bruker Impact II Q-TOF mass spectrometer (Bruker, Germany); the samples were dissolved in MeOH (c 0.001 mg/mL). HPLC separations were carried out on an Agilent 1100 Series chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a differential refractometer; Discovery C18 (5 µm, 250 × 10 mm, Supelco, Bellefonte, PA, USA) column was used. GC and GC-MS analyses were performed on a GC 2010 chromatograph with a flame ionization detector and a gas chromatograph couple to a mass spectrometer GCMS-QP5050, both from Shimadzu (Japan). Fused silica capillary columns Supelcowax 10 and MDN-5S (both columns 30 m, 0.25 mm ID, 0.25 µm film, Supelco, PA, USA) were used in the apparatus. Low-pressure liquid column chromatography was carried out with the Si gel KSK (50-160 µm, Sorbpolimer, Krasnodar, Russia). Sorbfil Si gel plates (4.5 × 6.0 cm, 5-17 µm, Sorbpolimer, Krasnodar, Russia) were used for thin-layer chromatography.

Extraction and Isolation
The fresh animals of C. patagonicus (3 kg, crude weight) were chopped into small pieces and extracted with CHCl 3 :MeOH (2:1) followed by further extraction with CHCl 3 :MeOH (1:1) and EtOH. The combined extracts were concentrated in vacuo to give a residue of 159.5 g. This residue was partitioned between H 2 O (1.5 L) and AcOEt:BuOH (2:1) (4.5 L), and the organic layer was concentrated in vacuo to give the less polar fraction (51.5 g), which was washed with cold acetone (1 L). The acetone-soluble part (28.5 g) was chromatographed over a silica gel column (19 × 4.5 cm) using

Methanolysis and Preparation of 4,4-Dimethyloxazoline Derivatives of Fatty Acids
Compounds 1-4 (1 mg) were heated with 1 N HCl in 80% aq MeOH (1.0 mL) at 80 • C for 4 h. The reaction mixtures were then extracted with n-hexane, and the extracts were concentrated in vacuo to yield FAME-1-FAME-4. The 4,4-Dimethyloxazoline derivatives of fatty acids of compounds 1 and 4 were prepared from FAME-1 and FAME-4 according to the procedure described previously [15].
FAMEs were analyzed on Supelcowax 10 columns at 200 • C. DMOX derivatives analyzed on a nonpolar MDN-5S column, where the temperature program ranged from 200 to 260 • C at 2 • C/min. Helium was used as the carrier gas at a linear velocity of 30 cm/s. Mass spectra were recorded at 70 eV. Mass spectra were compared with the NIST library and internet fatty acid mass spectra archive site. To determine the effect of compounds 1-4 on cell proliferation, the tested cell lines (8 × 10 3 cells/200 µL) were treated with tested compounds at concentrations of 1, 10, 50, and 100 µM or equivalent volume of DMSO (control) and incubated for additional 24, 48, and 72 h at 37 • C in 5% CO 2 . MTS reagent (20 µL) was added to each well, and the cells were incubated for additional 3 h in 5% CO 2 incubator. Absorbance was measured at 490/630 nm by microplate reader. All experimental conditions were assessed in triplicate.

Wound Healing Assay
MDA-MB-231 and HCT 116 cells (3 × 10 5 cells/mL) were seeded into six-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, MDA-MB-231 and HCT 116 cells were treated with equivalent volume of DMSO (control) or 1-4 at concentration of 20 µM and incubated for 48 h. All experiments were conducted in triplicate for each group. For the image analysis, cell migration into the wound area was photographed at the stages of 0 and 48 h using a Motic AE 20 microscope and ImageJ software. The cell migration distance was estimated by measuring the width of the wound and expressed as a percentage of each control for the mean wound closure area.

Statistical Analysis
All assays were performed using least three independent experiments. Results are expressed as the mean ± standard deviation (SD). Student's t-test was used to evaluate the data with the following significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001.

Conclusions
Four new steroidal conjugates, esters of polyhydroxysteroids with long-chain fatty acids (1-4), were isolated from the Far Eastern starfish C. patagonicus. Unusual compounds 1-4 contain the same 5α-cholestane-3β,6β,15α,16β,26-pentahydroxysteroidal moiety and differ from each other in fatty acid residues: 5 Z,11 Z-octadecadienoic (1), 11 Z-octadecenoic (2), 5 Z,11 Z-eicosadienoic (3), and 7 Z-eicosenoic (4) acid units. It should be noted that the isolated conjugates 1-4 have a shared steroidal part and differ in the composition of fatty acid residues. The question arises about the biological role of the extracted compounds. Previously we have been found that starfish polyhydroxylated steroids were presented mainly in digestive organs of starfishes during the whole year, and their maximum concentration coincided with periods of active nutrition of these animals [20,21]. Recently, we confirmed the digestive function of polyhydroxysteroids, when studying the distribution of polar steroids in various organs of the starfish Lethasterias fusca using the nLC/CSI-QTOF-MS method, and the highest level of polar steroids was found in the stomach and the pyloric caeca [22] Thus, it can be assumed that polyhydroxysteroids can bind food fatty acids and participate in their transport to peripheral tissues, like cholesterol of vertebrates and humans. This assumption is partially confirmed by the heterogeneous composition of fatty acids in compounds 1-4, since saturated, mono-and di-unsaturated C16, C18, and C20 fatty acids were found together with the main components. The isolation of conjugates of polyhydroxysteroids and fatty acids from the Far Eastern starfish C. patagonicus is a very interesting finding; to the best of our knowledge, hypotheses about the possible transport role of polyhydroxysteroids have not been put forward. At the same time, this assumption requires confirmation by experimental data.
We have expanded the data on the biological activity of these unique compounds-conjugates of polyhydroxysteroids and fatty acids. It was shown that tested compounds 1-4 possessed cytostatic activity against normal JB6 Cl41 cells and cancer MDA-MB-231 and HCT 116 cell lines. The compounds 1-4 at low concentration of 20 µM were able to suppress the colony formation in MDA-MB-231 and HCT 116 cells and almost completely prevent the migration of human breast and colorectal cancer cells. It should be noted that compound 2 was the most active in all experiments performed. This is likely due to the presence of an 11 Z-octadecenoic acid residue in its structure. Unfortunately, the lack of selectivity of the investigated compounds against normal and cancer cells was determined, which can be limit their possible practical use. However, the identification of these novel compounds and an initial characterization of their biological activity, performed for the first time, might be helpful to other researchers working on the development of conjugates of polyhydroxysteroids and fatty acids.  Figures S3, S23, S32, and S41), 1 H-NMR ( Figures S4-S8, S24, S33, and S42), 13 C-NMR ( Figures S9-S12, S25, S34, and S43), COSY (Figures S13, S14, S26, S35, and S44), HSQC (Figures S15, S16, S27, S36, and S45), HMBC (Figures S17, S18, S28, S37, and S46), and ROESY ( Figures S19, S20, S29, S38, and S47) spectra of compounds 1, 2, 3, and 4, respectively.
Author Contributions: T.V.M., the isolation and structure elucidation of metabolites, and manuscript preparation; A.A.K. and N.V.I., the analysis of the compounds and manuscript editing; V.M.Z. and I.P.K., the isolation and structure elucidation of metabolites; A.I.K., the acquisition and interpretation of NMR spectra; V.I.S., the analysis and identification of fatty acids; O.S.M., the determination of the metabolites' effects on the viability, proliferation, colony formation, and migration of tested cells; R.S.P., the acquisition and interpretation of mass spectra. All authors have read and agreed to the published version of the manuscript.