Disulfated Ophiuroid Type Steroids from the Far Eastern Starfish Pteraster marsippus and Their Cytotoxic Activity on the Models of 2D and 3D Cultures

New steroidal 3β,21-disulfates (2–4), steroidal 3β,22-disulfate (5), and the previously known related steroidal 3β,21-disulfate (1) were isolated from the ethanolic extract of the Far Eastern starfish Pteraster marsippus, collected off Urup Island in the Sea of Okhotsk. The structures of these compounds were determined by intensive NMR and HRESIMS techniques as well as by chemical transformations. Steroids 2 and 3 have an oxo-group in the tetracyclic nucleus at position C-7 and differ from each other by the presence of the 5(6)-double bond. The Δ24-22-sulfoxycholestane side chain of the steroid 5 has not been found previously in the starfish or ophiuroid steroids. The cytotoxic activities of 1, 4, 5, and the mixture of 2 and 3 were determined on the models of 2D and 3D cultures of human epithelial kidney cells (HEK293), melanoma cells (SK-MEL-28), small intestine carcinoma cells (HuTu80), and breast carcinoma cells (ZR-75-1). The mixture of 2 and 3 revealed a significant inhibitory effect on the cell viability of human breast carcinoma ZR-75-1 cells, but other tested compounds were less effective.


Introduction
Marine sulfated steroids are often found in representatives of two classes of marine echinoderms, namely ophiuroids and particularly starfish (the phylum Echinodermata), and in sponges (the phylum Porifera) [1,2]. These compounds have been reported to exhibit various biological activities, including anticancer, antimicrobial, cardiovascular, and antifouling properties [3]. Steroidal monosulfates, encountered in different species of starfish, are represented by sterol sulfates and polyhydroxysteroids, containing from four to nine hydroxy groups and a sulfate group at different positions of the tetracyclic core and side chains. In that position, the polyhydroxysteroids were found in both free and glycosylated forms with one to three monosaccharide units and also were found in sulfated form. Moreover, the most common steroidal oligoglycosides of starfish are known as classical asterosaponins and contain an oligosaccharide chain, attached to C-6 and including five or six monosaccharide residues and a sulfate group at C-3 [4][5][6][7][8][9][10]. On the other hand, characteristic secondary metabolites of ophiuroids are mainly steroidal disulfates that differ from other sulfated compounds of echinoderms in some structural peculiarities, namely in the presence of sulfoxy groups at 3α and 21 positions in 5β-, or ∆ 5 -, and very rarely 5α-cholestane cores. It is of interest, that similar steroidal disulfates, containing sulfoxy groups at 3β (or 3α) and 21 positions in 5α-, or ∆ 5 -cholestane nuclei were found in some species of the Pterasteridae family belonging to the Asteroidea class. From six species of starfish belonging to the Pterasteridae family, in particular Euretaster insignis [11], Pteraster sp. and Pteraster tessellatus [12,13],  13 C-NMR spectroscopic data attributable to the tetracyclic nucleus of 1 revealed the proton and carbon chemical shifts of two angular methyl groups CH 3 -18 (δ H 0.75 s, δ C 12.5) and CH 3 -19 (δ H 1.03 s, δ C 19.7), an oxygenated methine CH-3 (δ H 4.13 m, δ C 79.9), and the 5(6) double bond (δ H 5.38 m; δ C 141.7, 123.2). The proton and carbon resonances of CH 3 -18, CH 3 -19, CH-3, C-5, CH-6 and the broad multiplet of H-3 (∆W = 39.3 Hz) indicated a ∆ 5 -3β-sulfoxy steroidal nucleus in 1 [11].
An attempt to separate compounds 2 and 3 using repeated reversed-phase HPLC were failed. However, structures of 2 and 3 were established in the mixture by the thorough analysis of the 1D and 2D NMR spectra, including 1 H-and 13 C-NMR, 1D TOCSY, COSY, HSQC, HMBC, and ROESY experiments ( Figures S8-S13). The molecular formula of steroid 2 was determined to be C 28 13 C-NMR, mass spectra of 1, and the mixture of 2 and 3 clearly indicated that these compounds have the same ∆ 24(28) -21-sulfoxy-24-methylcholestane side chain, and steroids 2 and 3 differ from 1 by the existence of an additional oxo-group in tetracyclic pattern (Tables 1 and 2). Moreover, it followed from the chemical shifts and intensities of the proton signals in the 1 H-NMR spectrum that 2 unlike 3 has a supplementary double bond in the steroidal nucleus, which agreed with the molecular mass difference of 2 amu between 2 and 3 in the mass-spectra.
Previously reported feeding experiments labeled with deuterium precursors have shown that polyhydroxysteroids and related steroidal glycosides of starfish are biosynthesized from dietary cholesterol and cholesterol sulfate [28]. Obviously, the precursors of the biosynthesis of steroidal disulfates 1-5 are presumably cholesterol or cholesterol sulfate. The biosynthesis of these compounds takes place with the participation of enzymatic systems such as oxygenases, NAD and NADP-dependent dehydrogenases, SAMmethyltransferase, etc. The following hypothetical pathways for the biosynthesis of compounds 2-4 are proposed. Compound 1 undergoes changes only in the steroidal side chain in comparison with cholesterol sulfate by oxidation at CH 3 -21 followed by sulfation and introduction of a methylene group by SAM-methyltransferase at C-24 with loss of a proton. The introduction of a hydroxyl group at C-7 of ring B of the steroidal nucleus of 1 gives an intermediate. Oxidation of the hydroxyl group at C-7 in the intermediate leads to the formation of steroid disulfate 2, and reduction of the 5(6)-double bond leads to the formation of 4. The end product, obviously, is the steroid disulfate 3, which can be obtained from both compounds 2 and 4 (by oxidation or reduction). In compound 5, as well as in 1, there are no changes in the steroid nucleus, and only the side chain is modified by oxidation with the following sulfation at the C-22 position.

In Vitro Anticancer Activity of Compounds 1-5
Currently, the main cellular model of cell biology is a two-dimensional (2D) monolayer. However, the cell growth in a monolayer does not reflect the true picture of tumor growth in a living organism by many parameters, where interactions not only between the cells of the tumor but also with the surrounding extracellular matrix are of great importance in its progression. The three-dimensional (3D cell culture) model is represented by spheroids, and proved to be the most effective system that is as close as possible in properties and organization to a natural tumor, which is used for screening the potential anticancer drugs [29]. So, the cytotoxic activity of 1, 4, and 5 and the mixture of 2 and 3 was determined on the models of 2D and 3D cultures of human epithelial kidney cells (HEK293), melanoma cells (SK-MEL-28), small intestine carcinoma cells (HuTu80), and breast carcinoma cells (ZR-75-1) using the MTS method.
Mar. Drugs 2022, 20, x 11 of 18 was determined on the models of 2D and 3D cultures of human epithelial kidney cells (HEK293), melanoma cells (SK-MEL-28), small intestine carcinoma cells (HuTu80), and breast carcinoma cells (ZR-75-1) using the MTS method. The investigated compounds 1-5 were determined to possess moderate cytotoxic activity against normal and cancer cells with the greater impact of the mixture of 2 and 3. It was found that this mixture inhibited the cell viability of 2D HEK293, SK-MEL-28, HuTu80, and ZR-75-1 by 28, 33, 34, and 55%, respectively, at a concentration of 100 µM after 24 h of treatment ( Figure 4A-D). The concentration of the mixture of 2 and 3, which caused inhibition of 50% cell viability (IC50) was established against more sensitive breast carcinoma cells ZR-75-1 as 90.4 µM ( Figure 4D). The IC50 of doxorubicin (Doxo), used as a positive control, was 35.7, 40.0, 11.2, and 19.2 µM against 2D HEK293, SK-MEL-28, Hu-Tu80, and ZR-75-1, respectively ( Figure 4A-D). The investigated compounds insignificantly affect the size of the spheroids but inhibit their viability to varying degrees ( Figure 5A-C). It was determined that the mixture of 2 and 3 inhibited viability of SK-MEL-28, HuTu80, and ZR-75-1 spheroids by 16, 36, and 51%, respectively, at 100 µM after 24 h of treatment. As in the case of 2D culture cells, ZR-75-1 spheroids were the most sensitive to the cytotoxic action of the mixture of 2 and 3. IC50 of Doxo was 30.9 µM and 21.9 µM against HuTu80 and ZR-75-1, respectively. The investigated compounds insignificantly affect the size of the spheroids but inhibit their viability to varying degrees ( Figure 5A-C). It was determined that the mixture of 2 and 3 inhibited viability of SK-MEL-28, HuTu80, and ZR-75-1 spheroids by 16, 36, and 51%, respectively, at 100 µM after 24 h of treatment. As in the case of 2D culture cells, ZR-75-1 spheroids were the most sensitive to the cytotoxic action of the mixture of 2 and 3. IC 50 of Doxo was 30.9 µM and 21.9 µM against HuTu80 and ZR-75-1, respectively.
It should be noted that 3D cell cultures were more resistant to the action of compounds than 2D cultures, which can be explained by dynamic cellular interactions between neighboring cells in spheroids. Moreover, the increased resistance of 3D spheroids may be associated with limited diffusion of the tested substances into the spheroid and hypoxia of cells within the spheroid, which leads to the activation of genes involved in cell survival and the formation of drug resistance [30].
In summary, the results of the present study described the significant inhibiting effect of the mixture of compounds 2 and 3 on the cell viability of human breast carcinoma cells ZR-75-1 in 2D and 3D cell culture models and may contribute to the development of effective chemotherapeutic methods for cancer treatment. It should be noted that 3D cell cultures were more resistant to the action of compounds than 2D cultures, which can be explained by dynamic cellular interactions between neighboring cells in spheroids. Moreover, the increased resistance of 3D spheroids may be associated with limited diffusion of the tested substances into the spheroid and hypoxia of cells within the spheroid, which leads to the activation of genes involved in cell survival and the formation of drug resistance [30].
In summary, the results of the present study described the significant inhibiting effect of the mixture of compounds 2 and 3 on the cell viability of human breast carcinoma cells ZR-75-1 in 2D and 3D cell culture models and may contribute to the development of effective chemotherapeutic methods for cancer treatment.

Solvolysis of the Mixture of 2 and 3
A solution of the mixture of 2 and 3 (5.0 mg) in 2 mL of dioxane/pyridine (1:1) was heated at 100 • C for 4 h. The reaction mixture was evaporated under reduced pressure and separated by HPLC on a YMC-Pack Pro C18 column with 80% aq. MeOH (0.7 mL/min) as an eluent system to give pure desulfated derivatives 2a (0.5 mg, t R 40.6 min) and 3a (0.4 mg, t R 39.6 min).
(20R)-7-Oxo-24-methyl-5α-cholest-24 (28) Table 3). in a humidified 5% CO 2 incubator. The culture medium was supplemented with 10% of fetal bovine albumin (FBS), 100 mg/mL streptomycin, and 100 U/mL penicillin. At 90% confluence, cells were rinsed with PBS, detached from the tissue culture flask by 0.25% trypsin/0.5 mM EDTA, and 10-20% of the harvested cells were transferred to a new flask containing fresh complete appropriate medium. The passage number was carefully controlled and the mycoplasma contamination was monitored on a regular basis.

Preparation of Compounds for the Determination of Cytotoxic Activity
Compounds 1, 4, and 5 and the mixture of 2 and 3 were dissolved in sterile dimethyl sulfoxide (DMSO) to prepare stock concentrations of 20 mM. Cells were treated with serially diluted 1-5 (10, 50, 100 µM) (culture medium used as diluent) (final concentration of DMSO was less than 0.5%).
The vehicle control is the cells treated with the equivalent volume of DMSO (final concentration was less than 0.5%) for all of the presented experiments.

Formation of 3D Spheroids by Liquid Overlay Technique (LOT)
SK-ME-28, HuTu80, and ZR-75-1 spheroids were formed by the liquid overlay technique (LOT) method with slight modifications. Briefly, to create non-adherent surfaces for the efficient spheroids' formation, 50 µL of preheated (60 • C) agarose (1.5%) was overlaid the bottom of 96-well plates and left to solidify for 1 h at room temperature under sterile conditions. SK-MEL-28 cells (5.0 × 10 3 ), HuTu80 (3.0 × 10 3 ), and ZR-75-1 (3.0 × 10 3 ) were inoculated in an agarose layer and cultured in 200 µL of a complete appropriate culture medium for 96 h at 37 • C in a 5% CO 2 incubator. An image of each spheroid was made with a ZOE™ Fluorescent Cell Imager (Bio Rad, Hercules, CA, USA). ImageJ software bundled with 64-bit Java 1.8.0_112 (NIH, Bethesda, MD, USA) was used to measure the spheroid integrity, diameter, and volume. . The concentration at which the compounds exert half of its maximal inhibitory effect on cell viability (IC 50 ) was calculated by the AAT-Bioquest ® online calculator [31].

Statistical Analysis
All of the assays were performed in at least three independent experiments. Results are expressed as the mean ± standard deviation (SD). The 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
Three new 3β,21-disulfated steroids and one new 3β,22-disulfated steroid, along with a previously known related compound, were isolated from the Far Eastern starfish P. marsippus, and their chemical structures were established. Two steroids have an oxogroup at position C-7 in steroid nucleus; moreover, one of them additionally includes the conjugated 5,6-double bond. The ∆ 24 -22-sulfoxycholestane side chain, indicated in another new steroid, has not been earlier found in starfish and ophiuroid steroidal compounds.
Thus, in one more species of starfish, P. marsippus, belonging to the Pterasteridae family, like the previously studied six species of starfish of the same family, disulfated steroids of «the ophiuroid type» were found. It should be noted that the polyhydroxylated compounds and asterosaponins common in starfish were absent in the P. marsippus as well as in the previously studied species of this family. This fact once again confirms the assumption about a closer phylogenetic relationship between Asteroidea and Ophiuroidea classes compared to other classes of Echinodermata. The mixture of two steroids, having an oxogroup at position C-7 in steroid nucleus, was found to possess the highest cytotoxic activity against 2D and 3D human breast carcinoma cells ZR-75-1 among other investigated by us compounds and can be a candidate for further examination of the molecular mechanism of its anticancer action.