Polar Steroids Research Articles

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.

Polyhydroxy steroids isolated from starfish (Asterina pectinifera) and their embryotoxicity

Many marine organisms possess an essential capacity to produce secondary metabolites that exhibit toxic characteristics. A new polyhydroxy steroid, 24-methyl-5α-cholestane-24(28)-ene-3β, 4β, 6α, 7α, 8, 15β, 16β, 26-octol-6-O-sodium sulphate (1) was isolated from starfish (Asterina pectinifera), along with five polar steroid compounds (2–6) that were previously identified. NMR (1H and 13C NMR, 1H-1H COSY, HSQC, HMBC, and NOESY) and HR-ESI-MS were employed for structure elucidations. The embryotoxicity and teratogenicity of the isolated compounds were assessed using embryos of marine medaka (Oryzias melastigma). Compound 5 exhibited moderate embryotoxicity (96h-LC50: 65 μM).

Rare Ophiuroid-Type Steroid 3β,21-, 3β,22-, and 3α,22-Disulfates from the Slime Sea Star Pteraster marsippus and Their Colony-Inhibiting Effects against Human Breast Cancer Cells.

Two new steroid 3β,21-disulfates (1, 2) and two new steroid 3β,22- and 3α,22-disulfates (3, 4), along with the previously known monoamine alkaloid tryptamine (5) were found in the ethanolic extract of the Far Eastern slime sea star Pteraster marsippus. Their structures were determined on the basis of detailed analysis of one-dimensional and two-dimensional NMR, HRESIMS, and HRESIMS/MS data. Compounds 1 and 2 have a Δ22-21-sulfoxy-24-norcholestane side chain. Compounds 3 and 4 contain a Δ24(28)-22-sulfoxy-24-methylcholestane side chain, which was first discovered in the polar steroids of starfish and brittle stars. The influence of substances 1-4 on cell viability, colony formation, and growth of human breast cancer T-47D, MCF-7, and MDA-MB-231 cells was investigated. It was shown that compounds 1 and 2 possess significant colony-inhibiting activity against T-47D cells, while compounds 3 and 4 were more effective against MDA-MB-231 cells.

Evaluation of control of additive concentration in gradient analysis of supercritical fluid chromatography-coupled to tandem mass spectrometry

Mobile phase additives are used to improve retention behavior in chromatography. In supercritical fluid chromatography (SFC), for which supercritical fluid carbon dioxide (SF-CO2) is used as the main mobile phase, additives can only be added into the modifier. For that reason, when gradient analysis is performed by changing the modifier ratio to SF-CO2, the additive concentration in the mobile phase increases in parallel with the modifier ratio. In a preliminary study performed using the conventional SFC system, ammonium acetate was necessary to improve the peak shape of a polar steroid, dehydroepiandrosterone sulfate (DHEA-S), while the peak intensity of a non-polar steroid, progesterone, decreased by 78% compared to that in the absence of the additive in mobile phase when gradient elution was performed. Since ammonium acetate had both favorable and unfavorable effects on sensitive and simultaneous analysis of these two steroid compounds, a compromise between these effects had to be sought. A three-pump configuration of SFC was developed by adding a pump unit to SFC instrument, which enabled control of the additive concentration independently of the modifier ratio, for the purpose of investigating the additive effect in detail using both steroids as model compounds. The putative cause of the decrease in peak intensity of progesterone was excessively elevated additive concentration in gradient analysis. When the additive concentration in the mobile phase was controlled to ensure that it did not increase during gradient analysis, the peak intensities of progesterone, cortisol, corticosterone, and testosterone were 55%, 40%, 25%, and 17% higher than when the additive concentration was not controlled, respectively. On the other hand, the peak intensity of DHEA-S was almost identical between the conditions, with an increase of 2% with three-pump instrument. The three-pump configuration showed the potential to solve problems relating to the use of modifier additives by keeping their concentration constant in gradient SFC analysis.

Sources of sedimentary organic matter in Arctic fjords: Evidence from lipid molecular markers

The identification of the origin of organic matter in Arctic fjord systems is especially challenging due to complex and highly variable environmental settings, which are impacted by ongoing rapid climate changes. In this study we use a multiproxy approach based on various lipid biomarkers to investigate the provenance of organic matter deposited in surface sediments in the four high-Arctic fjords located in the western part of Spitsbergen (Hornsund, Adventfjorden, Kongsfjorden, and Raudfjorden). This work focuses on the analyses of several classes of lipid biomarkers such as n-alkanes, saturated cyclic hydrocarbons (hopanes, steranes), highly branched isoprenoid (IP25 – ice proxy), alkenones and polar steroids, including sterols and stanols. The findings of this research indicate the occurrence of sedimentary organic matter derived from multiple marine and land-based sources in the studied fjords. The analysis of biomarkers reveals pronounced differences between fjords located in southern and northern parts of western Spitsbergen with regard to contributions of sedimentary organic matter derived from biogenic sources (phytoplankton, animal-derived material as well as terrestrial vascular plants), relative abundances of specific phytoplankton genera (diatoms vs. dinoflagellates), and intensity of bio-advection (Emiliania huxleyi) related to inflows of warm Atlantic Water. The uncommon n-alkane profiles, especially in the short-chain range, reported for investigated sediments are likely to reflect probable contributions related to ancient organic matter and/or microbial sources/activities. To our knowledge, this is the first such comprehensive research on the origin of organic matter deposited in surface sediments from the western Spitsbergen fjords, based on lipid molecular biomarkers.

Application of MS-Based Metabolomic Approaches in Analysis of Starfish and Sea Cucumber Bioactive Compounds.

Today, marine natural products are considered one of the main sources of compounds for drug development. Starfish and sea cucumbers are potential sources of natural products of pharmaceutical interest. Among their metabolites, polar steroids, triterpene glycosides, and polar lipids have attracted a great deal of attention; however, studying these compounds by conventional methods is challenging. The application of modern MS-based approaches can help to obtain valuable information about such compounds. This review provides an up-to-date overview of MS-based applications for starfish and sea cucumber bioactive compounds analysis. While describing most characteristic features of MS-based approaches in the context of starfish and sea cucumber metabolites, including sample preparation and MS analysis steps, the present paper mainly focuses on the application of MS-based metabolic profiling of polar steroid compounds, triterpene glycosides, and lipids. The application of MS in metabolomics studies is also outlined.

Biogenic and anthropogenic sources of sedimentary organic matter in marine coastal areas: A multi-proxy approach based on bulk and molecular markers

The investigation of organic matter sources is an especially challenging issue for marine coastal areas influenced by both natural and anthropogenic inputs. This study focuses on the provenance of sedimentary organic matter deposited in the Gulf of Gdańsk (southern Baltic) and Oslofjord/Drammensfjord (southern Norway) based on the analysis of multiple specific proxies in recent sediments (0–5 cm layer) sampled at 12 sites in 2014. Bulk markers (total organic carbon/total nitrogen [TOC/TN] and stable organic carbon isotopes [δ13CTOC]) and various molecular geochemical markers of organic matter origin (n-alkanes, branched alkanes, unresolved complex mixture [UCM], polycyclic aromatic hydrocarbons [PAHs], hopanes, steranes, and polar steroids including sterols and stanols) were used to comprehensively assess the origin of sedimentary organic matter in the studied basins. The results of this study indicate that the combined use of TOC/TN and δ13CTOC may lead to ambiguous outcomes, and anthropogenic input may affect the sedimentary TOC/TN ratio. The analysis of molecular proxies allowed the identification of locations most impacted by anthropogenic organic matter derived from petrogenic sources and/or sewage effluents and the classification of the studied sites based on the contribution of marine/terrestrial biogenic matter. This study demonstrates that key issues, such as the forms of sedimentary chemical markers, post-depositional changes, and specific diagenetic products, although neglected by some researchers, must be considered for the reliable assessment of organic matter sources. To the best of our knowledge, this is the first such extensive study on surface sediments from the Gulf of Gdańsk and Oslofjord/Drammensfjord in terms of the use of both bulk and molecular markers to investigate organic matter sources.

PSX-A-27 Late-Breaking: The sulfatase mediated regeneration of androstenone from androstenone sulfate in boar fat

Abstract Boar taint is an off-odour or off-flavour that develops in heated pork products from entire male pigs, which is caused by the accumulation of androstenone, a sex pheromone, in the fat. However, we have previously demonstrated that a significant amount of androstenone undergoes sulfoconjugation upon synthesis in the Leydig cells and circulates in the plasma primarily as a polar steroid sulfate. Therefore, the purpose of this study was to determine if androstenone sulfate can be deconjugated within the adipose tissue by the sulfatase enzyme to return free androstenone and indirectly contribute to the development of boar taint. Backfat was obtained from 6-month-old terminal cross [Duroc x (Landrace x Yorkshire)] boars that had high (n=4) or low (n=4) sulfatase expression as determined by RT-PCR. Sulfatase activity in the fat was measured by quantifying the conversion of androstenone sulfate to free androstenone. Backfat was homogenized and the supernatant was incubated with [3H]-androstenone sulfate for 24-hours. Androstenone was extracted from the incubation using ether and steroid conversion was quantified using high-performance liquid chromatography (HPLC). Additionally, fat androstenone concentrations were quantified using an established HPLC procedure. Statistical analysis was conducted using a Student’s t-test. There was a significant difference (p=0.04) in the expression of sulfatase between the high (2.99 ± 0.67) and low (1.21 ± 0.19) sulfatase boars and the percentage of androstenone sulfate that was converted to free androstenone was proportional to the expression of sulfatase. Interestingly, the expression of sulfatase was positively related to the concentration of androstenone in the fat in boars with high sulfatase expression; however, this relation was not as strong in animals with low sulfatase expression. These preliminary results suggest that the development of boar taint may occur indirectly through the deconjugation of androstenone sulfate in boars with high expression of sulfatase in the fat.

Polar steroid compounds from the Arctic starfish Asterias microdiscus and their cytotoxic properties against normal and tumor cells in vitro

Two new natural compounds, sulfated polyhydroxysteroid, microdiscusol G (1), and polyhydroxysteroid bioside, microdiscusoside A (2), along with eight previously known polar steroid substances 3–10, were isolated from the Arctic starfish Asterias microdiscus. The structures of 1 and 2 have been elucidated by extensive 1D and 2D NMR spectroscopy and HRESIMS techniques. The 28-sulfooxy-24-methylcholestane side chain in 1 has been found among starfish steroid metabolites for the first time. Steroid saponins 9 and 10 exhibited cytotoxic effects against normal cells JB6 Cl41 and cancer cells HT-29, MDA-MB-231, THP-1, and Raji and effectively suppressed cell proliferation and colony formation of cancer cells HT-29 and MDA-MB-231 in non-toxic concentrations. Compounds 5, 7, and 8 were inactive or less active in the same experiments.

Marine Invertebrate Extracts Induce Colon Cancer Cell Death via ROS-Mediated DNA Oxidative Damage and Mitochondrial Impairment.

Marine compounds are a potential source of new anticancer drugs. In this study, the antiproliferative effects of 20 invertebrate marine extracts on three colon cancer cell models (HGUE-C-1, HT-29, and SW-480) were evaluated. Extracts from two nudibranchs (Phyllidia varicosa, NA and Dolabella auricularia, NB), a holothurian (Pseudocol ochirus violaceus, PS), and a soft coral (Carotalcyon sp., CR) were selected due to their potent cytotoxic capacities. The four marine extracts exhibited strong antiproliferative effects and induced cell cycle arrest at the G2/M transition, which evolved into early apoptosis in the case of the CR, NA, and NB extracts and necrotic cell death in the case of the PS extract. All the extracts induced, to some extent, intracellular ROS accumulation, mitochondrial depolarization, caspase activation, and DNA damage. The compositions of the four extracts were fully characterized via HPLC-ESI-TOF-MS analysis, which identified up to 98 compounds. We propose that, among the most abundant compounds identified in each extract, diterpenes, steroids, and sesqui- and seterterpenes (CR); cembranolides (PS); diterpenes, polyketides, and indole terpenes (NA); and porphyrin, drimenyl cyclohexanone, and polar steroids (NB) might be candidates for the observed activity. We postulate that reactive oxygen species (ROS) accumulation is responsible for the subsequent DNA damage, mitochondrial depolarization, and cell cycle arrest, ultimately inducing cell death by either apoptosis or necrosis.

The Distribution of Asterosaponins, Polyhydroxysteroids and Related Glycosides in Different Body Components of the Far Eastern Starfish Lethasterias fusca.

Glycoconjugated and other polar steroids of starfish have unique chemical structures and show a broad spectrum of biological activities. However, their biological functions remain not well established. Possible biological roles of these metabolites might be indicated by the studies on their distribution in the organism–producer. In order to investigate the localization of polar steroids in body components of the Far Eastern starfish Lethasterias fusca, chemical constituents of body walls, gonads, stomach, pyloric caeca, and coelomic fluid were studied by nanoflow liquid chromatography/mass spectrometry with captive spray ionization (nLC/CSI–QTOF–MS). It has been shown that the levels of polar steroids in the studied body components are qualitatively and quantitatively different. Generally, the obtained data confirmed earlier made assumptions about the digestive function of polyhydroxysteroids and protective role of asterosaponins. The highest level of polar steroids was found in the stomach. Asterosaponins were found in all body components, the main portion of free polyhydroxysteroids and related glycosides were located in the pyloric caeca. In addition, a great inter-individual variability was found in the content of most polar steroids, which may be associated with the peculiarities in their individual physiologic status.

Effects of Polar Steroids from the Starfish Patiria (=Asterina) pectinifera in Combination with X-Ray Radiation on Colony Formation and Apoptosis Induction of Human Colorectal Carcinoma Cells.

Despite significant advances in the understanding, prevention, and treatment of cancer, the disease continues to affect millions of people worldwide. Chemoradiation therapy is a rational approach that has already proven beneficial for several malignancies. However, the existence of toxicity to normal tissue is a serious limitation of this treatment modality. The aim of the present study is to investigate the ability of polar steroids from starfish Patiria (=Asterina) pectinifera to enhance the efficacy of radiation therapy in colorectal carcinoma cells. The cytotoxic activity of polar steroids and X-ray radiation against DLD-1, HCT 116, and HT-29 cells was determined by an MTS assay. The effect of compounds, X-ray, and their combination on colony formation was studied using the soft agar method. The molecular mechanism of the radiosensitizing activity of asterosaponin P1 was elucidated by western blotting and the DNA comet assay. Polar steroids inhibited colony formation in the tested cells, and to a greater extent in HT-29 cells. Asterosaponin P1 enhanced the efficacy of radiation and, as a result, reduced the number and size of the colonies of colorectal cancer cells. The radiosensitizing activity of asterosaponin P1 was realized by apoptosis induction through the regulation of anti- and pro-apoptotic protein expression followed by caspase activation and DNA degradation.

Structural Characterization of Polar Steroid Compounds of the Far Eastern Starfish Lethasterias fusca by Nanoflow Liquid Chromatography Coupled to Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Nanoflow liquid chromatography coupled with a captive spray ionization time-of-flight tandem mass spectrometer (nLC/CSI-QTOF-MS/MS) was used in the structural determination of polar steroid compounds of starfish Lethasterias fusca. A total of 207 compounds including 106 asterosaponins, 81 glycosides of polyhydroxysteroids, and 14 polyhydroxylated steroids were detected and characterized by MS and MS/MS. Twenty compounds among them were unambiguously identified using authentic standard compounds, isolated earlier from this and other starfish species. The other compounds were tentatively characterized by accurate mass measurement and comparing retention times and characteristic MS/MS fragmentation patterns with reference standards. Moreover, fragmentation behaviors of a series of pure standards of starfish polar steroids and polyhydroxysteroid compounds detected in L. fusca have been extensively investigated and characteristic fragmentation pathways were described and used for the characterization of unknown compounds.

Rationalizing Steroid Interactions with Lipid Membranes: Conformations, Partitioning, and Kinetics.

Steroids have numerous physiological functions associated with cellular signaling or modulation of the lipid membrane structure and dynamics, and as such, they have found broad pharmacological applications. Steroid–membrane interactions are relevant to multiple steps of steroid biosynthesis and action, as steroids are known to interact with neurotransmitter or membrane steroid receptors, and steroids must cross lipid membranes to exert their physiological functions. Therefore, rationalizing steroid function requires understanding of steroid–membrane interactions. We combined molecular dynamics simulations and isothermal titration calorimetry to characterize the conformations and the energetics of partitioning, in addition to the kinetics of flip–flop transitions and membrane exit, of 26 representative steroid compounds in a model lipid membrane. The steroid classes covered in this study include birth control and anabolic drugs, sex and corticosteroid hormones, neuroactive steroids, as well as steroids modulating the lipid membrane structure. We found that the conformational ensembles adopted by different steroids vary greatly, as quantified by their distributions of tilt angles and insertion depths into the membrane, ranging from well-defined steroid conformations with orientations either parallel or normal to the membrane, to wide conformational distributions. Surprisingly, despite their chemical diversity, the membrane/water partition coefficient is similar among most steroids, except for structural steroids such as cholesterol, leading to similar rates for exiting the membrane. By contrast, the rates of steroid flip–flop vary by at least 9 orders of magnitude, revealing that flip–flop is the rate-limiting step during cellular uptake of polar steroids. This study lays the ground for a quantitative understanding of steroid–membrane interactions, and it will hence be of use for studies of steroid biosynthesis and function as well as for the development and usage of steroids in a pharmacological context.

Granulatosides D, E and other polar steroid compounds from the starfish Choriaster granulatus. Their immunomodulatory activity and cytotoxicity

Two new steroid glycosides, granulatosides D (1) and E (2), belonging to the group of bi- and monoglycosides of polyhydroxysteroids, respectively, were isolated from the ethanolic extract of the starfish Choriaster granulatus along with thirteen previously known glycosides of polyhydroxysteroids (3–15) and one steroid heptaol (16). The structures of the new compounds were elucidated by extensive NMR and ESIMS techniques. Cytotoxic and immunomodulatory activities of compounds 1, 3–8, and 10–16 using murine splenocytes and peritoneal macrophages were studied. At a dose of 0.1 μM new glycoside 1 showed immunomodulatory properties, increasing the intracellular ROS (reactive oxygen species) level in peritoneal murine macrophages by 20% and decreasing intracellular ROS level by 21% in pre-treated with endotoxic lipopolysaccharide from E. coli (LPS) peritoneal macrophages.

Micellar solubilization of poorly water-soluble drugs: effect of surfactant and solubilizate molecular structure

Objective: This study aims to clarify the role of surfactant and drug molecular structures on drug solubility in micellar surfactant solutions.Significance: (1) Rationale for surfactant selection is provided; (2) the large data set can be used for validation of the drug solubility parameters used in oral absorption models.Methods: Equilibrium solubility of two hydrophobic drugs and one model hydrophobic steroid in micellar solutions of 19 surfactants was measured by HPLC. The drug solubilization locus in the micelles was assessed by UV spectrometry.Results: Danazol is solubilized much more efficiently than fenofibrate by ionic surfactants due to ion–dipole interactions between the charged surfactant head groups and the polar steroid backbone. Drug solubilization increases linearly with the increase of hydrophobic chain length for all studied surfactant types. Addition of 1–3 ethylene oxide (EO) units in the head group of dodecyl sulfate surfactants reduces significantly the solubilization of both studied drugs and decreases linearly the solubilization locus polarity of fenofibrate. The locus of fenofibrate solubilization is in the hydrophobic core of nonionic surfactant micelles and in the palisade layer of ionic surfactant micelles.Conclusions: Highest drug solubility can be obtained by using surfactants molecules with long chain length coupled with hydrophilic head group that provides additional drug–surfactant interactions (i.e. ion–dipole) in the micelles.

A New Steroidal Glycoside Granulatoside C from the Starfish Choriaster granulatus, Unexpectedly Combining Structural Features of Polar Steroids from Several Different Marine Invertebrate Phyla

A new unique steroidal glycoside, granulatoside C (1), was isolated from the ethanol extract of the starfish Choriaster granulatus. The structure of 1 was elucidated by extensive NMR and ESI-MS techniques as (20 R)-16,21- O-di-(P-D-fucopyranosyl)-24-methyl-cholesta-5,24(28)-diene-3p,7a,16a,21-tetraol (1). This glycoside is unusual one, because combines some characteristic structural peculiarities of steroids from several different phyla of marine invertebrates, it has a range of structural features that not previously found in starfish polyhydroxysteroidal glycosides, such as Δ5-3β,7α,16α-trihydroxysteroidal nucleus, Δ24(28)-21-hydroxy-24-methyl-cholestane side chain and 16,21-diglycosylation by D-fucopyranosyl residues, but also characteristic of sponges, ophiuroids and soft corals, respectively.

Synthesis of Deuterium-Labeled Steroid 3,6-Diols

A facile synthesis of a set of deuterium-labeled steroid 3,6-diols with different steroid A/B ring fusion, unsaturations, and configurations of hydroxyl groups at C-3 and C-6 is described. Reduction and deuteration, based on deuterium-exchange of the obtained the cholest-4-ene-3,6-dione from cholesterol using sodium borodeuteride and deuterium water, were used. The obtained steroid diols are intended to be used as precursors in the studies on biosynthesis of some marine polar steroids.

Polar steroid derivatives from the Vietnamese starfish Astropecten polyacanthus

Five polar steroid derivatives, including one new glycosylated polyhydroxysteroid namely polyacanthoside A (1), were isolated from the water-soluble materials from the MeOH extract of the Vietnamese starfish Astropecten polyacanthus using various chromatographic separations. The structure elucidation was confirmed by spectroscopic experiments such as HR-ESI-MS, 1D and 2D NMR. Among the isolated compounds, (20R,24S)-3β,6α,8,15β,24-pentahydroxy-5α-cholestane (3) showed significant cytotoxic effect against five human cancer cell lines as HepG2, KB, LNCaP, MCF7 and SK-Mel2 with the IC50 values from 18.03 ± 2.63 to 21.59 ± 3.23 μM.

Characterizing the distribution of steroid sulfatase during embryonic development: when and where might metabolites of maternal steroids be reactivated?

All vertebrate embryos are exposed to maternally derived steroids during development. In placental vertebrates, metabolism of maternal steroids by the placenta modulates embryonic exposure, but how exposure is regulated in oviparous vertebrates is less clear. Recent work in oviparous vertebrates has demonstrated that steroids are not static molecules, as they can be converted to more polar steroid sulfates by sulfotransferase enzymes. Importantly, these steroid sulfates can be converted back to the parent compound by the enzyme steroid sulfatase (STS). We investigated when and where STS was present during embryonic development in the red-eared slider turtle, Trachemys scripta We report that STS is present during all stages of development and in all tissues we examined. We conclude that STS activity may be particularly important for regulating maternal steroid exposure in oviparous vertebrates.