Four New Sulfated Polar Steroids from the Far Eastern Starfish Leptasterias ochotensis: Structures and Activities

Three new sulfated steroid monoglycosides, leptaochotensosides A–C (1–3), and a new sulfated polyhydroxylated steroid (4) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The structures of compounds 1–4 were established by extensive nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESIMS) analyses and chemical transformations. Although the isolated compounds did not show any apparent cytotoxicity against melanoma RPMI-7951 and breast cancer T-47D cell lines, leptaochotensoside A (1) demonstrated inhibition of T-47D cell colony formation in a soft agar clonogenic assay at nontoxic doses. In addition, this compound decreased the epidermal growth factor (EGF)-induced colony formation of mouse epidermal JB6 Cl41 cells. The cancer preventive action of 1 is realized through regulation of mitogen-activated protein kinase (MAPK) signaling pathway.


Introduction
Starfish (Echinodermata, Asteroidea) are characterized by a diversity of polar steroids, including polyhydroxylated steroids, structurally related mono-, bi-and triosides, and steroid oligoglycosides (asterosaponins) with carbohydrate chains comprising five or six sugars [1][2][3][4]. Free polyhydroxysteroids and their glycosides are highly oxygenated steroid compounds, having three to nine hydroxyl groups, and often occur in sulfated forms. Starfish polar steroids have been reported to show a wide spectrum of biological activities, including hemolytic, cytotoxic, antiviral, antibacterial, antifouling, neuritogenic, and antifungal effects [1][2][3][4]. Recently, some data on the cancer preventive and anticancer activities of starfish polar steroids were obtained. Several asterosaponins and steroid mono-or biosides were found to have anticancer properties showing strong in vitro cytotoxicity against different tumor cells [5]. For example, effective cytostatics, asterosaponin 1 and novaeguinoside II from the starfish Culcita novaeguineae induced apoptosis of human glioblastoma U87MG cells through several signaling transduction pathways [6,7]. Moreover, asterosaponin 1 inhibited the proliferation of A549 human lung cancer cells through induction of endoplasmic reticulum stress-associated apoptosis [8]. Polyhydroxysteroid glycosides isolated from the starfish Anthenea chinensis exhibited significant activity against promotion of tubulin polymerization in vitro inhibiting the proliferation of glioblastoma cells [9]. Leviusculoside G, steroid biglycoside from the starfish Henricia leviuscila, demonstrated anticarcinogenic action by the induction of p53-dependent apoptosis and inhibition of activator protein 1 (AP-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and extracellular-signal-regulated protein kinases (ERKs) activities in human leukemia HL-60, THP-1, and mouse epidermal JB6 Cl41 cells [10]. Archasteroside B from the starfish Archaster typicus induced basal AP-1-and p53-, but not NF-κB-transcriptional activations in JB6 Cl41 cells [11]. Some asterosaponins and other steroid glycosides from the starfish Hippasteria kurilensis, Asteropsis carinifera and Lethasterias fusca exhibited a significant suppression of the human tumor HT-29, HCT-116, RPMI-7951, and T-47D cell colony formation in a soft agar clonogenic assay [12][13][14]. All these results indicate that further studies of the anticancer properties of polar steroids from starfish are necessary to be conducted.
Recently, we have established structures of six new asterosaponins, leptasteriosides A-F along with one new and one previously known asterogenins from the starfish Leptasterias ochotensis (order Forcipulatida, family Asteriidae) collected near Shantar Islands in the Sea of Okhotsk. Leptasteriosides A-C demonstrated a substantial suppression of colony formation of human melanoma RPMI-7951 and breast cancer T-47D cells [15].
Herein, we report the results of the structural elucidation of four new sulfated steroid compounds (1-4) from the fraction of sulfated polyxydroxysteroids and related glycosides from L. ochotensis. Moreover, we discuss the capabilities of 1-4 to inhibit colony formation of cancer RPMI-7951 and T-47D cells ex vivo, the suppressing influence of 1 on the epidermal growth factor (EGF)-induced colony formation of mouse epidermal JB6 Cl41 cells, and the molecular mechanism of cancer preventive effect of 1 implemented through regulation of mitogen-activated protein kinase (MAPK) signaling pathway.

Cytotoxic Activity of Leptaochotensosides A-C (1-3) and Sulfated Polyhydroxysteroid 4 ex Vivo
Ex vivo cytotoxicity tests [21] are necessary to define the concentration range for further and more detailed ex vivo experiments and to provide meaningful information on signal transduction pathway and molecular targets of compounds. In

The Effect of Leptaochotensosides A-C (1-3) and Sulfated Polyhydroxysteroid 4 on Colony Formation of Melanoma and Breast Cancer Cells
Next, we determined the effect of compounds 1-4 on colony formation of human melanoma RPMI-7951 and human breast cancer T-47D cells using soft agar method. Cells were treated with compounds 1-4 at concentration 50 µM and number of colonies was counted after culturing during 14 days. The results showed that compounds 2-4 did not inhibit colony formation of RPMI-7951 cells and have slight effect on colony formation of T-47D cells ( Figure 4A). The compound 1 also did not effect on colony formation of RPMI-7951 cells ( Figure 4A), but significantly reduced colony formation of T-47D cells (the percentage of inhibition of colony formation was 48%) compared with non-treated control ( Figure 4B).
Our data indicated that compound 1 at nontoxic doses inhibited the formation of breast cancer cell colonies ex vivo. were either treated or not treated with the compounds 1-4 in 1 mL of 0.3% Basal Medium Eagle (BME) agar containing 10% fetal bovine serum, 2 mM L-glutamine, and 25 µg/mL gentamicin. The cultures were maintained at 37 °C in an 5% CO2 incubator for 14 days and the cell colonies were scored using a microscope Motic AE 20 (Motic, Xiamen, China) and the Motic Image Plus computer program. All assays were performed in at 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.

The Leptaochotensoside A (1) Inhibits the EGF-induced Colony Formation of JB6 Cl41 Cells and Signal Transduction in JB6 Cl41 Cells
The carcinogenesis is multistage process, including initiation (cell transformation), promotion (colony formation), and progression (metastasis) [22]. One of the perspective approaches for cancer therapy is the search and development of nontoxic compounds, which are effective in preventing cancer initiation. The mouse epidermal JB6 Cl41 cells are known to respond irreversibly to tumor promoters such as epidermal growth factor (EGF) by induction of anchorage independent increase in a number of cell colonies in soft agar [23]. That is why this well-established culture system was used to identify the effect of leptaochotensoside A (1) on EGF-induced cell transformation.
To estimate the effect of 1, JB6 Cl41 cells were treated with EGF (10 ng/mL) in the absence or presence of 1 (50, 100, 200 µM) in a soft agar matrix as described in Experimental Section. Our results revealed that JB6 Cl41 cells treated with compound 1 at concentration 200 µM formed less colonies compared with control cells treated with EGF only. It decreased EGF-induced colony number on 44% of control ( Figure 5A). The inhibition of by the compound 1 was not due to its cytotoxicity because the effective concentration range for suppressing cell transformation did not affect cell viability of JB6 Cl41 cells ( Figure 5B). Since the anchorage-independent growth ability is an ex vivo indicator and a key characteristic of the transformed cell phenotype [24], these results suggest that 1 can reduce the malignant potential of JB6 Cl41 cells induced by EGF.
To elucidate cancer prevention molecular mechanism, we tested the effect of leptaochotensoside A (1) on activation of mitogen-activated protein kinases (MAPKs). MAPK pathways comprise three kinase modules, in which MAPK is activated upon phosphorylation by a mitogen-activated protein kinase kinase (MAPKK), which in turn is activated, when phosphorylated by MAPKKK [25]. The most thoroughly characterized subgroups of the MAP kinase family include ERKs, c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases, and p38 kinases [26]. MAPK pathways are evolutionarily conserved kinase modules that link extracellular signals to the machinery that controls fundamental cellular processes such as growth, proliferation, differentiation, migration and apoptosis [27]. Herein, we describe the effects of compound 1 on the phosphorylation of proto-oncogene serine/threonine-protein (c-Raf), mitogen-activated protein kinase 1/2 (MEK1/2), extracellular signal-regulated 1/2 (ERK1/2), and mitogen-and stress-activated protein (MSK-1) kinases in JB6 Cl41 cells. Compound 1 was shown to inhibit effectively the EGF-induced phosphorylation of ERK1/2 and MSK-1 kinases ( Figure 5C). These data suggest that the anticlonogenic effects of compound 1 might be mediated by regulating the activity of MAP kinases, and ERK1/2 and MSK-1 kinases play a key role in its cancer preventive action.
It is well known that steroids such as steroid hormones act through intracellular receptors on gene transcription and protein synthesis (genomic mechanism of action). On the other hand, the regulatory cascades, i.e., MAP kinases, the phosphatidylinositol 3-OH kinase (PI3K) and tyrosine kinases are modulated through non-transcriptional mechanisms by steroid hormones [28,29]. Therefore, we can assume that the action of starfish steroid glycosides on MAPK signal pathway is realized through non-genomic mechanism. The identification of target molecule of leptaochotensoside A (1) and elucidation of its binding peculiarity with a target protein or cellular receptor is an aim of further research.  1) inhibits EGF-induced anchorage-independent growth of mouse epidermal JB6 Cl41 cells. JB6 Cl41 cells (8 × 10 3 ) were exposed to EGF (10 ng/mL) and treated with 1 (0-200 µM) in 1 mL of 0.3% Basal Medium Eagle (BME) agar containing 10% fetal bovine serum, 2 mM L-glutamine, and 25 µg/mL gentamicin. The cultures were maintained at 37 °C in an 5% CO2 incubator for 14 days, and the cell colonies were scored using a microscope Motic AE 20 (Motic) and the Motic Image Plus computer program. Data are represented as the mean ± SD as determined from triplicate experiments and the asterisks indicate a significant (*** p < 0.001) decrease of the colony formation of the cells treated with leptaochotensoside A (1) compared with the phosphate buffered saline (PBS)-treated group; (B) The absence of cytotoxic effect of leptaochotensoside A (1) on JB6 Cl41 cells. An MTS assay was used after treatment of cells with 1 for 24 h. All the experiments were performed in triplicate, and the mean absorbance values were calculated. Data are represented as the mean ± SD as determined from triplicate experiments; (C) Leptaochotensoside A (1) inhibits MAPK signaling pathway in JB6 Cl41 cells. After cells (6 × 10 5 ) were cultured in a 10-cm dish overnight, they were treated with compound 1 (0-200 µM) for 24 h. Then, the cells were starved in serum-free medium for another 12 h and treated with EGF (10 ng/mL) for 15 min. Cells were harvested and protein levels were determined by Western blot analysis.
The chemiluminescence's detection kit ECL Plus was from Amersham (Pittsburgh, PA, USA). The Basal Medium Eagle (BME), Minimum Essential Medium (MEM), Roswell Park Memorial Institute medium (RPMI 1640), phosphate buffered saline (PBS), L-glutamine, gentamicin solution, trypsin, fetal bovine serum (FBS), sodium hydrocarbonate (NaHCO3), and agar were purchased from Sigma and Gibco (Carlsbad, CA, USA). All other common chemicals, solvents and reagents were of highest grade available from various commercial sources.

Acid Hydrolysis and Determination of Absolute Configuration of Monosaccharide
The acid hydrolysis of 1 (2.0 mg) was carried out in a solution of 2 M TFA (1 mL) in a sealed vial on a H2O bath at 100 °C for 2 h. The H2O layer was washed with CHCl3 (3 × 1.0 mL) and concentrated in vacuo. One drop of concentrated TFA and 0.5 mL of R-(−)-2-octanol (Aldrich) were added to the sugar mixture, and the sealed vial was heated on a glycerol bath at 130 °C for 6 h. The solution was evaporated in vacuo and treated with a mixture of pyridine/acetic anhydride (1:1, 0.6 mL) for 24 h at room temperature. The acetylated 2-octylglycosides were analyzed by GC using the corresponding authentic samples prepared by the same procedure. The following peaks of monosaccharide unit were detected in the hydrolysate of 1: D-xylose (tR 24.32, 24.55, and 24.80 min). The retention times of the authentic samples were as follows: D-xylose (tR 24.28, 24.57, and 24.78 min) and L-xylose (tR 24.07, 24.15, 24.71, and 24.92 min). The JB6 Cl41, RPMI-7951, and T-47D cells were cultured in MEM/5% FBS, MEM/10% FBS, and RPMI-1640/10% FBS media, respectively. The cell cultures were maintained at 37 °C in an 5% CO2 incubator (MCO-18AIC, Sanyo, Moriguchi, Osaka, Japan). The cells were grown for 3-4 days and after reaching 90% of confluence were harvested by exposure to 0.25% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) solution and then passed into new T-75 tissue culture flasks.

MTS Assay
To estimate cell viability, JB6 Cl41, RPMI-7951, and T-47D cells (8 × 10 3 /well) were seeded in 96-well plates for 24 h at 37 °C in 5% CO2 incubator. The attached cells were fed with fresh medium containing various concentrations of compounds 1-4 from L. ochotensis (0-200 µM) for additional 24 h. After that, the cytotoxicity of 1-4 was measured using an MTS assay kit according to the manufacturer's instructions. All the experiments were performed in triplicate, and the mean absorbance values were calculated. The results are expressed as the percentage of inhibition that produced a reduction in absorbance after treatment with polar steroids compared to the non-treated cells (control).
To estimate the effect of compound 1 on the EGF-induced growth of the cell colonies, mouse epidermal JB6 Cl41 cells (2.4 × 10 4 ) were exposed to EGF (10 ng/mL) and treated with 1 (50, 100, 200 µM) in 1 mL of 0.3% BME agar containing 10% FBS, 2 mM L-glutamine, and 25 µg/mL gentamicin. The cultures were maintained at 37 °C in a 5% CO2 incubator for 14 days, and the cell colonies were scored as described above.

Statistical Analysis
All assays were performed at least in triplicate. The results are expressed as the mean ± standard deviation (SD). A 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 sulfated polyhydroxysteroidal compounds (1-4) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis and their chemical structures were elucidated by extensive NMR and ESIMS techniques. The biological action of new leptaochotensosides A-C (1-3) and sulfated steroid (4) were examined using the EGF-induced colony formation of normal mouse epidermal cells and the growth and colony formation of human melanoma and breast cancer cells. Our results indicated that compounds 2-4 at non-toxic doses did not inhibit colony formation of RPMI-7951 cells and have slight cytotoxic effect on colony formation of T-47D cells. Leptaochotensoside A (1) was able to inhibit colony formation of T-47D cells and the EGF-induced colony formation of JB6 Cl41 cells. Compound 1 demonstrated its antiproliferative effects in part through the inhibition of phosphorylation of MAP kinases, namely through the inhibition of EGF-induced phosphorylation of ERK1/2 and MSK-1 kinases.