Pregnane-Type Steroids from the Formosan Soft Coral Scleronephthya flexilis

Three pregnane-type steroids, including a new metabolite, 3β-methoxy-5,20-pregnadiene (1) along with two known analogues, 3β-acetoxy-5,20-pregnadiene (2) and 5α-pregna-1,20-dien-3-one (3) were isolated from the soft coral Scleronephthya flexilis. Standard spectroscopic techniques were used to determine the structure of new steroid 1. The absolute stereochemistry of steroid 2 was confirmed by X-ray diffraction analysis. Steroid 3 exhibited potent activity against MOLT-4 tumor cells.

The cytotoxicities of steroids 2 and 3 towards human leukemia cells, including MOLT-4 (acute lymphoblastic leukemia), HL-60 (acute promyelocytic leukemia) and K-562 (chronic myelogenous leukemia) cells, were studied, and the results are shown in Table 2. MOLT-4 was the cell line most sensitive to the cytotoxic effects of 5α-pregna-1,20-dien-3-one (3). This encouraged us to expand our cytotoxicity study with the aim of revealing the mechanism of action of 3 against leukemia cancer cell lines, which we pursued in the current study. We determined the effects of 3 treatment on the cell growth of different leukemia cell lines. Initially, we determined the IC 50 values of 3 against MOLT-4, HL-60 and K-562 cells after 72 h and found that the cell growth of MOLT-4, HL-60 and K-562 cells were inhibited in dose-dependent manner with the IC 50 values of 2.15, 3.14 and 8.32 μg/mL, respectively ( Figure 3A and Table 2). HL-60 and K-562 cell lines are p53-negative cell lines [18]. MOLT-4 cells, originally derived from the same patient as MOLT-3 cells, are lymphoblastoid T cells and express normal p53 [19]. In addition, the cell growth of different leukemia cells was significantly suppressed by 3 treatment in a dose-dependent manner, regardless of p53 status ( Figure 3A). We then evaluated whether the cytotoxicity of 3 is associated with apoptosis by examining the effect of 3 on cells stained with annexin V-FITC and propidium iodide (PI). As shown in Figure 3B, treatment with 3 at concentrations of 0, 1.25, 2.5 and 5 μg/mL for 24 h increased the percentages of annexin-positive cells from 4.1% to 23.3%, 61.1% and 98.2% as compared with the control group in MOLT-4 cells, respectively. To determine whether the cytotoxic effect of 3 is specific for cancer cells, we examined the effect of 3 on the viability of normal rat peripheral blood mononuclear cells (PBMC). At the highest dose (10 μg/mL), 3 treatment caused 71.6% suppression in the viability of PBMCs, nevertheless, doxorubicin treatment induced 99.9% suppression for 24 h ( Figure 3C). Compared with PBMCs, 3 suppressed 99.9% of cell growth in MOLT-4 cells for 24 h. Thus, it is concluded that cytotoxic effect of 3 is more sensitive towards MOLT-4 cells compared to the normal rat PBMCs. In addition, our result suggested that growth inhibition of 3 is mediated with induction of apoptosis and operated independently in P53 pathway. This encouraged us to expand our cytotoxicity study with the aim of revealing the mechanism of action of 3-induced leukemia MOLT-4 cells apoptosis. Mitochondria are organelles which play an important role in the life and death of the cells. Their importance is mainly attributed to energy production in the form of ATP. Additionally, mitochondrial dysfunction participates in the induction of apoptosis [20]. To examine whether the antiproliferative and apoptotic effects of 3 are involved mitochondrial dysfunction in MOLT-4 cells, flow cytometric assays with various fluorescent dyes were utilized. Different concentrations of 3 (0, 1.25, 2.5 and 5 μg/mL) were used for 24 h, and the change in the mitochondrial membrane potential (MMP) was analyzed. Treatment with 3 (1.25, 2.5 or 5 μg/mL) led to 23.8%, 49.2% and 93.8% disruption of the MMP, respectively, as detected using JC-1 cation dye in MOLT-4 cells (Figure 4). The recent result evidenced that one of 3 targets as an apoptosis inducer is to disrupt the bioenergetic steps of the mitochondria-medicated pathway.

Animal Material
Specimens of the octocoral Scleronephthya flexilis were collected by hand using scuba equipment off the coast of Southern Taiwan in September 2012, and stored in a freezer (−20 °C) until extraction. A voucher specimen (NMMBA-TWSC-12009) was deposited in the National Museum of Marine Biology and Aquarium, Pingtung, Taiwan.

Extraction and Isolation
Specimens of the soft coral Scleronephthya flexilis (wet weight 1.5 kg, dry weight 562 g) were minced and extracted with ethyl acetate (EtOAc). The EtOAc extract remaining after removal of the solvent (6.7 g) was separated by silica gel and eluted using n-hexane/EtOAc in a stepwise fashion from 100:1-pure EtOAc to yield 10 fractions A-J. Fraction C (514 mg) was chromatographed on silica gel using a mixture of n-hexane and EtOAc in a stepwise fashion from 45:1 to 10:1 to obtain 10 subfractions C1-C10. Fraction C3 (100 mg) was purified by NP-HPLC using a mixture of n-hexane and acetone (100:1) to obtain 8 subfractions C3A-C3H. Fraction C3F (3.1 mg) was repurified by NP-HPLC using a mixture of n-hexane and dichloromethane (5:2, flow rate: 1.0 mL/min) to yield 3β-methoxy-5,20-pregnadiene (1) (0.6 mg, t R = 45 min). Fraction C4 (48 mg) was purified by NP-HPLC using a mixture of n-hexane and EtOAc (18:1) to obtain 10 subfractions C4A-C4J. Fraction C4F (38 mg) was repurified by RP-HPLC using a mixture of methanol and H 2 O (98:2, flow rate: 2.0 mL/min) to yield 3β-acetoxy-5,20-pregnadiene (2)  Intensity data were measured on a Bruker APEX-II CCD diffractometer equipped with a micro-focus Cu radiation source and Montel mirror up to θ max of 66.7° at 100 K. All 12,991 reflections were collected. The structure was resolved by direct methods and refined by a full-matrix least-squares procedure. The refined structure model converged to a final R1 = 0.0279, wR2 = 0.0717 for 3358 observed reflections (I > 2σ(I)) and 229 variable parameters. The absolute configuration was determined by Flack's method, with Flack's parameter determined to be 0.09 (6) [21,22].

MTT Antiproliferative Assay
MOLT-4 (human acute lymphoblastic leukemia), HL-60 (human acute promyelocytic leukemia) and K-562 (human chronic myelogenous leukemia) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Rat PBMCs were generated as described previously [23]. Cells were maintained in RPMI-1640 medium supplemented with 10% FCS, 2 mM glutamine, and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere of 5% CO 2 . Cells were seeded at 4 × 10 4 per well in 96-well culture plates before treatment with different concentrations of the tested compounds. The compounds were dissolved in DMSO (less than 0.02%) and made immediately to 1.25, 2.5, 5, 10 and 20 μg/μL prior to the experiments. After treatment for 72 h, the cytotoxicities of the tested compounds were determined using a MTT cell proliferation assay (thiazolyl blue tetrazolium bromide, Sigma-M2128). The MTT is reduced by the mitochondrial dehydrogenases of viable cells to a purple formazan product. The MTT-formazan product was dissolved in DMSO. Light absorbance values (OD = OD 570 − OD 620 ) were recorded at wavelengths of 570 and 620 nm using an ELISA reader (Anthos labtec Instrument, Salzburg, Austria) to calculate the concentration that caused 50% inhibition (IC 50 ), i.e., the cell concentration at which the light absorbance value of the experimental group was half that of the control group. These results were expressed as a percentage of the control ± SD established from n = 4 wells per one experiment from three separate experiments.

Annexin V/PI Apoptosis Assay
The externalization of phosphatidylserine (PS) and the membrane integrity were quantified using an annexin V-FITC staining kit (Strong Biotech Corporation, Taipei, Taiwan). In brief, 10 6 cells were grown in 35-mm-diameter plates and were labeled with annexin V-FITC (10 μg/mL) and PI (20 μg/mL) prior to harvesting. After labeling, all plates were washed with a binding buffer and harvested. Cells were resuspended in the binding buffer at a concentration of 2  10 5 cells/mL before analysis by a flow cytometer FACS-Calibur (Becton-Dickinson, San Jose, CA, USA) and CellQuest software. Approximately 10,000 cells were counted for each determination.

Determination of Mitochondrial Membrane Potential Disruption
These assays were performed as described previously. MMP disruption was detected with JC-1 cationic dye (5 μg/mL) [24]. In brief, the treated cells were labeled with a specific fluorescent dye for 30 min. After labeling, cells were washed with PBS and resuspended in PBS at a concentration of 1 × 10 6 cells/mL before analysis by flow cytometry.

Statistics
The results were expressed as mean ± standard deviation (SD). Comparison in each experiment was performed using an unpaired Student's t-test, and p values lower than 0.05 were considered to be statistically significant. (* p < 0.05; ** p < 0.01).

Conclusions
In the first study on the chemical constituents of soft coral Scleronephthya flexilis, three pregnane-type steroids, including a new metabolite, 3β-methoxy-5,20-pregnadiene (1), along with two known compounds, 3β-acetoxy-5,20-pregnadiene (2) and 5α-pregna-1,20-dien-3-one (3), were isolated. The structure of new steroid 1 was elucidated on the basis of spectroscopic methods. The absolute configuration of steroid 2 was further confirmed by X-ray diffraction analysis for the first time. Our results also suggest that the use of steroid 3 induced MMP disruption, as well as apoptosis in Molt-4 cells. The current work clearly supports the potential application of 3 for leukemia therapy. The soft coral Scleronephthya flexilis has been transplanted to culturing tanks located in the National Museum of Marine Biology and Aquarium, Taiwan, for extraction of additional natural products to establish a stable supply of bioactive material.