Background
Brown seaweeds such as
Fucus vesiculosus are widely consumed by the public due to their potential anti-cancer activities warranting the need for further studies to characterize their biological actions. We previously reported anti-estrogenic properties of
F. vesiculosus in human and animal studies [
1]. Specifically, in a small case study,
F. vesiculosus administered to pre-menopausal women with endometriosis led to a reduction in circulating estradiol (E2) levels, an increase in the length of the menstrual cycle, and diminished symptoms of hypermenorrhea and dysmenorrhea [
1]. Anti-estrogenic action was further demonstrated where dosing with a
F. vesiculosus extract (FVE) in primary human luteinized granulosa cell cultures led to marked reductions in E2 levels [
2]. Rats fed
F. vesiculosus exhibited increased estrous cycle lengths and reduced serum E2. FVE also exerted inhibitory effects on the binding of E2 to estrogen receptor (ER)α and ERβ [
2].
To further explore the anti-estrogenic activity of
F. vesiculosus, the effects of FVE were tested on the activation of the ER and inhibition of aromatase enzymatic activity. We also investigated other modes of action of FVE biologically relevant to cancer initiation and progression in breast, endometrial and ovarian cancer cell lines. Fucoidan is a major component of
F. vesiculosus and other brown seaweeds and exhibits anti-tumor and anti-metastatic activities in numerous cancers [
3]. Therefore, we included fucoidan purified from
F. vesiculosus in our in vitro studies to compare its effects with those of FVE.
Methods
Atlantic F. vesiculosus (Maine Coast Sea Vegetables, Inc., Franklin, ME) was ground into a fine powder, mixed with deionized water (5 g into 100 mL) and stirred 2 h at room temperature. The insoluble material was removed by centrifugation; the supernatant (70 mL) was sterilized by filtration through a 0.2 μM filter and stored in 1-mL aliquots at −20 °C. This constitutes the 100 % v/v stock extract. For all experiments, treatment concentrations of the extract are expressed as the % v/v. For example, a 100-fold dilution in cell culture medium is expressed as 1 %. Four aliquots were completely dried under a vacuum using a SpeedVac evaporator overnight yielding 35 mg of solid residue per mL of extract. In total, the 70-mL extract contained 2.45 g of water-soluble material extracted from 5 g of starting plant powder.
Antibodies and reagents
Antibodies to Akt, phospho-Akt (Ser473), phospho-Akt (Thr308), beclin-1, phospho-Beclin-1 (Ser15), phospho-PI3Kinase p85(Tyr458)/p55 (Tyr199), phospho-4E-BP1 (Thr37/46), p70S6K, phospho-p70S6K(Thr389), LC3B, poly(ADP-ribose) polymerase (PARP), cleaved PARP (D214) and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal β-Actin antibody and crude fucoidan from F. vesiculosus were obtained from Sigma-Aldrich (St Louis, MO, USA). DMEM (Dulbecco’s Modification of Eagle’s Medium) with 4.5 g/L glucose, L-glutamine and sodium pyruvate, trypsin-EDTA, penicillin-streptomycin-amphotericin B solution (50X), fetal bovine serum (FBS), phosphate buffer solution (PBS), and PBS with Tween 20 (PBST) were purchased from Life Technologies (Waltham, MA, USA).
Estrogenic activity of FVE measured by a reporter assay
The effect of FVE on E2 signaling was investigated using a chemically activated luciferase reporter (CALUX® assay) for ERα and ERβ. The ER activity reporter, T47D-KBluc cell line, was purchased from ATCC (Manassas, VA). This CALUX® assay cell line is permanently transfected with a plasmid reporter construct expressing luciferase under control of a promoter region containing several repeats of the cognate responsive element for ER. Cells grown in medium depleted of steroids (charcoal filtered FBS (5 %) in DMEM without Phenol-red) for 7 days to minimize background activity were seeded in opaque 96-well plates and allowed to attach overnight. Medium containing 0 to 25 pM E2 as the calibration standard, or FVE (0 to 2 %) either alone or in co-treatments with 12.5 pM E2, was added to the wells in triplicates. Fucoidan was also tested in this assay at a range of concentrations (0 to 0.50 mg/mL). After a 24-h incubation, cells were lysed and luciferase activity was measured with a microplate luminometer using the Promega Flash Luciferase Assay kit (Madison, WI). A clear 96-well plate was seeded and treated identically and was used to normalize the luminescence raw data for possible cell number variations (measured with the MTT assay) due to the 24-h exposure to the treatments. Estrogenic activity was expressed as pM E2 equivalents.
Effects of FVE on aromatase activity
Aromatase activity was measured using the CYP19/MFC High Throughput Inhibitor Screening Kit (Discovery Labware, Inc. Wolburn, MA) according to the manufacturer’s instructions. Briefly, recombinant human CYP19 enzymatic hydrolysis of the fluorescent substrate, 7-methoxy-4-trifluoromethyl coumarin, was used to measure aromatase activity in vitro in a 96-well plate. Ketoconazole was used as the positive marker of aromatase inhibition. FVE was tested in this assay at a 0 to 2 % range of concentrations.
Cell lines and culture conditions
Human MDA-MB-231 and MCF-7 breast cancer cells were kindly provided by Dr. Samant (University of Alabama, Birmingham, AL) and the breast carcinoma (T47D), ovarian carcinoma (OVCAR-3 and Caov-3), uterine endometrium carcinoma (HEC-1-B, RL95-2 and AN3CA) and uterine sarcoma (MES-SA) cell lines were obtained from ATCC. The MDA-MB-231/GFP-LC3 stable cell line was a gift from Dr. Xu [
4] (University of Alabama, Birmingham, AL). Cell lines were maintained in their respective ATCC recommended media at 37 °C, in 100 % humidity and 5 % CO
2.
Effects of FVE on cell viability
The effects of FVE on the viability of all nine cell lines were analyzed using the MTT assay (Promega Corp. Madison, WI). Specifically, cells were seeded at 105 per well in 12-well plates and after overnight attachment, FVE (0 to 2 %) was added to the medium in triplicate wells. After 72 h, MTT dye was added to the medium, incubated one hr at 37 °C. The formazan crystals were dissolved in Solubilization Solution/Stop Mix and the OD 570 nm was used as a measurement of viable cell number.
Toxicity assessment
The Mitochondrial ToxGlo™ Assay (Life Technologies, Carlsbad, CA) was used to assess cell toxicity, which relies on biomarkers associated with changes in cell membrane integrity and cellular ATP levels. Cell membrane permeability in necrotic cells is indicated by the presence of specific protease activity, using the fluorescent proteolytic product of the peptide substrate (bis-AAF-R110). Digitonin is a positive marker for cellular membrane permeabilization. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) is a positive marker of mitochondrial toxicity.
Gene expression profiling
As an initial screening tool, transcript levels were measured (nCounter® Analysis System, (NanoString Technologies, Seattle, WA) in a panel of 248 genes involved in cancer, immunity, hormonal regulation, protein phosphorylation, transcription, metabolism, and cellular structure (Additional file
1: Table S1) in a subset of 6 cell lines (MCF-7, T47D, MDA-MB-231, RL95, HEC-1-B, RL95-2 and OVCAR-3). Cells were treated with FVE (0, 0.25 and 1.0 %, up to 4 h) and mRNA transcript levels were measured using total RNA that was purified using the Qiagen RNeasy Mini Kit and quantified using a BioTek Take-3 UV spectrophotometer. The geometric mean of six housekeeping genes (
TUBB, G6DP, GAPDH, POLR2A, RPL19 and
TBP) was used as the reference for normalization of measured mRNA levels. To filter out genes expressed at low background levels, we removed those with expression levels below 4 in all samples after normalization (70 counts in the raw data). For the remaining genes, a two-way ANOVA model with cell line effect and treatment dose effect was fitted to the normalized and logarithm-transformed expression data for each gene. The permutation-based F-test was used to identify genes differentially expressed between treatment doses. Due to the screening characteristics, we used a nominal p-value of 0.05 as significance threshold. Analyses were conducted in R, version 3.0.0.
Assessment of FVE on apoptosis and autophagy with pan-caspase and autophagy inhibitors
The relative contribution of apoptosis and autophagy to the reduced viability of the cancer cells exposed to FVE was examined using co-treatment with specific inhibitors. The pan-caspase inhibitor, Z-VAD (OMe)-FMK (VAD, 60 μM) and the autophagy inhibitor, 3-methyladenine (3-MA, 8 mM; Cayman Chemicals, Ann Arbor, MI) were used in co-treatments with 1 % FVE -for 72 h.
Caspase activity assay
Caspase activity was determined using the EnzChek Caspase-3 Assay Kit #1 (Molecular Probes, Grand Island, NY). Briefly, estrogen-dependent MCF-7 and estrogen-independent MDA-MB-231 breast cancer cells were incubated 2 h with 1 % FVE. Cells were collected after trypsin digestion, washed in PBS, and lysed in 1x lysis buffer by freeze and thaw. After centrifugation (5,000 x g, 5 min), the supernatant containing 100 μg protein was incubated with substrate (Z-DEVD-AMC, 25 °C, 30 min) or pre-incubated with caspase-3/7 inhibitor (Ac-DEVD-CHO) (10 min) before addition of substrate (Z-DEVD-AMC). Released AMC fluorescence was measured with a microplate reader (Synergy-H1) at excitation/emission 342/441 nm and the net change of fluorescence was calculated by subtraction of the fluorescence of the sample pre-incubated with the inhibitor. Caspase-3/7 activities were determined by direct comparison to the level in the untreated control cells. For caspase inhibitor treatments, cells were pretreated with a synthetic cell-permeable pan-caspase inhibitor (Z-VAD (OMe)-FMK, 10 mM, 30 min) before the addition of 1 % FVE and incubated for an additional 2 h.
Fluorescence microscopy
MDA-MB-231 cells/GFP-LC3 were grown to 60 % confluence in six-well plates with cover slides in DMEM supplemented with 10 % FBS. Cells were treated with FVE (0 to 2 %, 16 h) and fixed in 4 % neutral paraformaldehyde in PBS for 15 min at room temperature and washed 3 times in PBS. Slides were mounted with antifade medium Dapi Fluoromount-G (Southern Biotech, Birmingham, AL) and the GFP-LC3 staining was observed using a fluorescence microscope.
Western blots
Immunoblots were used to study the effects of FVE on phosphorylation and activation of kinases in the PI3K-Akt-mTOR pathway. Cells grown in 6-well plates were treated with FVE (0 to 2 %, up to 8 h), then lysed in RIPA buffer supplemented with a cocktail of protease and phosphatase inhibitors. After the addition of 2X Laemmli loading solution containing 10 % β-mercaptoethanol, cell lysates were sonicated 5 s and denatured at 100 °C for 5 min. SDS-Page Gels (Mini-Protean TGX Precast Gels, Any kD, BioRad, Hercules, CA) were loaded with 30 μg of sample per lane, run at 200 V for 40 min and transferred to PVDF membranes using the rapid semi-dry Trans-Blot Turbo Transfer System (BioRad, Hercules, CA). Blots were blocked in 5 % non-fat dry milk at 37 °C for 30 min followed by overnight incubation at 8 °C with primary antibodies in blocking buffer. Secondary antibodies conjugated to HRP were imaged by enhanced chemoluminescence on a ChemiDoc MP imaging system (BioRad) and analyzed using BioRad’s Image Lab software. Blots were re-probed with different antibodies after treatment with Restore™ Plus Western Blot Stripping Buffer (Thermo Fisher Scientific, Rockford, IL).
Statistical analyses
Experimental data were analyzed by the Student’s t-test. Data are shown as means ± S. E. of values obtained from triplicate or quadruplicate experiments. A cut-off value of p < 0.05 was used to indicate statistical significance.
Conclusions
Our results provide further evidence of the anti-estrogenic activity exerted by FVE that involves inhibition of ER activation by E2, and inhibition of E2 synthesis. This activity suggests that FVE may provide a beneficial protective effect against estrogen-dependent breast, endometrial and ovarian cancers. Moreover, the reduced viability of ER-positive and -negative cancer cell lines treated with FVE through inactivation of the PI3K/Akt/mTOR pathway suggests that FVE may be useful in the treatment of some breast, endometrial and ovarian cancers regardless of ER status. MDA-MB-231 breast, HEC-1B endometrial and OVCAR-3 ovarian cells do not express the ER, but were sensitive to FVE; HEC-1B endometrial carcinoma cells were the most sensitive to FVE. Endometrial cancers are often aggressive, and therapeutic options are limited and sometimes ineffective. Therefore, the identification of new therapeutic agents is highly desirable. Studies to further characterize the chemoprotective and anti-tumorigenic actions of FVE are currently underway.
Abbreviations
ATCC, American Type Culture Collection; ATP, adenosine triphosphate; CCCP, carbonyl cyanide m-chlorophenyl hydrazine; E2, estradiol; EC50, half maximal effective concentration; ER, estrogen receptor; FVE, Fucus vesiculosus extract; FBS, fetal bovine serum; GFP, green fluorescent protein; LC3, light-chain 3; 3-MA, 3-methyladenine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly(ADP-ribose) polymerase; PBS, phosphate buffer solution; PBST, phosphate buffer solution with Tween 20; VAD, Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK).
Acknowledgements
The authors acknowledge Dr. Zhi-Xiang Xu, MD, PhD and Dr. Rajeev Samant, PhD from University of Alabama at Birmingham for supplying cell lines.