Ethical approval statements
This investigation was performed following relevant guidelines and regulations of the ethical committee of Shahid Sadoughi University of Medical Sciences (Permission number: IR.SSU.RSI.REC.1397.027).
A. marina collection and extractions
The leaves of A. marina were collected from the Bushehr subtidal region, shores of the Persian Gulf, Iran. A voucher specimen was kept in the Center of Marine Comparative and Experimental Medicine, Bushehr University of Medical Sciences with specimen code PGMBRC20190120. Authentication of collected A. marina was done by a specialist from the Persian Gulf Marine Biotechnology Research Center, Bushehr University of Medical Science. The leaves of A. marina were air-dried in the dark at a room temperature and were powdered. The powders were subjected to ethanol and ethyl acetate extractions.
For ethanol extraction, 40g of dried leaves powder was dissolved in 100 mL of 70% ethanol (Merck, Germany) solvent. The extraction process was continued by shaking the mixture continuously for 3 days at room temperature in the dark condition. Then, the ethanol was evaporated by a rotary evaporator (Laborota 4003-control, Heidolph Instruments GmbH & CO. KG, Germany). For ethyl acetate extraction, 40g of dried leaves powder was dissolved in 100 mL of ethyl acetate (Merck, Germany) for 3 days at room temperature and then ethyl acetate was evaporated by rotary evaporator (Laborota 4003-control, Heidolph Instruments GmbH & CO. KG, Germany). The extracts were stored at -70° C. Then, the obtained extracts were applied for further analysis.
Phytochemical analysis of A. marina extracts
Total phenolic content of A. marina extracts
The total amount of polyphenol contents of the extracts was measured by Folin–Ciocalteu method [26]. The calibration curve was determined based on gallic acid (Merck, Germany). In detail, 20 mg of gallic acid was dissolved in 100 mL of 50% methanol (200 µg/mL) and then diluted to 25, 50, 75, and 100 µg/mL. Afterward, 100 µL of each concentration, 0.5 mL of Folin–Ciocalteu reagent (Merck, Germany), and 1 mL of 20% sodium carbonate (Merck, Germany) were mixed and kept in the dark for 1 h. The total amount of polyphenols was determined by ultraviolet spectrophotometry (CECIL, England) at a wavelength of 765 nm. A similar procedure was adopted for both ethanol and ethyl acetate extracts. All determinations were measured in triplicate. The results of both ethanol and ethyl acetate extracts were separately compared with the calibration curve.
Total flavonoid content of A. marina extracts
The total flavonoid content was measured by the aluminum chloride colorimetric method [27]. At first, different concentrations of quercetin (Fluorochem, United Kingdom) solutions including 25, 50, 75, and 100 µg/mL were prepared by dissolving in methanol solution and then 100 µL of the standard agent (quercetin), 0.2 mL of the aluminum chloride (Merck, Germany) solution and 0.1 mL of 33% aqueous acetic acid were added in a tube and well stirred. Finally, the materials were mixed by adding 90% ethanol until the solutions’ volume reached 5 mL and it was kept at room temperature for 30 min. The total amount of flavonoid was determined by ultraviolet spectrophotometry (CECIL, England) at a wavelength of 415 nm. A similar procedure was performed for both extracts. Briefly, in the previous procedure, instead of a quercetin agent, the same concentrations of ethanol and ethyl acetate extracts were separately used for flavonoid content measurement. All determinations were measured in triplicate.
GC-MS analysis of A. marina extracts
The extracts were lyophilized and were subjected to the 7890B Agilent Gas Chromatography-Mass Spectroscopy (GC-MS). Electron ionization (EI) mass spectra (scan range, m/z 50–500) were obtained using electrons with an energy of 70 eV. The filament emission was 0.5 mA. The GC separations were performed using an HP-5MS UI column (30 m × 0.25 mm i.d., film thickness 0.5 µm). Helium, as the carrier gas, was used with the flow of 0.8 ml min− 1 for EI. The GC oven was temperature programmed at 5°C min− 1 from 80°C after 3 min since the sample injection and held at 250°C for 10 min. The injection port of the gas chromatograph, the transfer line, and the ion source of 5977MSD were maintained at 240°C, 250°C, and 220°C, respectively. Identification of the separated compounds was performed by comparing them with the compound data of the National Institute of Standards and Technology (NIST MS database, 2014) library. The relative percentage of each compound was measured by average peaks area in comparison with the total areas.
In vitro analysis of A. marina extracts
Cell culture
For in vitro study, human breast cancer cell line (MCF-7), human ovarian carcinoma cell line (OVCAR3), human cervical cancer cell line (HeLa), and normal kidney epithelial cell line (VERO, originated from adult African green monkey) were provided (PerciaVista Co., Iran). The cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Life Technologies Co., US) supplemented with 10% fetal bovine serum (FBS, Kiazist, Iran) and 1% penicillin-streptomycin (Pen-Strep, Gibco, Life Technologies Co., US). After seeding the cell lines, the cells were incubated at 37°C and 5% CO2.
MTT assay and calculation of cytotoxic concentration
The MTT assay protocol was performed based on a previous study with slight modifications [28]. The cell lines were seeded in 96-well cell culture plates and after 24 h, the ethanol and ethyl acetate extracts of A. marina leaves were separately added to all cell lines in the concentrations of 40, 80, 120, and 160 µg/mL. For the control group, the DMEM supplemented with 10% FBS and the 5% concentration of dimethyl sulfoxide (DMSO, Sigma-Aldrich Co., Darmstadt, Germany) were added to wells, separately. The stock solution of ethanol and ethyl acetate extracts, and DMSO was 3200 µg/mL of both extracts and 100% DMSO with 1.10 g/mL density (Sigma-Aldrich Co., Darmstadt, Germany). Moreover, methotrexate (MTX) was used as positive control with the concentrations of 120, and 160 µg/mL which were the most effective doses in the current study. The samples were incubated at 37°C and 5% CO2 for 72 h. Then, the media were removed. For cell vitality assessment, 100 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay kit (Sigma-Aldrich Co., Darmstadt, Germany) with the concentration of 5 mg/mL were added into each well and they were incubated for 4 h until the formation of the intracellular purple formazan crystals. After that, the DMSO was added to each well and incubated at 37°C and 5% CO2 for 20 min. The absorbance of cells in each plate was read at a wavelength of 573 nm using an ELISA plate reader machine (BioTek, USA). This step was repeated three times. Then, 50% cell cytotoxic concentration (CC50) of A. marina leaves for both extracts were calculated via nonlinear regression of “log (inhibitor) vs. normalized response” in Graph pad prism (v7.0a, GraphPad Software, Inc., San Diego, CA, USA). The CC50 values of extracts were classified based on previous studies [29, 30]. In details, the CC50 lower than 20 µg/ml and the CC50 = 21–200 µg/ml, 201–500 µg/ml, and higher than 500 µg/ml considered as highly, moderately, weakly and none-active (or potent) for anticancer activity, respectively.
Growth curve assay and population doubling time
The growth curve and population doubling time assay were performed based on previous studies with some modifications [31, 32]. The VERO, MCF7, OVCAR3, and HeLa cell lines were seeded in 24-well cell culture plates at a density of 4×104, 5×104, 2×104, and 5×104 cells per well, respectively [33–36]. Concentrations of 40, 80, 120, and 160 µg/mL of ethanol extract and ethyl acetate extract of A. marina leaves were prepared in 10 mL DMEM supplemented with 10% FBS and 10 mL of DMSO, respectively. The cells were treated separately with crude ethanol and ethyl acetate extracts for 24 h. After that, the number of cells in three wells was counted every 24 hours (each time three wells/group). This procedure was repeated for 7 days, and the mean number of cells on each day was obtained. In more detail, the culture medium supplemented with crude extracts was changed on day 3. The following formula was used to determine population doubling time (PDT): T×ln2/ln (Xe/Xb), where Xe, Xb, and T were defined as the final cell number, the initial cell number, and the incubation time in any unit, respectively.
Cell viability assessment
Trypan blue exclusion method was selected for the cell viability based on previous studies with slight modifications [37, 38]. In detail, the cell lines were seeded at the same densities previously described. The cells were treated with 40, 80, 120, and 160 µg/mL concentrations of ethanol extract and ethyl acetate extract of A. marina leaves and were incubated at 37°C in the presence of 5% CO2 for 72 h. After that, the cells were treated with 300 µL of 0.5% trypsin enzyme (Kiazist Co., Iran). Finally, 20 µL of medium contained detached cells and an equal volume of trypan blue was mixed and viable, and dead cells were counted by hemocytometer chamber. The cell viability was measured by the following formula: Cell viability (%) = (number of non-stained cells/number of total cells) × 100.
Cell cycle analyses
Based on the findings of cell MTT assay, cell viability test, and PDT assay, the most effective concentrations of the extracts were determined and used for cell cycle assay in each cell line based on previous studies [39]. The cells in the logarithmic growth phase were taken for experimental intervention. The cells were cultured in a T75 flask with 2.5×106 seeding density and after 72 h of cell exposure to extracts, cells were digested, washed, and prepared as the single-cell suspension. The cell density was provided into 1–5 ×105 cells/mL. Then, 500 µL of binding buffer was added to suspend cells and mixed gently. Afterward, 5 µL propidium iodide (PI) was added to the cells and they were incubated in the dark condition at room temperature for 15 min. Then, l h Flow Cytometry (FACS, Becton, Dickinson, USA) was performed. The experimental results were analyzed using BD Cell Quest software (BD Biosciences Co., USA).
Apoptosis assay by flow cytometry
The proportion of apoptotic cells was determined by flow cytometry [39]. Same as the cell cycle assay, the cells were incubated with the extracts for 72 h and after that the cells were digested and washed and provided as the single-cell suspension. The cell density was also provided the same as the cell cycle assay. After adding 500 µL of binding buffer, 5 µL of annexin-V-FITC and 5 µL PI were added to the cells and they were incubated in the dark condition at room temperature for 15 min. The procedure was followed by l h using Flow Cytometry (FACS, Becton, Dickinson, USA). The experimental results were analyzed using the same manner of cell cycle assay.
Western blot analysis
Western blot analysis was done based on standard procedures with slight modifications [40]. Based on the findings of the cell MTT assay, cell viability test, and PDT assay, the most effective concentrations of the extracts were determined and used for western blot analysis in each cell line. In details, same as cell cycle and apoptosis assays, the cells cultured in T75 flask and after 72 h cell exposure to extracts, cells were lysed by RIPA buffer including 50 mM Tris–HCl (pH = 8.0), 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, 80 µg/mL leupeptin, 3 mM NaF, and 1 mM DTT at 4°C for 20 min. The lysates were centrifuged at 12000 ×g for 20 min at 4°C, and the protein concentration was measured by a Bradford protein assay. Proteins were then transferred to a microporous polyvinylidene difluoride membrane (Millipore, France). Membranes were incubated in 5% BSA (Sigma, USA) blocking buffer for 1 h at room temperature. After blocking, the membranes were incubated with the corresponding primary antibodies separately overnight at 4°C. Immunoblotting was performed with rabbit anti-β-actin, anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-caspase-1, -3, and − 7 antibodies (1:200) (Cell Signaling Technology, Danvers, MA). Membranes were washed three times (10 min each) in tween buffer before incubating with HRP-conjugated goat anti-mouse or rabbit secondary antibodies. To remove excess antibodies, membranes were washed four times before HRP activities were detected using ECL Plus Chemiluminescence Reagent (Amersham, Chalfont, UK) according to the protocol supplied with the kit.
Computational details
Ligand and Receptor preparation
Thirty-three compounds derived from the Persian Gulf A. marina and five proteins were selected for the docking process. The three-dimensional (3D) structure of ligands and receptors was downloaded from the PubChem database and the Protein Database Bank (PDB), respectively. HyperChem software version 8.0.10 was used to optimize the geometry of compounds. Chimera 1.15 was used to prepare receptors. The receptor preparation process includes removing all non-standard residues, water, and original hydrogens, the addition of polar hydrogen, charges and bond orders, and capping N and C termini. Finally, the format of all outputs was converted to a suitable format for the docking process.
Generation of grid
The generation of the grid box is an important step in the docking process. To generate a grid box, the position of the active site of each receptor was found using available structures with a co-crystallized ligand for BAX, BCL-2, Caspase-1, Caspase-3 and Caspase-7 with PDB code of 4zie, 6O0K, 6f6r, 4quj and 2ql9, respectively. Then, a grid box with a dimension of 35×35×35 Å3 and a spacing of 0.375 Å was generated at the active-site of each receptor using AUTOGRID.
Study of target proteins-marine derived compounds interactions
To study interactions of target proteins and selected compounds, the docking process was performed using Autodock Vina 1.1.2 after generating the grid box. Among different conformations suggested by the software for each ligand, the best one was selected according to the binding affinity score and RMSD given by the software. To validate the accuracy of the docking process, the co-crystallized ligand (1,2-ethanediol, LBM, CVE, MPD, and CIT for 4zie, 6O0K, 6f6r, 4quj and 2ql9, respectively) were re-docked. The best conformations of the co-crystallized ligands were reasonably located at the original active-site with RMSD ≤ 2 Å.
Statistical analysis
IBM SPSS Statistics 26 software (SPSS for Windows, version 26, SPSS Inc, Chicago, Illinois, USA) was used for the statistical analysis of data. The results were presented as mean ± standard error of the mean. The measurement results received the homogeneity test of variance. Comparison between groups was done using one-way ANOVA and post hoc LSD test (for comparing the MTT and cell proliferation and viability assays, these assays were performed in triplicate two times) or chi-square test (for comparing the cell cycle analysis and flow cytometry). The p < 0.05 was considered the statistically significant difference. The graphs were drawn by Graph pad prism (v7.0a, GraphPad Software, Inc., San Diego, CA, USA).