In order to characterize the compounds contained in Pg extract, Fourier-transform infrared spectroscopy and thermal analysis were employed.
Fourier-transform infrared spectroscopy - FT-IR
The FT-IR spectra of each resultant compound were recorded in the range from 4000-400 cm−1 on KBr pellets, under reduced pressure. A Prestige-21 spectrometer (Shimadzu, Duisburg, Germany) operating with a peak resolution of 4 cm−1, at room temperature conditions, was employed to perform the spectra.
Based on the presence of a peak and at a specific wavenumber, FT-IR investigations were employed to identify the functional groups of the main active compounds which are present in the dried Pg extract. FT-IR technique it’s a qualitative analysis which can be considered, like HPLC analysis, a chemical fingerprint . The difference between the two analyses is based on the fact that FT-IR is a chemical fingerprint for the functional groups contained by the extract molecules, while the HPLC is a chemical fingerprint for each component contained in the extract. In addition, the HPLC technique quantifies each component after identification.
Thermogravimetry-Differential scanning calorimetry (TG-DSC) is an analytical technique which was used to assess the stability and composition of organic compounds contained in dried extract based to Pg, thus predicting biological stability prior to in vitro and in vivo application . TG-DSC analysis was performed using a STA 449C instrument (Netzsch, Selb, Germany) in air atmosphere at a flow rate of 20 mL/min. The curves were recorded in the range 25–1000°C with a heating rate of 10 K/min, using alumina crucibles.
In vitroantimicrobial effects
Another aim of this study was to screen this extract for its antimicrobial properties against eight microorganisms (Thermo Scientific, USA) (Staphylococcus aureus, ATCC code 25923; Streptococcus mutans, ATCC code 35668; Streptococcus pyogenes, ATCC code 19615; Enterococcus faecalis, ATCC code 51299; Escherichia coli, ATCC code 25922; Pseudomonas aeruginosa, ATCC code 27853; Candida albicans, ATCC code 10231; Candida parapsilosis, ATCC code 22019). Were selected these eight strains because they are the most common pathogens responsible for most of the healthcare problems. The susceptibility of microorganisms to the tested compounds was determined using disk diffusion method and dilution method, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST), Clinical Laboratory and Standard Institute (CLSI) and based on previous similar studies [20, 21].
Disk diffusion method
The antimicrobial activity of the extract was evaluated by the agar disk diffusion method, as previously described [22, 23]. The standardized suspension was prepared in normal saline to a concentration of 0.5 McFarland. The un-supplemented Mueller Hinton (MH) agar with 5% defibrinated horse blood and β-NAD (MH-F) was inoculated with 100 µL of this suspension. Were added 10 µL from each sample to a 6 mm diameter blank paper disk (BioMaxima, Lublin, Poland), placed on the surface of the MH or MH-F agar. After 24h at 35-37°C, we measure the inhibition areas in millimeters. For all microbial strains these assays were done in triplicate and the average values were registered. The antibacterial activity of this extract was established according to the standardized value of the positive control, for gentamycin a diameter of the inhibition area ≥ 15 mm was considered as susceptibility, while for fluconazole the diameter was ≥ 19 mm. For negative control, were used a disk impregnated only with DMSO.
Standardized suspension was adjusted, by dilution, obtaining a microbial suspension of approximately 105 colony forming units (CFU)/mL. For each compound tested, serial dilutions were prepared in MH-F broth: 5, 2.5, 1.25, 0.625, 0.312, 0.156 mg/mL. One milliliter from each dilution of tested compound and 1 mL inoculum were added in six test tubes, to a final microbial suspension of 0.5×105 CFU/mL and a final compound dilution between 2.5 and 0.078 mg/mL. After incubating at 37°C for 24h, the MIC was analyzed. To determine the MBC a volume of 1 µl from the test tubes with no visible growth was inoculated on Columbia agar supplemented with 5% blood. After incubation at 37°C for 24h, the lowest concentration that kills 99.9% of the microorganisms was established .
Cell culture and cell preparation
The MCF7 human breast adenocarcinoma (ATCC® HTB-22TM) was purchased from the American Type Culture Collection (ATCC). MCF7 cells were cultured in RPMI-1640 medium (ATCC® 30-2001™). The cell line was supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, Germany) and 1% penicillin/streptomycin mixture (Pen/Strep, 10,000 IU/mL; Sigma-Aldrich, Germany). The cells were maintained in standard conditions (humidified atmosphere with 5% CO2 and 37°C). Human Dendritic cells were differentiated from isolated PBMCs from buffy coats according to Nair et al. . In brief, density centrifugation was performed using Ficoll (GE Healthcare, Uppsala, Sweden). Isolated PBMCs have been plated in 6-well plates and supernatant has been discarded upon 2 h of plastic adherence. Subsequently, cells were differentiated in RPMI 1640 GlutaMax medium (Thermo Fisher Scientific, Massachusetts, USA) supplemented with 10% autologous serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES (Sigma–Aldrich, Steinheim, Germany), 1 mM sodium pyruvate and 50 µM 2-β-ME (Thermo Fisher Scientific, Massachusetts, USA) supplemented with 40 ng/ml recombinant human GM-CSF (Peprotech, NJ, USA) and human IL4 (Peprotech, NJ, USA) for 6 days. Differentiated cells were stimulated with the Pg extract in different concentrations (1-100 µg/ml) or 1% DMSO as a control. In parallel, 250 nM LPS stimulation was used to generate inflammatory dendritic cells. After stimulation, cells were harvested by cell scraping for further analysis and supernatant has been collected and snapfrozen for further analysis.
Antiproliferative MTT assay
The Pg extract was evaluated for possible in vitro anticancer activity against the MCF7 human breast cancer cell line. The effect of black poplar bud extracts on MCF7 breast cancer cells viability was evaluated by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The method was conducted as previously described . Briefly, 1x104 cells/well were seeded in 96-well culture plates and allowed to adhere overnight. On the second day, the cells were stimulated with different concentrations of poplar bud extracts (10, 25, 50, 75, 100 and 150 µg/mL) and were incubated for 72h. The Control group is represented by cells treated with the solvent dimethyl sulfoxide (DMSO, Sigma-Aldrich). After the 72h incubation period, the cells were treated with 10 µL of 5 mg/mL MTT solution from the MTT kit (Sigma-Aldrich) and incubated for an additional 3h. The obtained formazan crystals were dissolved in 100 µL of lysis solution provided in the MTT kit. The absorbance was determined at 570 nm with a microplate reader (BioRad, xMark Microplate Spectrophotometer).
Determination of the cytotoxic potential by Lactate Dehydrogenase (LDH) assay
The cytotoxic effect of Pg on MCF7 tumor cells was determined by LDH assay (Cytotoxicity detection kit, 11644793001, Roche). Briefly, 5x103 cells/well were seeded in 96-well culture plates and allowed to adhere overnight. The second day, the cells were stimulated with different concentrations of poplar bud extract (10, 25, 50, 75, 100 and 150 µg/mL) and incubated for 72h. After the incubation period, 100 µL from each well was transferred into a 96-well culture plate and mixed with 100 µL of reaction mixture (prepared according to the manufacturer's instructions) and incubated at room temperature for 30 min. The level of LDH release in the medium was measured at 490 nm and 680 nm using a microplate reader (BioRad, xMark Microplate Spectrophotometer). Untreated cells (low control) and cells treated with 1% (v/v) Triton- X100 (high control) were used to determine spontaneous and maximum release of LDH, respectively.
The scratch assay was performed in order for the assessment of the regressive effect of Pg extracts on the invasion capacity of MCF7 breast cancer cells. Several 2x105 cells/well were seeded onto 12-well culture plates until 90% confluence was reached. After that, the attached cells were scratched following the diameter of the well using a sterile pipette tip. The detached cells and cellular debris were removed by gently washing the wells with Phosphate Buffer Saline (PBS). Furthermore, the cells were stimulated with different concentrations of poplar bud extracts (10, 25, 50, 75, 100 and 150 µg/mL). Wells were captured on images at 0 h and 24 h, in order to compare the cell growth of the stimulated and the control cells (no stimulation) in early stages and at consistent times. Images were taken with Olympus IX73 inverted microscope provided with DP74 camera (Olympus, Tokyo, Japan) and cellSense Dimension software was used for analyzing the cell growth. The scratch closure rate was calculated as previously described by Moacă et al. :
where: At0 is the scratch area at time 0; At is the scratch area at 24h.
DAPI (4’,6-diamidino-2- phenylindole) staining
In order to investigate the apoptotic potential of Pg at the selected concentration, MCF7 cells were plated to an initial density of 5x105 cells/well onto 6-well plates overnight. On the following day, the medium was removed, and the cells were stimulated with a fresh one containing the testing compound to a final concentration of 10, 25 50, 75, 100 and 150 µg/mL, respectively, for 72 h. At the end of stimulation period, the medium was removed from the wells and the cells were washed with ice-cold PBS twice and were fixed with 4% paraformaldehyde in PBS, permeabilized with 2% Triton- X/PBS for 30 min, followed by a blocking step with 30% FCS/0,01% Triton-X. Finally, the cells were washed with PBS and stained with DAPI (300 nM) in a dark chamber for 15 min. Fluorescent images were taken at a magnification of 40X, with a fluorescence inverted microscope Olympus IX73, equipped with an integrated DP74 digital camera (Olympus, Tokyo, Japan).
Cell cycle analysis
To characterize cell cycle distribution, the DNA content analysis of the cells was determined by FACSCalibur flow cytometer (Becton‑Dickinson, Franklin Lakes, NJ, USA). The MCF7 human breast cancer cells were seeded into 6 well plates and treated with poplar bud extracts. After 48h the cells were collected and fixed with cold 70% ethanol (1000 µl) and stored for 30 min at 4˚C. After centrifugation (1500 RPM, 5 min), cold PBS was used to wash the cells. Afterwards, 50 µl of propidium iodide (BD Pharmingen, BD Biosciences) were added to the cells and then incubated for 10 minutes at 4°C. The untreated cells and the cells treated with 0.15% DMSO were used as a control and solvent control respectively. As a final step the percentage of cells present in the different cell cycle (G0, G1, S, G2) phases was determined using Flowing software 2.5.1.
Annexin V-FITC apoptosis assay
The Pg extract was screened for the flow cytometric analysis with Annexin V-FITC apoptosis detection kit (Sigma-Aldrich) following the manufacturer’s protocol. A number of 104 cells/well were seeded into a 6-well plate and left overnight. After 24 h, the media was removed and were added fresh medium treated with Pg extracts (10, 25, 50, 75, 100, 150 µg/mL). After 72 h the cells were trypsinized and were washed two times with Binding Buffer, centrifuged at 1500 RPM for 7 minutes, resuspended in Binding Buffer and incubated with 5 µL of Annexin V-FITC for 15 min at room temperature. After cell washing, the cell pellet was resuspended in a 190 µL Binding Buffer with 10 µL PI solution (propidium iodide). As control was used the untreated cells, and as a solvent control was used cells treated with 0,15% DMSO. Cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson) using the fluorescence channel FL1 for annexin and FL2 for PI. The results were analyzed using Flowing Software 2.5.1.
Angiogenesis evaluation using the chick chorioallantoic membrane (CAM) assay
In order to investigate the effects of the Pg on the process of angiogenesis we conducted an in vivo experiment using as biological material the chorioallantoic membrane of fertilized chicken (Gallus gallus domesticus) eggs under development. The eggs were incubated at 37°C and 50% humidity and were prepared according to the basic protocol with some modifications [25, 27]. On the third day of incubation 5-6 ml of albumen were removed, and subsequently an opening was cut on the upper side of the eggs. The eggs were further incubated until day 7 of incubation, when 10 µl of test (Pg 150 µg/mL) and control (0.1% DMSO v/v in double distilled water) samples were inoculated inside plastic rings on the developing vascular plexus of the CAM. Application of the samples was repeated daily (24h, 48h) and was followed by stereomicroscopic monitoring, capturing relevant in ovo images (Discovery 8 Stereomicroscope, Zeiss, coupled to Axio CAM 105 color, Zeiss digital camera). The photographs were further processed by Zeiss ZEN software, ImageJ and GIMP.
Cell viability and apoptosis assay
Cytotoxic effects of the Pg extract on dendritic cells were analyzed by FACS staining with DAPI for cell viability and Annexin V/7AAD for apoptosis.
Additionally, to the stimulation of the differentiated inflammatory dendritic cells with the Pg extract in different concentrations, 1 µM Staurosporin (LC Laboratories, MA, USA) was used as a positive control for apoptosis. After 24 h cells were harvested and washed twice with Annexin Binding Buffer (0,1M NaCl, 25mM CaCl2, 0,1M Hepes), resuspended in 500 µl Annexin Binding Buffer and stained with 5 µl Annexin V FITC (Immunotools, Friesoythe; Germany) per tube for 15 minutes in the dark. After this incubation 5 µl of 7AAD (Invitrogen, MA, USA) was added and stained for further 5 minutes in the dark. The cells were pelleted and resuspended in 400µl Annexin Binding Puffer containing 100 ng/ml DAPI (Sigma, Merck, Darmstadt) for cell viability staining. During the next hour the samples were measured using FACS Canto II, the data were analyzed with FlowJo software 7.6.5.
The supernatants were analyzed by ELISAs for IL-10 and IL-23 (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s manual. The cell pellet was harvested, and total RNA was extracted using the peqGOLD Total RNA Kit (peqlab, Erlangen, Germany) as recommended by the manufacturer. RNA concentration was measured using the Nano-Drop 1000 analyzer (Thermo Scientific) and adjusted to 1 µg/µL for cDNA synthesis using the high-capacity cDNA reverse transcription kit (Life Technologies, Carlsbad, CA). TaqMan® gene expression assays (Thermo Fisher, Dreieich, Germany) were applied for all genes of interest and for the housekeeping genes gapdh and fbxo38, (Primer Design, Southampton, UK). The Precision FAST Mastermix (Primer Design) was used and quantitative real-time PCR was run according to manufactures’ recommendations (7500 Fast Real-Time PCR System, Applied Biosystems). Data were evaluated using the mean of the two housekeeping genes as a reference.