Plant material and preparation of açaí hydroalcoholic extracts
The specimen was collected at Juçara’s park, an environmental protection area located at São Luís County. The plant material was authenticated by the Rosa Mochel Herbarium from School of Biological Studies, State University of Maranhão, where a voucher specimen was deposited (reference number of 30).
Fruits were harvested and stored at -20°C until use. After defrosting at room temperature, fruits were separated into three portions: bark, seed and total fruit (bark + seed). The procedures used to prepared the hydroalcoholic extracts have been described previously . Briefly, about 360 g of each different portion were washed in running water and boiled in distilled water for 5 minutes. After, they were macerated separately in 400 ml of ethanol PA for 2 hours with intermittent shaking and then kept in dark bottles at 4°C for 10 d. The extracts were filtered thought Whatman no. 1 filter paper, and the ethanol was evaporated under low pressure at 40°C. The extracts were then lyophilized (LIO-TOP model 202; Fisatom Equipamentos, São Paulo, Brazil) and frozen at -20°C until use.
Determination of total polyphenols in açaí extracts was performed by the Folin-Ciocalteau colorimetric method determined on a Spectrophotometer UV/VIS by monitoring the absorbance at 700 nm using gallic acid as a reference standard (50, 100, 150, 250 and 1000 mg/mL). Values were evaluated as the mg equivalent of gallic acid per g of extract .
Cell culture and treatments
The cell lines Caco-2 (ATCC, # HTB-37, Rockville, MD, USA) and HT-29 (ATCC, # HTB-38), both derived from human colon adenocarcinoma, and MCF-7 (ATCC, # HTB-22) and MDA-MB-468 (ATCC, # HTB-132, Rockville, MD, EUA), derived from human mammary adenocarcinoma, were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum, penicillin G (60 mg/l), and streptomycin (100 mg/l) at 37°C in a humidified atmosphere of 5% CO2.
The lyophilized extracts were dissolved in Milli-Q water, and the solution was filtered through a 0.2-μm pore syringe filter and stored at -20°C until use. The cultured cells were treated with 10, 20, and 40 μg/ml of the extracts for 24 and 48 h.
MTT viability assay
Cells (1 × 104 cell/ml) were cultured in 96-well plates in the presence or absence of the extracts for 24 and 48 h. The supernatant was removed, and 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in DMEM medium was added to each well. Cells were incubated in a CO2 chamber for 3 h with protection from light. The absorbance at 538 nm was measured with a Spectra Max 190 spectrophotometer (Molecular Devices, Sunnyvale, CA, EUA).
Cells were cultured in 96-well plates in the presence or absence of the extracts for 24 and 48 h. After treatment, the morphological features of the cells were evaluated by phase-contrast microscopy using an inverted microscope Axio Observer Z1, equipped with a camera, AxiocamHRc Ver.3 (Carl Zeiss Inc., Germany).
For transmission electron microscopy analysis, cells were grown in culture flasks treated with açaí extracts for 24 h. Subsequently cells were washed with PBS and fixed for 1 h with Karnovsky fixer (2.5% glutaraldehyde, 4% paraformaldehyde, and 0.1 M cacodylate buffer). Post-fixation was carried out with 1% osmium tetroxide in cacodylate buffer containing 0.8% potassium ferrocyanide and 5 mM CaCl2 for 45 min at 4°C. Subsequently, cells were dehydrated in a graded series of acetone and embedded in epoxy resin. Ultrathin sections (60 nm) were stained with uranyl acetate and lead citrate and examined using a Zeiss EM 906 (Jena, Germany) transmission electron microscope.
Nuclear staining and Caspase-Glo® 3/7 luminescent assay
The nuclear and chromosome counterstain, DAPI (4′,6-diamidino-2-phenylindole), was used for nuclear morphologic observations of treated cells with açaí extracts. To evaluate possible apoptotic activity of the extracts, we used a luminescent kit that measures caspase-3/7 activity (Caspase-Glo™ 3/7, Promega, WI, USA). After treatment with the extracts, 10 μl of Caspase-Glo™ 3/7 reagent was added to the culture, and after 90 min, the luminescence was analyzed in an illuminometer, Veritas Turner Biosystems (Madison, USA).
Total cell lysate preparation and western blot analysis
Total cell lysates were obtained by incubating cells in lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 150 mM NaCl, 2 mM EDTA, 10 mM Hepes [pH 7.4], 20 mM NaF, 1 mM orthovanadate, and a protease-inhibitor cocktail [1:100 dilution]), for 30 min at 4°C. After centrifugation at 10,000 × g for 10 min at 4°C, the supernatant was removed and stored at -20°C for subsequent analysis.
Equal amounts of cell protein (40 μg/lane), quantified using the BCA protein assay kit (BioRad, Hercules, CA, USA), were electrophoretically separated by SDS-PAGE on 13% gels and transferred to nitrocellulose membranes. Membranes were blocked and incubated overnight with the primary antibody, anti-LC3B (1:1500) (Sigma Chemical Co., St. Louis, MO, USA), and for 1 h with peroxidase-conjugated goat anti-rabbit (1:10,000). Proteins were visualized using an enhanced chemiluminescence kit (GE Healthcare). α-Tubulin was used as the loading control for each protein.
Statistical analysis was performed using GraphPad Prism 4.0 version for Windows (GraphPad Software, San Diego, CA). The data were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc tests, as appropriate. Results were considered statistically significant for p values < 0.05.