Reagents and antibodies: Media for cell culture were from Gibco BRL Laboratories (Rockville, MD). Fetal bovine serum (FBS) was from GBO (Buenos Aires, Argentina). Transwell chambers were purchased to Corning Inc. (New York, NY) and Matrigel was obtained from BD Biosciences (Bedford, MA). Monoclonal anti-LC3II and anti-actin were purchased from BD Biosciences (San Diego, CA). Acrylamide, horseradish peroxidase conjugated anti-mouse antibodies, were obtained from Sigma (St. Louis, MO). Hybond-P membranes and chemiluminescence reagents (ECL) were from GE Healthcare Bio-Sciences (Little Chalfont, UK). Other reagents for polyacrylamide gel electrophoresis and zymography were obtained from Bio-Rad (Richmond, CA) and Annexin-V Kit was purchased from Thermo Fisher Scientific (Waltham, MA).
Compounds: Preparation of levoglucosenone (compound 1), and the synthetic derivatives: 3-bromolevoglucosenone (compound 2), 4-(phenylthio)-dihydrolevoglucosenone (compound 3) and 4-(p-methyl-phenylthio)-dihydrolevoglucosenone (compound 4) has been described elsewhere [17].
Cell lines and culture conditions: LM3, MCF-7 and MDA-MB-231 cell lines were used in this study. The LM3 cell line was previously established in our laboratory from spontaneous BALB/c mammary adenocarcinoma with tumorigenic and metastatic capacities [18]. The human MDA-MB-231 (CRM-HTB-26) and MCF-7 (HTB-22) cell lines were obtained from ATCC. LM3 cells were grown in minimum essential medium (MEM) supplemented with 10% FBS and 80 µg/ml gentamycin. MDA-MB-231 and MCF-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM/F12) with the same supplement. All cell lines were cultured at 37°C in a humidified air atmosphere with 5% CO2. Serial passages were performed treating the monolayers with 0.25% trypsin (Invitrogen, Carlsbad, CA) and 0.02% EDTA in Ca2+ free and Mg2+ free phosphate buffered saline (PBS) twice a week.
Cell viability assay: To assess the effect of the compounds on cell viability, cells were seeded onto 96 well plates in standard culture conditions. Twenty-four hours later cells were treated with different concentrations of the compounds (range 1–40 µM) during 48 h. Cell proliferation was evaluated using the MTS assay (Celltiter 96TM Non-Radioactive Proliferation Assay, Promega Madison, WI), as described by the manufacturer. Data were analyzed using the R package “ic50” [19] in order to determine the half maximal inhibitory concentration (IC50) values for each compound.
Apoptosis detection: Cells growing on glass coverslips were treated with the IC50 value of the compounds or vehicle alone for 24 h and then stained with Acridine orange (10 µg/ml) and Ethidium bromide (10 µg/ml). Visualization was performed with a Nikon, Eclipse E400 epifluorescence microscope. Uniform green nuclei with organized structure were considered as live cells, orange and red nuclei were classified as early or late apoptotic cells respectively.
Apoptosis induction was further evaluated by Annexin-V staining and flow cytometry. Cells treated or not for 24 h with the IC50 obtained for the different compounds were collected and apoptosis was quantified as described by the manufacturer. Briefly, 1x106 cells were washed and resuspended in 100 µl of 1X binding buffer. Then, cells were incubated 15 min in darkness at room temperature with 5 µl of Alexa488-conjugated Annexin-V. Cells were washed with 1X binding buffer and finally 5 µl of propidium iodide was added. Cells were mixed in darkness at room temperature for 10 min, then 300 µl of 1X binding buffer was added, and cells were mixed in an ice bath at dark. Cell suspension was examined under 488 nm excitation wavelength by flow cytometry using an Epics Elite ESP coulter cytometer (Beckman Coulter).
Loss of mitochondrial potential: Cells growing on glass coverslips were treated for 48 h with the IC50 of the compounds or vehicle alone as control. Then cells were washed with PBS and stained with 150 nM of tetramethylrhodamine methyl ester (TMRM) fluorescent dye (Image-iT™ TMRM Reagent, Molecular Probes, Thermo Fisher Scientific, Waltham, MA) in PBS. After 30 min incubation cells were washed again and mounted in PBS-Glycerol (1:1). Photographs were taken using a Nikon Eclipse E400 epifluorescence microscope equipped with a digital Coolpix Nikon camera (Tokyo, Japan).
Autophagy: Cells treated or not for 48 h with the different compounds IC50 were lysed and autophagy was assessed by determining LC3-I and LC3-II expression levels by Western blot as previously described [20].
Western blot: Semiconfluent monolayers treated for 48 h with the IC50 of the compounds, or vehicle as control, were washed twice with ice-cold PBS and then lysed with 1% Triton X-100 in PBS by scrapping with a Teflon scrapper. Samples were denatured by boiling in sample buffer with 5% β-mercaptoethanol and run in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Fifty µg of protein was loaded in each lane and gels were blotted to Hybond-P membranes. Membranes were blocked by incubation during 1 h with PBS containing 5% skim-milk plus 0.1% Tween-20. Then membranes were incubated with the first antibody overnight at 4°C, and finally with a secondary antibody coupled to horseradish peroxidase. Detection was performed by chemiluminescence. Bands were digitalized with a Photo/Analyst Express System (Fotodyne Inc. Hartland, WI) and signal intensity was quantified with Gel-Pro Analyzer software.
Senescence: To detect senescence-associated β-galactosidase activity, a detection kit was used following the manufacturer’s instructions (Biovision, Milpitas, CA, USA). Briefly, cells were seeded onto 12 well-plates and treated with different doses of the compounds up to 72 h. After the incubation, cells were rinsed with PBS and fixed with 2% formaldehyde and 0.2% glutaraldehyde for 15 min at room temperature. Next, 470 µl of staining solution, 5 µl supplement for staining and 25 µl of a 20 mg/ml X-gal solution were added to each well. After overnight incubation at 37°C, cells were rinsed again with PBS and mounted in PBS-Glycerol (1:1). Photographs were taken using a Nikon Eclipse E400 epifluorescence microscope equipped with a digital Coolpix Nikon camera (Tokyo, Japan).
Preparation of conditioned media (CM): Secreted metalloproteases (MMPs) activity was evaluated in CM. Briefly, subconfluent cell monolayers growing in 6-well plates were treated or not with the IC50 dose of the compounds for 24 h. Then, cells were extensively washed with PBS and subsequently incubated in FCS-free medium for another 24 h. Finally, CM were individually harvested, the remaining monolayers were lysed using a buffer containing 0.1 N NaOH and 2% Ca2CO3, and cell protein content was determined. CM samples were aliquoted, stored at -40ºC and used once after thawing.
Quantification MMPs secreted activity by zymography: MMPs enzymatic activity was determined by quantitative zymography [21]. Briefly, CM samples were run on a 9% SDS-PAGE gels co-polymerized with gelatin (1 mg/ml) under non-reducing conditions. After electrophoresis, gels were washed for 30 min with 2% Triton X-100 and subsequently incubated at 37°C for 48 h in a buffer containing 0.25 M Tris-HCl, 1 M NaCl, and 25 mM CaCl2 (pH 7.4) for activity detection. Finally, gels were fixed and stained with Coomassie Brilliant Blue (0.1% Coomassie R-250 in 10% acetic acid and 30% methanol). Gelatinolytic bands were visualized as negative staining and quantified using the Gel Pro Analyzer program. Data were expressed in arbitrary units and was relativized to cell lysates protein content.
Migratory capacity: Wounds of approximately 400 µm width were performed in LM3 and MDA-MB-231 subconfluent monolayers, and cells were then allowed to migrate into the cell-free area for a period of 18 h in the presence or not of the IC50 dose of the compounds and low FCS concentration (1%). The same spot was photographed at different times using an inverted microscope (Eclipse TE2000-S, Nikon) equipped with a digital camera and the migratory area was determined using the Image-ProPlus 4.5 software. Cell migration was expressed as the percentage of the area occupied by the migratory cells in the original cell-free wounded area.
Invasion assay: The effect of the compounds on invasive capacity was analyzed employing transwell culture cameras (Corning, Lowell, MA). Filters (8 µm membrane pores) were coated with 0.1% gelatin on the lower side and with a thin layer (250 µg/ml) of Matrigel (Becton Dickinson Labware, Bedford, MA) on the upper side. The lower chamber contained 0.5 ml of culture media supplemented with human fibronectin (16 µg/ml) (Sigma, St. Louis, MO) as chemoattractant. Cells (1x105) pretreated or not for 24 h with the IC50 dose of compounds were seeded in the upper chamber in culture medium without FCS. Eighteen hours later, filters were removed, fixed with Carnoy and the upper surface was wiped with a cotton swab to remove non-invasive cells. Finally, the filters were stained with DAPI. Cells that invaded Matrigel, passed through the pores, and reattached the lower surface of the filters were considered as invasive and their fluorescent nuclei were counted using an epifluorescence microscope (Eclipse TE2000-S, Nikon). The number of cells per field was determined in 10 randomly selected fields.
Animals: For in vivo assays, randomized inbred female BALB/c mice, 2–4 months old, obtained from the Animal Care Division of the Institute of Oncology “Angel H. Roffo” were employed. Mice were housed 6 per cage, kept under an automatic 12 h light/12 h darkness schedule, and provided with sterile pellets (Cooperacion, SENASA N° 04-288/A) and tap water ad libitum. For housing, rectangular polycarbonate cages (20 x 29 x 14 cm) with irradiated pine wood (15 KGy) as bed were employed. All animal studies were conducted in accordance with the standards of animal care as outlined in the NIH and ARRIVE Guide for the Care and Use of Laboratory Animals. Besides, protocols have the approval of the Committee for the Use and Care of laboratory Animals (CICUAL) of the Institute of Oncology “A. H. Roffo”, University of Buenos Aires. Animals were euthanized by CO2 inhalation at the experimental endpoint. We established the human endpoint when mice show one of the following signs: Loss of > 20% of the initial weight, lethargy, bristling coat and/or hemorrhagic diarrhea.
Lung colonization assay: To study the effect of the compounds on lung colonization, an experimental metastasis assay was performed as previously described [22]. Briefly, 2x105 LM3 cells pretreated or not with compounds for 24 h were injected in the lateral tail vein of syngeneic BALB/c mice using a 27-gauge needle. Cell viability was higher than 95% as determined by trypan blue exclusion test, and the order of injection of different groups was randomized to eliminate any difference that may bias the outcome. Mice were monitored daily and sacrificed 21 days later. Lungs were removed, fixed in Bouin’s solution and the number of superficial lung nodules was counted under a dissecting microscope.
Tumor growth: Exponentially growing LM3 cell monolayers were harvested with trypsin-EDTA, washed with MEM and resuspended in the same medium at a final concentration of 1x106 cells/ml. Then a 0.2 ml cell suspension was subcutaneously injected in mice left flank. Cell viability was higher than 95% as determined by trypan blue exclusion test. Animals were randomly divided into three groups and once the tumor was detected, they were treated twice a week with an intraperitoneal injection of 60 µg/kg of the compounds 1 or 2 or with vehicle in physiological solution as control. Mice were monitored daily and tumor-perpendicular diameters were measured with a sliding caliper twice a week. Tumor volume was calculated using the following formula V= ¾ (π x L x 2 W), where L is the longest and W is the shortest diameter. Animals were necropsied 21 days after cell inoculation.
Statistical Analysis: All assays were performed in triplicate, and independent experiments repeated at least twice. Statistical differences between groups were calculated by applying ANOVA, Chi Square or Kruskal-Wallis tests, as indicated. A value of p < 0.05 was considered to be significant.