Cell Culture
The murine macrophages cell line P388/D1 (ATCC® CCL-46™) was obtained from the lymphoma site of the mouse. Cells were cultured as a monolayer in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) mixed with RPMI-1640 Medium (Sigma-Aldrich, St. Louis, MO, USA) in 1:1 ratio. The medium was supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and an antibiotic (streptomycin/penicillin). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were characterized by adherent lymphoblastic morphology. When needed, the cells were washed with PBS and removed by trypsinization (0.025% trypsin and 0.02% EDTA; Sigma-Aldrich).
Drug preparation
Betulin (BE) and betulinic acid (BA) were purchased from Sigma company (Sigma, Poznan, Poland). Betulin derivatives were synthesized by prof. Marcin Drąg and Marcin Poręba on Wrocław University of Technology according to the already published paper [25]. Dexamethasone was used in the form of the injection drug (Dexaven®, 4 mg/ml, Bausch Health, Ireland). Due to the fact that Dexaven® is composed of dexamethasone sodium phosphate, the in vitro experiments were carried out after 12 h incubation in plasma containing culture medium, which is greater than the time required for drug hydrolysis in plasma (according to a paper by Samtani et. al) [46].
MTT Viability Assay
Cells’ viability after 48 h incubation with test compounds was analyzed with mitochondrial activity assay (MTT). The culture medium was removed from each well, and 100 µL of 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliµM bromide, Sigma-Aldrich) solution in PBS buffer was added. After 2 h of incubation at 37°C, acidified isopropanol (100 µL, 0.04 M HCl in 99.9% isopropanol) was added to dissolve the formazan crystals. The samples were fully dissolved by the pipet mixing technique. The absorbance of each well was measured at 570 nm using a multiplate reader (GloMax, Promega, Walldorf, Germany). The results were expressed as the percentage of viable cells relative to untreated control cells.
Fluorescent Studies
Anti-HSP-70 antibody (sc-32239, Santa Cruz, USA) was applied to assess the level of HSP-70 in P388D1 cells after incubation with BA, BE, and its derivatives. Anti-COX-2 antibody (C6827, Thermo Fisher Scientific, Waltham, MA, USA) was applied to assess the level of COX-2 in P388D1 cells after incubation with BA, BE, and its derivatives. The cells were incubated on in Petri dishes for 24 h to attach. Afterward, the culture medium was replaced with a medium containing the analyzed compounds. 24 h incubation was performed. Afterward, the cells were washed with PBS and fixed with 4% formalin. As soon as three replicates were performed, the single staining procedure was performed to obtain similar fluorescence between the same samples from different replicates. The samples were initially treated with Triton-X100 for 5 minutes to permeabilize the membranes. Following, the incubation with FBS was performed for 1 h. Next, the cells were washed with Triton-X100, and a primary antibody was added for 1 h incubation. Following, the cells were washed with PBS and the secondary antibody was added for 1 h incubation. In the end, the samples were washed with PBS. The samples were observed on the Olympus IX53 microscope (40x, Olympus, Tokyo, Japan) after blue and green laser excitations (depending on the fluorophore). The fluorescence of cells on each sample was analyzed in the CellProfiler software. Each sample consisted at least three photographs. All the data was plotted and statistically analyzed.
Immunocytochemical staining
Immunocytochemical staining aimed to assess the expression and distribution of HSP-70 in P388PD1 cells after the incubation with BA, BE and its derivatives. The cells were incubated on the 10-well microscopy slides overnight. Afterward, the media was replaced with culture media containing tested compounds in 0.5 and 2 µM concentrations. After 24 h the cells were washed with PBS. Then, cells were fixed in 4% formalin. Afterward the cells were washed in PBS once again. Hydrogen Peroxide Block was added for 10 minutes. After the time, the cells were washed in PBS with 1% Triton X. Protein Block agent was incubated with the cells for 10 minutes Further, the cells were incubated with the first order antibodies for 24 h in 4oC. Afterward, the cells were washed with PBS and 1% Triton X. Mouse Complement agent was added for 10 minutes. Afterward, the cells were washed with PBS with 1% Triton X, and Dab mixture (1 Dab : 50 Dab substrate) was applied for 10 minutes in the dark. Next, the cells were washed for 10 minutes in distilled water. After that, the samples were stained with hematoxylin for 1 minute. The excess of the stain was washed with water for 30 minutes. The cells were dehydrated by incubation for 5 minutes in each of ethanol solutions: 50%, 60%, 70%, 80%, 90%, and 96%. Xylene I and II were used in the last step of the preparation of the samples. In the end, DPX was used to mount the glass top of the sample. The samples were observed on the light microscope (Olympus BCX43, Tokyo, Japan). Plan-Apochromat 40x and 100× objectives (Olympus) were used to capture the images.
Confocal Microscopy Studies of IFNGR1
Phospho-IFNGR1 staining (1:500, PA5-38504, Thermo Fisher) was performed to visualize the distribution of the receptor in the murine macrophages following the incubation with BA, BE, and its derivatives. The cells were incubated on cover glasses in Petri dishes overnight to attach. After that, 48 h incubation with test compounds was performed, then cells were fixed in 4% formalin solution. The samples were treated with Triton-X100 for 5 minutes to permeabilize the membranes. Following, the incubation with FBS was performed for 1 h. Next, the cells were washed with Triton-X100 and a primary antibody was added for 1h incubation. Following, the cells were washed with PBS, and the secondary antibody (Alexa Fluor 488) was added for 1 h incubation. In the end, the samples were washed with PBS. Fluorshield™ with DapI (4,6-diamidino-2-phenylindole) was applied to visualize the nuclei and to mount the cells. The samples were observed on the Olympus FluoView FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).
ELISA assay for IL-6 release quantification
Secretory properties of the P388D1 cells after 48 h incubation 0.5 µM with BA, BE, and its derivatives were analyzed with Mouse IL-6 ELISA Kit (ab100712, Abcam). Cells were seeded in 20 000 cells / well density on the 96-well plate. After 24 h adhesion-required time, the cells were treated with BA, BE, and its derivatives for 48 h. Afterward, the culture medium was collected from each well and stored at -20oC, until three replicates of the experiment were performed. When the whole material was gathered, the medium was transferred to a 96-well plate coated with antibodies specific for Mouse IL-6. The wells were washed afterward, and a biotinylated anti-mouse IL-6 antibody was added. Next, after washing away, the unbound antibody, the HRP = conjugated streptavidin, was pipetted to the wells. In the end, the stop solution was added to each well, so the color changed from blue to yellow – proportionally to the total amount of IL-6 in each well. The absorbance was measured at 450 nm, and the data was plotted.
COX-2 (Ovine) Colorimetric Inhibitor Screening Assay
COX peroxidase activity was measured by the colorimetric method where the oxidized form of N, N, N ', N'-tetramethyl-p-phenylenediamine (TMPD), which is the substrate for most enzymes with peroxidase activity, was chanded. COX peroxidase component converts arachidonic acid to the reduction form PGG2 (prostaglandin G2) and then to corresponding alcohol PGH2 causing the oxidation of TMPD and resulting in a change in color measured at 590 nm. 10 µL of the test substance at a concentration of 20 µM and 100 µM was added to the incubation mixture consisting of 150 µL of Tris-HCl buffer (0.1 M, pH 8), 10 µL of heme and 10 µL of the COX-2 enzyme. The mixture was incubated for 5 min at 25°C, 20 µL of colorimetric substrate solution (TMPD) was added, followed by 20 µL of 1.1 mM arachidonic acid, the reaction was incubated for 2 min at 25°C, and the absorbance was measured at 590 nm using BioTek spectrophotometer microplate reader MQX200 (BioTek, Winooski, VT, USA).
All of the test samples were dissolved in DMSO. DMSO alone was used as a blank. The inhibitory activity was expressed as percentage inhibition calculated as (absorbance of the control minus absorbance of the test drug) / (absorbance of the control) x 100%. Experiments were done in triplicate.
Molecular Docking Studies
Molecular docking was performed with the CB-dock online server [47]. This is a blind docking server, which predicts binding sites of proteins and also calculates docking scores using Autodock Vina docking software [48]. Protein structures were downloaded from RCSB PDB database [49]. Structures of compounds were either downloaded from the PubChem database or were created with Avogadro Software [50]. Cyclooxygenase-2 (COX-2) with PDB code 4COX was chosen for docking. Crystal structures 4COX was available in complex with co-crystallized ligand- Indomethacin. First, the co-crystallized ligand was re-docked, and the enzyme's binding site was identified. Additionally, dexamethasone was docked for a comparison of the binding mode. The results of the molecular docking were anaLysed, and 2D interaction diagrams of the best-docked pose were created with BIOVIA Discovery Studio Visualiser [51]. For the generation of 3D visualization of protein-ligand complexes of chosen compounds, Pymol software was used [52]., The supplementary materials section included the results of all docking studies.
Statistical Analysis
Viability experiments were performed in at least 3 biological replicates. IFNGR1 was examined independently in immunofluorescence (confocal and fluorescent microscopy) and immunocytochemical studies. ELISA for IL-6 assessment was performed in 3 biological replicates. HSP70 staining studies were performed in at least 3 replicates, involving immunofluorescence and immunocytochemical staining. Data presented in the paper shows the mean of the obtained photographs. In the paper, the authors presented the staining, which was the most consistent with other data.
Data were expressed as mean ± SD and analyzed by two-way ANOVA (in GraphPad Prism 8), with p < 0.05 being considered statistically significant.