Materials
5-FluoroUracil (5-FU) was purchased from AK Scientific (Union City, CA). Pancreatic cancer MiaPaCa2 (ATCC® CRL1420™) and Panc-1 (ATCC® CRL1469™) were bought from American Type Culture Collection (ATCC) (Manassas, VA). All other solvents and reagents for analysis were purchased from Sigma Aldrich (St. Louis, MO).
Synthesis of MFU
The synthesis of MFU followed a method described by Zasada et al., with some modifications [14]. To a solution of 5-FU (2.1 g, 16 mmol), tetrahydrofuran-2-yl acetate (4.2 g, 32 mmol) in dimethylformamide (DMF, 15 mL) was added to 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU; 5 g, 32 mmol) at room temperature while stirring at 450rpms (Fig. 1). The resulting solution was heated and stirred for 24 h. Most of the DMF was removed in vacuo. The residue was diluted with EtOAc (400 mL) and washed with brine (200 mL). The Organic layer was dried and filtered. The filtrate was concentrated and followed by separation using chromatography on silica gel using EtOAc/Hexane (50%) to obtain MFU. The compound obtained was then crystallized from EtOAc/Hexane as white solid pellets.
The MFU synthesized with the method mentioned earlier was analyzed by proton and carbon-13 Nuclear Magnetic Resonance (NMR) spectroscopy, Micro-elemental analysis, High Performant Liquid Chromatography (HPLC), and High-Resolution Mass Spectrometry (HRMS). The melting point for the solid MFU compound was determined. The purity of MFU was calculated to be 99.6%, as determined by NMR and elemental analysis. Elemental analysis was performed by an independent lab (Atlantic Microlab, Inc., Norcross, GA.). HPLC and HRMS were formed by an independent laboratory to confirm NMR findings.
MFU: 1H NMR (CDCl3, 300 MHz): 7.36 (1H, d, JH-F = 6.0 Hz), 6.60–6.67 (1H, m), 5.97–5.99 (1H, m), 4.29–4.36 (1H, m), 4.20–4.26 (1H, m), 3.93–4.04 (2H, m), 2.44–2.54 (1H, m), 2.33–2.42 (2H, m), 2.20–2.28 (1H, m), 2.03–2.13 (2H, m), 1.86–1.96 (2H, m).
13C NMR (CDCl3, 151 MHz) δ (a mixture of two rotamers): 157.28 (d, J = 25.4 Hz), 157.17 (d, J = 25.4 Hz), 148.2, 148.66, 139.94 (d, J = 234.3 Hz), 139.87 (d, J = 234.3 Hz), 121.75 (d, J = 34.2 Hz), 121.58 (d, J = 34.2 Hz), 87.97, 87.87, 85.20, 84.99, 70.75, 70.74, 70.27, 70.21, 33.04, 32.92, 28.78, 28.73, 26.51, 26.47, 23.76, 23.70. MP = (114-115 °C), Rf value = 0.36 (100% ethyl-acetate). HPLC (H20/ACN/TEAA (80:15:5), % purity = 100%, RT = 1.85 min) MS (M + H) + = 270.08. Molecular formula: C12H15FN2O4, Mol wt: 270.02. Elemental analysis: Calculated for C12H15FN2O4: C 53.33, H 5.59, N 10.37; Found: C 53.20, H 5.61, N 10.40.
Cytotoxicity in 2 and 3 dimensional (2D and 3D) cultures
2D MiaPaca-2 and Panc-1 cells
Both cell lines were incubated in Dulbecco’s modified Eagle medium (DMEM) with high glucose and l-glutamine, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PenStrep) [2] and 2.5% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). MiaPaca-2 and Panc-1 cells were seeded into 96-well plates at the density of 1 × 103 cells per well in quintuplicate for each drug concentration level and incubated at 5% CO2 and a temperature of 37 °C. At 80–85% confluence, both MiaPaca-2, and Panc-1 cells were treated with MFU. A stock solution of MFU was prepared with distilled water and serially diluted with the growth medium to prepare varied concentrations: thus 80, 40, 20, 10, and 5 μM. Both cells were treated with 200 μL of each drug concentration in quintuplicate and incubated for 48 h. At termination, 20 μL of 0.05% resazurin sodium salt (Alamar blue®) was added and incubated at optimum conditions (5% CO2, 37 °C) for four h [3]. Fluorimetric analysis was determined at an excitation wavelength of 560/580 nm and emission wavelength of 590/610 nm, and the percent viable cells per concentration was calculated.
3D spheroid of Mia Paca-2 and Panc-1 cells
MiaPaca-2 and Panc-1 cells were plated in 3D formation 96-well plates, Nunclon Sphera® at seeding density of 5.0 × 104 and 2.0 × 104 in 100 μL/well of the growth medium, respectively, the volume of cell suspension in each well plate was then made up to 200 μL with growth medium and centrifuged at 1500rpms and incubated for 24 h to allow for spheroid formation. At treatment, 100 μL of supernatant was replaced with the drug in the growth medium prepared as described under 2D viability studies of Mia Paca-2 and Panc-1 cells and incubated for 48 h. At termination, 50 μL of 0.05% resazurin sodium salt (Alamar blue®) was added to each well and gently dispersed by pipetting and incubated for 4 h. Fluorometric analysis was measured as described above.
MFU inhibition of spheroid formation
MiaPaCa-2 and Panc-1 cell suspensions (5.0 × 104 and 2.0 × 104 in 100 μL/well) in treatment media were seeded into the wells of a 96 U Nunclon Sphera plate (Thermo Scientific, Waltham, MA). After 24 h, equivalent amounts of varying concentrations of MFU (0–10 μM) were supplemented to each well of the 96 U plate. The ability of the cells to form viable spheroids was examined at 48 h post-exposure to MFU. The viabilities of the spheroids were determined by staining them with Acridine Orange (AO)/Ethidium Bromide (EB) (5 μg/mL) solution and then capturing fluorescent and bright-field images. The ratios of the fluorescent intensities of AO over EB for the respective MFU concentrations used were computed, and the data was graphed using GraphPad Prism 5.0.
Stability of MFU
Human liver microsome (HLM) stability of MFU
The incubation of MFU (concentration 50 μm) with HLMs was performed in phosphate buffer (pH 7.4) at 37 °C. Then, the incubated samples were shaken for a specified time at 150 rpm. The optimization of conditions for carrying out the process included the incubation time (0–120 min), the concentration of HLM (20 mg/mL), and the reaction buffer consisting of an enzymatic reaction cofactor NADP/NADPH. The blank samples were of the same composition as the test samples; however, they did not contain any of the studied biologically active compounds. The process was terminated with 200 μL of ice-cold methanol. Thereafter, samples were immediately prepared and analyzed with the use of HPLC-UV/MS. All samples were prepared in triplicate. All samples were incubated and treated as described above and as also described in detail as per the manufacturer’s protocol (ThermoFisher Scientific).
In vitro stability of MFU
Cells (Mia Paca-2 and Panc-1) were cultured in Dulbecco’s modified Eagle medium (DMEM) with high glucose and l-glutamine, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PenStrep) and 2.5% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Additionally, MFU was added to the culture media at the concentrations 12.5 μm, 25 μm, and 50 μm for 24 h and 48 h. Cells were cultured at 37 °C in humidified atmosphere of 5% CO2. Cells were plated in T25 culture flask at the density of 1.5 × 105 cells per cm2 and grown for 48 h. Then the medium was aspirated at 24 or 48 h. Finally, MFU and potential metabolites were analyzed after filtration from the culture medium.
MFU induced apoptosis (flow cytometry)
In the morphologic analysis, Annexin V/propidium iodide staining was used as per the manufacturer’s instructions to study the mode of cancer cell death upon exposure to MFU. Mia Paca-2 and Panc-1 cells were seeded into a 25 mL culture flask at 2.0 × 104 cells/mL and left at 37 °C for 24 h to attach. Cells were exposed to MFU (0–10 μM) for 48 h, followed by washing in PBS and labeling with FITC-conjugated Annexin V for 20 min in the dark. Cells were then washed and analyzed using a Becton Dickinson FACSort flow cytometer with CellQuest software (Mansfield, MA).
Cell cycle analysis
The percentage of cells in the G1 (apoptotic cells), S, and G2/M phases was determined by DNA flow cytometry. In brief, Mia Paca-2 and Panc-1 cells were plated at 1 × 105 cells in a 25 mL flask and allowed to grow until 75–80% confluence. Cells were incubated with different concentrations of GemHCl and MFU (0–10 μM) for 48 h at 37 °C and 5% CO2. After treatment, cells were collected by trypsinization, fixed gently in 70% ethanol drop-wisely, and then stored at -20 °C overnight. Then cells were washed in PBS and stained with 0.5 mL PI/RNase for 30 min at room temperature in the dark. Cells were then collected and analyzed using a Becton Dickinson FACSort flow cytometer with CellQuest software (Mansfield, MA).
Western blot
Panc-1 cells were plated in a T25 flask and cultured for 2 days, respectively, and different concentrations of MFU were added for 48 h. The corresponding cells were collected, washed with PBS, and lysed with RIPA cell lysis buffer to extract the total proteins. The extracted proteins were loaded and subjected to electrophoresis, transferred to a PDVF membrane, the blots were then cut at appropriate molecular sizes prior to hybridization and incubated in the primary antibody overnight. The following day, the primary antibody was recovered, and the corresponding secondary antibody incubated. The immune complexes were detected using enhanced chemiluminescence solution (Biorad) and visualized using the ChemiDoc™ XRS+ imaging system (Bio-Rad).
Animal studies
Ethics statements
Eight-week-old mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The mice were housed in a virus-free environment with indoor light and temperature controlled and provided access to food and water ad libitum for 1 week before treatment started. All procedures with mice were in strict accordance with the National Institutes of Health Guide for the care and Use of Laboratory Animals and the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines. This was approved by the Florida A&M University Animal Care and Use Committee.
Tumor transplantation
Tumor tissue was surgically implanted in the left flank of immune-compromised mice as previously described [15]. A viable portion of resected tissue was isolated immediately following resection of primary PCa specimens to minimize critical ischemia time. The PCa tissue was then implanted subcutaneously into 8-week-old mice (n = 12). Xenografts were allowed to grow to a maximum of 1.5 cm before implantation to the flank of the new host.
Tumor efficacy studies
In this study, mice bearing surgically implanted tumors were randomized into groups as control, GemHCl and MFU (n = 5/group) once tumor volumes became palpable and reached a range of 70–100 mm [3]. Baseline tumor volumes were established, and dosing initiation began with intravenous administration of 40 mg/kg GemHCl and MFU (with Gem equivalent doses) twice weekly for 6 weeks. Tumor measurements were performed every other day. Tumor volumes were measured using calipers and calculated using the following equation: V = (L*(W)2)/2, where V is volume (mm3), W (width) is the smaller of two perpendicular tumor axes and the value L (length) is the larger of two perpendicular axes. Tumor growth volumes were calculated for each treatment group.
Euthanization
Carbon dioxide (CO2) flow to the chamber was adjusted to 3 l per minute for 2 to 3 minutes and observed each mouse for lack of respiration and faded eye color. The CO2 flow was maintained for a minimum of 1 minute after respiration was.
ceased and followed by decapitation with scissors. The tumors were then incised and prepared for immunohistochemistry studies.
Immunohistochemistry (IHC) analysis
Tumor samples excised from the mice, washed with PBS, placed in 10% buffered formalin for 24 h, and transferred to 70% ethanol for histopathological analysis. Immunohistochemistry was performed by Histowiz Inc. (histowiz.com) using a Standard Operating Procedure and fully automated workflow. Samples were processed, embedded in paraffin, and sectioned at 4μm [16]. Immunohistochemistry was performed on a Bond Rx autostainer (Leica Biosystems) with enzyme treatment (1:1000) using standard protocols. Antibodies used were rat monoclonal F4/80 primary antibody (eBioscience, 14–4801, and 1:200) and rabbit anti-rat secondary (Vector, 1:100) [17,18,19]. Bond Polymer Refine Detection (Leica Biosystems) was used according to the manufacturer’s protocol. After staining, sections were dehydrated, and film cover slipped using a Tissue-Tek Prisma and Cover-slipper (Sakura). Whole slide scanning (40×) was performed on an Aperio AT2 (Leica Biosystems).
Statistical analysis
All results were presented in the form of means ± SEM. The difference between GemHCl and MFU treatment groups was analyzed using ANOVA, and where necessary, significance was considered for p values < 0.05. All experiments were performed in at least triplicates, and analysis was done using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA). When necessary, results were presented in simple tables, graphs, and bar charts.