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 modifications14. 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 Liquid Chromatography-Mass Spectrometry (LC-MS). 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 LCMS 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= (114-115oC), 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 two and three 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). Mia Paca-2 and Panc-1 cells were seeded into 96-well plates at the density of 1x103 cells per well in quintuplicate for each drug concentration level and incubated at 5% CO2 and a temperature of 37oC. At 80–85% confluence, both Mia Paca-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 5μM. 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
Mia Paca-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, 100μL 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, 50μL 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
Mia Paca-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 96U 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 96U 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) (5μg/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 50μm) 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 (0-120mins), the concentration of HLM (20mg/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 200μL 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 12.5μm, 25μm, and 50μm 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 25mL 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 1x105 cells in a 25mL 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 37oC and 5% CO2. After treatment, cells were collected by trypsinization, fixed gently in 70% ethanol drop-wisely, and then stored at -20oC overnight. Then cells were washed in PBS and stained with 0.5mL 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 two 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, 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 ChemiDocTM XRS+ imaging system (Bio-Rad).
Immunohistochemistry (IHC) analysis
Patient-derived xenograft mice with tumors were euthanized, and tumor samples were excised from the mice, washed with PBS, placed in 10% buffered formalin for 24 h, and transferred to 70% ethanol for histopathological analysis. Histology 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 4μm15. 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)16-18. 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). For hematoxylin and eosin staining (H&E staining), the tumor sections were incubated in hematoxylin solution and then counterstained with eosin.
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.