The animals used in this study were housed and the experiments were carried out at an International Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at the National Health Research Institutes, Tainan, Taiwan R.O.C.. The Institutional Animal Care and Use Committees for Biotechnology and the National Health Research Institutes approved uses of animals in these studies (approval number: NHRI-IACUC-099070).
The Aurora-kinase inhibitor BPR1K653
Our previous structure-activity relationship studies (SAR) and X-ray co-crystallographic analysis had indentifed novel furanopyrimidine as Aurora kinase inhibitor 
. The pan-Aurora kinase inhibitor BPR1K653 () was synthesized from 4-chloro-6-phenylfuro[2,3-d]pyrimidine, which was originally obtained via a well-established 3-step process 
Human cervical carcinoma KB cells (this cell line was originally believed to be derived from an epidermal carcinoma of the mouth but has now been shown to have HeLa characteristics, purchased from ATCC®), nasopharyngeal carcinoma HONE-1 cells 
, colorectal carcinoma HT29 cells (purchased from ATCC®), oral squamous cell carcinoma OECM-1 cells 
, leukemia MV4-11 cells (purchased from ATCC®), myeloma IM9 cells 
were maintained in RPMI 1640 medium supplied with 5% fetal bovine serum. Human lung adenocarcinoma A549 cells and NTUB1 bladder cancer cells were maintained in RPMI supplied with 10% fetal bovine serum. KB-derived MDR1-expressing cell lines (i.e.
KB-VIN10 and KB-S15) and NTUB1-dervided MDR1-expressing cell line (i.e.
NTU0.017) were maintained in growth medium supplemented with 10 nM vincristine, 15 nM and 17 nM paclitaxel respectively. KB-VIN10 cells were generated in pervious study by vincristine selection and displayed over-expression of P-gp170/MDR1 
. KB-S15 and NTU0.017 cells were generated in previous studies by paclitaxel selection and also displayed over-expression of P-gp170/MDR1 
. KB-derived MRP1-expressing cell line, KB-7D, was maintained in growth medium supplemented with 7 µM VP-16. KB-7D cells were generated in pervious study by VP-16 selection and displayed over-expression of MRP1 
Kinase inhibition assay
Aurora-A and Aurora-B kinase - The recombinant GST-tagged Aurora-A (residues S123-S401) containing kinase domain was expressed in Sf9 insect cells. The recombinant full length His-tagged Aurora-B (residues M1~A344) was purchased from Invitrogen (PV3970). The kinase assay were carried out in 96-well plates with the tested compound at either 37°C (Aurora-A) for 90 min or 30°C (Aurora-B) for 120 min.
ALK – The recombinant His-tagged ALK (residues V1058-P1620) containing kinase domain was expressed in Sf9 insect cells. The kinase assay was carried out in 96-well plates with the tested compound at 30°C for 120 min.
CHK1/2 – The recombinant His-tagged CHK1 (residues M1-T476) or CHK2 (residues M1-L543) containing kinase domain were expressed in Sf9 insect cells. The kinase assay was carried out in 96-well plates with the tested compound at 30°C for 120 min.
c-Met – The recombinant GST-tagged c-Met (residues K956-S1390) containing kinase domain was expressed in Sf9 insect cells. The kinase assay was carried out in 96-well plates with the tested compound at 30°C for 120 min.
EGFR – The recombinant GST-tagged EGFR (residues G696-G1022) containing kinase domain was expressed in Sf9 insect cells. The kinase assay was carried out in 96-well plates with the tested compound at 37°C for 60 min.
FLT3 – GST-tagged FLT3-KDWT containing the FLT3 kinase catalytic domain (residues Y567~S993) were expressed in Sf9 insect cells. The FLT3WT Kinase-Glo assays were carried out in 96-well plates at 30°C for 4 h with the tested compound.
VEGFR1/2 – The recombinant GST-tagged VEGFR1 (residues R781-I1338) or VEGFR2 (residues V789-V1356) containing kinase domain were expressed in Sf9 insect cells. The kinase assay was carried out in 96-well plates with the tested compound at 30°C for 120 min.
Composition of the reaction buffers used in different kinase inhibitory assays is described in Figure S3
Two hundred cells in logarithmic growth phase were seeded in a 6-well plate. The cells were exposed to various concentrations of the test drugs for a three-generation period. At the end of the incubation period, cells were fixed and stained with 50% ethanol containing 0.5% methylene blue for 30 min. The plates were washed five times with water and allowed to air-dry. Colonies were countered manually. The IC50 value resulting from 50% inhibition of cell growth was calculated graphically as a comparison with the growth of the control group. Each value represents the average of at least three independent experiments run in triplicates.
Cell cycle analysis
Cell cycle progression was monitored using flow cytometry. After drug treatment, cells were trypsinized, washed with PBS and fixed in 80% ethanol at −20°C for 1 h. The fixed cells were stained with propidium iodide (containing RNase) at room temperature in the dark for 20 min. The DNA content was determined by a fluorescence-activated cell sorting IV flow cytometer (BD Biosciences, Franklin Lakes, NJ). For each analysis, 10,000 cells were counted and the percentage of cells in each phase was calculated using the ModFit LT software (Verity Software House, Topsham, ME). Experiments were repeated independently at least three times.
RT-PCR of MDR1
Total RNA was extracted with using TRIzol reagent (Invitrogen, Carlsbad, CA) and complementary DNA was synthesized from RNA with the SuperScript™ First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Polymerase chain reaction was performed with target-specific primers. MDR1 sense (forward) primer: 5′-GCCTGGCAGCTGGAAGACAAATRCACAAAATT-3′; MDR1 anti-sense (reverse) primer: 5′-CAGACAGCAGCTGACAGTCCRAGAACAGGACT-3′; GAPDH sense (forward) primer: 5′-ACCACAGTCCATGCCATCAC-3′ and GAPDH anti-sense (reverse) primer: 5′ TCCACCACCCTGTTGCTGTA-3′.
SDS-PAGE and Western Blot Analysis
Cells were lysed with ice-cold lysis buffer (10 mM Tris, 1 mM EDTA, 1 mM DTT, 60 mM KCl, 0.5% NP-40 and protease inhibitors). Total cell lysates were resolved on 10% and 12% polyacrylamide SDS gels under reducing conditions. The resolved proteins were electrophoretically transferred to PVDF membranes (Amersham Life Science, Amersham, U.K.) for Western blot analysis. The membranes were blocked with 5% non-fat milk at room temperature for two hours, washed twice with TBST (1% Tween) and then incubated with either anti-phosphorylated Aurora-A/-B/-C kinase antibody (#2914S, Cell Signaling Technology, Danvers, MA), anti-Aurora-A and -B kinase antibody (#ab1287 and #ab2254, Abcam, Cambridge, MA), anti-phosphorylated Histone H3 antibody (#9701, Cell Signaling Technology, Danvers, MA), anti-Histone H3 antibody (#9715, Cell Signaling Technology, Danvers, MA), anti-Cyclin B1 antibody or anti-Actin antibody (#sc-245 and #sc-130065, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Membranes were washed twice with TBST then subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature. Immunoreactivity was detected by Enhanced Chemiluminescence (Amersham International, Buckingham, U.K.) and autoradiography. Experiments were repeated independently at least two times.
Annexin V assay
Cells were cultured in chamber-slides, incubated with test agents for 48 h, and washed twice with PBS. Cells were labeled with Annexin-V-FLUOS reagent (catalog number #11858777001, Roche, Indianapolis, IN) for 30 min at room temperature. The cells were analyzed by fluorescence microscopy.
Real-time Caspase-3/-7 activity imaging
Caspase-3/-7 activity was analyzed with the MagicRed™ DEVD real-time caspase activity detection kit (catalog number #935, Immunochemistry Technologies LLC, Bloomington, MN). Briefly, cells were cultured in chamber-slides and incubated with test agents for various durations. Cells were then incubated with the Caspase-3/-7 substrate MR-(DEVD2) in culture medium for 1 hour, and then co-incubated with Hoechst 33342 for 15 min. Cells were viewed with a UV-enabled inverted-microscope at an excitation wavelength of 540 nm–560 nm and emission at 610 nm. Experiments were repeated independently at least two times.
Visualization of apoptosis by the TUNEL assay
Under in vitro conditions, cells were seeded and cultured in 8-well chamber-slides, and treated with various compounds. The treated cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min on ice, and permeabilized with PBST at room temperature. Apoptotic cells were stained by the TUNEL agent using the TMR (red) In-Situ Apoptosis Detection Kit (catalog number #12156792910, Roche Diagnostic, Mannheim, Germany). Cells were counterstained with DAPI to detect the nucleus, and examined by fluorescence microscopy. Amount of red fluorescence labeled (DNA fragmented) cells were counted and percentage of apoptotic cells were calculated as follows: Amount of the red fluorescence labeled cells ÷ Total cells available×100. Experiments were repeated independently at least two times.
Under in vivo conditions, tumors were dissected from the euthanized mice and instantly stored under −80°C. Tumor tissue sections were prepared from the use of cryostats (Leica Microsystems, Buffalo Grove, IL), and subsequently fixed with ice-cold methanol. Tissue sections were stained by the TUNEL reagent using Fluorescent (green) In-Situ Apoptosis Detection Kit (catalog number #11684795910, Roche Diagnostic, Mannheim, Germany). Cells were counterstained with DAPI to detect nucleus, and examined by fluorescence microscopy. Amount of green fluorescence labeled (DNA fragmented) cells were counted and percentage of apoptotic cells were calculated as follows: Amount of green fluorescence labeled cells ÷ Total cells available×100. Experiments were repeated independently at least two times.
Animals and implantation of cancer cells
Male nude mice (5–6-weeks-old) were purchased from the National Laboratory Animal Centre (Taiwan R.O.C.). The animals were s.c. implanted with 5×105 KB cells or 1×106 KB-VIN10 cells mixed with equal volume of Matrigel (Becton Dickinson) in 0.1 mL at one flank per mouse via a 22-gauge needle. Tumor growth was examined twice a week after implantation, and the volume of tumor mass was measured with an electronic caliper and calculated as 1/2×length×width2 in mm3.
Drug treatments and monitoring of the in vivo anti-tumor activity
BPR1K653 was dissolved completely in a vehicle mixture of DMSO/cremophor/saline (1
7). Selected dose of BPR1K653 was decided base on the following conditions: 1/2 of the dosage that caused noticeable body weight loss (>10%) in the treated mice during toxicity study. In the KB xenograft study, when the size of a growing tumor reached ≥75 mm3
, the xenograft tumor-bearing nude mice were treated with either BPR1K653 or VX680 i.p.
(5 mice per treatment group) at a dosage of 15 mg/kg or 30 mg/kg, respectively, for 5 days/week for 2 consecutive weeks. In KB-derived MDR1-overexpressing KB-VIN10 xenograft study, mice were treated with either BPR1K653 or VX680 (5 mice per treatment group) at a dosage of 15 mg/kg or 30 mg/kg respectively for 5 days/week for 3 consecutive weeks. The control group (5 mice) was treated with vehicle mixture only. Tumor size and animal body weight were measured every three days after drug treatment. Toxicity was evaluated based on the body weight reduction. At the end of the experiments (tumor size of the control >2000 mm3
), animals were euthanized with carbon dioxide.
Tumors were harvested and instantly stored at −80°C. Frozen cryostat sections were fixed with ice-cold methanol for 10 min. After washing with PBS, endogenous peroxidase was blocked using 3% hydrogen peroxide in TBS for 5 min. Immunostaining process was carried out according to the user's manual of the ABC Peroxidase Staining Kit (Pierce Biotechnology, Rockford, IL). Briefly, the tissues were incubated with a protein-blocking solution for 20 min, and subsequently stained with an anti-phosphorylated Histone H3 (Ser10) polyclonal antibody for 1 hour at room temperature. Then, the samples were incubated with the ABC reagent for 30 min, and subsequently incubated with the metal enhanced DAB substrate. The sections were counterstained with hematoxylin.
Pharmacokinetic studies of BPR1K653 in rats
Male Sprague-Dawley rats weighing 300–400 g each (8–12 weeks old) were obtained from BioLASCO, Taiwan Co., Ltd., Ilan, Taiwan. Animals were surgically prepared with a jugular-vein cannula one day prior to dosing and fasted overnight (~18–20 h) prior to dosing. Water was available ad libitum throughout the experiment. Single 5 mg/kg dose of BPR1K653, as a DMA/PEG (20/80, v/v) solution, was separately administered to groups of 3 rats each intravenously by a bolus injection via the jugular-vein cannula. At 0 (prior to dosing), 2, 5, 15 and 30 min, and at 1, 2, 4, 6, 8 and 24 h after dosing, a blood sample (0.15 mL) was collected from each animal via the jugular-vein cannula and stored in ice (0–4°C). Plasma was separated from the blood by centrifugation (14,000 g for 15 min at 4°C in a Beckman Model Allegra™ 6R centrifuge) and stored in a freezer (−20°C). All samples were analyzed for the parent drug by LC-MS/MS. LC/MS/MS conditions: The chromatographic system consisted of an Agilent 1200 series LC system and an Agilent ZORBAX Eclipse XDB-C8 column (5 µm, 3.0×150 mm) was connected to a MDS Sciex API3000 tandem mass spectrometer, which was equipped with a Turbo V™ ESI in the positive scanning mode at 600°C. Data was acquired via the multiple reactions monitoring (MRM) system. The MS/MS ion transitions were monitored at m/z of 541.4/106.4 for BPR1K653. The collision energy of 58.0 V was used for the analyst, BPR1K653. A gradient HPLC method was employed for the separation. Mobile phase A consisted of water containing 0.1% formic acid, and mobile phase B consisted of acetonitrile. The gradient profile was shown as follows (min/%B): 0.0–1.2/5, 1.3–3.9/95, 4.0–5.0/5. The flow rate was set to be 1.5 mL/min. The auto-sampler was programmed to inject 15 µL sample aliquots in every 5 min. The retention time of BPR1K653 was 2.39 min. Plasma concentration data were analyzed with non-compartmental method.
For all statistical analysis, values were expressed as mean ± SD. Values were compared using Student's t-test. P<0.05 was considered significant.