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Supplementary Materials and Methods
Real-time RT-PCR
Genomic DNA was isolated from washed cell pellets of eight biliary tract cancer cell lines (SNU-245, 308, 478, 869, 1079, 1196, HuCCT1, and
TFK-1) and MKN45 cells using Qiagen QIAamp DNA kit (Qiagen, Hilden, Germany). The primers used in the PCR
reaction were as follows: MET, forward primer 5’-TGTTGCCAAGCTGTATTCTGTTTAC-3’ and reverse primer 5’TCTCTGAATTAGAGCGATGTTGACA-3’; β-actin, forward primer 5’-TCATCACCATTGGCAATGAG-3’ and reverse primer 5’CACTGTGTTGGCGTACAGGT-3’.
Amplification reactions were conducted in 20 µL volumes for 94 ℃ for 5 min, 25 cycles at 94 ℃ for 30 s, 53 ℃ for 30 s, and 72 ℃ for 30 s,
followed by a final extension for 10 min at 72 ℃. The levels of MET gene mRNA were quantitatively analyzed via real-time RT-PCR assays
with
SYBR Green I (Molecular Probe) using an Cycler instrument (Bio-Rad) with more than duplet reactions. Each relative mRNA expression level
was calculated by normalization to the mean volume of β-actin.
Cell Growth Inhibition Assay
Tetrazolium dye (MTT; Sigma-Aldrich) assays were used to evaluate the growth inhibitory effect of PF00299804. The cells were seeded on 96well plates, incubated for 24 h, and then treated with PF00299804 for 72 h at 37℃. After drug treatment, MTT solution was added to each well
and incubated for 4h at 37℃ before the medium was removed. Then, DMSO was added and shaken for 30 min at room temperature. Cell
viability was determined by measuring absorbance at 540 nm in a microplate reader (Versa Max, Molecular Devices). Six replicate wells were
used for each analysis, and at least three independent experiments were conducted. Data points shown represent the mean while bars represent
the SE.
To evaluate the effects of PF00299804 administered in conjunction with 5-FU or cisplatin, cells were treated with serial dilutions of each drug
individually and with both drugs simultaneously at a fixed ratio. Two BTC cell lines (SNU308 and SNU478) were exposed to
PF00299804 with 5-FU or cisplatin at a ratio of 1:10 (0.005, 0.01, 0.05, 0.1, 0.5 μmol/L) or 1:100 (0.001, 0.002, 0.004, 0.008,
0.016 μmol/L). The other BTC cell lines (SNU-245, 869, 1079, 1196, HuCCT-1, and TFK-1) were exposed to PF00299804 with
5-FU or cisplatin at a ratio of 1:1(0.05, 0.25, 0.5, 2.5, 5 μmol/L) or 1:10 (0.01, 0.05, 0.1, 0.5, 1 μmol/L).
After 72 h of exposure, cell viability was measured using an MTT assay. The methods described by Chou and Talalay were then used to
determine if a synergistic effect existed. Analysis of the median effect was conducted using the Calcusyn software (Biosoft) to
determine a combination index value (CI > 1, antagonistic effect; CI = 1, additive effect; CI <1, synergistic effect).
Western Blot Analysis
Cells were incubated with PF00299804 in 10% FBS media. After 24 h, the cells were treated with lysis buffer. The same amount of protein was
then obtained from each suspension and subjected to SDS-PAGE, after which it was transferred to nitrocellulose membranes. After blocking with
buffer, the membrane was incubated with primary antibodies at 4℃ overnight. Antibodies against RRM1 were purchased from Abcam
(Cambridge, UK), and TS, and MET antibodies were obtained from Cell Signaling Technology (Beverley, MA, USA), and anti-actin antibody
was acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Supplementary figure 1
SNU 478
SNU 869
PF00299804
C
PF00299804
C
RRM1
Actin
Supplementary Figure 1. RRM1 downregulation by PF00299804 treatment in SNU478 cells. SNU478 and SNU869 cell
lines were treated with increasing doses of PF00299804 (0.01, 0.1, 1 μmol/L) for 24h, after which the extracts were
immunoblotted with RRM1 antibodies.
Supplementary figure 2
MET
Actin
35
Fold MET amplification
30
25
20
15
10
5
0
Supplementary Figure 2. Fold MET amplification in BTC cell lines. Protein expression level of MET was analyzed by immunoblotting in
BTC cell lines and MET amplified MKN45 gastric cancer cell line (positive control; upper). Fold MET amplification was analyzed in
genomic DNA isolated from BTC cell lines and MKN45 cells by real-time PCR(bottom).
Supplementary figure 3
SNU 308
SNU 478
PF00299804
C
SNU1079
PF00299804
C
PF00299804
C
TS
Actin
Supplementary Figure 3. TS downregulation by PF00299804 treatment. SNU308, SNU478 and SNU1079 cell lines were treated with
increasing doses of PF00299804 (0.01, 0.1, 1 μmol/L) for 24h, after which the extracts were immunoblotted with TS antibodies.
Supplementary Table 1. Combination of PF00299804 with 5-FU in biliary tract cancer cell lines
IC50 (μmol/L)
Combination Index
Cell Line
PF00299804
5-FU
ED50
SNU308
0.01
>10
0.062
SNU478
0.02
>10
<0.001
SNU245
>10
>10
0.016
SNU869
2.51
>10
0.863
SNU1079
1.50
>10
0.886
SNU1196
>10
>10
1.874
HuCCT1
4.72
>10
8.83
TFK-1
>10
>10
>10
NOTE: Shown are the IC50 values of PF00299804 or 5-FU using MTT assay. Combination index for the combination of
PF00299804 with 5-FU at the 50% fraction affected was calculated by the Chou and Talalay method in biliary tract cancer cell
lines (CI > 1: antagonistic effect, CI = 1: additive effect, CI <1: synergistic effect).
Supplementary Table 2. Combination of PF00299804 with cisplatin in biliary tract cancer cell lines
IC50 (μmol/L)
Combination Index
Cell Line
PF00299804
cisplatin
ED50
SNU308
0.01
1.96
0.57
SNU478
0.02
1.25
1.69
SNU245
>10
>10
5.5
SNU869
2.51
6.69
0.805
SNU1079
1.50
3.6
3.94
SNU1196
>10
4.49
0.207
HuCCT1
4.72
7.78
4.49
TFK-1
>10
>10
>10
NOTE: Shown are the IC50 values of PF00299804 or cisplatin using MTT assay. Combination index for the combination of
PF00299804 with cisplatin at the 50% fraction affected was calculated by the Chou and Talalay method in biliary tract cancer cell
lines (CI > 1: antagonistic effect, CI = 1: additive effect, CI <1: synergistic effect).