Supporting Information
Wiley-VCH 2011
69451 Weinheim, Germany
The Calcium-Chelating Capability of Tetrahydrofuranic Moieties
Modulates the Cytotoxicity of Annonaceous Acetogenins**
Chih-Chuang Liaw,* Wen-Ying Liao, Chien-Sheng Chen, Shu-Chuan Jao, Yang-Chang Wu,
Chia-Ning Shen,* and Shih-Hsiung Wu*
anie_201100717_sm_miscellaneous_information.pdf
Supporting Information
for
Calcium-chelating capability of tetrahydrofuranic moieties
modulates cytotoxicity of annonaceous acetogenins
Chih-Chuang Liaw1,2,*, Wen-Ying Liao2,3, Chien-Sheng Chen4, Shu-Chuan Jao4,
Yang-Chang Wu5, Chia-Ning Shen3,*, and Shih-Hsiung Wu4,*
1
Department of Marine Biotechnology and Resources, and Asia-Pacific Ocean Research Center, National
Contents
Supplementary Methods
2
Tables
Table S1. Cytotoxicity (IC50, µM) of AGEs against various cancer cell lines
3
Figures
Figure S1. 1H-NMR spectra of MAA-1 (1.5 mM) with various ratios of MAA-1/calcium.
4
Figure S2. 1H-NMR spectra of NAA-1 (1.5 mM) with various ratios of NAA-1/calcium.
4
1
Figure S3. H-NMR spectra of BAA-1 (1.5 mM) with various ratios of BAA-1/calcium.
5
Figure S4. Binding isotherms for the titration of Ca(ClO4)2 with the mono-THF AGE uvariamicin-I (MAA-2),
6
the non-adjacent bis-THF AGE bullatanocin (NAA-2), and the adjacent bis-THF AGE bullatacin (BAA-2).
Figure S5. Binding isotherms for the titration of Ca(ClO4)2 (46 µM) with the linear AGE (+)-monhexocin
6
(LAA), and the mono-THF AGE flanking a hydroxyl group, muricin H (MAA-3, 1.72 mM, B) at 25 °C.
Figure S6. ITC control injections for MAA-1, NAA-1, and BAA-1.
7
Figure S7. Analysis of apoptotic cells
8
Figure S8. Determination of average Flouo-4 AM fluorescence intensity that co-localized with Mitotracker
9
fluorescence.
Figure S9. Determination of average Rhod-2 fluorescence intensity
10
Figure S10. Analysis of mitochondrial membrane potential
11
Supplementary Methods
1. NMR spectroscopy of the complex formation of AGEs with calcium ions.1 1H-NMR spectroscopy
was carried out on a Bruker Avance NMR spectrometer (400 MHz) at 300 K. Chemical shifts of 1H NMR
spectra were reported relative to acetonitrile-d3 (δH, 1.94 ppm). A 0.4-mL aliquot of 1.5 mM AGE in
acetonitrile-d3 was transferred to a 5-mm NMR tube, and relative amounts of 20 mM Ca(ClO4)2 were
then added. For each pulse interval, 128 scans were recorded to obtain an acceptable signal-to-noise ratio.
2. Cell culture. HepG2 cells were obtained from Bioresource Collection and Research Center (BCRC,
Hsin-Chu, Taiwan) and were maintained in Dulbecco's modified Eagle's medium/Ham's F12 medium
(DMEM/F12 1:1, containing 1.05 mM CaCl2) supplemented with 2.5 mM L-glutamine (Sigma), 15 mM
HEPES, 0.5 mM sodium pyruvate, and 1200 mg/L sodium bicarbonate, 0.5 u/mL penicillin, and 500
ng/mL streptomycin (Sigma), and 10% fetal bovine serum (FBS) (Invitrogen).
3. Determination of cell apoptosis. To measure the apoptotic cells, cells were pre-treated with NAA-1,
BAA-1, or MAA-1 for 6 hours. The cells were then collected, washed with PBS, and co-stained with
FITC-conjugated annexin V antibodies (BD Biosciences) and Propidium Iodide(PI) (Sigma) for 15 min.
The fluorescence of FITC and PI was analyzed using a FACS Calibur (BD Biosciences).
4. Determination of mitochondrial calcium uptake.2 HepG2 cells were pre-treated with NAA-1,
BAA-1, or MAA-1 at 25 °C for 10 mins and then stained with 2 µM Rhod-2/AM at 25 °C for 20 min.
The cells were then washed with Hank's Buffered Salt Solution (HBSS) containing 2% FBS and 10 mM
HEPES. Fluorescent images were obtained on a temperature-controlled stage using confocal microscope.
5. Analysis of mitochondrial membrane potential.3 JC-1 is a lipophilic carbocyanine that exists as a
monomeric (green color) form and accumulates in depolarized mitochondria. In the presence of a high
mitochondrial membrane potential, JC-1 can reversibly form aggregates (orange/red color). HepG2 cells
were pre-treated with NAA-1, BAA-1, or MAA-1 and then stained with 10 µg ml−1 JC-1 in PBS at room
temperature for 15 min. The fluorescence of JC-1 monomer and J-aggregate was recorded using a FACS
Calibur (BD Biosciences). Valinomycin, used as a positive control, is an ionophore that causes a collapse
of the mitochondrial membrane potential by uncoupling mitochondrial respiration.
1. Peyrat, J.-F., Mahuteau, J., Figadère, B. & Cavé, A. J. Org. Chem. 62, 4811-4815 (1997)
2. Hawkins et al. Mol. Cell. Biol. 27 (21): 7582-7593 (2007)
3. Johnson L.V., Walsh M.L., Bockus B.J., Chen L.B. J. Cell Biol., 88, 526-535 (1981).
Table S1. Cytotoxicity (IC50, µM) of AGEs against various cancer cell lines[a]
Cytotoxicity (µ
µM)
Cell line
Compound
MDA-MB-231 MCF-7
A549
Hep 3B
Ca9-22
HepG2
Annonacin (MAA-1)
3.0
0.74
6.2
14.8
9.7
7.4× 10-3
Uvariamicin-I (MAA-2)
-
-
4.0
-
6.9
2.4× 10-1
Muricin H (MAA-3)
23.2
19.6
14.4
12.7
18.1
7.0× 10-2
Squamostatin A (NAA-1)
-
0.2
0.83
0.65
8.5
1.4× 10-3
Bullatanocin (NAA-2)
-
22.8
-
-
-
2.8× 10-2
Squamocin (BAA-1)
12.2
10.8
13.3
11.0
9.7
0.057× 10-3
Bullatacin (BAA-2)
-
19.4
12.3
24.8
13.3
0.31× 10-3
(+)-Monhexocin (LAA)
-
1.0
-
4.2
4.0
2.3× 10-1
Doxorubicin
0.26
0.28
0.67
0.74
0.30
0.037
[a] MDA-MB-231: human Caucasian breast adenocarcinoma cells; MCF-7: human breast
adenocarcinoma cells; A549: human lung carcinoma cells; Hep G2: human hepatoma cells; Hep3B:
human negroid hepatocyte carcinoma cells; Ca9-22: human gingival squamous carcinoma cells; -, not
tested.
Figure S1. 1H-NMR spectra of MAA-1 (1.5 mM) with calcium at various ratios in CD3CN.
Figure S2. 1H-NMR spectra of NAA-1 (1.5 mM) with calcium at various ratios in CD3CN.
Figure S3. 1H-NMR spectra of BAA-1 (1.5 mM) with calcium at various ratios in CD3CN.
Time (min)
A
0
10
20
30
40
50
60
70
80
90
B0
Time (min)
10
20
30
40
50
60
70
80
90
C0
Time (min)
10
20
30
40
50
60
70
80
90
0.0
0.0
0.0
-0.4
µcal/sec
-0.5
-0.2
-0.8
-1.0
-0.4
-1.2
-1.5
-0.6
-1.6
-2.0
-0.8
0.0
0
kcal/mole of injectant
0
-0.5
-1
-1
Model: OneSites
-1.0
χ 2 = 2052
N
2.13
±0.0290 Sites
-1
K
6.08E4 ±3.74E3 M
-3638 ±68.77 cal/mol -1.5
∆H
∆S
9.68 cal/mol/deg
-2
-3
0
1
2
3
4
5
6
7
Model: OneSites
χ2 = 3553
N
3.30
±0.0541 Sites
-1
K
1.09E5 ±1.49E4 M
-1639 ±37.71 cal/mol
∆H
17.5 cal/mol/deg
∆S
0
1
2
Molar Ratio
3
4
5
6
Model: OneSites
χ 2 = 4863
N
2.15
±0.0268 Sites
-1
K
2.51E5 ±2.97E4 M
∆H
-3088 ±60.29 cal/mol
∆S
14.4 cal/mol/deg
-2
-3
7
0
1
2
3
4
5
6
7
8
Molar Ratio
Molar Ratio
Figure S4. Binding isotherms for the titrations of 46 µM Ca(ClO4)2 with (A) mono-THF AGE MAA-2
(uvariamicin-I, 1.86 mM); (B) non-adjacent bis-THF AGE NAA-2 (bullatanocin, 1.57 mM); (C) adjacent
bis-THF AGE BAA-2 (bullatacin, 1.77 mM) at 25 °C. Raw data were obtained from 33 automatic
injections (8 µL/injection); the integrated fitted curves show the experimental points with a one-site
fitting function.
µcal/sec
A
Time (min)
B
0.20
0
70
10
0.10
8
0.00
-0.10
0
10
20
30
40
50
60
Time (min)
10
20
30
40
50
60
70
80
90
6
4
-0.20
2
-0.30
0
Figure S5. Binding isotherms for the titrations of 46 µM Ca(ClO4)2 with (A) the linear AGE LAA
(monhexocin, 1.63 mM) and (B) the mono-THF AGE MAA-3 (muricin H, 1.72 mM) at 25 °C. Raw data
were obtained from 33 automatic injections (8 µL/injection).
0.40
0.30
0.40
A
0.30
0.20
0.10
0.10
µ cal/sec
µcal/sec
0.20
0.00
-0.10
0.00
-0.10
-0.20
-0.20
-0.30
-0.30
-0.40
0.00
0.40
0.30
10.00
20.00
30.00
40.00
50.00
60.00
70.00
-0.40
0.00
0.40
80.00
C
0.30
0.20
0.10
0.10
µ cal/sec
µcal/sec
0.20
0.00
-0.10
-0.20
-0.30
20.00
30.00
40.00
50.00
Time (min)
60.00
70.00
80.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
D
-0.10
-0.30
10.00
10.00
0.00
-0.20
-0.40
0.00
B
-0.40
0.00
Time (min)
Figure S6. Control injections for the titrations of (A) CH3CN with 1.5 mM MAA-1; (B) CH3CN with 1.5
mM NAA-1; (C) CH3CN with 1.5 mM BAA-1; (D) 65 µM Ca(ClO4)2 with CH3CN at 25 °C. Raw data
were obtained from 33 automatic injections (8 µL/injection), and a dilution (< 0.2 µcal s−1) result was
observed.
Figure S7 Brief treatment of AGEs induces apoptosis in HepG2 cells. (A) HepG2 cells were grown in
DMEM/F12 medium without or with pre-incubation with exogenously added 1 mM or 10 mM CaCl2 for
48 hours. Cells were co-stained with Annexin-V and propidium iodide and then analyzed using a FACS
Calibur for evaluating Annexin V-positive apoptotic cells (B). HepG2 cells were pre-treated with 10 µM
NAA-1, 10 µM BAA-1, or 10 µM MAA-1 in culture medium at 37 °C for 6 hours. Cells were co-stained
with annexin-V and propidium iodide and then analyzed using a FACS Calibur for evaluating Annexin
V-positive apoptotic cells.
Figure S8. Determination of average flouo-4 AM fluorescence intensity co-localizing with Mitotracker
fluorescence. HepG2 cells were pre-stained with 0.5 µM of MitoTracker® Red 580 FM for 45 min and
then incubated with 50 µM NAA-1, BAA-1, or MAA-1 for 10 min. Subsequently, 2 µM of fluo-4 AM
solution was added. Fluorescent images were observed by confocal microscopy (63×) and were analyzed
using Metamorph image analysis software. Bars denote standard deviations. The statistical significance
was set at ***p<0.001 (compared to control cells).
Figure S9. Determination of average Rhod-2 fluorescence intensity. HepG2 cells, which were either
pre-incubated with 5 mM EDTA or not for 30 min, were treated with 50 µM NAA-1, BAA-1, or MAA-1
for 10 minutes. Cells were then stained with 2 µM Rhod-2/AM at 25 °C for 20 min. The fluorescent
images were observed by confocal microscopy (63×) and were analyzed using Metamorph image analysis
software. Bars denote standard deviations. The statistical significance was set at ***p<0.001.
Figure S10. Mitochondrial depolarization induced by AGEs. HepG2 cells were pre-incubated without or
with 10 µM valinomycin, 50 µM MAA-1, 50 µM NAA-1, or 50 µM BAA-1, and then changes in
mitochondrial membrane potential were measured at 3 h post-treatment in untreated or AGE-treated
HepG2 cells using the JC-1probe. Exposure of HepG2 cells to valinomycin served as a positive control.
Cells in the upper left-hand quadrant exhibited a high mitochondrial membrane potential, while cells in
the lower right-hand quadrant exhibited a low mitochondrial membrane potential. (An increase in green
fluorescence was observed in the FL1 channel of the FACS, and a decrease in red fluorescence was
observed in the FL-2 channel of the FACS.)