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Supplementary data
Figure S1. HKH40A treatment increases nuclear pool of ATF4 and CHOP. HCT-116
cells treated with 100 nM HKH40A were collected and cytoplasmic/nuclear fraction was
carried out followed by Western blot analysis using ATF4 and CHOP specific antibodies.
-Actin and Histone H3 were used as cytoplasmic and nuclear markers respectively.
Figure S2. Inhibition of catalytic activity of topoisomerase1 and topoisomerase2. (a)
Inhibition of topoisomerase1 (top1) activity studied by the relaxation assay. pBR322
DNA (Sigma-Aldrich) was incubated in the presence of top1 and various concentrations
of tested drugs as indicated. Supercoiled pBR322 DNA was relaxed by incubation with
top1. WMC-79 and HKH40A inhibited DNA relaxation induced by top1 at concentration
as low as 1.2 M. Camptothecin, the well known top1 inhibitor, under identical
experimental conditions required much higher concentration (50 μM) to achieve the same
effect. (b) Inhibition of the catalytic activity of topoisomerase2 (top2) by
imidazoacridones as measured by decatenation of kinetoplast DNA (kDNA). Top2
catalytic activity was assayed by decatenation assay that based on the conversion of
catenated DNA to its decatenated form, which requires DNA double strand breakage
followed by strand rotation and ligation activities uniquely done by top2. The removal of
these kDNA by the enzyme can be seen in agarose gel. As shown by a progressive
increase in the amount of catenated DNA substrate remaining in the wells
(Supplementary Figure S2b), HKH40A inhibited top2-mediated decatenation at all tested
concentrations (from 0.6 to 10 M) in a dose dependent manner, whereas WMC-79 its
parent drug, required higher doses to achieve the same effect (2.5 M and more).
However, both test compounds were more potent in this assay than adriamycin, an
intercalating agent that binds to DNA with affinity similar to WMC-79. These
observations indicate that only WMC-79 and HKH40A but not WMC-26 inhibit both
top1 and top2 catalytic activity at relatively low concentration.
Figure S3. Time-dependent cytotoxicity of HKH40A against HCT-116 and HT-29 cells.
Cell cycle and apoptosis was monitored after treatment of cells with 100 nM HKH40A by
FACS analysis. (a) Adherent and floating cells were collected, fixed with 70% ethanol,
stained with propidium iodide and analyzed using a fluorescence-activated cell sorter.
The numerical data corresponds to the percentage values for the indicated stages of the
cell cycle. Histograms are from a single experiment that has been repeated twice with
similar results. (b) At indicated time points floating and adherent cells were combined,
incubated with PE Annexin V in a buffer containing 7-amino-Actinomycin (7-AAD) and
analyzed by flow cytometry. Population of Annexin V and 7-AAD was negligible in cells
untreated and treated for 24 h with HKH40A, indicating that they were viable. After 48 h
of treatment with HKH40A, there was a big population of cells that was PE-Annexin V
and 7-AAD positive, indicating that they were in end stage apoptosis or necrosis.
Figure S4. HKH40A induces paraptosis. (a) Effect of HKH40A in HCT-116 cells on cell
morphology and vacuolization observed under light microscopy. (b) Representative
confocal images of HCT-116 cells stained with mitotracker-red (Life Technologies)
shows prominent swollen and rounded mitochondria after HKH40A treatment compared
to normal rod-shaped mitochondria in untreated cells. (c) Time dependent
phosphorylation of ERK1/2 and SAPK/JNK upon treatment of HCT-116 and HT-29 cells
with 100 nM HKH40A evaluated by Western blot. (d) Activity of HKH40A on HCT-116
and HT-29 cells treated alone or in the presence of inhibitors of caspase (Z-VAD-FMK),
MAPK’s (U0126), autophagy (3-MA) and necroptosis (NEK-1). HCT-116 and HT-29
cells were seeded in 96 well plates. Next day the cells were treated with 100 nM
HKH40A alone or in the presence of inhibitors. Cytotoxicity was determined by MTT
assay. The data represent the mean and the standard deviation from three experiments.
Figure S5. Effect of HKH40A on mitochondrial potential. HCT-116 and HT-29 cells
were treated with vehicle or 100 nM HKH40A. Floating and adherent cells were
combined, stained with TMRE and analyzed by flow cytometry. Cells stained with 20
M FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) were used as
positive control. After 48 h treatment with HKH40A, the population of cells with low
mitochondrial potential increased to 24.8% and 7.2% in HCT-116 and HT-29
respectively.
Figure S6. HKH40A treatment doesn’t induce production of superoxide by
mitochondria. HCT-116 and HT-29 cells were treated with vehicle or 100 nM HKH40A.
Only adherent (a) or both floating and adherent (b) cells were stained with MitoSOXTM
Red mitochondrial superoxide indicator. Cells treated for 10 min with 10 mM H2O2 were
used as positive control. In adherent untreated and treated cells there was no indication of
superoxide production, however small population of MitoSOX positive cells was
detected when all cells (adherent and floating) were collected.
Figure S7. Effect of HKH40A on ER calcium levels. (a) Cytosolic calcium was assessed
using Rhod-4 AM ester, a calcium indicator dye. The release of calcium from the ER, by
thapsigargin (TG) was compared in HKH40A-treated cells versus control HCT116 cells.
Profile response of mean of 100 control cells and cells treated with HKH40A for 24 h are
presented in (a). TG-mediated increase in cytosolic calcium (Δ[Ca2+]) was quantified by
the difference in calcium concentration before and after TG treatment for these cells and
shown in (b). Error bars represent standard deviation and p value was <0.0001. Calcium
concentrations were measured as described under materials and methods (c). Synergistic
effect of HKH40A and Gemcitabine on the level of GRP78 in pancreatic cancer cells.
Total cell lysates from Capan2 and MiaPaca2 cells treated 24 h with 100 nM HKH40A or
10 M Gemcitabine alone or with combination of both agents were subjected to Western
blot analysis with antiGRP78/BiP antibody. -actin was used as a loading control.
Supplementary data
Materials and Methods
Preparation of cytosol and nuclear extracts.
HCT-116 cells were plated at 2x106 density in 10 cm tissue culture dish. Next day
cells were treated with 100 nM HKH40A or vehicle. At desired time, cells were gently
washed with PBS at room temperature, collected by scraping in 1ml of ice-cold PBS,
transferred into an Eppendorf tube and pelleted by centifugation at 1500 x g for 5 min at
4°C. The pellet was resuspended in 400 l cold buffer A (20 mM HEPES pH 7.9, 10 mM
KCl, 0.2 mM EDTA, 1 mM DTT, 0.25 mM PMSF) by gentle pipetting. The cells were
allowed to swell on ice for 15 min, after which 25 l of 10% Nonident P-40 (Sigma) was
added and the tube was vortexed vigorously for 10 sec. The homogenate was centrifuged
for 30 sec at 10 000 x g. Supernatants were collected as a cytoplasmic fraction and the
nuclear pellet was resuspended in 50 l ice-cold buffer B (20 mM HEPES pH 7.9, 0.4
mM NaCl, 10 mM EDTA, 1mM DTT, 0.25 mM PMSF) and tubes were rocked at 4°C for
15 min on a shaking platform, and supernatants were collected after centrifugation for 5
min at 10 000 x g in cold room.
Assaying DNA Topoisomerase 1 Relaxation Activity
0.5 g of supercoiled pBR322 plasmid DNA was incubated with 3 units of
topoisomerase 1 in reaction buffer (150 mM KCl, 10 mM Tris-HCl, pH 7.5, 1 mM DTT,
1mM EDTA, 0.1 mg/ml BSA, 15 mM MgCl2), in the presence of varying concentrations
of the test compounds. Reactions were carried out at 37°C for 30 min and then terminated
by adding 1/4 volume of 5x stop solution (2.5% SDS, 15% Ficoll-400, 0.05%
bromphenol blue, 0.05% xylene cyanol and 25 mM EDTA). The reaction mixture was
subjected to electrophoresis through a 1% agarose gel in TAE buffer at 100 V for 2 h.
Gels were stained 30 min in 0.5 g/ml ethidium bromide to visualize DNA and
photographed under UV light.
Decatenation of kinetoplast DNA (kDNA) by Topoisomerase 2
The in vitro inhibition of topoisomerase 2 decatenation of kDNA was performed
according to protocol provided by TopoGEN, Inc., with small modifications. In brief,
each reaction was carried out in a 0.5 ml microcentrifuge tube containing 12 μl of H2O,
0.5 μl 20 mM ATP, 2 μl of 10x assay buffer supplied with the kit (1x buffer consisted of
50 mM Tris-HCl, pH 8, 120 mM KCL, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM
dithiothreitol (DTT), 30 μg/ml Bovine Serum Albumin), 2.5 μl of kDNA (200 ng/sample)
and 1μl of 20x concentrated drug. The combination was mixed thoroughly and kept on
ice. Two units (2 μl) of topoisomerase2 were added immediately before incubation in a
water bath at 37°C for 1 h. The reaction was stopped by the addition of 4 μl of stop
solution buffer (5% Sarkosyl, 0.0025% bromophenol blue, 25% glycerol) and placed on
ice. DNA electrophoresis was carried out on a 1% gel in TBE buffer containing ethidium
bromide (0.5 μg/ml) at 100V for 2 h. After electrophoresis, the gel was destined and
photographed under UV light.
Cell Cycle Distribution by FACS
Cells were seeded at a density of 0.5 x 105 cells in 25 cm2 T flasks, allowed to
attach for 24 h and then treated with 100 nM of HKH40A. At appropriate intervals, drug
treated and control cells (attached and floating) were collected and prepared for analysis
as previously described 1.
Annexin staining
Annexin staining was performed using Annexin V-PE kit (BD Pharmingen, cat.#
556422). HCT-116 and HT-29 cells were plated at a density of 0.3-0.5x106 cells per 25
cm2 T flask, next day cells were treated with vehicle or the drug. At different time points
all cells (floating and adherent) were harvested, stained according to instructions
provided by the supplier of the kit and analyzed by flow cytometry.
Mitochondrial staining
Cells treated with vehicle or with 100 nM HKH40A were incubated with 150 nM
Mitotracker Red580 (Invitrogen) in phenol red free medium for 30 min under growth
conditions. After incubation, cells were rinsed with dye free medium then examined and
photomicrographed.
Mitochondrial Membrane Potential
Mitochondrial membrane potential was performed using TMRE Mitochondrial
Membrane Potential Assay Kit (Abcam, cat. # ab113852) according to instructions
provided by the suppliers of the kits.
Detection of superoxide.
Detection of superoxide was performed using MitoSOXTM Red mitochondrial
superoxide indicator for live cell imaging (Invitrogen, cat. #M36008) according to
instruction provided by the supplier of the kit.
Intracellular calcium concentration.
Effect of HKH40A on intracellular Ca2+ concentration ([Ca2+]i) in HCT116 cells
was measured by confocal Ca2+ imaging using the Ca2+-sensitive indicator dye, Rhod4
(Abcam,) as per manufacturer’s protocol. In brief, 1x105 cells were seeded in 35 mm
glass bottom dish and 24-48 h later, cells were treated with or without HKH40A (100
nM). After 24 h, cells were loaded with Rhod-4 calcium dye dissolved in 10X pluronic
acid, for 30 min at 37°C followed by incubation at room temperature for another 30 min.
The cells were then washed with Hanks balanced salt solution (no Ca2+ and Mg2+) and
imaged using a Zeiss LSM510 confocal microscope (Carl Zeiss). Images were acquired
every 7 sec. Fluorescence intensity was recorded over the entire surface of each cell and
intracellular calcium concentration was evaluated using the formula for single
wavelength dyes i.e., [Ca2+]free = Kd * [F-Fmin]/ [FMax-F], where F is the fluorescence of
the indicator at experimental calcium levels, Fmin is the fluorescence in the absence of
calcium and Fmax is the fluorescence of the calcium saturated probe. Cells loaded with
Rhod-4 were treated with ionomycin (10 M) with 10 mM EGTA in Ca2+-free HBSS to
obtain Fmin and with 10 mM CaCl2 in HBSS to obtain Fmax. For thapsigargin treatment,
cells were treated with HBSS containing 1 µM thapsigargin during live cell imaging
(after 105 sec).
XBP1 splicing
Total RNA from untreated or treated with 100 nM of HKH40A cells was
extracted using RNeasy Kit (Qiagen, Valencia, CA) according to the manufacturer’s
instructions. 1 g of RNA was reversely transcribed to single-stranded cDNA using oligo
d(T)20 primer with Superscript reverse transcriptase III (Invitrogen). The XBP1 and
GAPDH specific primers were synthesized by Integrated DNA Technologies Inc. XBP1
Forward: 5’-GAGAACCAGGAGTTAAGACA-3, Reverse: 5’TAAGACTAGGGGCTTGGTA-3’; GAPDH – Forward: 5’ACCACAGTCCATGCCATCAC-3’, Reverse: 5’- TCCACCACCCTGTTGCTGTA-3’.
The PCR conditions were as follows: denaturation at 94°C for 5 min, followed by 25
cycles of 94°C for 1 min, 60 °C for 1min and 72°C for 2 min then 72°C for 10 min and
4° C. The cDNA fragments were resolved at 100V for 5 h on 1.5% agarose gel in TBE
buffer containing ethidium bromide. Cells treated with 2.5 μM Tunicamycin for 7 h were
used as a positive control.
References
1.
Kosakowska-Cholody T, Cholody WM, Monks A, Woynarowska BA, Michejda
CJ. WMC-79, a potent agent against colon cancers, induces apoptosis through a
p53-dependent pathway. Mol Cancer Ther 2005 Oct; 4(10): 1617-1627.