Triggering apoptosis in cancer cells with an analogue of cribrostatin 6 that elevates intracellular ROS

Article abstract of DOI:10.1039/c6ob01591c

Elevation of reactive oxygen species (ROS) is both a consequence and driver of the upregulated metabolism and proliferation of transformed cells. The resulting increase in oxidative stress is postulated to saturate the cellular antioxidant machinery, leaving cancer cells susceptible to agents that further elevate their intracellular oxidative stress. Several small molecules, including the marine natural product cribrostatin 6, have been demonstrated to trigger apoptosis in cancer cells by increasing intracellular ROS. Here, we report the modular synthesis of a series of cribrostatin 6 derivatives, and assessment of their activity in a number of cell lines. We establish that placing a phenyl ring on carbon 8 of cribrostatin 6 leads to increased potency, and observe a window of selectivity towards cancer cells. The mechanism of activity of this more potent analogue is assessed and demonstrated to induce apoptosis in cancer cells by increasing ROS. Our results demonstrate the potential for targeting tumors with molecules that enhance intracellular oxidative stress.

Full text of DOI:10.1039/c6ob01591c

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Triggering apoptosis in cancer cells with an
analogue of cribrostatin 6 that elevates
Cite this: DOI: 10.1039/c6ob01591c
intracellular ROS†
D. J. Asby,‡a M. G. Radigois,‡a,b D. C. Wilson,‡a F. Cuda,a C. L. L. Chai,b,c A. Chen,b
A. S. Bienemann,d M. E. Light,a D. C. Harrowven*a and A. Tavassoli*a,e
Elevation of reactive oxygen species (ROS) is both a consequence and driver of the upregulated meta-
bolism and proliferation of transformed cells. The resulting increase in oxidative stress is postulated to
saturate the cellular antioxidant machinery, leaving cancer cells susceptible to agents that further elevate
their intracellular oxidative stress. Several small molecules, including the marine natural product cribro-
statin 6, have been demonstrated to trigger apoptosis in cancer cells by increasing intracellular ROS. Here,
we report the modular synthesis of a series of cribrostatin 6 derivatives, and assessment of their activity in
a number of cell lines. We establish that placing a phenyl ring on carbon 8 of cribrostatin 6 leads to
increased potency, and observe a window of selectivity towards cancer cells. The mechanism of activity
of this more potent analogue is assessed and demonstrated to induce apoptosis in cancer cells by
increasing ROS. Our results demonstrate the potential for targeting tumors with molecules that enhance
intracellular oxidative stress.
Received 26th July 2016,
Accepted 7th September 2016
DOI: 10.1039/c6ob01591c
www.rsc.org/obc
Aerobic glycolysis is a key characteristic of cancer cells that sig- which potentially makes them vulnerable to agents that cause
nificantly affects the metabolic pathways and metabolites of additional oxidative pressure, providing a mechanism by
transformed cells. While these changes enable the unregulated which cancer cells can be selectively targeted.6 In line with this
growth and survival of cancer cells, they may also be used for hypothesis, a range of chemotherapeutics have been proposed
selective chemotherapeutic targeting. One such defining to function by increasing the oxidative stress in cancer
change is the increased production of ROS and the subsequent cells,7–10 and several screens aimed at identifying compounds
additional oxidative stress placed on cancer cells,1 which is that selectively inhibit the growth of transformed cells have
thought to play a key role in the maintenance of the tumour converged on compounds that act to increase oxidative
phenotype.2–4 Adaptation to this increase in oxidative stress pressure by reducing glutathione levels.11–13 Compounds such
requires upregulation of the cellular antioxidant machinery, as phenyl isocyanate,12 2-methoxyoestradiol,14 and piperlongu-
which works to maintain the reducing environment necessary mine11 selectively kill cancer cells (at doses of 5–20 µM) by
for correct function of a variety of cellular processes.5 NADPH interfering with ROS homeostasis pathways.
and glutathione are key antioxidants whose levels are elevated
Cribrostatin 6 is an imidazo[5,1-a]isoquinoline isolated
in response to increased ROS. The antioxidant machinery of from the marine sponge Cribrochalina sp.,15 which has been
cancer cells is thought to operate at near capacity due to shown to be cytotoxic in several cancer cell lines, triggering
higher basal ROS and upregulated ROS-mediated signalling, apoptosis by elevating intracellular ROS.16 The mechanism of
this process is likely to be similar to other quinones, which
undergo bioreduction in cells to the corresponding semi-
aChemistry, University of Southampton, Southampton, SO17 1BJ, UK.
quinone, which then reacts with molecular oxygen to generate
E-mail: dch2@soton.ac.uk, ali1@soton.ac.uk
bInstitute of Chemical and Engineering Sciences, A*Star, 138665, Singapore
superoxide.8 Cancer cells treated with cribrostatin 6 also
cDepartment of Pharmacy, National University of Singapore, 117543, Singapore
dSchool of Clinical Sciences, University of Bristol, Southmead Hospital, Bristol,
BS10 5NB, UK
eThe Institute for Life Sciences, University of Southampton, Southampton, UK
†Electronic supplementary information (ESI) available. CCDC 1060587. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c6ob01591c
showed an elevation of antioxidant transcripts, including
heme oxygenase 1, several ferritins and proteins associated
with glutathione synthesis.16 Although cribrostatin 6 has been
shown to affect the viability of cancer cell lines, its selectivity
for cancer cells over normal cells has never been demon-
strated. Such selectivity is seldom explored, yet is increasingly
seen as a critical property of compounds proposed as potential
‡Authors contributed equally.
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cancer therapeutics. Given its mechanism of action and the aryllithium 9 to a cyclobutenedione 8 and thermolysis of the
demonstrated susceptibility of cancer cells to additional oxi- resulting adduct 7 with aerial oxidation. To achieve a synthesis
dative stress, we sought to generate a more potent analogue of of analogue A, in which the positions of the alkyl and alkoxy
cribrostatin 6, with the aim of improving both the potency and residues are swapped, required a subtle change of strategy. In
cancer cell selectivity.
this case the simple expedient of switching from organo-
There have been several reported total syntheses of cribro- lithium intermediate 9 to organoytterbium ate complex 11
statin 6.17–21 We chose to adapt a modular route that we had changed the course of nucleophilic addition in favor of
developed as it facilitated the rapid incorporation of a variety addition to the vinylogous ester carbonyl C3.22 Thermolysis of
of substituents on carbons 8 and 9 of the isoquinoline ring the resulting adduct 10 followed by aerial oxidation then gave
and was readily adapted to reverse the positioning of these analogue A.
alkyl and alkoxy residues (Scheme 1).20 Thus, the brominated
The effect of cribrostatin 6 and analogues A–K on the viabi-
core, imidazo[1,5-a]pyridine 4, was prepared in three steps lity of MCF7 breast cancer cells was measured by MTT (3-(4,5-
from 3-bromo-2-cyanopyridine 1 as previously described.20 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
Contemporaneously, a series of cyclobutenediones 8 were pre- (Table 1 and ESI Fig. 1–12†). The most potent molecule was
pared as surrogates for the quinone ring each bearing alkoxy analogue D (IC50 of 428 40 nM) with a phenyl ring on C8 in
and organyl residues at C3 and C4 respectively. Syntheses of place of the methyl residue found in the parent molecule
cribrostatin 6 and analogues B–K were then achieved by (Fig. 1A, from hereon referred to as 8PC6, for 8-phenylcribro-
halogen–lithium exchange of 4, addition of the resulting statin 6). This substitution resulted in a 30% increase in
Scheme 1 Synthesis of cribrostatin 6 and analogues A–K.
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Table 1 The effect of cribrostatin 6 and its synthetic analogues on the
viability of MCF7 breast cancer cells. R and R’ refer to structures in
Scheme 1
Table 2 The effect of 8PC6 and cribrostatin 6 on the viability of a
variety of cell lines
8PC6
IC50 (nM)
Cribrostatin
6 IC50 (nM)
Cell line
Tissue and type
Compound
R
R′
IC50 (nM)
MRC-5
Lung, normal
Colon, normal
1845 186
1798 429
502 76
428 40
505 55
2231 173
2189 587
726 99
628 67
1045 212
Cribrostatin 6
Analogue A
Analogue B
Analogue C
Analogue D
Analogue E
Analogue F
Analogue G
Analogue H
Analogue I
Analogue J
Analogue K
Et
Me
tBu
Et
Et
Et
Et
Et
Et
Et
Et
Et
Me
628 67
OtBu
2064 884
1546 243
1671 112
428 40
2024 329
1692 209
5064 2766
993 160
CCD841
MDA-MB-231
MCF7
Breast, adenocarcinoma
Breast, adenocarcinoma
Pancreas, epithelioid
carcinoma
Cervix, adenocarcinoma
Colon, colorectal
carcinoma
Me
tBu
PANC-1
Ph
Naphthyl
2,4,6-(OMe)3Ph
4-(Me3Si)Ph
4-(Me)Ph
4-(F3C)Ph
4-(Me3C)Ph
4-(2-Furanyl)
HeLa
HCT 116
418 36
548 69
659 51
748 75
1661 230
4275 1358
2393 476
cells (viability reduced to 69–87%) was less pronounced at this
dose (Fig. 1).
The IC50 of 8PC6 was next determined by MTT assay in
each of these cell lines (Table 2, and ESI Fig. 20–26†). The IC50
of 8PC6 was found to be ca. four-fold higher in non-cancer cell
lines (average IC50 of 1.8 µM) than that in cancer cell lines
(average IC50 of 480 nM). In addition, 8PC6 was more potent
than its parent molecule in every cell line (Table 2).
We next sought to determine the mechanism of cell death
induced by 8PC6. Flow cytometry was used to assess annexin V
and 7-aminoactinomycin D staining in cells treated with
increasing doses of 8PC6 (25 nM–2.5 µM) for 24 hours (Fig. 2A
and ESI Fig. 27†). In cells treated with <1 µM 8PC6 we observed
a dose-dependent increase in cells undergoing the earlier
potency against MCF7 breast cancer cells compared to cribro-
statin 6 (IC50 of 628 67 nM).
With a more potent analogue in hand, we assessed the
effect of 1 µM 8PC6 on a number of cancer and non-cancer
cell lines from a variety of tissue types (Fig. 1B). As a compari-
son, we also assessed the effect of 1 µM cribrostatin 6 in the
same panel of cells (Fig. 1). 8PC6 was more potent than cri-
brostatin 6 in all cases, with a greater effect on the viability of
the cancer cell lines (reduced to 16–28%) than normal cells
(reduced to 69–88%). The selectivity of cribrostatin 6 for the
cancer cell lines in our panel (reduced to 36–54%) over normal
Fig. 1 Selective inhibition of cancer cell viability by cribrostatin 6 and 8PC6. (A) the crystal structure of 8PC6 (CCDC deposition code 1060587).
(B) The effect of 1 µM cribrostatin 6 and 8PC6 on the viability of a panel of cancer and non-cancer cell lines.
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Fig. 2 The effect of 8PC6 on intracellular ROS, and the effect of increased intracellular ROS on the potency of 8PC6. (A) Flow cytometric analysis
of the effect of 8PC6 treatment on the cell population by annexin V and 7-aminoactinomycin staining. For scatter plots see ESI Fig. 27.† (B) Phase
and fluorescent micrographs of MCF7 cells treated with 100 µM DCDFA (upper panel), and MCF7 cells dosed with 100 µM DCDFA followed by
2.5 µM 8PC6 (lower panel). Intracellular ROS causes DCDFA to be Fluorescent at 529 nm. (C) Quantifying the increase in the fluorescence (at
529 nm) of MCF7 cells treated with 100 µM DCDFA and increasing doses of 8PC6. (D) The effect of TIGAR and scrambled siRNA on TIGAR mRNA in
MCF7 cells. (E) The effect of TIGAR and scrambled siRNA on TIGAR protein in MCF7 cells. (F) Knockdown of TIGAR increases the potency of 8PC6 by
2-fold in MCF7 cells.
stages of apoptosis, accompanied by a reduction in cell viabi- of 8PC6 (Fig. 2C), indicating that treatment with 8PC6 results
lity, and a small percentage of late apoptotic and/or necrotic in an increase in intracellular ROS.
cells (Fig. 2A). At doses higher than 1 µM of 8PC6, we observed
These data suggest that 8PC6 selectively targets cancer
a much greater proportion of cells in the latter stages of apop- cells, presumably as a result of their increased ROS content.
tosis, accompanied by a reduction in the number of cells Consequently, we next sought to establish a link between vari-
expressing phosphotidylserine and very low levels of total cell ations in intracellular ROS and the effect of 8PC6 on cell viabi-
viability (Fig. 2A). The observed pattern of annexin V/7-AAD lity in the same cell line. To increase intracellular ROS we
staining for 8PC6 is indicative of cell death via apoptosis and utilized TIGAR, a P53-inducible protein that regulates glyco-
agrees with previously reported data for cribrostatin 6.16
lysis and apoptosis and serves to protect cells from ROS.
The effect of 8PC6 on intracellular ROS levels was next Knockdown of TIGAR has been shown to result in an increase
probed using the ROS-sensing dye 2′,7′-dichlorofluorescin di- in intracellular ROS,24 therefore the effect of 8PC6 would be
acetate (DCFDA).3,23 No fluorescence was observed at 529 nm expected to be more pronounced in cells treated with TIGAR
in MCF7 cells treated with 100 µM DCFDA alone (Fig. 2B, siRNA. MCF7 cells treated with 50 nM TIGAR siRNA showed
upper panel), whereas a significant increase in 529 nm fluo- the expected knockdown in TIGAR mRNA and protein levels,
rescence was observed when cells were treated with 2.5 µM while 50 nM of scrambled control siRNA had no effect on
8PC6 prior to the addition of 100 µM DCFDA (Fig. 2B, lower TIGAR mRNA or protein levels (Fig. 2D and E). In line with our
panel). Quantification of this change in fluorescence showed a hypothesis, 1 µM of 8PC6 reduced the viability of MCF7 cells
dose-dependent increase in fluorescence with increasing doses with TIGAR knocked-down by over 90%, a 2-fold increase in
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potency compared to the effect of the same dose of 8PC6 in of 8PC6 on cancer cell viability. The protective effect of NAC
untreated MCF7 cells and/or MCF7 cells treated with was apparent when comparing micrographs of MCF7 cells pre-
scrambled siRNA (Fig. 2F). The effect of TIGAR knockdown on treated with NAC prior to administration of 8PC6, to those
the IC50 of 8PC6 was further quantified; the IC50 of 8PC6 was treated with 8PC6 only (Fig. 3A). The observed protective effect
478 76 nM in untreated MCF7 cells and 477 150 nM in of NAC on 8PC6-treated cells was quantified by MTT assay; pre-
MCF7 cells treated with scrambled siRNA, whereas the potency treatment of MCF7 cells with 5 mM NAC resulted in a 20-fold
of 8PC6 was increased by 1.5-fold, with an IC50 of 326 19 nM, loss in the potency of 8PC6, with the IC50 of 8PC6 increasing
in MCF7 cells treated with TIGAR siRNA (ESI Fig. 28–30†).
to 9086 95 nM in these cells (Fig. 3B). The above data demon-
To assess the effect of 8PC6 in MCF7 cells with reduced strates that the activity and potency of 8PC6 directly correlates
intracellular ROS we utilized N-acetylcysteine (NAC), a mole- with intracellular ROS levels.
cule that not only functions as a ROS-scavenger, but also acts
The mechanism of action of 8PC6 was next probed. The
as the precursor to glutathione (another ROS-scavenging mole- generation of superoxide by quinones requires molecular
cule).25 NAC-treatment has previously been shown to protect oxygen,8 and as 8PC6 is proposed to function through a
cells from the oxidative damage of other ROS-generating mole- similar mechanism, its potency is expected to be reduced in
cules,11 and would therefore be expected to reduce the effect cells pre-incubated in a hypoxic environment. It should be
Fig. 3 Probing the role of 8PC6 in MCF7 cells. (A) Micrographs of MCF7 cells treated with increasing doses of 8PC6 (top row) and MCF7 cells
treated with 5 mM NAC prior to dosing with 8PC6 (bottom row). (B) The effect of 5 mM of NAC on the potency of 8PC6 in MCF7 cells. (C) The effect
of a hypoxic microenvironment on the potency of 8PC6. (D) The effect of increasing doses of 8PC6 on the clonogenic survival of MCF7 cells (quan-
tified in ESI Fig. 31†).
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noted that while hypoxia leads to a short-term increase in 5 mM. For experiments involving the culture of cells under
basal ROS in cells,6,26 the decrease in molecular oxygen levels hypoxic conditions, cells were initially cultured and seeded
within these cells means that ROS generation by 8PC6 and its under normoxic conditions, and then transferred to a hypoxia
subsequent effect on cell viability would be expected to be workstation (H35 Hypoxystation, Don Whitley) and either pre-
reduced.
incubated for 24 h in hypoxic conditions (1% oxygen, 5%
MCF7 cells were pre-incubated in hypoxia for 24 hours, carbon dioxide, 37 °C) and then dosed and incubated with
treated with increasing doses of 8PC6 in hypoxia, and the 8PC6 in normoxic conditions for 24 h; or grown continuously
effect on cell viability measured by MTT assay after a further in hypoxic conditions, including 24 h of pre-incubation and
24 h of incubation in a hypoxic environment. In line with our 24 h of treatment with 8PC6.
hypothesis, the IC50 of 8PC6 was significantly raised in
hypoxic MCF7 cells to 19.6 7 µM (Fig. 3C). To further demon-
MTT cell viability assays
strate the link between the observed loss of 8PC6 potency in Cells were seeded in triplicate in 96-well plates at optimized
hypoxia and the reduction in molecular oxygen, MCF7 cells cell densities appropriate to the size and doubling times of the
were pre-incubated in hypoxia, treated with 8PC6 and then various cell lines used in the study to produce equivalent cell
switched to a normoxic environment for 24 h prior to asses- confluency prior to experiments (MCF7 8000 cells per well;
sing their viability by MTT assay. The IC50 of 8PC6 was HeLa 6000 cells per well; PANC-1 10 000 cells per well;
measured as 1.47 0.1 µM in these cells. This increase in the MDA-MB-231 12 000 cells per well; HCT116 20 000 cells per
potency of 8PC6 (compared to cells treated and incubated in well; MRC5 6000 cells per well; and CCD841 7000 cells per
hypoxia only) demonstrates the necessity of molecular oxygen well). Cells were incubated for 16 hours prior to dosing with
for the activity of this 8PC6.
test compounds in fresh medium. MTT-based cell proliferation
The effect of 8PC6 on the long-term proliferative potential assays were performed 24 hours post-dosing, as follows:
of cancer cells was probed using a colony forming assay. MCF7 culture medium was replaced with fresh culture medium
cells were treated with a single dose of 8PC6 (0.5 µM, 1 µM, without phenol red. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl
1.5 µM, 2 µM) and incubated for 10 days to assess their colony tetrazolium bromide (MTT; Sigma) was freshly prepared in
forming potential. We observed a dose-dependent reduction in sterile PBS and added to cells at a final concentration of 1 mM
the number and size of colonies formed in cells treated with (10% v/v). Cells were then incubated for up to 4 hours at 37 °C
8PC6 compared to untreated cells (Fig. 3D and ESI Fig. 30†), until intracellular punctate purple precipitates were clearly
with a 2 µM dose of 8PC6 causing a 2.7-fold reduction in the visible under the microscope. An equal volume of DMSO was
clonogenicity of MCF7 cells (ESI Fig. 31†).
then added to solubilize the resulting formazan and the cells
incubated for 5 minutes in the dark at room temperature with
agitation to further dissolve the insoluble formazan particles.
Absorbance was measured at 570 nm on a microplate reader
(Tecan Infinite M200 Pro) and corrected for 650 nm back-
ground. Each well was triplicated and each experiment
repeated at least three times. IC50 values quoted are mean
SEM.
Conclusions
Our findings demonstrate that 8PC6 and cribrostatin 6 selec-
tively inhibit the growth of cancer cells via elevation of intra-
cellular ROS. Work is currently underway in our laboratories
to further probe the mechanism and effect of 8PC6 in other
cell lines, and we are continuing to synthesize and assess
additional derivatives of this molecule.
Detection of ROS species using DCF-DA fluorophore
MCF7 cells were either seeded in duplicate at 20 000 MCF7
cells per well in collagen-coated 8-well chamber slides (NUNC
and/or LabTek2) for fluorescent microscopy or seeded in tripli-
cate at 10 000 MCF7 cells per well in a black 96-well plate for
quantitative fluorescent assays. In both cases cells were then
incubated for 16 hours in cell culture media without phenol
red prior to any treatments. Culture media was gently
removed, and the cells washed in warm PBS. Warm PBS con-
taining dichlorodihydrofluorescein diacetate (DCF-DA, Sigma)
was added to the cells to give a final concentration of 100 µM,
and the cells were incubated for 30 min with the dye. The PBS
Materials and methods
Synthetic chemistry
Please see ESI Materials and methods† for detailed experi-
mental procedures and spectroscopic data for all molecules
synthesized.
Cell treatments
Cells were grown in the presence or absence of test compounds containing DCF-DA was then gently removed, and replaced
for 24 hours unless stated otherwise. For all compounds, seri- with fresh cell culture media without phenol red. Cells were
ally diluted stock solutions were made up in DMSO and then then dosed with 8PC6 and incubated for 6 hours. For the fluo-
further diluted such that the final concentration of DMSO in rescent microscopy experiments, cells on chamber slides were
each well was 0.5% v/v. For treatment with N-acetylcysteine imaged using a fluorescent microscope (Ex 495 nm/Em
(NAC), an aqueous solution of NAC was added to cells for 1 h 529 nm) and the experiment was repeated three times with
prior to addition of 8PC6, with a final concentration of NAC of representative micrographs shown. For quantitative analyses of
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DCF-DA fluorescence, cells on black 96-well plates were then the expression of 18S and beta-actin. Mean values for fold
washed in PBS and resuspended in a final volume of 80 µL change SEM are given.
fresh PBS. Fluorescence was measured at Ex 495 nm/Em
Protein immunoblotting
529 nm using a microplate reader (Tecan Infinite M200 Pro)
and background fluorescence levels consisting of blank wells Cells were washed with ice-cold PBS and harvested on ice.
containing only DCF-DA in PBS subtracted. These experiments Cells were lysed by incubation on ice for 15 min with radio-
were repeated three times and data expressed as mean fold- immunoprecipitation assay (RIPA) buffer (50 mM Tris
change SEM.
[pH 7.4], 150 mM NaCl, 10% [v/v] sodium-deoxycholate, 10%
[v/v] IGEPAL CA-630; Sigma) containing protease inhibitor
cocktail. Lysates were sonicated for 1 min in a sonicating water
siRNA transfection
MCF7 cells were seeded at either 7000 cells per well on 96-well bath and then centrifuged at 10 500 rpm for 10 min at 4 °C.
plates for MTT assays, 250 000 cells per well on 6-well plates The protein concentration in the supernatant was quantified
for harvesting RNA for qPCR analysis, or 1 000 000 cells per by the Bradford assay. Proteins were separated on SDS-
6 cm dish for harvesting protein for performing immunoblot- polyacrylamide bis–tris gels under denaturing conditions,
ting. Cells were incubated for 16 h following seeding. Cell den- transferred to unsupported pure nitrocellulose membrane
sities were optimized so that cells reached 50–70% confluency (Amersham), and subjected to immunoblot analysis.
just prior to transfection with siRNA. All cells were transfected
A mouse monoclonal primary antibody raised against
with siRNA using Lipofectamine RNAiMax transfection reagent TIGAR (9C10; 1 : 100; Santa Cruz) was used for blotting.
(Life Technologies) according to the manufacturer’s instruc- Antibodies were diluted in phosphate-buffered saline contain-
tions for performing a ‘forward transfection’. Briefly, cell ing 5% non-fat powdered milk and 0.1% Tween 20, and incu-
culture media was removed from cells prior to transfection bated with the membrane overnight at 4 °C. Horseradish
and replaced with serum-free OptiMEM (Life Technologies) peroxidase-conjugated anti-mouse immunoglobulin G anti-
cell culture medium. siRNA was firstly diluted in a small body (NXA931, 1 : 4000; GE Healthcare) was used as the sec-
volume of OptiMEM equivalent to 10% of the final volume of ondary antibody, and monoclonal anti-beta-actin-peroxidase
cells and incubated at RT for 5 min. In parallel, Lipofectamine antibody (A3854, 1 : 50 000; Sigma) served as a loading control.
was diluted in a small volume OptiMEM equivalent to 10% of Bound immunocomplexes were detected using ECL prime
the final volume of cells and incubated at RT for 5 min. The western blot detection reagent (RPN2232; GE Healthcare) and
diluted oligonucleotides and Lipofectamine were then com- analyzed using Image Lab 4.0 computer software (Bio-Rad).
bined and mixed gently, and then incubated at RT for 10 min. Experiments were repeated three times and a representative
The siRNA-Lipofectamine complexes formed were added to blot shown.
cells, which were incubated at 37 °C for 24 h. The final concen-
tration of siRNA was 50 nM and the final amount of
Apoptosis assay
Lipofectamine was 0.2% v/v for all experiments. Cells were MCF7 cells were seeded at 8 × 104 cells per well on 12-well
either transfected with TIGAR siRNA (Silencer Select pre- plates and incubated for 16 hours prior to being dosed with
designed annealed human oligonucleotide duplex; s32680; different concentrations of 8PC6, as described above. Cells
Life Technologies), scrambled siRNA (Silencer Select negative were harvested after 24 hours of drug exposure. This was per-
control number 2; Life Technologies) or vehicle alone. formed by firstly removing the culture media from the treated
Following transfection, cells were either harvested for total cells, which was set aside and later pooled back together with
RNA extraction (as above); harvested for whole protein extrac- the treated cells, in order to retain any dead, apoptotic and
tion (as above); or fresh culture media was added and cells detached cells contained in the spent culture media. Then all
were dosed with 8PC6 for 24 h and then an MTT assay per- remaining adherent cells were gently detached from plates
formed (as above).
using StemPro Accutase cell dissociation solution (Life
Technologies), which helps maintain plasma membrane integ-
rity, and therefore phosphatidylserine (PS) expression. Cells
Quantitative RT-PCR
Total RNA was extracted from MCF7 cells transfected with were stained with 7-amino-actinomycin (7-AAD) and FITC-
either TIGAR siRNA or scrambled siRNA using RNeasy Mini annexin V, using a commercially available FITC-Annexin V
Kit (QIAGEN) and quantified using a Nanodrop ND-1000 Apoptosis Detection Kit (Biolegend, #640922) according to the
spectrophotometer. Complementary DNA was synthesised in a manufacturer’s instructions, with some cell line-specific
20 μl reaction containing 1 μg of total RNA, using iScript™ optimization. Briefly, 7-AAD and FITC-annexin V was diluted
cDNA synthesis kit (Bio-Rad) according to the manufacturer’s in binding buffer at a ratio of 1 in 25 and 1 in 400, respectively,
instructions. Quantitative real-time PCRs were performed and then added to cell suspensions at a ratio of 1 : 1. Cells
using Universal Taqman PCR master mix (Applied Biosystems) were incubated for 15 minutes at room temperature in the
and the TIGAR (C12orf15; Hs00608644_m1) Taqman Gene dark with gentle agitation. The stained cells were then ana-
Expression Assay (Applied Biosystems) on a CFX Connect Real- lyzed by flow cytometry on an ACEA Novocyte 1000 (ACEA
Time PCR system (Bio-Rad). Fold-change for each experiment Biosciences Inc.). Percentage of early apoptotic and late
was calculated and normalized using the geometric mean of apoptotic/necrotic cell populations were calculated using
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NovoExpress software (version 1.2.1, ACEA Biosciences Inc.)
and compared with appropriate controls.
inhibitors FK228 and m-carboxycinnamic acid bis-hydrox-
amide with proteasome inhibitors PSI and PS-341 against
gastrointestinal adenocarcinoma cells, Clin. Cancer Res.,
2004, 10, 3853–3862.
Colony forming assays
8 J. S. Bair, R. Palchaudhuri and P. J. Hergenrother,
Chemistry and biology of deoxynyboquinone, a potent
inducer of cancer cell death, J. Am. Chem. Soc., 2010, 132,
5469–5478.
9 P. S. Lecane, M. W. Karaman, M. Sirisawad, L. Naumovski,
R. A. Miller, J. G. Hacia, et al., Motexafin gadolinium and
zinc induce oxidative stress responses and apoptosis in
B-cell lymphoma lines, Cancer Res., 2005, 65, 11676–11688.
10 P. Perez-Galan, G. Roue, N. Villamor, E. Montserrat,
E. Campo and D. Colomer, The proteasome inhibitor borte-
zomib induces apoptosis in mantle-cell lymphoma through
generation of ROS and Noxa activation independent of p53
status, Blood, 2006, 107, 257–264.
MCF7 cells were seeded in duplicate at 800 cells per dish in
6 cm dishes 16 h prior to dosing with 8PC6 for 24 h. Cells were
then gently washed in PBS and fresh culture medium added.
Cells were cultured for up to 14 days until colonies were visible
with fresh medium added every 48–72 h. At the end of the
experiment cells were fixed by addition of methanol/acetic acid
(3 : 1) for 5 min, then stained in 0.5% (v/v) crystal violet solu-
tion (diluted in methanol) for 10 min, and colonies counted
using the colony area plugin for imageJ. The experiments were
repeated twice and representative images are shown.
Acknowledgements
11 L. Raj, T. Ide, A. U. Gurkar, M. Foley, M. Schenone, X. Li,
et al., Selective killing of cancer cells by a small molecule
targeting the stress response to ROS, Nature, 2011, 475,
231–234.
This manuscript is dedicated to Dr Peter Simmonds. The
authors thank Professor Adrian Harris and Dr Karim Bensaad
for helpful discussions. This work was funded by Cancer
Research UK (Career Establishment Award 10263 to AT), the
Engineering and Physical Sciences Research Council
(EP/H04986X/1 to AT) and the University of Southampton and
A*STAR (Ph.D. studentship for M. G. R. to D. C. H.,
C. L. L. C. and A. C.).
12 D. Trachootham, Y. Zhou, H. Zhang, Y. Demizu, Z. Chen,
H. Pelicano, et al., Selective killing of oncogenically trans-
formed cells through a ROS-mediated mechanism by beta-
phenylethyl isothiocyanate, Cancer Cell, 2006, 10, 241–252.
13 W. S. Yang, R. SriRamaratnam, M. E. Welsch, K. Shimada,
R. Skouta, V. S. Viswanathan, et al., Regulation of ferropto-
tic cancer cell death by GPX4, Cell, 2014, 156, 317–331.
14 P. Huang, L. Feng, E. A. Oldham, M. J. Keating and
W. Plunkett, Superoxide dismutase as a target for the selec-
tive killing of cancer cells, Nature, 2000, 407, 390–395.
References
1 T. P. Szatrowski and C. F. Nathan, Production of large
amounts of hydrogen peroxide by human tumor cells, 15 G. R. Pettit, J. C. Collins, J. C. Knight, D. L. Herald,
Cancer Res., 1991, 51, 794–798.
R. A. Nieman, M. D. Williams, et al., Antineoplastic agents.
485. Isolation and structure of cribrostatin 6, a dark blue
cancer cell growth inhibitor from the marine sponge
Cribrochalina sp., J. Nat. Prod., 2003, 66, 544–547.
2 Z. Chen, L. C. Trotman, D. Shaffer, H. K. Lin, Z. A. Dotan,
M. Niki, et al., Crucial role of p53-dependent cellular senes-
cence in suppression of Pten-deficient tumorigenesis,
Nature, 2005, 436, 725–730.
16 M. T. Hoyt, R. Palchaudhuri and P. J. Hergenrother,
Cribrostatin 6 induces death in cancer cells through a reac-
tive oxygen species (ROS)-mediated mechanism, Invest. New
Drugs, 2011, 29, 562–573.
3 Y. Hu, D. G. Rosen, Y. Zhou, L. Feng, G. Yang, J. Liu, et al.,
Mitochondrial
manganese-superoxide
dismutase
expression in ovarian cancer: role in cell proliferation and
response to oxidative stress, J. Biol. Chem., 2005, 280, 17 D. Knueppel and S. F. Martin, Total synthesis of cribro-
39485–39492. statin 6, Angew. Chem., Int. Ed., 2009, 48, 2569–2571.
4 D. C. Radisky, D. D. Levy, L. E. Littlepage, H. Liu, 18 D. Knueppel and S. F. Martin, Tandem Electrocyclic Ring
C. M. Nelson, J. E. Fata, et al., Rac1b and reactive oxygen
species mediate MMP-3-induced EMT and genomic
instability, Nature, 2005, 436, 123–127.
Opening/Radical Cyclization: Application to the Total
Synthesis of Cribrostatin 6, Tetrahedron, 2011, 67, 9765–
9770.
5 K. Hirota, M. Murata, Y. Sachi, H. Nakamura, J. Takeuchi, 19 M. D. Markey and T. R. Kelly, Synthesis of cribrostatin 6,
K. Mori, et al., Distinct roles of thioredoxin in the cyto- J. Org. Chem., 2008, 73, 7441–7443.
plasm and in the nucleus. A two-step mechanism of redox 20 M. Mohamed, T. P. Goncalves, R. J. Whitby, H. F. Sneddon
regulation of transcription factor NF-kappaB, J. Biol. Chem.,
1999, 274, 27891–27897.
6 P. T. Schumacker, Reactive oxygen species in cancer cells:
live by the sword, die by the sword, Cancer Cell, 2006, 10,
175–176.
and D. C. Harrowven, New insights into cyclobutenone
rearrangements: a total synthesis of the natural ROS-gener-
ating anti-cancer agent cribrostatin 6, Chem. – Eur. J., 2011,
17, 13698–13705.
21 S. Nakahara, A. Kubo, Y. Mikami and J. Ito, Synthesis of cri-
brostatin 6 and its related compounds, Heterocycles, 2006,
68, 515–520.
7 M. Adachi, Y. Zhang, X. Zhao, T. Minami, R. Kawamura,
Y. Hinoda, et al., Synergistic effect of histone deacetylase
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
22 E. Packard, D. D. Pascoe, J. Maddaluno, T. P. Goncalves
and D. C. Harrowven, Organoytterbium ate complexes
regulator of glycolysis and apoptosis, Cell, 2006, 126,
107–120.
extend the value of cyclobutenediones as isoprene equi- 25 M. Zafarullah, W. Q. Li, J. Sylvester and M. Ahmad,
valents, Angew. Chem., Int. Ed., 2013, 52, 13076–13079.
23 A. S. Keston and R. Brandt, The Fluorometric Analysis of
Molecular mechanisms of N-acetylcysteine actions, Cell.
Mol. Life Sci., 2003, 60, 6–20.
Ultramicro Quantities of Hydrogen Peroxide, Anal. 26 N. S. Chandel, E. Maltepe, E. Goldwasser, C. E. Mathieu,
Biochem., 1965, 11, 1–5.
24 K. Bensaad, A. Tsuruta, M. A. Selak, M. N. Vidal,
K. Nakano, R. Bartrons, et al., TIGAR, a p53-inducible
M. C. Simon and P. T. Schumacker, Mitochondrial reactive
oxygen species trigger hypoxia-induced transcription, Proc.
Natl. Acad. Sci. U. S. A., 1998, 95, 11715–11720.
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.