Carcinogenesis vol.30 no.3 pp.512–519, 2009
Advance Access publication January 9, 2009
Phosphoaspirin (MDC-43), a novel benzyl ester of aspirin, inhibits the growth of human
cancer cell lines more potently than aspirin: a redox-dependent effect
Wenping Zhao, Gerardo G.Mackenzie, Onika T.Murray,
Zhiquan Zhang and Basil Rigas?
Department of Medicine, Division of Cancer Prevention, Life Sciences
Building, Room 06, Stony Brook University, Stony Brook, NY 11794-5200,
?To whom correspondence should be addressed. Tel: þ1 631 632 9035;
Fax: þ1 631 632 1992;
Aspirin is chemopreventive against colon and probablyother can-
cers, but this effect is relatively weak and its chronic administra-
tion to humans is associated with significant side effects. Because
of these limitations, extensive effort has been exerted to improve
the pharmacological properties of aspirin. We have determined
the anticancer activity and mechanisms of action of the novel para
positional isomer of phosphoaspirin [P-ASA; MDC-43; 4-((dieth-
oxyphosphoryloxy)methyl)phenyl 2-acetoxybenzoate]. P-ASA in-
hibited the growth of 10 human cancer cell lines originating from
potently than conventional aspirin. P-ASA achieved this effect by
modulating cell kinetics; compared with controls, P-ASA reduced
cell proliferation by up to 68%, increased apoptosis 5.5-fold and
blocked cell cycle progressionin theG2/M phase. P-ASAincreased
intracellular levels of reactive oxygen species (ROS), depleted glu-
tathione levels and modulated cell signaling predominantly
through the mitogen-activated protein kinase (p38 and c-jun
N-terminal kinase), cyclooxygenase (COX) and nuclear factor-
kappa B pathways. P-ASA targeted the mitochondria, increasing
mitochondrial superoxide anion levels; this effect on ROS led to
collapsed mitochondrial membrane potential and triggered the
intrinsic apoptotic pathway. The antioxidant N-acetyl cysteine ab-
rogated the cell growth inhibitory and signaling effects of P-ASA,
underscoring the centrality of ROS in its mechanism of action.
Ourresults, establishingP-ASAas a potent inhibitorofthegrowth
of several human cancer cell lines, suggest that it may possess
broad anticancer properties. We conclude that the novel P-ASA
is a promising anticancer agent, which merits further evaluation.
The discovery that nonsteroidal antiinflammatory drugs (NSAIDs) are
effective chemopreventive agents against a variety of human cancers
represents a major breakthrough in the field of cancer prevention.
Based on extensive epidemiological data assessing NSAID use in
preventing major human cancers, aspirin appears to be one of the
most effective NSAIDs in cancer prevention (1). Although aspirin
has formally been proven to be chemopreventive against human colon
cancer (2), its effect is relatively weak and its chronic administration,
as would berequired forits usein chemoprevention,is associated with
significant side effects (3). Because of these limitations, extensive
effort has been exerted to improve the pharmacological properties
of aspirin (4,5).
Ongoing work in our laboratory evaluates a series of novel acyloxy
benzyl esters, which have anticancer properties. We have recently
reported our findings concerning a novel acyloxy benzyl ester-based
derivative ofaspirin (MDC-63;the metapositional isomer inFigure1),
rine model of cancer, achieving .60% reduction in tumor volume of
xenografted HT-29 human cancer cells, and (ii) no apparent toxicity,
evidenced, among others, by the absence of changes in body weight
during treatment and organ damage (6). The mode of action of this
compound includes, at the cytokinetic level, brisk induction of apo-
ptosis and some suppression of proliferation.
As can be appreciated from Figure 1, this molecule lends itself to
positional isomerism. The diethylphosphate group can occupy any of
three positions on the benzene ring, o-, m- or p- with respect to the
ester bond between aspirin and its linker molecule. Since positional
isomerism can at times have a profound effect on the pharmacological
properties of a drug (7), we decided to evaluate the effect of the para
isomer (MDC-43) on thegrowth of various human cancer cell lines; to
study its mechanism of action, we subsequently focused on SW480
human colon cancer cells.
We observed that the growth inhibitory effect of p-phosphoaspirin
(P-ASA; MDC-43), independent of the tissue origin of the cancer cell
lines, was mediated by elevated intracellular levels of reactive oxygen
species (ROS), which in turn activated relevant cell signaling. Our
results suggest the anticancer potential of P-ASAand indicate that this
class of compounds merits further evaluation.
Materials and methods
p-Phosphoaspirin (MDC-43) was a gift of Chem-Master International, East
Setauket, NY. Dihydroethidium (DHE), 2#,7#-dichlorodihydrofluorecein diac-
etate (DCFDA), 4-amino-5-methylamino-2#,7#-difluorofluorescein (DAF-
FM), MitoTracker Green FM, MitoSOX Red and Annexin V were purchased
from Invitrogen (Carlsbad, CA). Conventional aspirin and N-acetyl-L-cysteine
(NAC) were purchased from Sigma (St Louis, MO).
Cell culture and cell kinetic assays
Human colon (HT-29, LoVo, HCT116, HCT-15 and SW480), pancreatic
(BxPC-3 and MIA PaCa-2), breast (MCF-7), liver (Hep G2) and lung
(H838) cell lines (American Type Culture Collection, Manassas, VA) were
grown in media as per the instructions of American Type Culture Collection.
The cell viability/growth response to P-ASA was measured using the 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay (Roche Diag-
nostics Indinapolis, IN) or the trypan blue exclusion method.
To measure cell proliferation (i.e. cell renewal), SW480 cells, treated with
P-ASA for 24 h, were pulse labeled with 10 lM bromodeoxyuridine BD Bio-
science (San Jose, CA) 15 min prior to harvesting and analyzed by flow
cytometry. To measure apoptosis and necrosis, SW480 cells were treated with
P-ASA for 18 h, harvested by trypsinization, stained with fluorescein isothio-
cyanate-conjugated Annexin Vand propidium iodide (PI) as per the manufac-
turer’s protocol and subjected to flow cytometry analysis. For cell cycle
analysis, cells were stained with PI following standard protocols.
Detection of ROS
SW480 cells were pretreated with ROS probes (5 lM DCFDA, 5 lM DHE,
2 lM DAF-FM or 5 lM MitoSOX Red) in RPMI 1640 medium without fetal
bovine serum or phenol red for 1 h (30 min in the case of MitoSOX Red). This
was followed by treatment with 25 lM P-ASA for 1 h. Finally, cells were
washed and analyzed by flow cytometry. For ROS live imaging, SW480 cells
were pretreated with 5 lM MitoSOX Red for 30 min followed by MitoTracker
Green FM for 10 min. The cells were then treated with 25 lM P-ASA for 1 h.
Images were captured with a Zeiss LSM510 meta confocal microscope and
processed in Adobe Photoshop.
Determination of glutathione levels
The levels of glutathione (GSH) were determined by the GSH reductase-
coupled 5,5#-dithiobis-(2-nitrobenzoic acid) assay, based on the 5,5#-dithio-
bis-(2-nitrobenzoic acid)/enzymatic recycling procedure of Tietze (8). Briefly,
Abbreviations: BSO, D,L-buthionine(S,R)-sulfoximine; COX, cyclooxygenase;
DAF-FM, 4-amino-5-methylamino-2#,7#-difluorofluorescein; DCFDA, 2#,7#-
dichlorodihydrofluorecein diacetate; DHE, dihydroethidium; ERK, extracellular
signal-regulated kinase; GSH, glutathione; IC50, 50% inhibitory concentration;
JC-1, 5,5#,6,6#-tetrachloro-1,1#,3,3#-tetraethylbenzimidazolylcarbocyanine iodide;
JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NAC,
N-acetyl-L-cysteine; NF-jB, nuclear factor-kappa B; P-ASA, phosphoaspirin; PI,
propidium iodide; ROS, reactive oxygen species.
? The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com
50 ll of each working standardor dilutedsample extract was added to thewells
of a flat-bottomed 96-well microtiter plate. All standards and samples were run
in duplicate in adjacent wells. Fifty microliters of 5,5#-dithiobis-(2-nitroben-
zoic acid) 1.26 mM and 50 ll of GSH oxidoreductase 2.5 kU/l were then added
to each well and after 15 min at room temperature, the reaction was started by
the addition of 50 ll of 0.72 mmol/l of reduced nicotinamide adenine dinu-
cleotide phosphate to each well. Absorbance was measured at 410 nm using
a 96-well plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA).
Data were analyzed using SoftMax Pro v5 software.
Determination of mitochondrial membrane potential
The mitochondrial membrane potential was determined by flow cytometry
using the 5,5#,6,6#-tetrachloro-1,1#,3,3#-tetraethylbenzimidazolylcarbocya-
nine iodide (JC-1) cationic dye (Invitrogen). In healthy cells, the JC-1 dye
stains the mitochondria bright red, but in apoptotic cells, in which the mito-
chondrial membrane potential collapses, it remains in the cytoplasm in its
green fluorescence form. Briefly, SW480 cells were incubated with 1.5?
50% Inhibitory Concentration (IC50) P-ASA for 3 h, when cells were trypsi-
nized and washed once with phosphate-buffered saline. The supernatant was
discarded and cells were incubated with 5 lM JC-1 for 30 min at 37?C pro-
tected fromlightand analyzed byflowcytometryusingthe FL1andFL2(green
and red fluorescence, respectively).
Cell lysates (20 lg of total protein) were resolved in 10% sodium dodecyl
sulfate–polyacrylamide gel and transferred onto polyvinylidene fluoride
membranes. Membranes were probed with antibodies against p38, p-p38, c-jun
N-terminal kinase (JNK), p-JNK, extracellular signal-regulated kinase (ERK),
p-ERK,AKT,p-AKTandcaspase9Cell Signaling (Beverly, MA), procaspase 8
Santa Cruz (Santa Cruz, CA) or cyclooxygenase (COX)-2 Cayman Chemical
(Ann Arbor, MI). b-Actin (Sigma) was used as the loading control.
Electrophoretic mobility shift assay
After the indicated treatment of cells, nuclear fractions were isolated from
3 ? 106cells, as described previously (9,10). The oligonucleotide containing
the consensus sequence for nuclear factor-kappa B (NF-jB) was end labeled
with [c-32P]adenosine triphosphate using T4 polynucleotide kinase. Samples
were incubated with the labeled oligonucleotide (20 000–30 000 c.p.m.) for
20 min at room temperature in binding buffer [10 mM Tris–HCl buffer, pH 7.5,
containing 4% (vol/vol) glycerol, 1 mM MgCl2, 0.5 mM ethylenediaminetetra-
acetic acid, 0.5 mM dithiothreitol, 50 mM NaCl and 0.05 mg/ml poly(dI-dC)].
The reaction products were separated by electrophoresis in a 6% (wt/vol) non-
denaturingpolyacrylamidegel using 0.5? TBE (45 mM Tris–borate and 1 mM
ethylenediaminetetraacetic acid, pH 8.3) as the running buffer. The gels were
dried and the radioactivity was quantified.
Enzyme-linked immunosorbent assay
Trypsinized cells were suspended in lysis buffer to which Nonidet P-40 was
added in a subsequent step; nuclei were washed and centrifuged, followed by
resuspension in extraction buffer and centrifuged. Nuclear extracts were stored
at ?80?C until assayed. TransBinding NF-jB assay was performed using
an ELISA kit (Panomics, Fremont, CA) and following the manufacturer’s
Statistical evaluation of the data was performed by one-factor analysis of
variance followed by Tukey’s test for multiple comparisons. P , 0.05 was
regarded statistically significant. The data, obtained from at least three inde-
pendent experiments, were expressed as the mean ± SEM.
P-ASA inhibits the growth of various human cancer cell lines
We evaluated the growth inhibitory effect of P-ASA on human cancer
cell lines originating from the colon, pancreas, breast, lung and liver.
Cells plated at a density of 5.5 ? 104/cm2were treated with P-ASA
for 24 h and their IC50was determined.
As shown in Table I, the most sensitive cell line was HepG2
(IC505 13.8 lM), whereas the least sensitive was the pancreatic cell
line MIA PaCa-2 (IC505 113 lM), being ?8-fold higher than that of
HepG2. In general, the IC50values of these cell lines do not differ
greatly among themselves and in the case of the five colon cancer cell
lines, their range of values is even narrower (14.3–67.6 lM).
In agreement with previous findings (11), conventional ASA in
concentrations up to 2 mM failed to inhibit the growth of any of these
cell lines by 50% or more; thus, its IC50values could not be deter-
mined. In all 10 cell lines that we studied, P-ASA was more potent
than aspirin; the fold enhancement of potency ranged between
.18 and .144, being on average .66.8. Of note, para P-ASA is
more potent than its meta isomer [(6) and similar data not shown].
Cell kinetic effect of P-ASA on SW480 human colon cancer cells
To explore the mechanism of the growth inhibitory effect of P-ASA,
we determined its effect on cell renewal, cell death and cell cycle
Table I. P-ASA (MDC-43) inhibits the growth of human cancer cell lines
Cell linesIC50(lM) (mean ± SD)
42.6 ± 10.6
14.3 ± 6.7
23.1 ± 3.9
46.6 ± 7.3
67.6 ± 10.4
27.4 ± 6.3
113 ± 17.8
13.8 ± 5.8
54.0 ± 4.5
38.0 ± 5.1
m- Phosphoaspirin (MDC-63)
p- Phosphoaspirin (MDC-43)
Fig. 1. Chemical structures of aspirin, m-phosphoaspirin (MDC-63) and
p-phosphoaspirin (MDC-43). An aromatic linker molecule binds
diethylphosphate to the carboxyl group of conventional aspirin. The position
of diethylphosphate on the benzene ring of the linker moiety defines the two
Phosphoaspirin inhibits the growth of human cancer cell lines
SW480 cells were incubated overnight and then treated with P-
ASA as indicated. Cell renewal (proliferation) was evaluated by the
bromodeoxyuridine method. P-ASA reduced bromodeoxyuridine in-
corporation concentration-dependently, decreasing it by 68% at
50 lM, the highest concentration used. After 24 h of incubation, there
was cell cycle arrest in G2/M phase, evident at all drug concentrations.
Treatment with 25 lM P-ASA for 18 h increased the proportion of
apoptotic cells 5.5-fold compared withcontrols. Thegreatest increase,
8.4-fold over control, was in late apoptotic cells [Annexin V (þ)/
PI(þ)]; early apoptotic cells [Annexin V (þ)/PI(?)] were increased
only 3.1-fold. Finally, P-ASA increased 4-fold the number of purely
necrotic cells [Annexin V (?)/PI(þ)]. As detailed below, the antiox-
idant agent NAC prevented the growth inhibitory effect of P-ASA
P-ASA induces the production of ROS in SW480 cells
Previous work indicates that the induction of ROS by chemopreven-
tive and chemotherapeutic agents represents a critical early event in
their mechanism of action (12). Thus, we explored whether P-ASA
induces the production of ROS. To this end, we used the following
molecular probes: DCFDA, which reacts with nearly 10 individual
species and is considered a ‘general probe’ for RONS (13); DHE,
which detects intracellular superoxide anions; MitoSOX Red, which
specifically detects mitochondrial superoxide anions and DAF-FM,
which detects nitric oxide. SW480 cells were cultured and treated
with P-ASA 25 lM for 1 h and the levels of ROS were determined.
As shown inFigure 3,P-ASAincreased DCFDAand MitoSOX Red
fluorescence, but not that of DHE and DAF-FM. Compared with
controls, the ROS levels detected by DCFDA were increased by
33%. NO and cellular superoxide anion levels (detected by DHE)
were not altered by P-ASA. In sharp contrast, superoxide anion levels
specifically in the mitochondria were markedly elevated in response
to P-ASA. As the image overlay of the mitochondrial probes Mito-
Tracker Green FM and MitoSOX Red confirms, P-ASA 25 lM in-
creased mitochondrial superoxide anion levels by 112%, compared
To further assess the reliability of our detection, we employed the
antioxidant NAC, a precursor of intracellular GSH (14,15). Pretreat-
ment of SW480 cells with NAC 20 mM for 4 h reversed most of the
induction of superoxide anion in the mitochondria, as detected by
MitoSOX Red. Moreover, NAC reversed P-ASA-induced apoptosis
and necrosis (Figure 2D) and also abrogated the inhibitory effect of
P-ASA on cell number (Figure 2E).
P-ASA decreases thiol levels and induces intrinsic apoptosis: redox
We determined the effect of P-ASA on the intracellular levels of GSH,
one of the most important antioxidant systems in mammalian cells
Fig. 2. The cell kinetic effect of P-ASA on SW480 colon cancer cells. SW480 cells were grown overnight and treated with P-ASA (MDC-43) as shown. (A) Cell
proliferation assay based on bromodeoxyuridine (BrdU) incorporation into DNA during the S-phase of the cell cycle. The percentage of bromodeoxyuridine
positive cells is shown in the right upper corner of each panel. (B) Cell cycle analysis by PI staining for DNA content of cells treated with and without P-ASA.
Results,quantifiedin (C), demonstrate theinductionofa G2/Mto G0/G1blockbyP-ASA.(D) Flowcytometricanalysisof cellsstainedwithPI andAnnexinV(A).
A(?)/PI(?) cells are viable cells; A(þ)/PI(?) are early apoptotic; A(þ)/PI(þ) are late apoptotic and A(?)/PI(þ) are necrotic. The numbers inside each panel
represent the percentage of cells in each category. NAC 20 mM was used to pretreat the cells for 4 h. (E) The effect of pretreatment with NAC on cell viability in
response to P-ASA was determined by trypan blue staining and cell counting. Figures are representative of two experiments, whose results were within 10%.
W.Zhao et al.
(14). As shown in Figure 4, treatment of SW480 cells with P-ASA led
to a significant concentration-dependent decrease of GSH levels. In-
cubation of SW480 cells with 80 lM P-ASA for 4 h decreased GSH
levels by 35%. The GSH synthase inhibitor D,L-buthionine (S,R)-
sulfoximine (BSO) (16) decreased GSH levels by 80%, whereas pre-
treatment with NAC 20 mM for 4 h largely restored GSH levels.
GSH depletion, induced by BSO, enhanced the cell growth inhib-
itory effect of P-ASA (Figure 4C). P-ASA 80 lM inhibited the growth
of SW480 cells (IC5023 lM under our experimental protocol), but
pretreatment with 10 lM BSO for 24 h reduced the IC50to 11 lM.
These findings clearly indicate that ROS controls the growth of cancer
cells in response to P-ASA.
The two major pathways of apoptosis are the intrinsic, character-
ized by cytochrome c release and caspase 9 activation, and the extrin-
sic, involving activation of caspase 8 (17). To determine which
pathway is operative in response to P-ASA, we assayed the levels
of caspase 9 and 8 (Figure 4D). As indicated by the cleavage of
procaspase 9, caspase 9 became activated. In contrast, no procaspase
8 cleavagewas observed,indicatingthat the extrinsicpathway was not
activated by P-ASA.
Fig. 3. The effect of P-ASA on ROS levels in SW480 colon cancer cells. (A) SW480 cells were preloaded with a molecular probe for ROS as indicated and treated
with P-ASA (MDC-43) for 1 h. DCFDA is a general ROS probe; DHE detects superoxide anion in cells; MitoSOX Red detects specifically mitochondrial
superoxide anion and DAF-FM detects NO. (B) Superoxide anion levels in mitochondria detected by MitoSOX Red were decreased following pretreatment with
NAC. Values are the mean ± SEM offour independent experiments;?P , 0.05. (C) Cells were stained with MitoSOX Red and MitoTracker Green,a stain specific
for mitochondria. The overlay images (lower row) establish the mitochondrial origin of the increased superoxide anion levels in response to P-ASA.
Phosphoaspirin inhibits the growth of human cancer cell lines
To evaluatewhether the altered redox state of the cell plays a role in
the activation of caspase 9, we pretreated SW480 cells with NAC (18).
NAC almost completely blocked the cleavage of caspase 9 induced by
P-ASA at its IC50concentration (Figure 4E). Since the mitochondria
were involved in triggering cell death induced by P-ASA, we evalu-
ated the effect of P-ASA on the mitochondrial membrane potential by
using the JC-1 cationic dye. As shown in Figure 4F, incubation of
SW480 cells with P-ASA 1.5? IC50for 3 h increased green fluores-
cence by 70% compared with controls, indicating the collapse of the
mitochondrial membrane potential. As seen in the lower panel of
Figure 4F, the percentage of cells that display green fluorescence
(right half of each panel) increased from 4.6% in controls to 17.1%
in P-ASA-treated cells, i.e. it increased 3.7-fold.
Cell signaling effects of P-ASA in SW480 cells
To explore the effect of P-ASA on intracellular signaling pathways, we
analyzed in cells treated with P-ASA the status of mitogen-
As shown in Figure 5, P-ASA increased progressively in a time-
dependent manner the levels of phosphorylated (i.e. activated) p38.
Thisactivation started 15 min after treatment withP-ASAand reached
its highest level at 24 h, the last time point of observation. However,
over the same period of time the levels of p38 remained unchanged,
indicating an effect limited only to protein activation. JNK was sim-
ilarly activated by phosphorylation, but this was an effect limited to
a 7 h period, between 1 and 8 h post-treatment with P-ASA; at 24 h,
the levels of p-JNK were barely detectable. Similar to p38, no change
in the protein levels of JNK was noted, indicating again an effect
limited to the activation of a signaling protein. We also noted modest
changes in the levels of phosphorylated ERK1/2 (mainly at 3 h) and
AKT (mainly between 1 and 8 h); the protein levels of both remained
unchanged in response to P-ASA treatment. As shown in Figure 5C,
NAC abrogated the activation of p38, JNK and ERK brought about by
P-ASA, indicating its redox dependence.
COX-2 is a ROS-dependent enzyme (19). To investigate the effect
of P-ASA on COX-2 signaling, we used the HT-29 colon cancer cell
line because SW480 cells do not express COX-2. As shown in Figure
5D, P-ASA stimulated the expression of COX-2 in HT-29 cells in
a concentration-dependent manner. The redox dependence of this
effect was confirmed by its attenuation by pretreating these cells with
20 mM NAC.
Finally, we studied the effect of P-ASA on NF-jB activation. Both
the enzyme-linked immunosorbent assay method and electrophoretic
mobility shift assay gave concordant results, demonstrating that P-
ASA suppressed NF-jB activity by .50% (Figure 5E and F). Again,
as shown by the electrophoretic mobility shift assay study,
Fig. 4. P-ASA decreases thiol levels and induces intrinsic apoptosis in SW480 colon cancer cells. (A) SW480 cells were grown overnight and treated with various
concentrations ofP-ASA(MDC-43)for4 h.GSHlevels,determinedasin MaterialsandMethods,were decreased ina concentration-dependentmanner.Valuesare
the mean ± SEM of three independent experiments.?P , 0.05 compared with control. (B) Overnight pretreatment with 20 mM NAC restores GSH levels in
P-ASA-treated cells. BSO, an inhibitor of GSH synthase, was used as a control for GSH depletion. (C) SW480 cells were treated with or without BSO for 24 h,
followed by treatment with P-ASA for 18 h. Data (mean ± SEM of three experiments) are expressed as percent of control. (D) Inmunoblots for procaspase 8,
procaspase 9 and caspase 9 in SW480 cells treated for 18 h with 1?, 1.5? or 2? IC50P-ASA. Loading control: b-actin. (E) SW480 cells treated with P-ASA for
18 h following pretreatment with 20 mM NAC or vehicle for 4 h. Procaspase 9, caspase 9 and b-actin were detected by immunoblot. (F) SW480 cells were treated
with P-ASA 1.5? IC50for 3 h and their mitochondria membrane potential was determined by flow cytometry as described in Materials and Methods. Upper panel:
fluorescence histograms of control SW480 cells and cells treated with P-ASA; the latter show a shift to the right indicating increased green fluorescence and thus
collapsed mitochondrial membrane potential [the corresponding geometric means are as follows: control 5 152 ± 17, P-ASA 5 258 ± 27 (mean ± SEM)]. Lower
panel: flow cytometry of SW480 cells stained as in Materials and Methods for mitochondrial membrane potential. Abscissa, FL1 (green fluorescence); ordinate,
FL2 (red fluorescence). The shift toward green fluorescence indicates collapsed mitochondrial membrane potential.
W.Zhao et al.
pretreatment with 20 mM NAC restored NF-jB binding to its cognate
DNA sequence, inagreement with thewell-known redox sensitivity of
Our data establish the strong growth inhibitory effect of P-ASA on
human cancer cells. This effect (i) far exceeds that of aspirin, its
parent molecule; (ii) appears to be generalized, and (iii) is mediated
by changes in cellular redox homeostasis, which, through modulation
of cell-signaling cascades, lead to a profound cytokinetic effect that
engenders cell growth inhibition.
The cell lines used in this study originate from human colon, pan-
creatic, liver, breast and lung cancers and thus represent the major
human cancers, which in 2006 accounted for ?54% of all new cases
of cancer in the USA. Among them, the most sensitive to P-ASAwas
HepG2 (IC505 13.8 lM), whereas the least sensitive was the pan-
creatic cell line MIA-PaCa2 (IC505 113 lM). Thus, it is clear that
the tissue of origin of these carcinoma cell lines has limited influence
on their responsiveness to P-ASA.
The results from the colon cancer cell lines, the largest group of cell
lines from a single organ that we studied, revealed that P-ASA’s effect
was not significantly affected by individual variations in cell lines.
Rather,their IC50s fell within a relatively narrow range (14.3–67.6 lM),
suggesting perhaps a broadly applicable mechanism of action.
An important finding was the uniform enhancement of the potency
of P-ASA compared with conventional aspirin, whose precise IC50
could not be determined due to its limited solubility. In the 10 cell
lines evaluated, the fold enhancement ofpotencyranged between .18
and .144, being on average .66.8. The reasons for the enhanced
potency of P-ASA are not readily apparent, although they are clearly
associated with the modification of aspirin’s structure.
The growth inhibitory effect of P-ASA is brought about by a triple
cell kinetic effect consisting ofinhibition of proliferation, induction of
apoptosis and necrosis, as well as the induction of cell cycle block at
the G2/M phase. Specifically, P-ASA inhibited cell proliferation
concentration-dependently (this inhibition reached 68% at 50 lM
P-ASA) and induced both early and late apoptosis as well as pure
necrosis. Treatment with P-ASA 25 lM increased the amount of
apoptotic cells 5.5-fold and of necrotic cells 4-fold, compared with
controls. The greatest increase concerned late apoptosis. The relative
contribution of each effect to the overall growth inhibitory effect of P-
ASA cannot be quantified precisely, but it appears that the induction
of apoptosis is the dominant cytokinetic effect.
preventive agent is a mechanistic event that is both significant and
Fig. 5. Cell signaling effects of P-ASA in SW480 colon cancer cells. (A and B) SW480 cells were grown overnight and treated with 25 lM P-ASA (MDC-43) for
up to 24 h, and the proteins shown were detected by immunoblot in whole-cell extracts. Phosphorylated (p-) and total p38, JNK, ERK and AKT were assayed.
Loading control in A–D: b-actin. (C and D) Pretreatment with NAC 20 mM for 4 h reduces the activation of MAPKs and the induction of COX-2 in response to
P-ASA treatment. (E) NF-jB-DNA binding was detected by an enzyme-linked immunosorbent assay method in SW480 cells treated with several concentrations
of P-ASA for 4 h. Values are the mean ± SEM of three independent experiments;?P , 0.01 compared with control. (F) Electrophoretic mobility shift assay for
NF-jB in SW480 cells treated with 20 lM P-ASA for 4 h. NAC 20 mM was used to pretreat the cells for 4 h. To determine the specificity of the NF-jB-DNA
complex, the control nuclear fraction was incubated before the binding assay with 100-fold molar excess of unlabeled oligonucleotide containing the sequence for
NF-jB (lane labeled þNF-jB) or an unrelated transcription factor (lane labeled þSP-1).
Phosphoaspirin inhibits the growth of human cancer cell lines
early (12,21). This proved to be the case for P-ASA as well. As our
data clearly demonstrate, P-ASA enhanced the intracellular levels of
ROS assayed using a ‘general ROS probe’, which reacts with several
individual species. Although ROS measured by this probe increased
by 36.5% in response to P-ASA, the main redox effect of P-ASAwas
on the levels of superoxide anion in mitochondria. Superoxide anions
detected in the entire cell by DHE did not change in response to P-
ASA, and NO levels, which might have contributed to the DCFDA
response, were also unchanged. The most significant ROS increase
(112% over control) concerned the mitochondrial superoxide anion,
indicating that mitochondria are the most important target of P-ASA.
Indeed, mitochondrial superoxide anion plays a critical role in the
initiation of apoptotic cell death and this is what we observed in
response to P-ASA.
The intrinsic (mitochondrial) pathway of apoptosis is characterized
by cytochrome c release and activation of caspase 9 but not of caspase
8 (17). This form of apoptosis can be triggered by increased ROS,
which permeabilize the mitochondrial membrane and release proa-
poptotic factors from the mitochondrial intermembrane space into the
cytosol (22). Our data demonstrated that P-ASA activated caspase 9,
while leaving caspase 8 intact. P-ASA increased mitochondrial ROS
and led to the collapse of the mitochondrial membrane potential.
Collectively, these findings establish that P-ASA targets the mito-
chondria and that this effect triggers the intrinsic apoptotic pathway.
Further support that ROS are involvedin the induction of cell death by
P-ASA comes from the finding that the antioxidant agent NAC, which
raises intracellular GSH levels, blocked both the rise of super-
oxide anion levels in the mitochondria and the induction of cell death
The ROS levels in a cell represent the balance between ROS pro-
duction and ROS inactivation by an intricate system of antioxidant
mechanisms (23). GSH, one of the most important antioxidant mech-
anisms in mammalian cells, responds directly to intracellular redox
changes and is also used as a cofactor for antioxidant enzymes (15). P-
ASA depleted GSH stores in a concentration-dependent manner. That
P-ASA may have increased ROS levels, at least in part, through its
effect on GSH is evidenced by two manipulations of the system de-
signed to affect the levels of GSH. First, BSO, which decreased in-
tracellular GSH, reduced the IC50of P-ASA for cell growth by more
than half and second, supplementing the cells with NAC greatly at-
tenuated the apoptotic effect of P-ASA. These effects are similar to
those we obtained with nitroaspirin, a structurally similar compound,
which depletes GSH by forming a conjugate with it (24).
The increase in intracellular levels of ROS by P-ASA had important
repercussions for the fate of the cancer cell. Our findings make it clear
that ROS, elevated in response to P-ASA, modulated predominantly
ily of serine/threonine kinases, play an essential role in signal trans-
duction by modulating gene transcription in the nucleus in response to
changes in the cellular environment (25). MAPKs are required for
specialized cell functions controlling cell proliferation, cell differen-
tiation, as well as cell death and are deregulated in several malignan-
cies including colon cancer (26–28). We observed that P-ASA
activated (by phosphorylation) p38 and JNK, while its effect on
ERK1/2 was insignificant. It is noteworthy that the p38 and JNK
‘branches’ of the MAPK cascade are redox sensitive, whereas ERK,
the one that remained unchanged, is not redox responsive (29). AKT,
which inhibits apoptosis and is frequently altered in various human
malignancies (30), was activated by P-ASA, albeit quite modestly.
Interestingly, recent data suggest that antineoplastic compounds mod-
ulate this pathway and such effects may mediate their pharmacolog-
ical activity (31). This seems to also be the case for P-ASA. Indeed,
P-ASA could exert part of its pharmacological effect through modu-
lation of MAPKs. This is supported by our previous observations that
nitroaspirin, a structurally related compound, modulated MAPKs and
the cell growth inhibitory effect of nitroaspirin was prevented by
MAPK inhibitors and by silencing the p38 and JNK MAPKs (27).
Two signaling pathways that probably interact closely are COX and
NF-jB, both regulate cell death and are of great importance to carci-
nogenesis (10). In particular, NF-jB, a mediator of inflammatory
responses, is now emerging as a link between inflammation and can-
cer (32,33). P-ASA stimulated the expression of COX-2 and inhibited
NF-jB signaling; the effect of P-ASA on both was redox dependent,
as NAC reversed it. Although it is usually assumed that NF-jB acti-
vation induces COX-2, severalother signaling cascades convergeonto
the cox-2 promoter, including Sp-1, c-MYB, activator protein-1,
T-cell factor, cAMP responsive element and activator protein-2
(34). Moreover, de Moraes et al. (34) suggest two different scenarios
to explain COX-2 regulation. In the ‘inflammatory scenario’, p53
activated by DNA damage, recruits NF-jB to activate COX-2, result-
ing in antiapoptotic effects that contribute to cell expansion in in-
proliferation scenario’, oncogenic stress due to activation of growth
signaling cascades (e.g. those involving Wnt/b-catenin, K-ras or
c-Myb) upregulates cox-2 independent of NF-jB to promote cancer
progression (34). In agreement with the above, we have recently
shown that several chemopreventive agents increase ROS production
leading to COX-2 overexpression (35) and NF-jB inhibition (36).
These chemopreventive agents, including P-ASA, affect the thiore-
doxin system that is essential to maintain critical cysteine residues of
the p50 and p65 subunits in their reduced form, which is required for
NF-jB binding to itsconsensus sequence (36). Thus, we postulate that
P-ASA can activate COX-2, while inhibiting NF-jB binding.
Changes in cell signaling in response to pharmacological agents
can be both extensive and complex.As we and others have pointed out
(4), it is often difficult to discern the relevance to the final pharmaco-
logical result of each pathway that has been changed. This appears to
be the case with our data. Nevertheless, it is conceivable that one or
more of the effects of P-ASA on these signaling pathways could be
pivotal for its remarkable pharmacological action.
In conclusion, our data demonstrate that P-ASA (MDC-43), which
targets several types of cancer, possesses broad anticancer properties.
P-ASA is significantly more potent than conventional aspirin in in-
hibiting cancer cell growth. Underlying this growth inhibition appears
to be changes in cellular redox homeostasis, which activate significant
intracellular signaling pathways probably culminating in a major cy-
tokinetic effect. These findings make it clear that P-ASA is a promis-
ing novel agent for the control of cancer that merits further evaluation.
National Institutes of Health (2R01 CA92423, R01 CA101019).
Conflict of Interest Statement: None declared.
1.Baron,J.A. (2003) Epidemiology of non-steroidal anti-inflammatory drugs
and cancer. Prog. Exp. Tumor Res., 37, 1–24.
2.Baron,J.A. et al. (2003) A randomized trial of aspirin to prevent colorectal
adenomas. N. Engl. J. Med., 348, 891–899.
3.Rayyan,Y. et al. (2002) The role of NSAIDs in the prevention of colon
cancer. Cancer Invest., 20, 1002–1011.
4.Rigas,B. (2007) The use of nitric oxide-donating nonsteroidal anti-
inflammatory drugs in the chemoprevention of colorectal neoplasia. Curr.
Opin. Gastroenterol., 23, 55–59.
5.Lazzarato,L. et al. (2008) Searching for new NO-donor aspirin-like mole-
cules: a new class of nitrooxy-acyl derivatives of salicylic acid. J. Med.
Chem., 51, 1894–1903.
6.Rigas,B. et al. (2008) The novel phenylester anticancer compounds: study
of a derivative of aspirin (phoshoaspirin). Int. J. Oncol., 32, 97–100.
7.Kashfi,K. et al. (2005) Positional isomerism markedly affects the growth
inhibition of colon cancer cells by nitric oxide-donating aspirin in vitro and
in vivo. J. Pharmacol. Exp. Ther., 312, 978–988.
8.Tietze,F. (1969) Enzymic method for quantitative determination of nano-
gram amountsof totaland oxidizedglutathione:applications to mammalian
blood and other tissues. Anal. Biochem., 27, 502–522.
W.Zhao et al.
9.Mackenzie,G.G. et al. (2004) Epicatechin, catechin, and dimeric procyani- Download full-text
dins inhibit PMA-induced NF-kappaB activation at multiple steps in Jurkat
T cells. FASEB J., 18, 167–169.
10.Williams,J.L. et al. (2008) NO-donating aspirin inhibits the activation of
NF-kappaB in human cancer cell lines and Min mice. Carcinogenesis, 29,
11.Williams,J.L. et al. (2001) Nitric oxide-releasing nonsteroidal anti-
inflammatory drugs (NSAIDs) alter the kinetics of human colon cancer cell
lines more effectively than traditional NSAIDs: implications for colon
cancer chemoprevention. Cancer Res., 61, 3285–3289.
12.Rigas,B. et al. (2008) Induction of oxidative stress as a mechanism of action
of chemopreventive agents against cancer. Br. J. Cancer, 98, 1157–1160.
13.LeBel,C.P. et al. (1992) Evaluation of the probe 2#,7#-dichlorofluorescin as
an indicator of reactive oxygen species formation and oxidative stress.
Chem. Res. Toxicol., 5, 227–231.
14.Meister,A. (1991) Glutathione deficiency produced by inhibition of its
synthesis, and its reversal; applications in research and therapy. Pharmacol.
Ther., 51, 155–194.
15.Klaunig,J.E. et al. (2004) The role of oxidative stress in carcinogenesis.
Annu. Rev. Pharmacol. Toxicol., 44, 239–267.
16.Griffith,O.W. et al. (1979) Potent and specific inhibition of glutathione
synthesisby buthioninesulfoximine(S-n-butyl homocysteine sulfoximine).
J. Biol. Chem., 254, 7558–7560.
17.Watson,A.J. (2004) Apoptosis and colorectal cancer. Gut, 53, 1701–1709.
18.Burgunder,J.M. et al. (1989) Effect of N-acetylcysteine on plasma cysteine
and glutathione following paracetamol administration. Eur. J. Clin. Phar-
macol., 36, 127–131.
19.Pathak,S.K. et al. (2005) Oxidative stress and cyclooxygenase activity in
prostatecarcinogenesis: targets forchemopreventivestrategies. Eur. J. Can-
cer, 41, 61–70.
20.Bubici,C. et al. (2006) The NF-kappaB-mediated control of ROS and JNK
signaling. Histol. Histopathol., 21, 69–80.
21.Zhou,H. et al. (2009) Nitric oxide-donating aspirin inhibits the growth of
pancreatic cancer cells through redox-dependent signaling. Cancer Lett.,
22.Mukhopadhyay,P. et al. (2007) Simultaneous detection of apoptosis and
mitochondrial superoxide production in live cells by flow cytometry and
confocal microscopy. Nat. Protoc., 2, 2295–2301.
23.Halliwell,B. (2007) Oxidative stress and cancer: have we moved forward?
Biochem. J., 401, 1–11.
24.Gao,J. et al. (2005) Nitric oxide-donating aspirin induces apoptosis in
human colon cancer cells through induction of oxidative stress. Proc. Natl
Acad. Sci. USA, 102, 17207–17212.
25.Turjanski,A.G. et al. (2007) MAP kinases and the control of nuclear events.
Oncogene, 26, 3240–3253.
26.Chang,L. et al. (2001) Mammalian MAP kinase signalling cascades. Na-
ture, 410, 37–40.
27.Hundley,T.R. et al. (2006) Nitric oxide-donating aspirin inhibits colon
cancer cell growth via mitogen-activated protein kinase activation. J. Phar-
macol. Exp. Ther., 316, 25–34.
28.Manning,A.M. et al. (2003) Targeting JNK for therapeutic benefit: from
junk to gold? Nat. Rev. Drug Discov., 2, 554–565.
29.Pearson,G. et al. (2001) Mitogen-activated protein (MAP) kinase
pathways: regulation and physiological functions. Endocr. Rev., 22,
30.Michl,P. et al. (2005) Mechanisms of disease: PI3K/AKT signaling in
gastrointestinal cancers. Z. Gastroenterol., 43, 1133–1139.
31.Bode,A.M. et al. (2004) Targeting signal transduction pathways by chemo-
preventive agents. Mutat. Res., 555, 33–51.
32.Karin,M. et al. (2005) NF-kappaB: linking inflammation and immunity to
cancer development and progression. Nat. Rev. Immunol., 5, 749–759.
33.Zhang,Z. et al. (2006) NF-kappaB, inflammation and pancreatic carcino-
genesis: NF-kappaB as a chemoprevention target (review). Int. J. Oncol.,
34.de Moraes,E. et al. (2007) Cross-talks between cyclooxygenase-2 and tu-
mor suppressor protein p53: balancing life and death during inflammatory
stress and carcinogenesis. Int. J. Cancer, 121, 929–937.
35.Sun,Y. et al. (2008) Chemopreventive agents induce oxidative stress in
cancer cells leading to COX-2 overexpression and COX-2-independent cell
death. Carcinogenesis, in press.
36.Sun,Y. et al. (2008) The thioredoxin system mediates redox-induced cell
death in human colon cancer cells: implications for the mechanism of
action of anticancer agents. Cancer Res., 68, 8269–8277.
Received September 22, 2008; revised December 31, 2008;
accepted January 5, 2009
Phosphoaspirin inhibits the growth of human cancer cell lines