Amidization of doxorubicin alleviates doxorubicin-induced contractile dysfunction and reduced survival in murine cardiomyocytes

Article abstract of DOI:10.1016/j.toxlet.2008.03.010

Doxorubicin is an effective anthracycline used for cancer therapy. However, the clinical application of doxorubicin has been largely limited by its irreversible cardiotoxicity, which is mainly induced by the primary amine group. In this study, we structurally modified doxorubicin by converting the primary amine into an acid-labile amide before assessing the acute cardiac effect of doxorubicin (pristine or modified) on cardiomyocyte contractile function. Contractile properties of murine cardiomyocytes were analyzed including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR₉₀) and maximal velocity of shortening/relengthening (±dL/dt). Cell toxicity and survival rate were evaluated using the MTT assay. The doxorubicin-free base was amidized by reacting with 3,4,5,6-tetrahydophthalic anhydride (THPA) or 3,3,4,4-tetramethylsuccinic anhydride (TMSA) to yield doxorubicin-THPA or -TMSA. Acute exposure of pristine doxorubicin (10^-9-10^-5 M) for 30 min significantly prolonged TPS and TR₉₀ without affecting PS and ±dL/dt. Interestingly, doxorubicin-induced prolongation of TPS and TR₉₀ was significantly attenuated or abrogated by amidization of doxorubicin. Neither doxorubicin-THPA nor -TMSA affected PS and ±dL/dt. ROS and MTT assay revealed significantly reduced ROS production and cardiac cell toxicity from amidized doxorubicin compared with the pristine compound. Comparable cytotoxicity in human ovarian cancer SKOV-3 cells was observed between amidized and pristine doxorubicin compounds. These data provide evidence for the first time that structural modification of doxorubicin alleviates its cardiac toxicity.

Full text of DOI:10.1016/j.toxlet.2008.03.010

Toxicology Letters 178 (2008) 197–201
Contents lists available at ScienceDirect
Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
Amidization of doxorubicin alleviates doxorubicin-induced contractile
dysfunction and reduced survival in murine cardiomyocytes
Subat Turdia,b, Peisheng Xuc, Qun Lia, Youqing Shenc,∗∗, Parhat Kerramb, Jun Rena,∗
a Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071, USA
b Xinjiang Medical University, Urumqi, Xinjiang, PR China
c Department of Chemical and Petroleum Engineering, University of Wyoming, Laramie, WY 82071, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Doxorubicin is an effective anthracycline used for cancer therapy. However, the clinical application of
doxorubicin has been largely limited by its irreversible cardiotoxicity, which is mainly induced by the pri-
mary amine group. In this study, we structurally modified doxorubicin by converting the primary amine
into an acid-labile amide before assessing the acute cardiac effect of doxorubicin (pristine or modified)
on cardiomyocyte contractile function. Contractile properties of murine cardiomyocytes were analyzed
including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity
of shortening/relengthening ( dL/dt). Cell toxicity and survival rate were evaluated using the MTT assay.
The doxorubicin-free base was amidized by reacting with 3,4,5,6-tetrahydophthalic anhydride (THPA)
or 3,3,4,4-tetramethylsuccinic anhydride (TMSA) to yield doxorubicin-THPA or -TMSA. Acute exposure
of pristine doxorubicin (10−9–10−5 M) for 30 min significantly prolonged TPS and TR90 without affecting
PS and dL/dt. Interestingly, doxorubicin-induced prolongation of TPS and TR90 was significantly atten-
uated or abrogated by amidization of doxorubicin. Neither doxorubicin-THPA nor -TMSA affected PS and
dL/dt. ROS and MTT assay revealed significantly reduced ROS production and cardiac cell toxicity from
amidized doxorubicin compared with the pristine compound. Comparable cytotoxicity in human ovar-
ian cancer SKOV-3 cells was observed between amidized and pristine doxorubicin compounds. These
data provide evidence for the first time that structural modification of doxorubicin alleviates its cardiac
toxicity.
Received 17 January 2008
Received in revised form 14 March 2008
Accepted 14 March 2008
Available online 25 March 2008
Keywords:
Doxorubicin
Amidization
Cardiac myocytes
Shortening
Relengthening
ROS
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
(Taylor and Bulkley, 1982). Up-to-date, the precise mechanism(s)
of cardiac damages are largely unknown, making it somewhat
The anthracycline derivative doxorubicin (adriamycin) has been
widely used to treat patients with neoplastic diseases. How-
ever, its clinical value is greatly compromised by the overt
cardiac toxicity of doxorubicin which results in a late irreversible
congestive cardiomyopathy namely doxorubicin cardiomyopa-
thy usually refractory to common medications (Kalyanaraman
et al., 2002; Taylor and Bulkley, 1982). Clinical manifestations
of doxorubicin-induced cardiotoxicity encompass electrocardio-
graphic abnormalities such as Q-T prolongation and Q-T dispersion
followed by the life-threatening doxorubicin cardiomyopathy
(Chugun et al., 2000; Wang et al., 2001). Morphologically, dox-
orubicin exposure may result in acute and distinctive nuclear
alterations associated with mild cytoplasmic changes in the heart
difficult to enable development of therapies to prevent and/or
treat doxorubicin cardiomyopathy. Several mechanisms have been
postulated for the pathogenesis of this deadly cardiotoxicity
including free radical accumulation, vasoactive amine release,
myocyte damage induced by intracellular Ca2+ overload, inhib-
ited expression of cardiomyocyte-specific genes, impairment in
myocardial adrenergic signaling/regulation and cellular toxicity
of doxorubicin metabolites such as doxorubicinol (Chugun et
al., 2000; Mushlin and Olson, 1988; Olson and Mushlin, 1990;
Shan et al., 2003). In particular, generation of reactive oxy-
gen species (ROS) and subsequent cardiac DNA or membrane
damage are thought to be the most significant mechanisms under-
scoring the cardiac toxicity of doxorubicin (Myers et al., 1977;
Rajagopalan et al., 1988). It has been demonstrated that the
quinone moiety of doxorubicin is prone to form highly reactive
semiquinone radicals by one-electron reduction. These doxorubicin
semiquinone radicals may readily react with molecular oxygen
to generate superoxide anion and other reactive oxygen species
∗
Corresponding authors. Tel.: +1 307 766 6131; fax: +1 307 766 2953.
Corresponding authors.
∗∗
E-mail addresses: sheny@uwyo.edu (Y. Shen), jren@uwyo.edu (J. Ren).
0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2008.03.010

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S. Turdi et al. / Toxicology Letters 178 (2008) 197–201
en route to ultimate cell injury (Lown et al., 1982; Minotti et al.,
1999).
2.2. Murine cardiomyocyte isolation
The experimental procedure used in this study was approved by the Animal Use
and Care Committee at University of Wyoming and was in compliance with the pro-
visions of the Declaration of Helsinki in 1995. In brief, hearts were rapidly removed
from anesthetized mice and immediately mounted on a temperature-controlled
(37 ◦C) Langendorff system. After perfusion with modified Tyrode solution for
2 min, the heart was digested for 10 min with 0.9 mg/ml collagenase D (Boehringer
Mannheim Biochemicals, Indianapolis, IN) in modified Tyrode solution. The mod-
ified Tyrode solution (pH 7.4) contained the following (in mM): NaCl 135, KCl 4.0,
MgCl2 1.0, HEPES 10, NaH2PO4 0.33, glucose 10, butanedione monoxime 10, and the
solution was gassed with 5% CO2–95% O2. The digested heart was then removed
from the cannula and left ventricle was cut into small pieces in the modified Tyrode
solution. These pieces were gently agitated and the pellet of cells was resuspended
in modified Tyrode solution and allowed to settle for 20 min at room temperature
during which time extracellular Ca2+ was added incrementally back to 1.2 mM. Only
rod-shaped myocytes with clear edges were selected for mechanical study (Dong et
al., 2006).
Ample evidence has suggested that the primary amino group
in doxorubicin plays an important role in its cardiotoxicity. The
synthetic doxorubicin analog, hydroxyrubicin, where the primary
amino group is replaced by a hydroxyl group, displayed reduced car-
diac toxicity compared with doxorubicin, possibly due to decreased
affinity of hydroxyrubicin to cardiolipin and deamination at the 3ꢀ
position (Priebe et al., 1993). We thus hypothesized that amidiza-
tion of the amine group into an acid-labile amide may alleviate
the doxorubicin cardiotoxicity. The amidization procedure is ratio-
nal since amide is hydrolyzed upon internalization of doxorubicin
into lysosomes and a fully functioning doxorubicin can be regen-
erated. A number of studies have shown that ␤-carboxyamides of
primary and second amines are acid labile and quickly hydrolyze
to regenerate the amines at acidic pHs, although they are stable at
neutral pH (Kluger et al., 1979; Lee et al., 2007; Xu et al., 2007).
Thus the aim of this study was to determine the effect of amidiza-
tion (by converting the primary amine into to ␤-carboxyamides) on
doxorubicin-induced cardiomyocyte contractile dysfunction and
cell toxicity. Our goal was to alleviate doxorubicin-induced car-
diomyopathy so that prevention and management of cancer may
be optimized.
2.3. Cell shortening/relengthening
Mechanical properties of adult murine cardiac myocytes were assessed by an
IonOptix Myocam system (IonOptix Incorporation, Milton, MA, USA) as described
previously (Dong et al., 2006). Cells were placed in a chamber mounted on the stage
of an inverted microscope and superfused (at 30 ◦C) with a buffer containing (in
mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose and 10 HEPES at pH 7.4. The
cells were field stimulated at a frequency of 0.5 Hz. Cell shortening and relengthen-
ing were assessed using the following indices: peak shortening (PS) – indicative of
peak ventricular contractility, time-to-90% PS (TPS) – indicative of systolic duration,
time-to-90% relengthening (TR90) – indicative of diastolic duration, maximal veloc-
ities of shortening (+dL/dt) and relengthening (−dL/dt) – indicatives of maximal
velocities of ventricular pressure rise/fall. To test the acute effect of doxorubicin on
cardiac mechanical properties, cell shortening was recorded before and 5 min after
each individual concentration of pristine or amidized doxorubicin (10−9–10−5 M)
treatment.
2. Methods
2.1. Amidization of doxorubicin
Doxorubicin hydrochloride salt (DOX, 100 mg) was dissolved in 10 ml
of dimethylsulfoxide (DMSO) under constant stirring. Triethylamine (120 ␮l)
was added and stirred for another hour. The solution was extracted using
dichloromethane. The dichloromethane phase was collected and dried over anhy-
2.4. Generation of intracellular reactive oxygen species (ROS)
drous magnesium sulfate. The dichloromethane was removed with
a rotary
Production of cellular ROS was evaluated by analyzing changes in flu-
orescence intensity resulting from oxidation of the intracellular fluoroprobe
5-(6)-chloromethyl-2ꢀ,7ꢀ-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). In
brief, isolated cardiomyocytes following treatment with various concentrations of
pristine or amidized doxorubicin (10−8–10−6 M) were loaded with 1 ␮M of the
non-fluorescent dye 2ꢀ,7ꢀ-dichlorodihydrofluorescein diacetate (H2DCFDA, Molec-
ular Probes, Eugene, OR, USA) at 37 ◦C for 30 min. The myocytes were rinsed and the
fluorescence intensity was then measured using a fluorescent micro-plate reader at
an excitation wavelength of 480 nm and an emission wavelength of 530 nm (Molec-
ular Devices, Sunnyvale, CA, USA). Untreated cells showed no fluorescence and were
used to determine background fluorescence, which was subtracted from the treated
samples. The final fluorescent intensity was normalized to the protein content in
each myocyte group and was expressed as percent changes from the control value
(0 concentration of doxorubicin) (Privratsky et al., 2003).
evaporator and the doxorubicin-free base was obtained. The doxorubicin-free
base was dried under high vacuum for 8 h at room temperature. Doxorubicin-
free base solid (81 mg) was obtained and was amidized by reacting with
3,4,5,6-tetrahydophthalic anhydride (THPA) or 3,3,4,4-tetramethylsuccinic anhy-
dride (TMSA). In brief, the doxorubicin-free base (20 mg) was dissolved in 5 ml
of dried DMSO in a 25 ml flask. 3,4,5,6-tetrahydophthalic anhydride (6.14 mg) was
added and the mixture was stirred for 6 hrs at room temperature in the dark. The
product was purified by column chromatography. The column was first washed
with anhydrous diethyl ether to remove unreacted anhydride. The amidized DOX
was obtained by washing the column using DMSO. The solution was lyophilized
to yield
a solid (DOX-THPA, 12.5 mg). DOX amidized with TMSA (DOX-TMSA)
was synthesized similarly (Fig. 1). The final concentration of DMSO used in cell
culture was <0.1% which did not exert any effect on cell contractility or cell
survival.
Fig. 1. Structure of amidized doxorubicin-THPA and doxorubicin-TMSA.

S. Turdi et al. / Toxicology Letters 178 (2008) 197–201
199
relengthening (TR90) at the concentrations of 10−7 M or higher.
Interestingly, the threshold of doxorubicin-THPA-elicited prolon-
gation of TPS and TR90 was shifted to the right to 10−5 M while
doxorubicin-TMSA failed to prolong TPS and TR90 at the concen-
trations tested (Fig. 3). Given the known role of ROS production in
doxorubicin-induced cardiomyocyte contractile dysfunction (Wold
et al., 2005), ROS generation was measured in cardiomyocytes
treated with or without pristine or amidized doxorubicin using DCF
fluorescence. Results shown in Fig. 4 display that pristine doxoru-
bicin (at 10−7 and 10−6 M) significantly enhanced ROS generation.
Interestingly, amidized doxorubicin exhibited significantly reduced
potency in ROS generation, indicating a likely role of dampened
ROS production in reduced cardiomyocyte contractile aberration
following amidization of doxorubicin. Our further evaluation of
cell toxicity using the MTT assay revealed that amidization of
doxorubicin significantly reduced cytotoxicity in cardiomyocytes
in a comparable fashion with doxorubicin-TMSA being the least
cytotoxic. However, the cytotoxicity of modified doxorubicin was
not significantly different from that of the pristine doxorubicin in
human ovarian cancer SKOV-3 cells (Fig. 5).
2.5. Cytotoxicity assay
The cytotoxicity assay was carried out using the (3-(4,5-dimethylthiazolyl-2)-
2,5-diphenyltetrazolium bromide) (MTT) cell proliferation kit (ATCC, Manassas, VA)
according to the modified manufacturer’s protocol. Both murine cardiomyocytes
and human ovarian cancer SKOV-3 cells were seeded in 96-well plates at an ini-
tial density of 15,000 cells/well in 200 ␮l of RPMI medium. Murine cardiomyocytes
were used immediately and SKOV-3 cells were allowed to grow for 24 h. The original
medium in each well was replaced with 100 ␮l of fresh medium. The doxorubicin,
DOX-TMSA or DOX-THPA was added to the medium. Treated cells were incubated at
37 ◦C under a humidified air with 5% CO2 for 4 h. MTT reagent (10 ␮l) was added to
each well and the cells were incubated for 2 h at 37 ◦C or until purple crystals were
visible. Detergent reagent (100 ␮l) was added to each well and then the plates were
placed in a 37 ◦C incubator for 2 h, or until all the crystals dissolved. The absorbance
at 570 nm of the solution in each well was recorded using a microplate UV spec-
trometer (SpectraMax 384 Plus). Cell viability was calculated according to the
protocol.
2.6. Data analysis
A total of eight mice were used in this study. For each experimental series, data
are presented as mean S.E.M. Statistical significance (p < 0.05) for each variable
was estimated by two-way analysis of variance (ANOVA). A Dunnett’s test was used
for post hoc analysis when required.
3. Results
4. Discussion
Acute exposure (30 min) of pristine and amidized doxorubicin
(10−9–10−5 M) reduced resting cell length at the highest concen-
tration tested with no effects at lower concentrations. Similar to
our earlier report (Wold et al., 2005), pristine doxorubicin did
not affect peak shortening (PS) amplitude and maximal veloc-
ity of shortening/relengthening ( dL/dt). Similar response was
observed for the two amidized doxorubicin (doxorubicin-THPA and
doxorubicin-TMSA) (Fig. 2). Acute administration of pristine dox-
orubicin prolonged the duration of myocyte shortening (TPS) and
Our current work has provided evidence for the first time
that amidization of doxorubicin significantly lessened doxorubicin-
induced contractile dysfunction, ROS generation and cell survival
in adult murine cardiomyocytes. The major mechanical interrup-
tions in response to acute pristine doxorubicin exposure found
in our study were shown as prolonged duration of contraction
and relaxation, both of which considered the hallmarks of car-
diomyopathies originated from several kinds of cardiac morbidities
Fig. 2. Effect of acute treatment of pristine and amidized doxorubicin (10−9–10−5 M) on resting cell length (panel B), peak cell shortening (percent of resting cell length,
panel C), indicative of peak ventricular contractility, and maximal velocity of shortening/relengthening ( dL/dt, panel D), indicatives of maximal ventricular pressure rise/fall
in adult murine cardiomyocytes. Panel A depicts representative cell shortening traces from control, doxorubicin and doxorubicin-TMSA (10−5 M) groups. Mean S.E.M.,
n = 65–70 cells at each data point, *p < 0.05 vs. control (0 doxorubicin).

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S. Turdi et al. / Toxicology Letters 178 (2008) 197–201
Fig. 3. Effect of acute treatment of pristine and amidized doxorubicin (10−9–10−5 M)
on time-to-peak shortening (panel A) – indicative of systolic duration, and time-
to-90% relengthening (panel B) – indicative of diastolic duration in adult murine
cardiomyocytes. Mean S.E.M., n = 65–70 cells at each data point, *p < 0.05 vs. con-
trol (0 doxorubicin).
Fig. 5. Effect of acute treatment of pristine and amidized doxorubicin on cell sur-
vival in cardiomyocytes (panel A) and human ovarian cancer SKOV-3 cells (panel
B). Mean S.E.M., n = 5 independent assays using triplicates, *p < 0.05 vs. control (0
doxorubicin), #p < 0.05 vs. all other doxorubicin groups at the same concentration.
such as heart failure, ischemia-reperfusion injury, hypertension
and diabetes (Wold et al., 2001; Ye et al., 2003). Given compara-
ble cytotoxicity maintains for SKOV-3 human ovarian cancer cells,
our results suggested that structural modification of doxorubicin
may greatly alleviate its cardiotoxicity, a major limitation in the
anticancer therapy of the anthracycline agent.
Earlier study from our lab revealed that doxorubicin pro-
longs cardiomyocyte contraction and relaxation duration (TPS
and TR90) via p38 MAP kinase-dependent accumulation of oxida-
tive stress (Wold et al., 2005). Interestingly, data from our
current study displayed a rightward shift in the threshold of
doxorubicin-THPA-induced prolongation of TPS and TR90 and
abrogation of doxorubicin-TMSA-induced mechanical defects in
cardiomyocytes, compared with the pristine doxorubicin. Mean-
while, direct measurement of ROS generation revealed reduced
ROS generating capacity in amidized doxorubicin, supporting
the notion of ROS-induced oxidative damage as a major cul-
prit factor for doxorubicin-induced cardiomyopathy (Olson and
Mushlin, 1990). Recent evidence has indicated that peroxynitrite
formation resulted from dysregulated nitric oxide and activa-
tion of the nuclear enzyme poly(ADP-ribose) polymerase (PARP)
by oxidant-mediated DNA damage may contribute to cardiotox-
icity of doxorubicin (Pacher et al., 2002; Pacher et al., 2003;
Pacher et al., 2007). Our earlier evidence suggested that the
doxorubicin-induced prolongation of TR90 may be underscored by
prolongation in intracellular Ca2+ transient clearance (Wold et al.,
2005). Although this is beyond the scope of our current study, it
is quite possible that modification of doxorubicin may impose a
lesser detrimental effect on intracellular Ca2+ clearance. Several
lines of evidence have suggested the existence of an abnormal
Ca2+ homeostasis in cardiomyocytes, which may explain mechani-
cal abnormalities in pristine doxorubicin-induced cardiomyopathy.
Doxorubicin has been reported to prolong action potential dura-
tion, inhibit gene transcription of sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA), reduce sarcoplasmic reticulum (SR) Ca2+
load and subsequently Ca2+-induced Ca2+ release in isolated car-
diomyocytes (Arai et al., 2000; Wang et al., 2001). The observation
Fig. 4. Effect of acute treatment of pristine and amidized doxorubicin on ROS gen-
eration in cardiomyocytes. Mean S.E.M., n = 5–8 independent assays, *p < 0.05 vs.
control (0 doxorubicin), #p < 0.05 vs. pristine doxorubicin at the same concentration.

S. Turdi et al. / Toxicology Letters 178 (2008) 197–201
201
that acute doxorubicin exposure in our current experimental set-
References
ting failed to affect any other mechanical indices such as peak
shortening and maximal velocity of shortening/relengthening may
suggest a significant difference in sensitivity among various cardiac
contractile components upon doxorubicin exposure. For example,
proteins responsible for duration of cell shortening/relengthening
Arai, M., Yoguchi, A., Takizawa, T., Yokoyama, T., Kanda, T., Kurabayashi, M., Nagai, R.,
2000. Mechanism of doxorubicin-induced inhibition of sarcoplasmic reticulum
Ca(2+)-ATPase gene transcription. Circ. Res. 86, 8–14.
Chugun, A., Temma, K., Oyamada, T., Suzuki, N., Kamiya, Y., Hara, Y., Sasaki, T., Kondo,
H., Akera, T., 2000. Doxorubicin-induced late cardiotoxicity: delayed impairment
of Ca2+-handling mechanisms in the sarcoplasmic reticulum in the rat. Can. J.
Physiol Pharmacol. 78, 329–338.
(TPS and TR90
)
such as SR Ca2+ release channel, myosin
ATPase, sarco(endo)plasmic reticulum Ca2+-ATPase and Na+/Ca2+
exchanger may be more sensitive to acute doxorubicin exposure
than those proteins responsible for cardiac contractility such as
actin, myosin and SR Ca2+ load. It has been shown that late stage
doxorubicin-induced cardiac toxicity is associated with reduced
cardiac contractility, decreased velocity of contraction/relaxation
and intracellular Ca2+ overload, something that do not exist follow-
ing acute doxorubicin toxicity (Chugun et al., 2000). The decreased
contractility of individual myocytes may be attributed to their low
myosin content, which may result in decreased cardiac output
following doxorubicin treatment (Jones et al., 1990). It is worth
mentioning that doxorubicin-induced heart failure is not asso-
ciated with ␤-adrenoceptor desensitization, unlike heart failure
induced by pressure- or volume-overload (Jones et al., 1990).
Our data revealed significantly reduced cytotoxicity in response
to amidized doxorubicin exposure in cardiomyocytes but not
human ovarian cancer SKOV-3 cells. One possible explanation for
the lessened cardiac cytotoxicity may be that the amidized doxoru-
bicin does not localize in lysosomes and therefore cannot efficiently
regenerate pristine doxorubicin. Unlike the pristine doxorubicin
which carries a cationic charge and is easily sequestrated into
lysosomes (Gong et al., 2003), the amidized doxorubicin is nega-
tively charged and thus is not efficient in lysosome sequestration.
We are currently exploring to amizide the amine using a polymer
anhydride to ensure that the amidized doxorubicin is localized in
lysosomes for regeneration. It is possible that difference between
normal cardiomyocytes and cancer cells such as pH may contribute
to the discrepant cytotoxic response to amidized doxorubicin. As
a limitation of our study, we failed to measure the intracellular
doxorubicin concentration to validate ␤-carboxylix acid amide
hydrolysis in an acidic environment. It is expected that regenerated
doxorubicin following hydrolysis may quickly bind to DNA, making
it difficult to be quantitated. Further study is warranted to explore
the mechanism of action behind ␤-carboxylix acid amide hydrol-
ysis in cardiomyocytes and cancer cells following the amidization
procedure.
Dong, F., Fang, C.X., Yang, X., Zhang, X., Lopez, F.L., Ren, J., 2006. Cardiac overex-
pression of catalase rescues cardiac contractile dysfunction induced by insulin
resistance: Role of oxidative stress, protein carbonyl formation and insulin sen-
sitivity. Diabetologia 49, 1421–1433.
Gong, Y., Duvvuri, M., Krise, J.P., 2003. Separate roles for the Golgi apparatus and lyso-
somes in the sequestration of drugs in the multidrug-resistant human leukemic
cell line HL-60. J Biol. Chem. 278, 50234–50239.
Jones, S.M., Kirby, M.S., Harding, S.E., Vescova, G., Wanless, R.B., Dalla Libera,
L.D., Poole-Wilson, P.A., 1990. Adriamycin cardiomyopathy in the rabbit: alter-
ations in contractile proteins and myocyte function. Cardiovasc. Res. 24, 834–
842.
Kalyanaraman, B., Joseph, J., Kalivendi, S., Wang, S., Konorev, E., Kotamraju, S.,
2002. Doxorubicin-induced apoptosis: implications in cardiotoxicity. Mol. Cell
Biochem. 234–235, 119–124.
Kluger, R., Chin, J., Choy, W.W., 1979. Carboxylic acid participation in amide
hydrolysis. reactivity of intermediates in the internally catalyzed hydrolysis
of N-substituted 2,3-dimethylmaleamic acids. J. Am. Chem. Soc. 101, 6976–
6980.
Lee, Y., Fukushima, S., Bae, Y., Hiki, S., Ishii, T., Kataoka, K., 2007. A protein nanocarrier
from charge-conversion polymer in response to endosomal pH. J. Am. Chem. Soc.
129, 5362–5363.
Lown, J.W., Chen, H.H., Plambeck, J.A., Acton, E.M., 1982. Further studies on the
generation of reactive oxygen species from activated anthracyclines and the
relationship to cytotoxic action and cardiotoxic effects. Biochem. Pharmacol.
31, 575–581.
Minotti, G., Cairo, G., Monti, E., 1999. Role of iron in anthracycline cardiotoxicity:
new tunes for an old song? FASEB J. 13, 199–212.
Mushlin, P.S., Olson, R.D., 1988. Anthracycline cardiotoxicity: new insights. Ration.
Drug Ther. 22, 1–9.
Myers, C.E., McGuire, W.P., Liss, R.H., Ifrim, I., Grotzinger, K., Young, R.C., 1977. Adri-
amycin: the role of lipid peroxidation in cardiac toxicity and tumor response.
Science 197, 165–167.
Olson, R.D., Mushlin, P.S., 1990. Doxorubicin cardiotoxicity: analysis of prevailing
hypotheses. FASEB J. 4, 3076–3086.
Pacher, P., Beckman, J.S., Liaudet, L., 2007. Nitric oxide and peroxynitrite in health
and disease. Physiol. Rev. 87, 315–424.
Pacher, P., Liaudet, L., Bai, P., Mabley, J.G., Kaminski, P.M., Virag, L., Deb, A.,
Szabo, E., Ungvari, Z., Wolin, M.S., Groves, J.T., Szabo, C., 2003. Potent
metalloporphyrin peroxynitrite decomposition catalyst protects against the
development of doxorubicin-induced cardiac dysfunction. Circulation 107,
896–904.
Pacher, P., Liaudet, L., Bai, P., Virag, L., Mabley, J.G., Hasko, G., Szabo, C.,
2002. Activation of poly(ADP-ribose) polymerase contributes to develop-
ment of doxorubicin-induced heart failure. J. Pharmacol. Exp. Ther. 300,
862–867.
Priebe, W., Van, N.T., Burke, T.G., Perez-Soler, R., 1993. Removal of the basic cen-
ter from doxorubicin partially overcomes multidrug resistance and decreases
cardiotoxicity. Anticancer Drugs 4, 37–48.
Privratsky, J.R., Wold, L.E., Sowers, J.R., Quinn, M.T., Ren, J., 2003. AT1 blockade pre-
vents glucose-induced cardiac dysfunction in ventricular myocytes: role of the
AT1 receptor and NADPH oxidase. Hypertension 42, 206–212.
Rajagopalan, S., Politi, P.M., Sinha, B.K., Myers, C.E., 1988. Adriamycin-induced free
radical formation in the perfused rat heart: implications for cardiotoxicity. Can-
cer Res. 48, 4766–4769.
In conclusion, our study demonstrates that doxorubicin
cardiomyopathy characterized by prolonged duration of cell short-
ening/relengthening may be effectively alleviated by amidization
of doxorubicin. While the precise natures of acute doxorubicin
exposure-induced mechanical defects and cardiac protection from
structural modification are still not clear, future studies should
focus on the mechanism of action behind doxorubicin-induced
ROS-dependent or independent regulation on cardiac contractile
response. These approaches should be helpful to advance our
knowledge of the cellular effects and toxicological profiles of dox-
orubicin on heart function, in order to design better therapy against
doxorubicin-induced cardiomyopathy.
Shan, Y.X., Liu, T.J., Su, H.F., Samsamshariat, A., Mestril, R., Wang, P.H., 2003. Hsp10
and Hsp60 modulate Bcl-2 family and mitochondria apoptosis signaling induced
by doxorubicin in cardiac muscle cells. J. Mol. Cell Cardiol. 35, 1135–1143.
Taylor, A.L., Bulkley, B.H., 1982. Acute adriamycin cardiotoxicity: morphologic alter-
ations in isolated perfused rabbit heart. Lab Invest. 47, 459–464.
Wang, G.X., Wang, Y.X., Zhou, X.B., Korth, M., 2001. Effects of doxorubicinol on
excitation–contraction coupling in guinea pig ventricular myocytes. Eur. J. Phar-
macol. 423, 99–107.
Wold, L.E., Aberle, N.S., Ren, J., 2005. Doxorubicin induces cardiomyocyte dysfunction
via a p38 MAP kinase-dependent oxidative stress mechanism. Cancer Detect.
Prev. 29, 294–299.
Wold, L.E., Relling, D.P., Colligan, P.B., Scott, G.I., Hintz, K.K., Ren, B.H., Epstein,
P.N., Ren, J., 2001. Characterization of contractile function in diabetic hyperten-
sive cardiomyopathy in adult rat ventricular myocytes. J. Mol. Cell Cardiol. 33,
1719–1726.
Xu, P., Van Kirk, E.A., Zhan, Y., Murdoch, W.J., Radosz, M., Shen, Y., 2007. Targeted
charge-reversal nanoparticles for nuclear drug delivery. Angew. Chem. Int. Ed
Engl. 46, 4999–5002.
Ye, G., Metreveli, N.S., Ren, J., Epstein, P.N., 2003. Metallothionein prevents diabetes-
induced deficits in cardiomyocytes by inhibiting reactive oxygen species
production. Diabetes 52, 777–783.
Acknowledgments
The authors appreciate the skillful assistance of Ms. Leah Kyle.
The work was supported in part by and the National Institutes of
Health, University of Wyoming Northern Rockies Regional INBRE
(5P20RR016474) and the Center for Cardiovascular Research and
Alternative Medicine (C-CRAM) from University of Wyoming.