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Geranylgeranylacetone blocks doxorubicin-induced cardiac toxicity and reduces cancer cell
growth and invasion through RHO pathway inhibition
Polina Sysa-Shah1, Yi Xu1, Xin Guo1, Djahida Bedja1, Rachel Bartock1, Allison Tsao1, Angela
Hsieh1, Michael S. Wolin2, An Moens3, Venu Raman4, Hajime Orita5*, Kathleen L. Gabrielson1
1Johns Hopkins Medical Institutions, Department of Molecular and Comparative Pathobiology,
Baltimore, MD, USA
2 New York Medical College. Department of Physiology, Valhalla, NY, USA
3Johns Hopkins Medical Institutions, Department of Cardiology, Baltimore, MD, USA
4Johns Hopkins University, Department of Radiology, Baltimore, MD, USA
5 Johns Hopkins University School of Medicine, Department of Pathology, Baltimore, MD, USA
*Current address: Hajime Orita, Juntendo Shizuoka Hospital, Department of Surgery, Izunokuni,
Shizuoka, Japan
To whom correspondence should be addressed: Dr. Kathleen Gabrielson, Department of Molecular
and Comparative Pathobiology, Johns Hopkins Medical Institutions, MRB 807, 733 N. Broadway,
Baltimore, MD, USA, 21205. Tel.: 410 955 4584; Fax: 443-287-2954; E-mail: [email protected]
Running title: GGA exerts cardioprotective and anti-cancer effects
Keywords: Doxorubicin, cardiac toxicity, GGA, oxidative stress, breast cancer, MDA-MB-231,
RHO, ROCK, NOS
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This work is supported by the RO1HL088649 (K.L. Gabrielson),
Spore in Breast Cancer Developmental Research Award #P50CA88843-04 (K.L. Gabrielson),
Department of Defense BC030727 (K.L. Gabrielson),
AHA SDG 0335273N (K.L. Gabrielson).
Word count:
Abstract - 250 words
Introduction- discussion – 4326 words
Figures legends - 893 words
67 references
5 figures
We report no conflict of interest. The authors had full access to the data and take responsibility for
its integrity. All authors have read and agree to the manuscript as written.
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Abstract
Doxorubicin is a widely used chemotherapy for solid tumors and hematological malignancies, but
its use is limited due to cardiotoxicity. Geranylgeranylacetone (GGA), an anti-ulcer agent used in
Japan for thirty years, has no significant adverse effects, and unexpectedly reduces ovarian cancer
progression in mice. Since GGA reduces oxidative stress in brain and heart, we hypothesized that
GGA would prevent oxidative stress of doxorubicin cardiac toxicity and improve doxorubicin’s
chemotherapeutic effects. Nude mice implanted with MDA-MB-231 breast cancer cells were
studied after chronic treatment with doxorubicin, doxorubicin/GGA, GGA, or saline. Trans-thoracic
echocardiography was used to monitor systolic heart function and xenografts evaluated. Mice were
euthanized and cardiac tissue evaluated for reactive oxygen species generation, TUNEL assay and
RHO/ROCK pathway analysis. Tumor metastases were evaluated in lung sections. In vitro studies
using Boyden chambers were performed to evaluate GGA effects on RHO pathway activator
lysophosphatidic acid (LPA) induced motility and invasion. We found that GGA reduced
doxorubicin cardiac toxicity, preserved cardiac function, prevented TUNEL-positive cardiac cell
death and reduced doxorubicin-induced oxidant production in a NOS-dependent and independent
manner. GGA also reduced heart doxorubicin induced ROCK1 cleavage. Remarkably, in xenograft
implanted mice, combined GGA/doxorubicin treatment decreased tumor growth more effectively
than doxorubicin treatment alone. As evidence of anti-tumor effect, GGA inhibited LPA-induced
motility and invasion by MDA-MB-231 cells. These anti-invasive effects of GGA were suppressed
by geranylgeraniol suggesting GGA inhibits RHO pathway through blocking geranylation. Thus,
GGA protects the heart from doxorubicin chemotherapy-induced injury and improves anti-cancer
efficacy of doxorubicin in breast cancer.
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Introduction
Doxorubicin (Adriamycin) is one of the most widely used anticancer agents and it is currently a first
choice chemotherapeutic drug for the treatment of primary, recurrent and metastatic breast cancer
(1-4). Unfortunately, the use of doxorubicin in breast cancer chemotherapy is frequently limited
due to its severe cumulative cardiac toxicity(5). Clinical signs of cardiac toxicity may occur during,
weeks, months or even years after chemotherapy. Strategies that reduce cardiac toxicity could
potentially allow higher dosages of doxorubicin to be used with an improved long-term survival in
patients with improved quality of life.
A prevention strategy that reduces oxidative stress, a common mechanism of doxorubicin cardiac
toxicity(5), led us to consider potential benefits of geranylgeranylacetone (GGA), an acylic
polyisoprenoid (Selbex®), which has been used since 1984 as an anti-ulcer drug in Japan with no
adverse reactions(6, 7). In multiple animal models of ischemia and reperfusion, GGA prevents
oxidative stress in liver, heart, brain, kidney and retina(8-14). A second mechanism linked to
GGA’s mechanism of action is its ability to inhibit the activation of the RHO family of GTPases
through reduction of protein geranylgeranylation, a post-translational modification required for
RHO family membrane targeting and signaling(15, 16). Targeting the RHO/ROCK pathway with
specific inhibitors is beneficial in a number of cardiovascular diseases including hypertension,
angina, ischemia-reperfusion injury, cardiac hypertrophy, chronic heart failure (17-23), all
conditions with some level of oxidative stress. Interestingly, this molecular activity of GGA might
also make it useful as a cancer therapeutic since RHO GTPases, and their downstream target, RHO-
associated kinases (ROCKs), are implicated in a variety of physiological functions associated with
cancer-related changes in the actin cytoskeletal assembly, such as cell adhesion, motility, and
migration (24).
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Since GGA may reduce a variety of molecular functions important for cancer cell growth and
migration, and at the same time, may reduce oxidative stress in the heart, we undertook an
investigation to determine whether GGA can inhibit the cardiac adverse effects of doxorubicin
while simultaneously inhibiting cancer cell growth. Specifically, we developed a breast cancer
mouse model of chronic doxorubicin injury to the heart and investigated cellular and molecular
responses to various treatments involving GGA and doxorubicin.
Materials and Methods
Reagents and materials: GGA (Lot # 17022802) was provided by Eisai Co Ltd. (Tokyo, Japan).
GGA was dissolved in 100% ethanol for in vitro studies. 1-Oleoyl LPA (18:1 LPA) (Cat. # 857130)
was obtained from Avanti Polar Lipids (Alabaster, AL). Doxorubicin (Cat. # NDC 55390-238-01)
was from Bedford Laboratories (Bedford, OH). Matrigel (Cat. # 354234) and Cell culture inserts
(Cat. # 353182) for invasion and motility assay were obtained from BD Biosciences (Franklin
Lakes, NJ). ROCK1 primary antibody (Cat. # A300-457A) was from Bethyl Laboratories
(Montgomery, TX). RPMI 1640 (Cat. # 11835-030) was from Gibco (Life Technologies, Grand
Island, NY), fetal bovine serum (FBS, Cat. # SH30088.03) was from HyClone (Thermo Fisher
Scientific, Pittsburg, PA), Penicillin-Streptomycin Solution (Cat. # 30-001-CI) was from Cellgro
(Manassas, VA).
Cell line: The human breast cancer cell line MDA-MB-231 (ATCC) was obtained in 2010, and was
used both for xenograft in vivo studies and in cell culture for in vitro experiments. Cells were grown
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in RPMI medium 1640, supplemented with 10% (v/v) fetal bovine serum, penicillin (10 U/ml)-
streptomycin (10 U/ml) at 37�C in humidified 5%CO2 atmosphere.
Animal studies: 5-6 weeks old female athymicnude-Foxn1nu mice (Harlan Laboratories,
Indianapolis, IN) were exposed to 500 cGy of radiation. The next day the mice were anesthetized
and an incision was made near the right flank to expose the mammary fat pad. 1x106 cells (MDA-
MB-231 breast cancer cells) were injected using a Hamilton syringe into the mammary fat pad.
Tumor development was followed and when xenografts averaged 4 mm in each dimension (length,
width and height, approximately 3 weeks after the implantation), mice with comparable sized
tumors were randomly divided between the four treatment groups: DOX 9mg/kg, DOX 9mg/kg and
GGA, GGA and saline. Doxorubicin has been reported to induce cardiotoxicity in a wide range of
dosages (4mg/kg to 25mg/kg)(25-29), we selected an intermediate dosage which would allow for
gradual cardiotoxicity development with multiple doxorubicin injections(25, 26). Doxorubicin was
administered via tail vein injection every 2 weeks for a total of 4 injections. GGA treatment (1mg /g
body weight) was given 48 hour before doxorubicin per os method (pipette). GGA was previously
shown to elicit a protective response when given 24-48 hours prior to stressor(30-35) in dosages
from 200mg/kg to 1000mg/kg (orally or intraperitoneally)(34-36).
Tumor progression was evaluated by palpation and tumor size measurements with calipers. Tumor
volumes were calculated by the following formula: [1/2xLxWxH] (37), where L-length, W-width,
H-height. All mice were housed under a 12 hours light-dark cycle with free access to food and
water. This study was performed in accordance with the “Guide for the Care and Use of Laboratory
Animals” (2011) of the National Institutes of Health. The protocol was approved by the Animal
Care and Use Committee of the Johns Hopkins Medical Institutions (Animal Welfare Assurance #
A-3273-01).
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Echocardiography: Trans-thoracic echocardiography was performed on conscious mice using
Acuson Sequoia C256 ultrasound machine (Siemens Corps, Mountain View, CA) equipped with the
15MHz linear-array transducer. The mouse heart was imaged in a two-dimensional mode followed
by M-mode using the parasternal short axis view at a sweep speed of 200 mm/sec. Measurements
were acquired using the leading-edge method, according to the American Echocardiography Society
guidelines(38). Left ventricle wall thickness and left ventricle chamber dimensions were acquired
during the end diastolic and end systolic phase including: inter-ventricular septum (IVSD), left
ventricular posterior wall thickness (PWTED), left ventricular end diastolic dimension (LVEDD)
and left ventricular end systolic dimension (LVESD). Three to five values for each measurement
were acquired and averaged for evaluation. The LVEDD and LVESD were used to derive fractional
shortening (FS) to measure left ventricular performance by the following equation: FS (%) =
[(LVEDD – LVESD)/LVEDD] X 100.
Necropsy: Mice were euthanized (CO2) and received postmortem examination and weighed. The
hearts were immediately excised, rinsed in cold PBS, weighed and sectioned at the level of
attachment of papillary muscles. Sections of left ventricle, right ventricle and septum were frozen in
liquid nitrogen for molecular studies. The remainder of the heart was fixed in 10% formalin for
histopathology.
Western blot: The tissue was homogenized in RIPA buffer (25mM Tris-HCl; 150mM NaCl; 1%
NP-40; 1% sodium deoxycholate; 0.1% SDS; 1x proteinase inhibitor (Roche, cat.#11697498001);
1x phosphatase inhibitors (Sigma, cat.#P5726 and P0044)) and centrifuged at 12,000 × g at 4°Cfor
15 min. Protein measurements were performed using a Bio-Rad protein assay (BioRad, Hercules,
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CA); equal amounts of total protein (40 μg) were used per lane. Proteins were denatured in SDS
gel-loading buffer (0.125 M Tris, pH 6.8, 20% glycerol, 5% mercaptoethanol, 4% SDS, and0.002%
bromophenol blue) at 100°C for 5 min and separated on a4-12% SDS-PAGE gradient gel using an
XCell SureLock™ Mini-Cell Electrophoresis System with Kaleidoscope prestained molecular
weight standards (Bio-Rad, Hercules, CA). After electrophoresis, the proteins were transferred to a
PVDF membrane. Blots were blocked with 5% nonfat milk in 0.1% TBST (50 mM Tris-HCl, pH
7.4, 150 mM NaCl, and 0.1% Tween 20) and probed with primary antibody (diluted in 5% nonfat
milk-TBST). Immunoblots were processed with horseradish peroxidase-conjugated anti-rabbit IgG;
bound antibodies were detected using a Western blot chemiluminescence reagent kit (Pierce,
Rockford, IL).
Histology: Histopathology was assessed in each treatment group as described previously (39). The
hearts were fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned at a
thickness of 5 μm, and stained with hematoxylin-eosin (H&E). Lungs were inflated with 10%
formalin after euthanasia, allowed to fix for 48 hours, embedded in paraffin, sectioned at a thickness
of 5 μm, and stained with hematoxylin-eosin (H&E). Lung sections with 5 lobes were scanned by
Aperio and evaluated for the presence of tumor metastases.
TUNEL: Staining was performed using DeadEnd Fluorometric TUNEL system (Promega,
Madison, WI) according to the manufacturer's instructions, as described previously(40).
Counterstaining with DAPI aided in the morphological evaluation of normal and apoptotic nuclei,
in which normal nuclei were stained as blue and apoptotic nuclei as green. The number of TUNEL-
positive cells within a 2.5-mm2 field in LV free wall was counted, and eight randomly selected
fields per slide and five sections per hearts were averaged for statistical analysis.
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Measurement of myocardial superoxide generation: Fresh frozen left ventricular myocardium
was homogenized on ice and sonicated. After centrifugation (30 sec, 4000 RPM), the supernatant
was added to a lucigenin (5 μM) solution containing NADPH (100μM). Superoxide generation was
measured using lucigenin-enhanced chemiluminescence (Beckman LS6000IC) and corrected for the
baseline value. Superoxide generation was expressed as counts per minute (cpm)/mg tissue. NOS-
dependent superoxide generation was measured by adding L-NAME (100μM) to the solution(41).
Invasion and motility assay: Invasion studies were performed as described previously(16). MDA-
MB-231 cells were plated in serum-free medium (2 x 104 /well ) onto 12-well Transwell plates
containing Matrigel. Complete medium (10% FBS) was added into the bottom well and the cells
were incubated at 37�C. Lysophosphatidic acid (LPA) (25μM) was applied to stimulate the invasion
of the cancer cells through Matrigel and LPA-induced invasiveness was compared to the basal level
of cell invasiveness. 72 hours later, the number of cells in the bottom well was counted. Motility
assays were performed similar to invasion assay, but no Matrigel was used.
Cell viability/proliferation quantification with MTT assay: MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5 diphenyl tetrazolium bromide) assay is based on the MTT conversion into formazan crystals by
live cells, proportionally to mitochondrial activity, thus allowing to estimate the number of viable
cells. MDA-MB-231cells were plated in 96 well plates (0.3 x 104/well) and treated with specified
concentrations of doxorubicin, GGA or both drugs for 24 hours. MTT stock solution was prepared
(5mg of MTT (Sigma M-5655) to 1mL of the growth media), further diluted 1:10 with growth
media and the cells were incubated in MTT solution for 2-3 hours, until visible formation of blue
formazan crystals was confirmed under light microscope. After that, the formazan crystals were
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solubilized with MTT solubilization solution (10% Triton X-100, 0.1 N HCL in 200 ml
isopropanol) and absorbance was read at 570nM.
Statistical methods: GraphPad Prism software (GraphPad, La Jolla, CA) was used to perform
statistical analysis. After determining means and standard deviations, the unpaired Student’s t-test
or one-way analysis of variance (ANOVA) were performed to compare two or three or more
unrelated groups as appropriate, with a P-value of <0.05 deemed significant.
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RESULTS
GGA prevents cardiac dysfunction induced by doxorubicin.
To our knowledge, GGA has not been used previously in the setting of doxorubicin therapy.
However, since GGA is protective in models of oxidative stress (8-14), we questionedwhether GGA
would be cardioprotective in mice with oxidative stress in the heart induced by doxorubicin. To
evaluate cardiac function, echocardiography was performed in conscious mice from four treatment
groups: saline control, GGA alone, doxorubicin (9mg/kg) alone and doxorubicin combined with
GGA. We chose to use the dosing schedule, where GGA treatment is initiated 48 hours before the
oxidative stress, since a 48 hour pretreatment likely induces the induction of gene expression, and
pretreatment is required for maximal protective effect (33, 34). Echocardiography of animals at 6
weeks after initiation of doxorubicin treatment demonstrated significantly decreased systolic
function, as evidenced by lower fractional shortening (FS, %) (Figure 1A, B). Remarkably,
however, pretreatment with GGA significantly protected animals from the doxorubicin-induced
decrease in cardiac function. This protective effect of GGA was unlikely due to a direct stimulatory
effect on cardiac function, since GGA treatment alone had no effect on systolic function compared
to saline treatment. GGA did not have a significant effect on the weight loss seen with doxorubicin
treatment (Figure 1C)
GGA blocks doxorubicin-induced cell death in the heart.
Extending these findings of GGA-induced protection of cardiac function to a cellular level, we
investigated whether GGA can reduce doxorubicin-induced cell death in the heart, using TUNEL
staining to evaluate heart sections. The percentage of TUNEL-positive nuclei in left ventricles was
compared among four treatment groups: saline control, GGA alone, doxorubicin (9mg/kg) alone
and doxorubicin combined with GGA. We found that doxorubicin treatment resulted in significant
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increase in cell death in the hearts, while GGA pre-treatment significantly reduced the doxorubicin-
induced cell death (Figure 2A,B), consistent with the protective effects seen with measurements of
systolic function (Figure 1) in corresponding treatment groups. H&E sections of heart from each
treatment group reveal that GGA blocks the characteristic cytoplasmic vacuolization induced by
doxorubicin (Figure 2C).
GGA blocks doxorubicin-induced ROCK1 cleavage in the heart.
GGA is known to inhibit RHO activation in cancer cells (15, 16), although the role of GGA on
RHO inactivation and ROCK1 (RHO-associated coiled-coil protein kinase 1) cleavage in the heart
has not been previously addressed. Normally ROCK1 is folded and auto-inhibited, but RHO-GTP
binding prevents that folding and auto-inhibition, thus RHO activates ROCK1.ROCK1 cleavage
occurs during apoptosis and is mediated by activated caspase-3(42). To explore the effects of GGA
on the downstream effectors of RHO signaling, we evaluated the ROCK1 cleavage in all four
treatment groups by western blotting. The cleaved product was reported previously to be absent in
normal hearts, while it is present in myocardium from heart failure patients (42). ROCK 1 cleavage
has been induced by doxorubicin in cell culture, yet it is not known if ROCK1 cleavage product is
present in the myocardium after doxorubicin treatment in vivo. A representative blot shows that
cleaved ROCK1 (130 kDa) levels were increased in doxorubicin treatment group, accompanied by
decreased full-length ROCK1 (top band) (160 kDa), compared to saline group. The combination of
GGA with doxorubicin treatment significantly reduced the level of ROCK1 cleavage comparable to
saline treatment. Hearts of mice treated with GGA alone showed further decrease of cleaved
ROCK1 (Figure 2 D,E).
GGA reduces doxorubicin-induced oxidative stress in the heart.
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One of the described mechanisms of doxorubicin cardiac toxicity is the generation of highly
reactive oxygen species (ROS), such as superoxide radical (5, 43).GGA treatment is known to
reduce oxidative stress in various animal models (8-14), yet GGA has never been used in the setting
of doxorubicin toxicity. To evaluate the contribution of ROS generation in doxorubicin-induced
cardiac toxicity and whether GGA pretreatment would reduce oxidative stress, lucigenin
chemiluminescence assay was performed to measure superoxide in freshly frozen mouse cardiac
tissue(41). In doxorubicin treated mice, superoxide production from the heart was significantly
increased, as compared to saline controls. GGA treatment reduced the basal level of superoxide
production and when given prior to doxorubicin, significantly abolished the doxorubicin induced
superoxide production (Figure 3).
Doxorubicin induces uncoupling of nitric oxide synthase (NOS) enzymes so that doxorubicin
undergoes redox activation by the enzyme to form a doxorubicin semiquinone and superoxide(44).
Endothelial NOS (eNOS) deficient mice are less susceptible to doxorubicin induced cardiac toxicity
suggesting a role of eNOS in the mechanism of oxidant generation and cell damage(43). To
elucidate the role of GGA in inhibiting NOS and doxorubicin-induced superoxide production, we
used L-NAME, a potent inhibitor of NO synthases. L-NAME did not have a significant effect on
basal superoxide generation in controls or in GGA only treated animals, but significantly reduced
production of superoxide in doxorubicin treated animals. L-NAME did not cause further significant
change in superoxide production in doxorubicin-GGA treated hearts, providing a NOS- mediated
mechanism for GGA’s protection against doxorubicin toxicity. L-NAME treatment did not result in
a complete reduction of superoxide production in the doxorubicin treated mice, suggesting that
other sources of superoxide production exist in doxorubicin treated hearts not related to NOS
(Figure 3).
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GGA enhanced doxorubicin-induced chemotherapy effects in breast cancer xenografts.
In light of the cardioprotective effects of GGA during doxorubicin therapy, we next asked whether
GGA would also attenuate or enhance the anti-neoplastic effects of doxorubicin. Previously,
inactivation of the RHO pathway by GGA was found to inhibit ovarian tumor cell growth and
invasion(15, 16), leading us to hypothesize that GGA might actually contribute to anti-neoplastic
activity during doxorubicin chemotherapy, while at the same time have a cardioprotective effect. To
test this hypothesis, MDA-MB-231 breast cancer cells (45) (with a high level of RHO expression)
were implanted in fat pad of athymic nude female mice. When xenograft tumors became palpable,
tumors were measured and mice were randomly assigned to treatment groups. After treatment,
compared to saline controls, GGA control mice did not have a significant difference in tumor
volumes. But remarkably, GGA given with doxorubicin significantly enhanced the anti-tumor effect
of doxorubicin compared to doxorubicin treatment alone (Figure 4A).
Additionally, we compared metastatic lung tumors occurrence in all treatment groups, using
H&E stained lung sections evaluation, evaluating the average number of tumor nodules per lung
section (Figure 4B). Representative lung sections are provided (Figure 4C). GGA alone slightly
inhibited the number of tumor nodules per lung section; although, the differences did not reach
statistical significance (p=0.2446) due to high individual variability of the number of tumor nodules
per lung section. Furthermore, these tumors were highly responsive to doxorubicin alone, and thus
any added effect of GGA on decreasing tumor growth or metastasis could not be readily appreciated
at the doses of doxorubicin used in our experiments.
GGA inhibited LPA-induced tumor cell motility and invasion in breast cancer cells.
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To explore possible cellular mechanisms for the anti-cancer effects of GGA, we examined effects of
GGA on tumor cell migration and invasiveness using lysophosphatidic acid RHO signaling
stimulus. Lysophosphatidic acid (LPA) exerts its biological effects through the LPA receptors in
cancer cells (46) and is associated with breast cancer cell migration and invasiveness(47, 48). LPA
as an inducer of RHO signaling and tumor invasion is used in in vitro systems to study cancer cells
invasion (49, 50). GGA reduced invasion of ovarian cancer cells in vitro(16);although its role in
motility and invasion in breast cancer cells has not been evaluated. Therefore we performed in vitro
experiments to assess GGA effects on motility and invasiveness MDA-MB-231 breast cancer cells
that express a high level of RHO A (45). In motility experiments, GGA reduced cell motility in the
presence of LPA (Figure 5A). GGA treatment alone reduced the basal level of invasion, while
GGA in combination with LPA, further reduced invasion of MDA-MB-231 cells (Figure 5B).
Since GGA inhibits RHO pathway in ovarian cancer, we hypothesized that GGA, would inhibit
RHO activation in breast cancer cells by inhibiting the RHO family member activation through
reduced geranylgeranylation. To confirm the role of RHO family geranylgeranylation in GGA-
induced inhibition of invasion of MDA-MB-231 cells, we applied GGOH (geranylgeraniol) to a
subset of experiments testing the mechanism of GGA’s inhibition on LPA-induced migration.
GGOH, added to the cells before migration, is metabolized to geranylgeranylpyrophosphate, which
subsequently aids RHO geranylgeranylation, to rescue the inhibitory effects of GGA (16). In our
experiments, GGOH reduced GGA anti-invasive effect, providing evidence that GGA inhibits RHO
family geranylgeranylation in MDA-MB-231 cells suggesting a mechanism for the role of GGA in
reducing tumor cell invasion (Figure 5B). GGOH also reduced but did not completely eliminate
GGA anti-invasive effect in LPA-treated cancer cells (similarly to the findings in ovarian cancer
cells), suggesting additional mechanisms in LPA-induced invasiveness.
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GGA decreases cell viability/proliferation in breast cancer cells.
To evaluate the effects of GGA alone or in combination with doxorubicin on cancer cells viability
in vitro, we performed MTT assay with various concentrations of GGA and doxorubicin. GGA
alone significantly reduced MTT absorbance in MDA-MB-231 cells treated for 24 hours, while
GGA in combination with doxorubicin did not result in a significant change compared to
doxorubicin alone (Figure 5C).
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Discussion
The optimal cardiac toxicity prevention strategy for doxorubicin would include an agent that
improves the efficacy of the doxorubicin-based cancer therapy and prevents cardiac toxicity. In our
experiments, pre-treatment with geranylgeranylacetone (GGA) blocked doxorubicin cardiac toxicity
by maintaining systolic function and decreasing cell death in the heart. Most remarkably, GGA also
contributed to doxorubicin’s chemotherapy efficacy in MDA-MB-231 xenografts in parallel with
protecting the heart. GGA’s anti-neoplastic effect is likely due to its inhibition of RHO family
proteins in both the heart and cancer cells, and we selected MDA-MB-231 for these experiments
because of the endogenous high RHO activity in these cells. Since GGA has been used in Japan
since 1984 to prevent stomach ulcers and has a long history of safety and lack of adverse effects, we
suggest that this novel approach to prevention of doxorubicin toxicity should be further
investigated.
By comparison, dexrazoxane, an iron chelator used to reduce superoxide formation in doxorubicin
toxicity(51) currently has limited use due to European Medicines Agency and Food and Drug
Administration restrictions. In 2011, dexrazoxane was restricted for use only in breast cancer
patients when doxorubicin dose exceeds 300mg/m2 or epirubicin exceeds 540 mg/m2. This
restriction was based on clinical trials that reported cases of acute myeloid leukemia and
myelodysplastic syndrome in children receiving dexrazoxane(52, 53). Furthermore, there are
anecdotal reports of reduced anti-cancer therapeutic efficacy for dexrazoxane, GGA, by contrast,
offers an attractive alternative to dexrazoxane, with molecular activity that both prevents cardiac
toxicity and increases effectiveness of doxorubicin chemotherapy.
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The efficacy of cardiac protection by GGA may be related to multiple mechanisms. First, GGA
induced a reduction in NOS-dependent superoxide production with overall reduction in oxidative
stress in the heart, an important mechanism of doxorubicin-induced cardiomyocyte cell death and
toxicity. Second, GGA also prevented doxorubicin-induced ROCK1 cleavage, thus blocking pro-
apoptotic RHO/ROCK1 pathway in the heart with resulting in less TUNEL positive cells in the
myocardium. In the heart, the RHO/ROCK pathway is currently emerging as a potential target for
inhibition in cardiovascular disease(17-23). In multiple settings of cardiac disease (17), specific
RHO/ROCK inhibitors have shown promise in disease prevention yet RHO/ROCK inhibitors have
never been used to prevent doxorubicin cardiac toxicity, although, ROCK1was found to be
activated with doxorubicin treatment in vitro(42). This finding suggested to us that GGA treatment
may be beneficial to induce RHO/ ROCK inhibition in the heart and thus reduce cardiac toxicity.
We hypothesized that ROCK activation occurs in hearts in mice treated with doxorubicin. In the
current study, we demonstrate that ROCK activation does occur in the heart of mice treated with
doxorubicin and this provides rationale to use GGA as a cardioprotective agent. In total, our study
demonstrates that GGA can inhibit doxorubicin-induced RHO activation in the heart, oxidative
stress and cardiac toxicity.
The connection between oxidative stress and ROCK1 activation has recently been studied in
erythroid cells (54), and we observed that GGA reduced both in the current study. Doxorubicin
treatment induced an elevation of ROCK1 cleavage product, a downstream effect or protein
indicative of RHO pathway activation. Additionally, doxorubicin induced an increase of superoxide
formation. The L-NAME experiments demonstrated that a portion of superoxide induced by
doxorubicin treatment was NOS-dependent. We suggest that the non-NOS dependent superoxide is
related to RHO activation in the doxorubicin-treated mice, since GGA further inhibited the extent of
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superoxide production when comparing doxorubicin versus doxorubicin and GGA treatment
groups. GGA may also inhibit uncoupled eNOS-dependent superoxide production, since L-NAME
did not decrease superoxide production neither in GGA only treatment group, nor in
doxorubicin+GGA treatment group. This mechanism may be possible due to positive GGA effects
on HSP90 expression (55). RHOA-RHO kinase is also a well-documented inducer of oxidative
stress and oxidative stress is known to induce RHO family activation(56-58).
Inhibiting the RHO family of proteins has a different role in cancer cells, which might explain how
GGA could have divergent effects on cardiac cells (non-motile, non-dividing cells) and cancer cells.
In ovarian cancer cells, GGA was shown to inhibit both RHO and RAS activation, possibly through
inhibiting geranylation(15, 16). Multiple types of human cancers (including breast cancer) have a
high activity of RHO family proteins, which likely contributes to the invasive malignant phenotype
and tumor metastasis (59, 60). Since the RHO family is responsible for this phenotype, this
subgroup of cancers will be more susceptible to GGA-induced RHO pathway inhibition. We
selected MDA-MB-231 due to high RHO activity and found that in xenograft implanted nude mice,
GGA+doxorubicin treatment also significantly reduced tumor mass versus doxorubicin treatment
alone. The numbers of metastases in the lung were reduced in GGA versus control groups, although
these differences did not reach statistical significance. The numbers of metastases were significantly
reduced in both doxorubicin groups with and without GGA. Due to the high effectiveness of
doxorubicin (a high dose needed to produce cardiac toxicity) in this xenograft model, any added
effect of GGA was masked. However, in our in vitro studies, GGA inhibited lysophosphatidic acid
(LPA)-induced Matrigel invasion by MDA-MB-231 cells suggesting that RHO pathway inhibition
is an important target of GGA in breast cancer cell motility and invasion. GGA anti-invasive effect
was suppressed by GGOH demonstrating that RHO geranylation is inhibited by GGA.
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Our finding of GGA enhancing doxorubicin anti-tumor effect in vivo can be attributed to GGA
negative effect on cancer cells viability, as shown in our in vitro study and in previously published
studies(61). We did not see an additive effect in combined GGA-doxorubicin treatment groups in
vitro, however, in vivo multiple factors contribute to the tumor progression, which may not be
accounted for with in vitro assays on isolated cancer cells, but which may be affected by GGA
treatment. For example, angiogenesis, an important factor which regulates tumor development, may
be affected by GGA. It was shown that inhibition of geranylgeranylation interferes with
angiogenesis, evaluated by tube formation by human dermal microvascular endothelial cells(62).
RHO GTPases, and RHO-associated kinases (ROCKs) inhibition is also reported to affect
angiogenesis (63). In addition, Rho/ROCK1 pathway is involved in cancer cells survival and
propagation through various functions, including cell adhesion, motility, and migration (24).
ROCK1 is found in MDA-MB-231 cells, although in low quantities, and GGA negative effects on
RHO/ROCK1 pathway may also contribute to overall GGA effects on cancer progression, observed
in our study(64).
Our studies demonstrate that GGA has both important cardioprotective properties and anti-
neoplastic properties, which together can be therapeutically effective when GGA is used in
combination with doxorubicin. Since GGA has been used for thirty years without adverse effects,
we suggest that this novel approach to prevention of doxorubicin cardiac toxicity should be
considered for clinical testing in patients undertaking doxorubicin treatment for cancer.
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LEGENDS
Figure 1.Effect of GGA on cardiac function in murine model of doxorubicin-induced
cardiotoxicity
A. Representative M-mode echocardiograms of the mice from saline, GGA, doxorubicin and
doxorubicin in combination with GGA treatment groups. Doxorubicin (9mg/kg) given once every
two weeks for 4 cycles induces contractility deficit at 6 wks from initial injection. GGA
administration (48 hours before doxorubicin) preserves cardiac function in doxorubicin treatment
group, GGA alone does not have an effect. B. Scatter plot of fractional shortening percentage (FS,
%). Doxorubicin causes reduction of FS, % (p<0.0001), and addition of GGA to the treatment
improves FS, % (p<0.0001), GGA alone had no effect on FS, %. C. Histogram of mouse body
weights in grams. The data are presented as means ± SD, n=9-12 mice per group. *** p<0.001.
Figure 2. Effects of GGA treatment on cardiac cell death, ROCK1 cleavage, and superoxide
production in the hearts of doxorubicin-treated mice.
A. Histogram and B. Representative images of TUNEL-positive nuclei. Mice were euthanized at 6
weeks after the initial injection of doxorubicin. Doxorubicin (9mg/kg) was given once every two
weeks for 4 cycles. GGA administration was given 48 hours before doxorubicin. Total number of
cells per x20 field was quantified by DAPI staining and TUNEL-positive nuclei were expressed as a
percentage of total cells (nuclei) in each heart with five fields per heart analyzed. Cell death in the
hearts of the animals treated with doxorubicin was significantly higher than in controls (p=0.0491).
In the group which received combinatory therapy, cell death was significantly lower than in
doxorubicin group (p=0.0124). Cell death in GGA group was not significantly different from
control group. The data are presented as means ± SD, n= 3-8 mice per group.* p<0.05.C.
Representative H&E stained heart sections from mice treated with saline, GGA, DOX or
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combination DOX and GGA. DOX induces fine vacuolization with cardiomyocytes cytoplasm and
GGA/DOX combination hearts have fewer vacuoles. The presence of cytoplasmic vacuoles is used
in human and animal studies to assess degree of cardiac toxicity(65-67).
D. Western blot of ROCK1. ROCK1 is cleaved in the hearts of doxorubicin-treated mice, but not in
the hearts of mice treated both with doxorubicin and GGA. ROCK1 cleavage is further reduced in
the hearts of GGA-treated mice. ROCK1 full length � 160 kDa, cleaved ROCK1 � 130 kDa. E.
Densitometry of cleaved ROCK1, expressed as a fold change from the cleaved ROCK1 levels in
saline-treated mice hearts. Doxorubicin treatment significantly increased cleaved ROCK1 levels
(p=0.0054), while addition of GGA to doxorubicin therapy reduced ROCK1 cleavage (p=0.0002).
GGA treatment did not affect cleaved ROCK1 levels. The data are presented as means ± SD, n=3
mice per group. ** p<0.01, *** p<0.001.
Figure 3. Effects of doxorubicin and GGA treatment on total and eNOS-dependent superoxide
generation. Doxorubicin treatment significantly increased superoxide levels in murine left
ventricles (p=0.0159). L-NAME had no effect at superoxide generation in controls, but significantly
reduced doxorubicin-mediated overproduction of superoxide in treated animals(p=0.0159). GGA
had no effect at superoxide production by itself, but abolished doxorubicin-induced superoxide
production(p=0.0077). L-NAME did not result in further reduction of superoxide production in
doxorubicin-treated compared to controls. The data are presented as means ± SD, n=3-5 mice per
group.* p<0.05, ** p<0.01.
Figure 4.Effects of GGA on xenograft tumor volumes in doxorubicin-treated mice.
A. Effects of doxorubicin and GGA treatment on the average xenograft tumor volume. The mice
were injected with MDA-MB-231 breast cancer cells into the mammary fat pad. Tumor
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development was followed and the mice were divided into four treatment groups: DOX 9 mg/kg,
DOX 9 mg/kg and GGA, GGA and saline. Doxorubicin (9mg/kg) was given once every two weeks
for 4 cycles. GGA administration was given 48 hours before doxorubicin. Mice were euthanized at
6 wks after the initial injection of doxorubicin. GGA alone did not affect tumor volumes, but
significantly enhanced anti-tumor effect of doxorubicin(p=0.0012).The data are presented as means
± SD, n=5-7 mice per group.** p<0.01. B, C. Lung metastases were evaluated using H&E stained
lung sections, comparing the average number of tumor nodules (black arrows) per lung
section.GGA treatment (alone or in combination with doxorubicin) did not significantly change the
number of metastatic nodules. The data are presented as means ± SD, n=9-13 per group.
Figure 5.Effects of GGA on invasiveness of MDA-MB-231 breast cancer cells in vitro.
Invasion studies were performed with MDA-MB-231 cells. Lysophosphatidic acid (LPA) was
applied to stimulate the motility or invasion of the cancer cells and LPA-induced motility and
invasiveness was compared to the basal level of cell motility and invasiveness. GGOH treatment
was used in invasion studies to counteract the GGA inhibitory effect on cancer cells invasion. A.
GGA does not affect basal motility of MDA-MB-231 breast cancer cells, but reduces motility in
LPA presence (p=0.0195). The data are presented as means ± SD, n=3 wells per group. B. GGA
treatment decreases invasiveness of MDA-MB-231 cells in vitro. GGA treatment reduced invasion
(p=0.0423), and GGA in combination with LPA further reduced invasion of MDA-MB-231 cells
(p=0.0121). GGOH abolishes GGA anti-invasive effect (p=0.0550). Bar graph represents invasion
of MDA-MB-231 cells as a total number of cells used in the assay. The data are presented as means
± SD, n=3 wells per group.* p<0.05, ** p<0.01. C. GGA decreases MDA-MB-231 cells viability in
vitro, while not significantly altering doxorubicin effects in MTT assay. The data are presented as
means ± SD, n=7 wells per group. **** p<0.0001.
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Published OnlineFirst April 15, 2014.Mol Cancer Ther Polina Sysa-Shah, Yi Xu, Xin Guo, et al. RHO pathway inhibitiontoxicity and reduces cancer cell growth and invasion through Geranylgeranylacetone blocks doxorubicin-induced cardiac
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