geranylgeranylacetone blocks doxorubicin-induced cardiac...

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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

on July 3, 2019. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 15, 2014; DOI: 10.1158/1535-7163.MCT-13-0965

http://mct.aacrjournals.org/

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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.

References




21

1. Senkus E, Kyriakides S, Penault-Llorca F, Poortmans P, Thompson A, Zackrisson S, et al. Primary breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 2013;24 Suppl 6:vi7-23. 2. Cardoso F, Harbeck N, Fallowfield L, Kyriakides S, Senkus E, Group EGW. Locally recurrent or metastatic breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 2012;23 Suppl 7:vii11-9. 3. Blum RH, Carter SK. Adriamycin. A new anticancer drug with significant clinical activity. Annals of internal medicine. 1974;80:249-59. 4. http://www.cancer.gov [Internet]. Bethesda: National Cancer Institute. c2013-2014 [Updated 2014 February 21, cited 2014 February 28]. Breast Cancer Treatment (PDQ®): Stage I, II, IIIA, and Operable IIIC Breast Cancer. Available at: http://www.cancer.gov/cancertopics/pdq/treatment/breast/healthprofessional/Page6#Section_859. 5. Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S, Crijns HJ, Moens AL. Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol. 2012;52:1213-25. 6. Niwa Y, Nakamura M, Miyahara R, Ohmiya N, Watanabe O, Ando T, et al. Geranylgeranylacetone protects against diclofenac-induced gastric and small intestinal mucosal injuries in healthy subjects: a prospective randomized placebo-controlled double-blind cross-over study. Digestion. 2009;80:260-6. 7. http://www.eisai.com [Internet]. Tokyo: Eisai Co. Selbex®. c2013-4 [Updated January 4, cited 4 February 28]. Available at: http://www.eisai.jp/medical/products/di/EPI/SLX_C-FG_EPI.pdf. 8. Ohkawara T, Takeda H, Nishiwaki M, Nishihira J, Asaka M. Protective effects of heat shock protein 70 induced by geranylgeranylacetone on oxidative injury in rat intestinal epithelial cells. Scand J Gastroenterol. 2006;41:312-7. 9. Tanito M, Kwon YW, Kondo N, Bai J, Masutani H, Nakamura H, et al. Cytoprotective effects of geranylgeranylacetone against retinal photooxidative damage. J Neurosci. 2005;25:2396-404. 10. Suzuki S, Maruyama S, Sato W, Morita Y, Sato F, Miki Y, et al. Geranylgeranylacetone ameliorates ischemic acute renal failure via induction of Hsp70. Kidney Int. 2005;67:2210-20. 11. Hirota K, Nakamura H, Masutani H, Yodoi J. Thioredoxin superfamily and thioredoxin-inducing agents. Ann N Y Acad Sci. 2002;957:189-99. 12. Hirota K, Nakamura H, Arai T, Ishii H, Bai J, Itoh T, et al. Geranylgeranylacetone enhances expression of thioredoxin and suppresses ethanol-induced cytotoxicity in cultured hepatocytes. Biochem Biophys Res Commun. 2000;275:825-30. 13. Yoda M, Sakai T, Mitsuyama H, Hiraiwa H, Ishiguro N. Geranylgeranylacetone suppresses hydrogen peroxide-induced apoptosis of osteoarthritic chondrocytes. J Orthop Sci. 2011;16:791-8. 14. Nagano Y, Matsui H, Shimokawa O, Hirayama A, Nakamura Y, Tamura M, et al. Bisphosphonate-induced gastrointestinal mucosal injury is mediated by mitochondrial superoxide production and lipid peroxidation. J Clin Biochem Nutr. 2012;51:196-203. 15. Hashimoto K, Morishige K, Sawada K, Ogata S, Tahara M, Shimizu S, et al. Geranylgeranylacetone inhibits ovarian cancer progression in vitro and in vivo. Biochem Biophys Res Commun. 2007;356:72-7. 16. Hashimoto K, Morishige K, Sawada K, Tahara M, Shimizu S, Sakata M, et al. Geranylgeranylacetone inhibits lysophosphatidic acid-induced invasion of human ovarian carcinoma cells in vitro. Cancer. 2005;103:1529-36. 17. Shimokawa H, Rashid M. Development of Rho-kinase inhibitors for cardiovascular medicine. Trends Pharmacol Sci. 2007;28:296-302. 18. Lin G, Craig GP, Zhang L, Yuen VG, Allard M, McNeill JH, et al. Acute inhibition of Rho-kinase improves cardiac contractile function in streptozotocin-diabetic rats. Cardiovasc Res. 2007;75:51-8. 19. Kobayashi N, Horinaka S, Mita S, Nakano S, Honda T, Yoshida K, et al. Critical role of Rho-kinase pathway for cardiac performance and remodeling in failing rat hearts. Cardiovasc Res. 2002;55:757-67.




22

20. Bao W, Hu E, Tao L, Boyce R, Mirabile R, Thudium DT, et al. Inhibition of Rho-kinase protects the heart against ischemia/reperfusion injury. Cardiovasc Res. 2004;61:548-58. 21. Wolfrum S, Dendorfer A, Rikitake Y, Stalker TJ, Gong Y, Scalia R, et al. Inhibition of Rho-kinase leads to rapid activation of phosphatidylinositol 3-kinase/protein kinase Akt and cardiovascular protection. Arterioscler Thromb Vasc Biol. 2004;24:1842-7. 22. Noma K, Oyama N, Liao JK. Physiological role of ROCKs in the cardiovascular system. Am J Physiol Cell Physiol. 2006;290:C661-8. 23. Surma M, Wei L, Shi J. Rho kinase as a therapeutic target in cardiovascular disease. Future Cardiol. 2011;7:657-71. 24. Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998;279:509-14. 25. Pacher P, Liaudet L, Bai P, Mabley JG, Kaminski PM, Virag L, et al. Potent metalloporphyrin peroxynitrite decomposition catalyst protects against the development of doxorubicin-induced cardiac dysfunction. Circulation. 2003;107:896-904. 26. Kang YJ, Sun X, Chen Y, Zhou Z. Inhibition of doxorubicin chronic toxicity in catalase-overexpressing transgenic mouse hearts. Chemical research in toxicology. 2002;15:1-6. 27. D'Alessandro N, Candiloro V, Crescimanno M, Flandina C, Dusonchet L, Crosta L, et al. Effects of multiple doxorubicin doses on mouse cardiac and hepatic catalase. Pharmacological research communications. 1984;16:145-51. 28. Riad A, Bien S, Westermann D, Becher PM, Loya K, Landmesser U, et al. Pretreatment with statin attenuates the cardiotoxicity of Doxorubicin in mice. Cancer Res. 2009;69:695-9. 29. Doroshow JH, Locker GY, Ifrim I, Myers CE. Prevention of doxorubicin cardiac toxicity in the mouse by N-acetylcysteine. The Journal of clinical investigation. 1981;68:1053-64. 30. Ishii Y, Kwong JM, Caprioli J. Retinal ganglion cell protection with geranylgeranylacetone, a heat shock protein inducer, in a rat glaucoma model. Investigative ophthalmology & visual science. 2003;44:1982-92. 31. Uchida S, Fujiki M, Nagai Y, Abe T, Kobayashi H. Geranylgeranylacetone, a noninvasive heat shock protein inducer, induces protein kinase C and leads to neuroprotection against cerebral infarction in rats. Neuroscience letters. 2006;396:220-4. 32. Fujiki M, Hikawa T, Abe T, Uchida S, Morishige M, Sugita K, et al. Role of protein kinase C in neuroprotective effect of geranylgeranylacetone, a noninvasive inducing agent of heat shock protein, on delayed neuronal death caused by transient ischemia in rats. Journal of neurotrauma. 2006;23:1164-78. 33. Abe E, Fujiki M, Nagai Y, Shiqi K, Kubo T, Ishii K, et al. The phosphatidylinositol-3 kinase/Akt pathway mediates geranylgeranylacetone-induced neuroprotection against cerebral infarction in rats. Brain research. 2010;1330:151-7. 34. Nagai Y, Fujiki M, Inoue R, Uchida S, Abe T, Kobayashi H, et al. Neuroprotective effect of geranylgeranylacetone, a noninvasive heat shock protein inducer, on cerebral infarction in rats. Neuroscience letters. 2005;374:183-8. 35. Zhu Z, Takahashi N, Ooie T, Shinohara T, Yamanaka K, Saikawa T. Oral administration of geranylgeranylacetone blunts the endothelial dysfunction induced by ischemia and reperfusion in the rat heart. Journal of cardiovascular pharmacology. 2005;45:555-62. 36. Zhang K, Zhao T, Huang X, Liu ZH, Xiong L, Li MM, et al. Preinduction of HSP70 promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain. Cell stress & chaperones. 2009;14:407-15. 37. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol. 1989;24:148-54. 38. Sahn DJ, DeMaria A, Kisslo J, Weyman A. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation. 1978;58:1072-83.




23

39. Gabrielson KL, Hogue BA, Bohr VA, Cardounel AJ, Nakajima W, Kofler J, et al. Mitochondrial toxin 3-nitropropionic acid induces cardiac and neurotoxicity differentially in mice. Am J Pathol. 2001;159:1507-20. 40. Gabrielson KL, Mok GS, Nimmagadda S, Bedja D, Pin S, Tsao A, et al. Detection of dose response in chronic doxorubicin-mediated cell death with cardiac technetium 99m annexin V single-photon emission computed tomography. Mol Imaging. 2008;7:132-8. 41. Moens AL, Takimoto E, Tocchetti CG, Chakir K, Bedja D, Cormaci G, et al. Reversal of cardiac hypertrophy and fibrosis from pressure overload by tetrahydrobiopterin: efficacy of recoupling nitric oxide synthase as a therapeutic strategy. Circulation. 2008;117:2626-36. 42. Chang J, Xie M, Shah VR, Schneider MD, Entman ML, Wei L, et al. Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis. Proc Natl Acad Sci U S A. 2006;103:14495-500. 43. Neilan TG, Blake SL, Ichinose F, Raher MJ, Buys ES, Jassal DS, et al. Disruption of nitric oxide synthase 3 protects against the cardiac injury, dysfunction, and mortality induced by doxorubicin. Circulation. 2007;116:506-14. 44. Vasquez-Vivar J, Martasek P, Hogg N, Masters BS, Pritchard KA, Jr., Kalyanaraman B. Endothelial nitric oxide synthase-dependent superoxide generation from adriamycin. Biochemistry. 1997;36:11293-7. 45. Pille JY, Denoyelle C, Varet J, Bertrand JR, Soria J, Opolon P, et al. Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo. Mol Ther. 2005;11:267-74. 46. Choi JW, Herr DR, Noguchi K, Yung YC, Lee CW, Mutoh T, et al. LPA receptors: subtypes and biological actions. Annu Rev Pharmacol Toxicol. 2010;50:157-86. 47. Panupinthu N, Lee HY, Mills GB. Lysophosphatidic acid production and action: critical new players in breast cancer initiation and progression. Br J Cancer. 2010;102:941-6. 48. Liu S, Umezu-Goto M, Murph M, Lu Y, Liu W, Zhang F, et al. Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases. Cancer Cell. 2009;15:539-50. 49. Stahle M, Veit C, Bachfischer U, Schierling K, Skripczynski B, Hall A, et al. Mechanisms in LPA-induced tumor cell migration: critical role of phosphorylated ERK. J Cell Sci. 2003;116:3835-46. 50. Jeong KJ, Cho KH, Panupinthu N, Kim H, Kang J, Park CG, et al. EGFR mediates LPA-induced proteolytic enzyme expression and ovarian cancer invasion: inhibition by resveratrol. Mol Oncol. 2013;7:121-9. 51. Jones RL. Utility of dexrazoxane for the reduction of anthracycline-induced cardiotoxicity. Expert Rev Cardiovasc Ther. 2008;6:1311-7. 52. Salzer WL, Devidas M, Carroll WL, Winick N, Pullen J, Hunger SP, et al. Long-term results of the pediatric oncology group studies for childhood acute lymphoblastic leukemia 1984-2001: a report from the children's oncology group. Leukemia. 2010;24:355-70. 53. Tebbi CK, London WB, Friedman D, Villaluna D, De Alarcon PA, Constine LS, et al. Dexrazoxane-associated risk for acute myeloid leukemia/myelodysplastic syndrome and other secondary malignancies in pediatric Hodgkin's disease. J Clin Oncol. 2007;25:493-500. 54. Vemula S, Shi J, Mali RS, Ma P, Liu Y, Hanneman P, et al. ROCK1 functions as a critical regulator of stress erythropoiesis and survival by regulating p53. Blood. 2012;120:2868-78. 55. Fujimura N, Jitsuiki D, Maruhashi T, Mikami S, Iwamoto Y, Kajikawa M, et al. Geranylgeranylacetone, heat shock protein 90/AMP-activated protein kinase/endothelial nitric oxide synthase/nitric oxide pathway, and endothelial function in humans. Arterioscler Thromb Vasc Biol. 2012;32:153-60. 56. Soliman H, Gador A, Lu YH, Lin G, Bankar G, MacLeod KM. Diabetes-induced increased oxidative stress in cardiomyocytes is sustained by a positive feedback loop involving Rho kinase and PKCbeta2. Am J Physiol Heart Circ Physiol. 2012;303:H989-H1000.




24

57. Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, et al. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol. 2012;303:L939-52. 58. Aghajanian A, Wittchen ES, Campbell SL, Burridge K. Direct activation of RhoA by reactive oxygen species requires a redox-sensitive motif. PLoS One. 2009;4:e8045. 59. Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 2000;406:532-5. 60. Itoh K, Yoshioka K, Akedo H, Uehata M, Ishizaki T, Narumiya S. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat Med. 1999;5:221-5. 61. Okada S, Yabuki M, Kanno T, Hamazaki K, Yoshioka T, Yasuda T, et al. Geranylgeranylacetone induces apoptosis in HL-60 cells. Cell structure and function. 1999;24:161-8. 62. Park HJ, Kong D, Iruela-Arispe L, Begley U, Tang D, Galper JB. 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of RhoA. Circulation research. 2002;91:143-50. 63. Bryan BA, Dennstedt E, Mitchell DC, Walshe TE, Noma K, Loureiro R, et al. RhoA/ROCK signaling is essential for multiple aspects of VEGF-mediated angiogenesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2010;24:3186-95. 64. Escudero-Esparza A, Jiang WG, Martin TA. Claudin-5 is involved in breast cancer cell motility through the N-WASP and ROCK signalling pathways. Journal of experimental & clinical cancer research : CR. 2012;31:43. 65. Mettler FP, Young DM, Ward JM. Adriamycin-induced cardiotoxicity (cardiomyopathy and congestive heart failure) in rats. Cancer Res. 1977;37:2705-13. 66. Billingham ME, Mason JW, Bristow MR, Daniels JR. Anthracycline cardiomyopathy monitored by morphologic changes. Cancer treatment reports. 1978;62:865-72. 67. Chatterjee K, Zhang J, Honbo N, Karliner JS. Doxorubicin cardiomyopathy. Cardiology. 2010;115:155-62.




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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




26

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




27

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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