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  • Small Molecule Therapeutics

    Geranylgeranylacetone Blocks Doxorubicin-InducedCardiac Toxicity and Reduces Cancer Cell Growth andInvasion through RHO Pathway Inhibition

    PolinaSysa-Shah1, Yi Xu1, XinGuo1, Scott Pin1, DjahidaBedja1, Rachel Bartock1, AllisonTsao1, AngelaHsieh1,Michael S. Wolin5, An Moens2, Venu Raman3, Hajime Orita4, and Kathleen L. Gabrielson1

    AbstractDoxorubicin is a widely used chemotherapy for solid tumors and hematologic malignancies, but its use is

    limited due to cardiotoxicity. Geranylgeranylacetone (GGA), an antiulcer agent used in Japan for 30 years, has

    no significant adverse effects, and unexpectedly reduces ovarian cancer progression in mice. Because GGA

    reduces oxidative stress in brain and heart, we hypothesized that GGA would prevent oxidative stress of

    doxorubicin cardiac toxicity and improve doxorubicins chemotherapeutic effects. Nudemice implantedwith

    MDA-MB-231 breast cancer cells were studied after chronic treatment with doxorubicin, doxorubicin/GGA,

    GGA, or saline. Transthoracic 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 nitric oxide synthasedependent 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 antitumor 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 doxoru-

    bicin chemotherapy-induced injury and improves anticancer efficacy of doxorubicin in breast cancer.

    Mol Cancer Ther; 13(7); 171728. 2014 AACR.

    IntroductionDoxorubicin (Adriamycin) is one of the most widely

    used anticancer agents and it is currently a first-choicechemotherapeutic drug for the treatment of primary,recurrent, and metastatic breast cancer (14). Unfortu-nately, the use of doxorubicin in breast cancer chemo-therapy is frequently limited due to its severe cumulativecardiac toxicity (5). Clinical signs of cardiac toxicity mayoccur during weeks, months, or even years after chemo-

    therapy. Strategies that reduce cardiac toxicity couldpotentially allow higher dosages of doxorubicin to beused with an improved long-term survival in patientswith improved quality of life.

    A prevention strategy that reduces oxidative stress, acommon mechanism of doxorubicin cardiac toxicity (5),led us to consider potential benefits of geranylgeranyla-cetone (GGA), an acylic polyisoprenoid (Selbex), whichhasbeenused since 1984as anantiulcerdrug in Japanwithno adverse reactions (6, 7). In multiple animal models ofischemia and reperfusion, GGA prevents oxidative stressin liver, heart, brain, kidney, and retina (814). A secondmechanism linked to GGAs mechanism of action is itsability to inhibit the activation of the RHO family ofGTPases through reduction of protein geranylgeranyla-tion, a posttranslational modification required for RHOfamilymembrane targeting and signaling (15, 16). Target-ing the RHO/ROCK pathway with specific inhibitors isbeneficial in a number of cardiovascular diseases, includ-ing hypertension, angina, ischemia-reperfusion injury,cardiac hypertrophy, chronic heart failure (1723),all conditions with some level of oxidative stress.

    Authors' Affiliations: Departments of 1Molecular andComparative Patho-biology, and 2Cardiology, Johns Hopkins Medical Institutions; 3Depart-ment of Radiology, Johns Hopkins University; 4Department of Pathology,Johns Hopkins University School of Medicine, Baltimore, Maryland; and5Department of Physiology, New YorkMedical College, Valhalla, New York

    Current address for HajimeOrita: Juntendo ShizuokaHospital, Departmentof Surgery, Izunokuni, Shizuoka, Japan.

    Corresponding Author: Kathleen L. Gabrielson, Johns Hopkins MedicalInstitutions, MRB 807, 733 N. Broadway, Baltimore, MD 21205. Phone:443-287-2953; Fax: 443-287-2954; E-mail: kgabriel@jhmi.edu

    doi: 10.1158/1535-7163.MCT-13-0965

    2014 American Association for Cancer Research.

    MolecularCancer

    Therapeutics

    www.aacrjournals.org 1717

    on July 14, 2018. 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

    Published OnlineFirst April 15, 2014; DOI: 10.1158/1535-7163.MCT-13-0965

    http://mct.aacrjournals.org/

  • Interestingly, this molecular activity of GGA might alsomake it useful as a cancer therapeutic because RHOGTPases, and their downstream target, RHO-associatedkinases (ROCK), are implicated in a variety of physiologicfunctions associated with cancer-related changes in theactin cytoskeletal assembly, such as cell adhesion, motil-ity, and migration (24).

    Because GGA may reduce a variety of molecular func-tions important for cancer cell growth andmigration, andat the same time, may reduce oxidative stress in the heart,we undertook an investigation to determine whetherGGAcan inhibit the cardiac adverse effects of doxorubicinwhile simultaneously inhibiting cancer cell growth. Spe-cifically, we developed a breast cancer mouse model ofchronic doxorubicin injury to the heart and investigatedcellular and molecular responses to various treatmentsinvolving GGA and doxorubicin.

    Materials and MethodsReagents and materials

    GGA (Lot # 17022802) was provided by Eisai Co Ltd..GGAwas dissolved in 100% ethanol for in vitro studies. 1-Oleoyl LPA (18:1 LPA; Cat. # 857130) was obtained fromAvanti Polar Lipids. Doxorubicin (Cat. # NDC 55390-238-01) was from Bedford Laboratories. Matrigel (Cat. #354234) and cell culture inserts (Cat. # 353182) for invasionand motility assay were obtained from BD Biosciences.RHO-associated coiled-coil protein kinase 1 (ROCK1)primary antibody (Cat. # A300-457A) was from BethylLaboratories. RPMI 1640 (Cat. # 11835-030) was fromGibco (Life Technologies), FBS (Cat. # SH30088.03) wasfrom HyClone (Thermo Fisher Scientific), penicillinstreptomycin solution (Cat. # 30-001-CI)was fromCellgro.

    Cell lineThe human breast cancer cell lineMDA-MB-231 (ATCC)

    was obtained in 2010, and was used both for xenograftin vivo studies and in cell culture for in vitro experiments.Cells were grown in RPMI medium 1640, supplementedwith 10% (v/v) FBS, penicillin (10 U/mL)streptomycin(10 U/mL) at 37C in humidified 5% CO2 atmosphere.

    Animal studiesFive- to six-week-old female athymic nude-Foxn1nu

    mice (Harlan Laboratories) were exposed to 500 cGy ofradiation. The next day themicewere anesthetized and anincision was made near the right flank to expose themammary fat pad. A total of 1 106 cells (MDA-MB-231 breast cancer cells) were injected using a Hamiltonsyringe into the mammary fat pad. Tumor developmentwas followedandwhenxenografts averaged4mmin eachdimension (length, width, and height, approximately 3weeks after the implantation), mice with comparablesized tumors were randomly divided between the fourtreatment groups: DOX 9 mg/kg, DOX 9 mg/kg andGGA, GGA, and saline. Doxorubicin has been reportedto induce cardiotoxicity in awide range of dosages (4 to 25

    mg/kg; refs. 2529); we selected an intermediate dosage,which would allow for gradual cardiotoxicity develop-ment with multiple doxorubicin injections (25, 26). Doxo-rubicin was administered via tail vein injection every 2weeks for a total of 4 injections. GGA treatment (1 mg/gbodyweight)was given 48hour before doxorubicin per osmethod (pipette). GGA was previously shown to elicit aprotective response when given 24 to 48 hours beforestressor (3035) in dosages from 200 to 1,000 mg/kg(orally or intraperitoneally; refs. 3436).

    Tumor progression was evaluated by palpation andtumor size measurements with calipers. Tumorvolumes were calculated by the following formula:(1/2 L W H; ref. 37), in which L is the length,W is the width, and H is the height. All mice werehoused under a 12-hour light-dark cycle with free accessto food and water. This study was performed in accor-dance with the "Guide for the Care and Use of Labora-tory Animals" (2011) of the NIH. The protocol wasapproved by the Animal Care and Use Committee ofthe Johns Hopkins Medical Institutions (Baltimore, MD;Animal Welfare Assurance # A-3273-01).

    EchocardiographyTransthoracic echocardiography was performed on

    conscious mice using Acuson Sequoia C256 ultrasoundmachine (Siemens Corps) equipped with the 15-MHzlinear array transducer. The mouse heart was imaged ina two-dimensional mode followed byM-mode using theparasternal short axis view at a sweep speed of 200mm/sec. Measurements were acquired using the lead-ing-edge method, according to the American Echocar-diography Society guidelines (38). Left ventricle wallthickness and left ventricle chamber dimensions wereacquired during the end diastolic and end systolicphase, including interventricular septum (IVSD), leftventricular posterior wall thickness (PWTED), left ven-tricular end diastolic dimension (LVEDD), and leftventricular end systolic dimension (LVESD). Three tofive values for each measurement were acquired andaveraged for evaluation. The LVEDD and LVESD wereused to derive fractional shortening (FS) to meas

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