metronomic docetaxel in print nanoparticles and ezh2 ... · conducted in well-established...

Small Molecule Therapeutics

Metronomic Docetaxel in PRINT Nanoparticles and EZH2Silencing Have Synergistic Antitumor Effect in OvarianCancer

Kshipra M. Gharpure1,5, Kevin S. Chu8, Charles J. Bowerman7,11, Takahito Miyake1, Sunila Pradeep1,Selanere L. Mangala4, Hee-Dong Han1,4,16, Rajesha Rupaimoole1,6, Guillermo N. Armaiz-Pena1,Tojan B. Rahhal8, Sherry Y. Wu1, J. Christopher Luft7,11, Mary E. Napier12, Gabriel Lopez-Berestein2,4,Joseph M. DeSimone7,9,10,11,13,14,15, and Anil K. Sood1,3,4

AbstractThe purpose of this studywas to investigate the antitumor effects of a combination ofmetronomic doses of a

novel delivery vehicle, PLGA-PRINT nanoparticles containing docetaxel, and antiangiogenic mEZH2 siRNA

incorporated into chitosan nanoparticles. In vivo dose-finding studies and therapeutic experiments were

conducted in well-established orthotopic mouse models of epithelial ovarian cancer. Antitumor effects were

determined on the basis of reduction in mean tumor weight and number of metastatic tumor nodules in the

animals. The tumor tissues from these in vivo studies were stained to evaluate the proliferation index (Ki67),

apoptosis index (cleaved caspase 3), andmicrovessel density (CD31). The lowest dose of metronomic regimen

(0.5 mg/kg) resulted in significant reduction in tumor growth. The combination of PLGA-PRINT-docetaxel

and CH-mEZH2 siRNA showed significant antitumor effects in the HeyA8 and SKOV3ip1 tumor models (P <0.05). Individual as well as combination therapies showed significant antiangiogenic, antiproliferative, and

proapoptotic effects, and combination therapy had additive effects. Metronomic delivery of PLGA-PRINT-

docetaxel combinedwithCH-mEZH2 siRNAhas significant antitumor activity in preclinicalmodels of ovarian

cancer. Mol Cancer Ther; 13(7); 1750–7. �2014 AACR.

IntroductionConventional chemotherapeutic regimens involve

high doses of a cytotoxic agent followed by a 3- to 4-week rest period. Such scheduling was believed to beimportant to balance the drug’s toxic effects on thetumor and on the host. However, recent studies usingthis approach have demonstrated a rapid acquisition ofchemoresistance in certain patient populations after an

initial response (1). Furthermore, conventional chemo-therapy drugs have been associated with many toxicadverse effects that often impair quality of life and limitcontinuation of therapy (2).

Angiogenesis, or the formation of new blood vessels,has been shown to be vital for cancer growth and dissem-ination, thus making it an attractive target for anticancertherapies. Although standard chemotherapies target thehighly proliferating cancer cells, they have little effect onthe slowly dividing endothelial cells (2–4). In addition, theresting period between chemotherapy cycles allows thetumor endothelium to continue forming new vessels (2).However, the advent of metronomic dosing, whichinvolves more frequent administration of low chemother-apeutic doses, has demonstrated the potential for morepronounced antitumor and antiangiogenic effects thanuse of standard maximum tolerated dose (MTD) chemo-therapy (5).

Particle replication in nonwetting templates (PRINT) isa recently developed method of fabricating nanocarriersto efficiently deliver therapeuticmoieties. This soft lithog-raphy process allows control of various parameters ofthe nanoparticles, such as size, shape, and compositionand thus allowsmore flexibility than do other approaches(6). Previous reports have shown that docetaxel incorpo-rated in Poly(D,L-lactide-co-glycolide) (PLGA)-PRINTnanoparticles has higher plasma exposure, greater intra-tumor accumulation than docetaxel alone (7).

Authors' Affiliations: Departments of 1Gynecology Oncology, 2Experi-mental Therapeutics, and 3Cancer Biology; 4Center for RNA Interferenceand Non-Coding RNAs, The University of Texas MD Anderson CancerCenter (MDACC); 5Experimental Therapeutics Program and 6Cancer Biol-ogy Program, The University of Texas Graduate School of BiomedicalSciences at Houston, Houston, Texas; Departments of 7Chemistry, 8Phar-maceutical Sciences, and 9Chemical and Biomolecular Engineering; and10Department of Pharmacology, Eshelman School of Pharmacy; 11Line-berger Comprehensive Cancer Center; 12Frank Hawkins Kenan Institute ofPrivate Enterprises; 13Carolina Center of Cancer Nanotechnology Excel-lence; 14Institute for Nanomedicine; and 15Institute for AdvancedMaterials,The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina;and 16Department of Immunology, School of Medicine, Konkuk University,South Korea

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Anil K. Sood, The University of Texas MD Ander-son Cancer Center, 1155 Pressler St, Unit 1362, Houston, TX 77030.Phone: 713-745-5266; Fax: 713-792-3643; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-0930

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 13(7) July 20141750

on October 30, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst April 22, 2014; DOI: 10.1158/1535-7163.MCT-13-0930

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Enhancer of Zeste Homolog 2 (EZH2), found to beoverexpressed in tumor endothelial cells, methylates, andinactivates vasohibin-1 (VASH1), a potent antiangiogenicfactor (8). We recently demonstrated that silencing EZH2with siRNA incorporated into chitosan (CH) nanoparti-cles resulted in decreased angiogenesis and significantreduction in tumor growth (9). Here, we examined thebiological effects of combinedmetronomic PLGA-PRINT-docetaxel and CH-mEZH2 siRNA in ovarian cancermodels.

Materials and MethodsCell lines and cultureHeyA8, SKOV3ip1, and HeyA8-luciferase cells were

maintained as described previously (9). Briefly, the cellswere maintained in 5% CO2 at 37

�C in RPMI-1640 mediasupplemented with 15% FBS and 0.1% gentamycin sul-fate. The cell lines were obtained from ATCC and wereroutinely tested for absence of Mycoplasma.

CH-mEZH2 siRNAmEZH2 siRNAwas incorporated into CHnanoparticles

as described previously (8). The nanoparticles were for-mulated based on ionic gelation of anionic tripolypho-sphate (TPP) and siRNA with cationic CH. Briefly, CHwasdissolved in 0.25% acetic acid. The nanoparticleswerespontaneously formed after adding TPP (0.25% w/v) andsiRNA (1 mg/mL) to the CH solution under constant stir-ring at room temperature. The nanoparticles were thenincubated at 4�C for 40 minutes and then centrifuged at12,000 rpm for 40 minutes at 4�C. The pellet was washedthree times to remove any unbound chemicals or siRNA.Thenanoparticles thus formedwere storedat4�Cuntiluse.

PLGA-PRINT-docetaxelPLGA-PRINT nanoparticles containing docetaxel were

formulatedasdescribedpreviously (6).Briefly,150mLof10mg/mLPLGA and 10mg/mLdocetaxel were spread on a600 � 1200 sheet of poly (ethylene terephthalate) (PET) usinga Mayer Rod (R. D. specialties). The solvent was evapo-rated with heat. The PET sheet was then placed in contactwith themold, and themoldwas separated from the sheetby passing the mold and sheet through a hot laminator at130�C and 80 psi (ChemInstruments Hot Roll Laminator).The mold was then placed in contact with another PETsheet coated with 2,000 g/mol polyvinyl alcohol (PVOH).The nanoparticles were then transferred from the mold tothe PET sheet by passing through a hot laminator andfinally removed from the PET sheet by passing the sheetthrough rollers and applyingwater to dissolve the PVOH.The nanoparticleswere purified and later concentrated bytangential flow filtration (Spectrum Labs).

Particle characterizationThe nanoparticles were characterized with use of a

scanning electron microscope (SEM). Briefly, a 50-mLnanoparticle sample was pipetted onto a glass slide. Thesample was then dried and coated with a 3-nm thick gold

palladium alloy using a Cressington 108 auto-sputtercoater. Images were obtained with use of a Hitachi modelS-4700 SEM at an accelerating voltage of 2 kV. Size andpolydispersity index were measured by using dynamiclight scattering (Malvern Instruments Nano-ZS).

Stability of PLGA-PRINT nanoparticles in plasmaNanoparticles in phosphate-bufferred saline (PBS)

were mixed with plasma at a 1:1 ratio and shaken at37�C. At set time points, nanoparticles were removedand washed with water 20 times by tangential flowfiltration to remove plasma proteins (mPES hollow fiberfilter with 0.05 mm pore size; Spectrum Labs). Thenanoparticles were then collected on a filter andimaged. The nanoparticles were imaged by SEM (Hita-chi S-47000 Cold Cathode Field Emission ScanningElectron Microscope).

Drug loadingDocetaxel loading was measured with use of high-per-

formance liquid chromatography (HPLC) with C18reverse phase column (Agilent Technology series 1200).All of the samples and standards of docetaxel and PLGAwere prepared in acetonitrile. The nanoparticle sampleswere prepared by diluting a sample at a ratio of 1 to 10 inacetonitrile. A linear gradient of 100% water to 100%acetonitrile was run over for more than 10 minutes fol-lowedby100%acetonitrile for 5minutes.Theflowratewas1 mL/min, and the wavelength of detection was 205 nm.

In vitro drug releaseWe placed 100 mL of nanoparticle solution (200 mg/mL

docetaxel) into a miniature dialysis unit with a 20kmolecular weight cutoff. The solution was dialyzedagainst a constantly stirred 1 L bath of 1� PBS at 37�C.At each time point, the nanoparticle solution from eachunit was removed and centrifuged. The nanoparticlepellet was analyzed for the amount of docetaxel remain-ing in the nanoparticles. By comparing this amount withthe initial amount of docetaxel present in the system, thepercentage of drug release could be calculated.

Orthotopic model of ovarian cancerFemale athymic nude mice were purchased from

Taconic Farms and housed in pathogen-free conditions.They were cared for in accordance with the guidelinesof the American Association for Accreditation of Lab-oratory Animal Care and the U.S. Public Health ServicePolicy on Human Care and Use of Laboratory Animals.All in vivo experiments and protocols were approved byMD Anderson’s Institutional Animal Care and UseCommittee.

For all of the in vivo experiments, cells were trypsinizedat 60% to 80% confluence, centrifuged at 1,200 rpm for6 minutes at 4�C, washed twice with PBS, and reconsti-tuted in Hanks balanced salt solution (HBSS; Cellgro) to adesired concentration (1.5 � 106 cells/mL for HeyA8and 5 � 106 cells/mL for SKOV3ip1 for intraperitoneal

Effect of Metronomic Docetaxel in PRINT Particles and siEZH2

www.aacrjournals.org Mol Cancer Ther; 13(7) July 2014 1751




injections). For intraovarian injections, HeyA8-luciferasecells (1.5� 106 cells/mL)were resuspended in 1:1mixtureof BDmatrigel and HBSS). Mice were anesthetized withketamine and an incision was made just above the leftovary. A 1-mL tuberculin syringe with a 30-gaugeneedle was used to inject cells directly into the ovary.The incision was then closed using surgical clips. Themice were returned to cages after full recovery. Theclips were removed after a week when the incision wasfully healed.

The dose-finding studies for PLGA-PRINT-docetaxelwere performed with five treatment groups (10 mice/group) divided into a control (saline) group, MTD (20mg/kg) group, and three groups at 0.5, 1.0, and 2.0 mg/kg of docetaxel injections. Each mouse was injectedintraperitoneally (i.p.) with 200 mL of cell suspensioncontaining 3 � 105 HeyA8 cells. Treatment was started 1week after the injections of tumor cells. An MTD dose of20 mg/kg was given once in 2 weeks during the entire invivo treatment. Saline and metronomic doses of doce-taxel were given as 200 mL i.p. injections three times perweek. The mice were monitored daily for any signs oftoxic adverse effects. All the mice were sacrificed whenthe mice in any group seemed to be moribund. Mouseweight, tumor weight, and the number of nodules wererecorded.

For the therapeutic experiment, mice were divided intofour groups (10 mice/group): CH-control siRNA, CH-mEZH2 siRNA, PLGA-PRINT-docetaxel, and a combina-tion of PLGA-PRINT-docetaxel and CH-mEZH2 siRNA.Themicewere injected i.p.with a 200mL cell suspension ofeitherHeyA8 (3� 105 cells/mouse) or SKOV3ip1 cells (1�106 cells/mouse). Treatment was started 1 week after thetumor cell injections. The siRNAwas injected at a dose of3.5 mg in 100 mL of saline intravenously (i.v.) twiceweekly.The PLGA-PRINT-docetaxel was injected at a dose of 0.5mg/kg in 125mLof saline i.p. three timesweekly. Themicewere sacrificed when the mice in any group becamemoribund. Mouse weight, tumor weight, and the numberof tumor nodules were recorded. Tumors were collectedand the tissues were formalin-fixed or snap-frozen inoptimal cutting medium for immunohistochemicalstaining.

We performed additional therapeutic experiment withHeyA8-luciferese intraovarian mouse model. The micewere injected with 100 mL suspension of HeyA8 cells (3 �105 cells/mouse). The mice were treated as mentionedearlier. Bioluminescence imaging was conducted to mon-itor metastasis after initiation of treatments. Imaging anddata acquisition were performed with IVIS spectrum sys-tem coupled to the Living Image Software (Xenogen). Themice were anesthetized in an acrylic chamber with amixture of 1% isoflurane in air. They were then injectedintraperitoneallywith luciferin potassium salt (15mg/mL)in PBS at a dose of 150 mg/kg body weight. A digitalgrayscale image was initially acquired which was thenoverlaidwith a pseudocolor image representing the spatialdistribution of detected photons emerging from active

luciferase present within the animal. Signal intensity wasexpressed as a sum of all photons detected per second.

Immunohistochemical stainingFor immunohistochemical analysis, paraffin sections

(5 mm) of tumor tissues were sectioned and used for thedetection of a proliferation marker (Ki67) and an apo-ptosis marker (cleaved caspase 3). Frozen sections wereused to detect the expression of an endothelial cellmarker (CD31). Formalin-fixed paraffin sections weredeparaffinized by sequential washing with xylene,100% ethanol, 95% ethanol, 80% ethanol, and PBS. Asuitable antigen retrieval method was used. For CD31staining, frozen-cut sections were fixed by incubatingfor 5 minutes in cold acetone, acetone þ chloroform(1:1), and then cold acetone in a sequential manner. Noantigen retrieval procedure was necessary for CD31staining. For all of the slides, endogenous peroxide wasblocked by incubating the slides with 3% hydrogenperoxide in PBS. To prevent nonspecific binding of theprimary antibody, the slides were incubated in 5%normal horse serum þ 1% normal goat serum (for Ki67and CD31 staining) or in 4% fish gelatin in PBS (forcleaved caspase 3 staining) This was followed by anovernight incubation at 4�C with a primary antibodydiluted in the protein block solution. The primary anti-bodies used were Ki67-rabbit polyclonal anti-humanantibody from Thermo/lab vision, cleaved caspase 3-rabbit polyclonal anti-human, mouse, rat from BiocareMedical, and CD31 rat anti-mouse antibody from Phar-mingen/Invitrogen. After the slides were incubatedwith primary antibodies, they were washed with PBSand incubated with suitable secondary antibodies. ForKi67 and CD31 staining, goat anti-rabbit HRP antibodydiluted in protein block solution was used, and theslides were incubated in this solution for 1 hour at roomtemperature. For cleaved caspase 3 staining, the slideswere first incubated with biotinylated 4 þ goat anti-rabbit secondary antibody for 20 minutes, after whichthey were washed with PBS and later incubated withstreptavidin 4 þ goat anti-rabbit secondary antibody for20 minutes. After the slides were incubated with sec-ondary antibody, they were washed with PBS followedby development with DAB (Research Genetics). Thenuclei were stained with Gill’s hematoxylin solution,and the slides were then mounted with paramountmounting media.

For Ki67 and cleaved caspase staining, the numbers oftumor cells that were positive for expression werecounted. For CD31 staining, microvessel-like structurescontaining endothelial cells that were positive for CD31staining were counted. Expression was quantified byusing 15 random fields of slides at �100 magnification.

Statistical analysisContinuous variables were analyzed with use of the

Student t test or the Mann–Whiney U test. A P value lessthan 0.05 was considered to be significant.

Gharpure et al.

Mol Cancer Ther; 13(7) July 2014 Molecular Cancer Therapeutics1752




ResultsPLGA-PRINT nanoparticle characterizationBefore carrying out in vivo experiments, we first

examined the PRINT nanoparticles, which were of uni-form size (80 � 320 nm) and shape as shown in Fig. 1A.The percentage of docetaxel loading, calculated byHPLC, was 17.1% (Fig. 1B). When incubated with plas-ma, the nanoparticles were stable up to 3 days (Fig. 1C).The nanoparticles exhibited a "burst release profile," inwhich 100% of the drug is released within 24 hours (Fig.1D). We performed cell viability experiments after incu-bation with PLGA-PRINT nanoparticles. There was noeffect on cell viability after incubation with PRINTnanoparticles (data not shown). We also conductedstability assays of the nanoparticles. The nanoparticleswere stable at �20�C up to 3 months, whereas thelyophilized nanoparticles were stable at 4�C up to6 months (Supplementary Table S1).

Internalization and stability assays of CHnanoparticlesWe evaluated intracellular delivery of CH nanoparti-

cles using HeyA8 cells and Cy3-labeled CH-controlsiRNA. There was efficient internalization of these nano-particles into the cancer cells (Supplementary Fig. S1A).We also checked the stability of CH-mEZH2 siRNA inpresence of 10%FBS.Naked siRNAwasdegraded rapidlyupon incubation with serum, whereas siRNA protectedby CH nanoparticles was not degraded (SupplementaryFig. S1B). To evaluate for any possible effects of the

nanoparticles alone, we tested the effect of CH nanopar-ticles on SKOV3ip1 cells. There was no effect on cellviability after 48 hours of incubation with CH nanopar-ticles (Supplementary Fig. S1C).

Determining the optimal metronomic dose forPLGA-PRINT-docetaxel nanoparticles

Next, we conducted a dose-finding study to determinethe lowest docetaxel dose that is biologically effective. Allof the doses were significantly effective in reducing thetumor burden (P < 0.05 for MTD and P < 0.01 for metro-nomic doses). The lowest dose at 0.5 mg/kg reduced thetumor weight by approximately 80% and was, therefore,chosen for subsequent therapeutic studies (Fig. 2A).

Therapeutic studies with metronomic dosesWe next determined whether a combination of PLGA-

PRINT-docetaxel nanoparticles with CH-mEZH2 siRNAwould result in a strong antitumor effect. The mice weredivided into the following groups: (i) CH-control siRNA,(ii) CH-mEZH2 siRNA, (iii) metronomic dose of PLGA-PRINT-docetaxel (0.5 mg/kg), and (iv) metronomic doseof PLGA-PRINT-docetaxel and CH-mEZH2 siRNA.PLGA-PRINT-docetaxel was injected i.p. three timesweekly, whereas siRNA was injected i.v. twice weekly.CH-mEZH2 siRNA treatment reduced aggregate tumorweight by approximately 70% in both HeyA8 and SKO-V3ip1 models (P < 0.01 for HeyA8 and P < 0.05 forSKOV3ip1). PLGA-PRINT-docetaxel treatment reducedtumor weight by approximately 80% (P < 0.01).

Figure 1. Particle characterization,drug loading, and release profile.A, 80 � 320 nm PLGA-PRINT-docetaxel nanoparticles SEMimage. B, size, polydispersity index(PDI), and drug loading profile.C, stability of PLGA-PRINTnanoparticles after incubation withplasma. SEM images are shown.Nanoarticles before (top left) andafter (top right) plasma exposurefor 1 day. Nanoparticles after3 days in plasma (bottom left).Nanoparticles after 7 days inplasma (bottom right).D, in vitro drug release profile.






Combination therapy showed the greatest therapeuticbenefit with approximately 95% reduction in tumorweight in both models (P < 0.001 in HeyA8 and

P < 0.01 in SKOV3 ip1). In the HeyA8 model, individualtherapies resulted in reduction in the number of nodules,and combination therapy showed a significant effect

Figure 2. Dose determination andtherapeutic in vivo experiments.A, dose-finding study. Effect ofmetronomic (0.5, 1, and 2 mg/kg)versus MTD doses (20 mg/kg) ofPLGA-PRINT-docetaxel therapyon HeyA8 orthotopic mousemodel. Average of tumor weightand number of nodules are shown.All the metronomic doses showedsignificant tumor reduction. Errorbars, standard error of the mean.�, P < 0.05; ��, P < 0.01 comparedwith saline. Tumor growthinhibition with CH-mEZH2 siRNAand PLGA-PRINT-docetaxel,alone, and with combinationtherapy in HeyA8 (B) andSKOV3ip1 (C) orthotopic mousemodel. Average of tumor weightsand number of nodules are shown.Combination therapy showed thegreatest therapeutic benefit in bothmouse models. There wassignificant reduction in tumorweight as well as in tumor nodules.Error bars, standard error of themean. �, P < 0.05; ��, P < 0.01; and��, P < 0.001 compared withCH-control siRNA group.

Gharpure et al.





(P<0.01). In the SKOV3ip1model, combination therapyaswell as individual therapies resulted in significant reduc-tion of tumor nodules (P < 0.05; Fig. 2B and C). For theobjective evaluation of reduction of metastasis, we con-

ducted an additional in vivo experiment with luciferase-labeled HeyA8 cells injected directly into the ovary. Bio-luminescence imaging revealed reduced metastasis afterindividual and combination therapies (Fig. 3A and B). No

Figure 3. Biologic effects of the treatments. A, noninvasive images of metastasis and tumor growth in orthotopic mouse model of HeyA8. Representativeimages show reduction in metastasis after the treatment of CH-mEZH2 siRNA, PLGA-PRINT-docetaxel, and combination therapy. The effect is pronouncedin the combination group. Quantification of luciferin signal intensity is shown in the adjoining graph. Error bars, standard error of the mean �, P < 0.05;��, P < 0.01. B, representative images of sites of metastasis and tumor burden in orthotopic mouse model of HeyA8. The data show reduction in metastasisafter individual and combination therapies. C, proliferation index (Ki67), apoptosis index (cleaved caspase 3), andmicrovessel density (CD31) inHeyA8 tumorstreated with CH-control siRNA, CH-mEZH2 siRNA, PLGA-PRINT-docetaxel, and combination therapy. A graphic representation of the average number ofKi67 or cleaved caspase 3–positive cells and CD31-positive vessels are shown in the adjoining graphs. Combination therapy showed significantantiproliferative, proapoptotic, and antiangiogenic effects. Error bars, standard error of the mean �, P < 0.05; ��, P < 0.01; and ��, P < 0.001 compared withCH-control siRNA group. Scale bar, 100 mm.






significant effects of therapywere noted onmouseweightor activity. Following siRNA delivery, downregulation ofmurine EZH2 in endothelial cells was confirmed atmRNA level by qRT-PCR and at protein level by immu-nohistochemistry (Supplementary Fig. S1D and S1E).

Biologic effects of metronomic PLGA-PRINT-docetaxel and CH-mEZH2 siRNA therapy

To determine the biologic effects of combined thera-py, in vivo samples were subjected to immunohisto-chemical analysis for Ki67, cleaved caspase 3, and CD31(Fig. 3C). Individual as well as combination therapiesresulted in a significant reduction in proliferation(P < 0.001). Individual metronomic dosing of PLGA-PRINT-docetaxel and CH-mEZH2 siRNA increasedthe apoptotic index significantly (P < 0.05), whereasthe combination therapy had an additive effect (P <0.001). Previous studies have suggested that metronom-ic chemotherapy affects endothelial cell proliferation.To test this hypothesis, frozen tumor sections from thein vivo studies were stained for CD31. CH-mEZH2siRNA as well as metronomic PLGA-PRINT-docetaxeltherapy showed a significant effect on tumor vascula-ture (P < 0.01). Combining CH-mEZH2 siRNA withmetronomic docetaxel also resulted in a significantreduction in microvessel density (P < 0.01).

DiscussionThe key finding from this study is the pronounced

antitumor effect of metronomic delivery of PLGA-PRINT-docetaxel therapy alongwith CH-mEZH2 siRNA.Wehavepreviously shown that themajor biologic effect ofmetronomic therapy is through its antiangiogenic effects(10, 13, 14). Concordant with those studies, we saw asignificant antiangiogenic effect and downstream anti-proliferative and proapoptotic effects.

Metronomic chemotherapy has been proposed to serveas an alternative to the standard dosing schedule toovercome the problems associated with the latter.Long-term administration of standard chemotherapies islimited by their toxic effects on normal cells, whereasmetronomic therapy has minimal adverse effects (2–5, 11). Metronomic dosing has also been shown to beeffective against chemoresistant models (12, 13). Whereasstandard therapy has little effect on tumor vasculature,studies have shown that metronomic therapy has signif-icant antiangiogenic effects (2–5, 11–14). Our findingsfrom this study also suggest that endothelial cells arequite sensitive to low doses of docetaxel therapy in ametronomic schedule. Moreover, the low toxicity profileof metronomic dosing makes it a suitable candidate forcombination therapies. Several studies have demonstrat-ed that a combination of metronomic dosing with anantiangiogenic therapy has significant antitumor effectsin ovarian cancer (10, 13, 15, 16). Here, we provide strongevidence that metronomic docetaxel dosing with novelPRINT nanoparticles has strong antiproliferative, antia-poptotic, and antiangiogenic effects, and that combining it

withCH-mEZH2siRNAproved tobehighly efficacious inreducing tumor burden.

Angiogenesis is one of the key processes that promotecancer growth and metastasis. The process is regulatedby several pro- and antiangiogenic factors in the bodythat become deregulated in cancer (17). EZH2 wasfound to be overexpressed in tumor-associated endo-thelial cells (8). Previous studies in our laboratory (9)have shown that VEGF directly increases EZH2 expres-sion on endothelial cells, which in turn methylates andinhibits an antiangiogenic factor, VASH1. Several otherstudies have shown that EZH2 plays a critical role inseveral oncogenic pathways (18–21). Hence, silencingEZH2 in endothelial cells is a promising therapeuticapproach to target angiogenesis.

With increasing understanding of toxicity profiles andbecause of the poor pharmacokinetic properties of thecurrent chemotherapeutics, there is a need to develop adelivery vehicle with improved drug efficacy and reducedtoxicity. Docetaxel is a commonly used chemotherapeuticagent but is often associatedwith systemic toxicities.Whencompared with docetaxel alone, PLGA-PRINT nanoparti-cles loaded with docetaxel showed higher plasma expo-sure, higher tumor concentration, and lower volume ofdistribution in a subcutaneous ovarian cancermousemod-el (6). It also exhibited a characteristic "burst release pro-file," inwhich 100% of the drug is releasedwithin 24 hours.This property is ideal for metronomic drug delivery.

In summary, we have demonstrated that combinationtherapy of metronomic PLGA-PRINT-docetaxel and CH-mEZH2 siRNA is highly effective in reducing tumorburden. This approach holds great potential for newtreatment strategies for ovarian and other cancers.

Disclosure of Potential Conflicts of InterestJ.C. Luft is a consultant/advisory board member for Liquidia Technol-

ogies, Inc. M.E. Napier has ownership interest in and is a consultant forLiquidia Technologies, Inc. J.M.DeSimone received a commercial researchgrant from Liquidia Technologies, Inc., is a consultant for Liquidia Tech-nologies, Inc., has ownership interest in patents held by Liquidia Tech-nologies, Inc. and University of North Carolina, Chapel Hill, and is afounder of Liquidia Technologies, Inc. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: K.M. Gharpure, S.Y. Wu, J.C. Luft, M.E Napier,G. Lopez-Berestein, J.M DeSimone, A.K. SoodDevelopment of methodology: K.M. Gharpure, S.L. Mangala, H.-D. Han,S.Y. Wu, M.E Napier, G. Lopez-Berestein, A.K. SoodAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.M. Gharpure, K.S. Chu, C.J. Bowerman,T. Miyake, S.L. Mangala, R. Rupaimoole, J.C. Luft, G. Lopez-Berestein,A.K. SoodAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): K.M. Gharpure, T. Miyake, R. Rupaimoole,G.N. Armaiz-Pena, J.C. Luft, G. Lopez-Berestein, A.K. SoodWriting, review, and or revision of the manuscript: K.M. Gharpure,C.J. Bowerman, T. Miyake, S.L. Mangala, R. Rupaimoole, G.N. Armaiz-Pena, J.C. Luft, G. Lopez-Berestein, J.M DeSimone, A.K. SoodAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K.M. Gharpure, S. Pradeep,M.E Napier, G. Lopez-Berestein, J.M DeSimone, A.K. SoodStudy supervision: K.M. Gharpure, J.C. Luft, M.E Napier, G. Lopez-Berestein, J.M DeSimone, A.K. SoodOther (provided nanoparticle formulations): K.S. ChuOther (conducted study of PRINT particle stability): T.B. Rahhal


Gharpure et al.




AcknowledgmentsThe authors thank D. Reynolds and Dr. R. Langley for technical

assistance and helpful discussions.

Grant SupportK.M. Gharpure is supported by Altaman Goldstein Discovery Fel-

lowship. S.Y. Wu is supported by Ovarian Cancer Research Fund,Foundation for Women’s Cancer, and Cancer Prevention ResearchInstitute of Texas training grants (RP101502, RP101489, and RP110595).Portions of this work were supported by the NIH (U54 CA151668,CA016672, CA109298, P50 CA083639, P50 CA098258, CA177909,U54 CA151652, and UH2 TR000943-01), the DOD (OC120547 and

OC093416), the Ovarian Cancer Research Fund (Program Project Devel-opment Grant), the Blanton-Davis Ovarian Cancer Research Program,Meyer and Ida Gordon Foundation #2, the RGK Foundation, theChapman Foundation, the Gilder Foundation, and the Betty AnneAsche Murray Distinguished Professorship.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 30, 2013; revised March 25, 2014; accepted April 8,2014; published OnlineFirst April 22, 2014.

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2014;13:1750-1757. Published OnlineFirst April 22, 2014.Mol Cancer Ther Kshipra M. Gharpure, Kevin S. Chu, Charles J. Bowerman, et al. Have Synergistic Antitumor Effect in Ovarian CancerMetronomic Docetaxel in PRINT Nanoparticles and EZH2 Silencing

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