hsp70 inhibition synergistically enhances the effects of ......models and technologies hsp70...

Models and Technologies

HSP70 Inhibition Synergistically Enhances theEffects of Magnetic Fluid Hyperthermia inOvarian CancerKarem A. Court1, Hiroto Hatakeyama2,3, Sherry Y.Wu2, Mangala S. Lingegowda2,Cristian Rodríguez-Aguayo4, Gabriel L�opez-Berestein4,5, Lee Ju-Seog6,Carlos Rinaldi7,8, Eduardo J. Juan9, Anil K. Sood2,5, and Madeline Torres-Lugo1

Abstract

Hyperthermia has been investigated as a potential treatment forcancer. However, specificity in hyperthermia application remainsa significant challenge. Magnetic fluid hyperthermia (MFH) maybe an alternative to surpass such a challenge, but implications ofMFH at the cellular level are not well understood. Therefore, thepresent work focused on the examination of gene expression afterMFH treatment and using such information to identify targetgenes thatwhen inhibited could produce an enhanced therapeuticoutcome after MFH. Genomic analyzes were performed usingovarian cancer cells exposed to MFH for 30 minutes at 43�C,which revealed that heat shock protein (HSP) genes, includingHSPA6, were upregulated. HSPA6 encodes the Hsp70, and itsexpression was confirmed by PCR in HeyA8 and A2780cp20ovarian cancer cells. Two strategies were investigated to inhibit

Hsp70-related genes, siRNA and Hsp70 protein function inhibi-tion by 2-phenylethyenesulfonamide (PES). Both strategiesresulted in decreased cell viability following exposure to MFH.Combination index was calculated for PES treatment reporting asynergistic effect. In vivo efficacy experiments with HSPA6 siRNAand MFH were performed using the A2780cp20 and HeyA8ovarian cancer mouse models. A significantly reduction in tumorgrowth rate was observed with combination therapy. PES andMFH efficacy were also evaluated in the HeyA8 intraperitonealtumor model, and resulted in robust antitumor effects. This workdemonstrated that HSP70 inhibition combination with MFHgenerate a synergistic effect and could be a promising targetto enhance MFH therapeutic outcomes in ovarian cancer.Mol Cancer Ther; 16(5); 966–76. �2017 AACR.

IntroductionHyperthermia is the application of heat to tissues as a thera-

peutic tool using temperatures between 41�C and 47�C. Hyper-thermia has been successfully employed as an adjuvant for thetreatment of several cancers and is known to enhance the effects ofchemotherapy and radiotherapy (1–3). Clinically, heat can beapplied locally, regionally or in the whole-body, but currentmodalities are not site specific (1, 4). Challenges such as temper-ature homogeneity during application, achieving an efficient heat

dose, patient discomfort, and hot spots limit the use of thistherapy (2, 4). The use of nanotechnology could help overcomesuch challenges. Magnetic fluid hyperthermia (MFH) is an attrac-tive alternative as it can deliver heat to the desired area usingmagnetic nanoparticles under the influence of a magnetic field(1, 2, 5).

There is evidence that MFH can deliver heat more efficientlythan conventional hyperthermia (6–12). Aspects such as thermalchemosensitization, membrane permeabilization, and sensitiza-tion of drug-resistant cancer cells have been observed as responsestoMFH in vitro (2, 7–9). Thesefindings demonstrate thatMFHhaspotential as an adjuvant cancer treatment, but there is still a dearthof knowledge in thefield regarding the implications ofMFHat themolecular level and how these can be exploited to enhance theeffects of MFH in cancer treatment.

Previous work with conventional hyperthermia demonstratedthat molecular and cellular responses depend on the cell line,thermal dose, and recuperation time (13–16). In the clinic,intraperitoneal administration of hyperthermia (HIPEC), in com-binationwith chemotherapeutic drugs, have been investigated formore than three decades as an attractive adjuvant to cytoreductivesurgery in ovarian cancer (17). Currently, there are more than 30clinical trials that have recently started or concluded using thistechnology (18). A recent meta-analysis of clinical trials per-formed by Huo and colleagues (19), demonstrated that thecombination of HIPEC, cytoreductive surgery, and chemotherapyshowed significant patient survival. Still, a challenge remains: theability to reach a therapeutic temperature without causing toxicityto healthy tissue and risk complications (20–24). Furthermore,

1Department of Chemical Engineering, University of Puerto Rico-Mayag€uez,Mayag€uez, Puerto Rico. 2Department of Gynecologic Oncology, MD AndersonCancer Center, Houston, Texas. 3Faculty of Pharmaceutical Sciences, ChibaUniversity, Chiba, Japan. 4Department of Experimental Therapeutics, MDAnder-son Cancer Center, Houston, Texas. 5Center for RNA Interference and Non-Coding RNA, MD Anderson Cancer Center, Houston, Texas. 6Department ofSystems Biology, MD Anderson Cancer Center, Houston, Texas. 7J. CraytonPruitt Family Department of Biomedical Engineering, University of Florida,Gainesville, Florida. 8Department of Chemical Engineering, University of Florida,Gainesville, Florida. 9Department of Electrical and Computer Engineering,University of Puerto Rico-Mayag€uez, Mayag€uez, Puerto Rico.

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

Corresponding Author: Madeline Torres-Lugo, University of Puerto Rico-Mayag€uez, PO Box 9000, Mayag€uez, 00680, Puerto Rico. Phone: 787-832-4040, ext. 2585; Fax: 787-834-3655; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0519

�2017 American Association for Cancer Research.

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intrinsic biological aspects such as the inherent thermo-toleranceproperties of tumors are a challenging aspect that has beenneglected to this point. Nanoscale heat generation systems suchas iron oxide nanoparticles represent an attractive alternative asone could generate heat within the system, only under high-frequency and moderate amplitude magnetic fields that can beconstrained to the tumor region. Therefore, the goal of thiswork was to investigate gene expression profiles after MFH inovarian cancer cell lines to elucidate cellular response and selectmolecular targets to enhance its effect in vitro and in vivo. It washypothesized that upregulated genes and proteins resultingfrom MFH treatment, when inhibited, could potentiallyenhance MFH outcome. Results indicated the upregulation ofHSPA6, which encodes for heat shock 70 kDa protein 6(Hsp70), several heat shock proteins (HSP), and ubiquitinC. Hsp70-related genes and proteins were selected as potentialtargets. Two strategies were investigated to inhibit Hsp70 genesand proteins (i) siRNA-based therapy; and, (ii) HSP70 proteinfunction inhibition by 2-phenylethyenesulfonamide (PES;ref. 25). Both strategies resulted in enhanced cell death invarious ovarian cancer cell lines in vitro and in vivo.

Materials and MethodsCell culture

Ovarian cancer cell lines HeyA8, HeyA8 MDR, A2780, A2780cp20, and SKOV3 were grown in RPMI1640 (Sigma-Aldrich),15% FBS (Life Technologies), and 0.1% gentamicin sulfate (Sig-ma-Aldrich). Cells weremaintained at 37�C, 95% relative humid-ity and 5% CO2. Cell lines were obtained from the institutionalCell Line Core Laboratory MD Anderson Cancer Center in 2012.Authentication was performed within 6 months of the currentwork by the short tandem repeat method using the PromegaPower Plex 16HS Kit (Promega).

Magnetic nanoparticlesCarboxymethyl dextran coated iron oxide nanoparticles

(CMDx-IO) were prepared using the co-precipitation method aspreviously described (8, 26). Briefly, an aqueous solution of ferricchloride hexahydrate, ferric chloride tetrahydrate, and ammoni-um hydroxide were mixed at a pH 8.0 under nitrogen at 80�C.Nanoparticles were peptized with tetramethyl ammoniumhydroxyl and dried. They were functionalized in dimethylsulf-oxide, 3-aminopropyltriethoxysilane, DI water, and acetic acid.Then washed with ethanol and dried. Later they were exposed tocarboxymethyl dextran, N,N-(3-dimethylaminopropyl)-N0-ethyl-carbodiimide hydrochloride, and N-hydroxysuccinimide, at pH4.5–5.0. Particles were centrifuged and washed with ethanol anddried. Particles characterization is presented in SupplementaryFig. S1.

In vitro MFHAttached cells. Cells were seeded in 35-mm cell culture dishes.New culture media and CMDx-IO nanoparticles (0.5 mgFe/mL)were added the following day. The dish was placed inside themagnetic induction coil of an Easy Heat 8310 LI (Ambrell)induction heater and exposed to a magnetic field intensity thatranged between 24 and 36 kA/m (245 kHz) to achieve 41�C or43�C for 30 minutes. The copper coil had five turns and an innerdiameter 5 cm. Temperature was measured with an NI-9211thermocouple signal conditioner and acquisition board (Nation-

al Instruments) with type T thermocouples (Omega Engineering).After 48 hours, cell viability was assessed by Trypan Blue (Sigma-Aldrich). For PES (EMD Chemicals)-treated cells, the concentra-tion was 5 mmol/L for A2780cp20 and 10 mmol/L for HeyA8 andSKOV3. PES was added to cells 2.5 hours before MFH and notremoved until the end of the treatment. Cells were incubated for48 hours in the presence of PES.

Suspended cells.Cells were suspended in 2.5mLofmedia in a glassvial with CMDx-IO nanoparticles (0.5 mgFe/mL). An inductionheater (RDO Induction) was used to generate a magnetic fieldintensity that ranged between 29 and 35 kA/m. The copper coilhad 4 turns and an inner diameter of 2 cm. Cells were exposed toMFH for 30minutes. After treatment, cellswere seeded in a 25 cm3

flask and placed in an incubator. After 48 hours, cell viability wasassessed with Trypan Blue (Sigma-Aldrich).

Microarray analysisOvarian cancer cells were exposed to MFH for 30 minutes at

43�C. RNA was prepared after treatment using a mirVana RNAisolation labeling kit (Life Technology). Three hundred nano-grams of RNA were used for labeling and hybridization on aHuman HT-12 v4 Beadchip (Illumina) following manufacturer'sprotocols. The bead chipwas scannedwith an Illumina BeadArrayReader (Illumina). Microarray data were normalized using thequantile normalization method in the Linear Models for Micro-array Data (LIMMA) package using R language. The expressionlevel of each gene was determined by the fold change in theuntreated control group, and MFH treated group (43�C and 30minutes) transformed into a log2 base for analysis. The originalmicroarray data were uploaded to GEO with accession numberGSE92990.

Quantitative real-time PCRRNAwas extracted from cells and tumor tissue using Direct-zol

RNA MiniPrep (Zymo Research). cDNA was synthesized from1,000 ng of RNA with the Thermo Verso cDNA Synthesis Kit(Thermo Fisher). Quantitative Real-Time PCR was performedusing SYBR Green (Thermo Fisher) on a 7500 Fast Real-TimePCR System and primers forHSPA6 andHSPA7 (Integrated DNATechnologies). Specific primers for HSPA6 F-CGCTGCGAGT-CATTGAAATA, R-GAGATCTCGTCCATGGTGCT and HSPA7F-CAACCTGCTGGGGCGTTTTGA, R-CCGGCCCTTGTCATTGGT-GATCTT, 18s were used as an endogenous control.

Western blot analysisCells were lysed with RIPA (50 mmol/L Tris-HCl, 150 mmol/L

NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS)incorporating the Protease Inhibitor sigmaFAST (Sigma-Aldrich).Protein concentration was determined with Bradford Reagent(Sigma-Aldrich). Equal amounts of protein were separated with4% to 20%Mini-PROTEAN TGX Gel (Bio-Rad Laboratories) andtransferred to a nitrocellulose membrane (Bio-Rad Laboratories).The membrane was incubated with HSP70 antibody, 1:1,000(Novus Biologicals) and b-actin rabbit mAb 1:1,000 (Cell Sig-naling Technology) overnight. Primary antibody was detectedwith anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Tech-nology) and developed with SuperSignal WestPico Chemilumi-nescent Substrate (Thermo Scientific) in Chemidoc XRS (Bio-RadLaboratories).

HSP70 Inhibition and Magnetic Fluid Hyperthermia in Cancer

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PES cytotoxicity in ovarian cancer cellsCells were seeded in 96-well plates in 200 mL of media and

allowed to attach overnight. Cellswere exposed to a concentrationof PESbetween5and30mmol/L for 24and48hours. Cell viabilitywas measured with EZU4 assay (ALPCO) following the manu-facturer's protocol.

Determination of synergismCombination index (CI) was evaluated using the proposed

model of Chou–Talay with the CompuSyn software (27). Com-puSynuses themedian–drug effect analysis. Concentration–effectcurves were assumed to be sigmoidal for the single drug and thecombination treatment. Calculation of doses was performed bythe median–effect equation. The parameters used for the calcu-lation were the fraction affected and PES andMFH exposure time.Equations are described in the Supplementary Material.

In vitro siRNA delivery and MFHCells were transfected with Lipofectamine RNAiMax (Invitro-

gen) at 2.5 mL of the reagent: 1 mg siRNA. HSPA6 siRNA fromRosetta Prediction Human (Sigma-Aldrich) was selected as targetgene, sequence 1: SASI_Hs01_00244829, sequence 2: HSPA7 1andHSPA7 2, sequence 3: SASI_Hs01_00244824 and sequence 4:SASI_Hs01_00244827 and SASI_Hs01_00244829 and controlsiRNA was MISSION siRNA Universal Control #1 (Sigma-Aldrich) at a concentration 60.2 or 100 nmol/L. A total 36 hoursof transfectionwasperformed.MFHwas applied, and cell viabilitywas measured following 48 hours of incubation as previouslydescribed.

Liposomal nanoparticle preparation (siRNA-DOPC)HSPA6 and control SiRNAs for in vivo delivery were incor-

porated into 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine(DOPC) nanoliposomes as previously described (28). DOPCand siRNA were mixed in the presence of excess tertiary butanolat a ratio of 1:10 (w/w) siRNA/DOPC. Tween 20 was added to themixture in a ratio of 1:19 Tween 20:siRNA/DOPC. The mixturewas vortexed, frozen in an acetone/dry ice bath and lyophilized.Before in vivo administration, this preparation was hydrated withPBS.

Subcutaneous MFH and HSPA6 siRNA deliverySubcutaneous tumors were generated in an athymic nude

mouse model. Studies were performed according to the protocolapproved by the MD Anderson Cancer Center Institutional Ani-mal Care and Use Committee. HeyA8 or A2780cp20 cells (1 �106) suspended in 50 mL of Hank's balance salt solution wereinjected subcutaneously in themice right flank on day 0, to obtainsubcutaneous tumors on day 7. Tumor volumes were 35–113mm3. siRNA liposomes injection started on day 7, 5 mg ofHSPA6siRNA or control siRNA liposomes were injected intraperitone-ally. ThreeMFH treatments and siRNA deliveries were performed.The firstMFH treatment was applied on day 9. Tumor volumewascalculated as (largest diameter) � (smallest diameter)2 � p/6.

MFH startedwith iron oxide nanoparticles injected directly intothe tumor (5 mgFe/cm3 tumor volume). Nanoparticles wereinjected for 30 seconds. The tip needle was removed from thetumor after 5 minutes. MFH was applied 4 hours after nanopar-ticle injection to allow particle distribution (29). Mice wereanesthetized and placed inside the magnetic induction coil of anEasyHeat 8310 LI (Ambrell) induction heater for 30 minutes at a

magnetic field intensity of 23 kA/m. The tumor was positioned inthe center of the coil. The copper coil consisted of 7.5 turns and aninner diameter of 3.16 cm. Warm water was circulated through aplastic tubing to maintain the environmental temperature insidethe coil at 37�C. Body temperature was measured in the anus toguarantee mice safety. The external temperature of the tumor andthe surrounding wall was also measured with an NI-9211 ther-mocouple signal conditioner and acquisition board (NationalInstruments) and type T thermocouples (Omega Engineering).

Intraperitoneal tumor model and MFHThe intraperitoneal tumor was generated in an athymic nude

mouse model. Studies were performed according to the protocolapproved by the M.D. Anderson Cancer Center InstitutionalAnimal Care and Use Committee. 2.5 � 10 5 HeyA8 cells sus-pended in 200 mL of Hank's balance salt solution were injecteddirectly into the peritoneal cavity (IP) ofmice. Treatment started 2weeks later after cells IP injection. PES was injected a 10 mg/Kg 2days before the first MFH. CMDx-IO nanoparticles were injectedintraperitoneally at 2 mg Fe/mouse in 200 mL of PBS the nightbefore treatment. Next daymice were anesthetized with ketamineandMFHapplied for 30minutes,with the same coil configurationpreviously described. The temperature was also measured in themouse belly.

HistologyTumors were embedded in paraffin and cut (8-mm sections).

Slides were deparaffinized, and rehydrated. The Prussian Blue,Iron Stain Kit (Sigma) was used following the manufacturer'sprotocol.

Statistical analysisStatistical analyzes were performed using the Student t test

(two-tailed distributions, two samples with unequal variables).For values thatwere not normally distributed, theMann–Whitneyrank sum test was used. A P value of

were used. This temperaturewas chosen since the aforementionedresults indicated a substantial decrease in cell viability when thetemperature was increased from 41�C to 43�C. Compared withcontrols, 60 genes were upregulated (by �1.5-fold) in treatedcells. The top 20 upregulated genes are presented Fig. 1C. Sup-plementary Tables S2 and S3 present descriptions of each of thedominant upregulated and downregulated genes.

Data were further analyzed using the Database for Annotation,Visualization and Integrated Discovery (DAVID) and enrichmentof Gene Ontology (GO) terms for significantly expressed genes.From this analysis, three main biological functions were found tobe affected by MFH at 43�C, including response to unfoldedproteins, response to protein stimulus, and protein folding(Table 1). Top genes related to the aforementioned functionsaffected by MFH were HSPs, Hsp70 (HSPA6/HSPA7, HSPA1A,HSPA1B, HSPA1L, HSPA4L), Hsp60 (LOC643300), Hsp40(DNBAJ family), Hsp20 (CRYAB) and Hsp27 (SERPINH1), andBAG3 (modulator of Hsp70). Besides HSPs, the gene ubiquitin C(UBC)was also in the top ten upregulated genes afterMFH. Figure1B presents the connection of HSPA6 with upregulated genesfrom the microarray.

To validate the results from the microarray, qRT-PCR wasperformed for HSPA6 and HSPA7. Hsp70 was selected becauseit has a critical function in cancer cells and hyperthermia. Hsp70 is

overexpressed in various human cancers, playing different roles asan inhibitor of apoptosis (31). HSPA6 and HSPA7 expressionwere confirmed by qRT-PCR, and expressionwasmeasured for upto 8 hours following heat shock treatment (Fig. 2A). A2780cp20showed significant expression at 1 hour for both genes. In HeyA8cells, gene expression was sustained for almost 4 hours at 41�Cand 8 hours at 43�C.

Hsp70 protein level was also evaluated according to Westernblot analysis. At 41�C, the highest expression level was observedafter 6 hours of MFH treatment. At 43�C, protein expression forA2780cp20 cells increased after 1 hour of recovery time, and thehighest expression was observed at 6 hours after MFH (Fig. 2B).HeyA8 cells showed no significant differences in Hsp70 proteinexpression via Western blot analysis (Supplementary Fig. S2C).HSPA6 (Hsp70) was selected as the target gene because it was themost upregulated (32). Two approaches were performed toinhibit Hsp70 including gene silencing by siRNA and proteinfunction inhibition using PES.

HSPA6 siRNA enhanced the effects of MFHA2780cp20 and HeyA8 cell lines were selected to further assess

the efficacy of HSPA6 inhibition along with MFH (Fig. 2).A2780cp20 and HeyA8 were selected according their capabilityto create subcutaneous tumor models. First, several siRNAsequences were investigated to evaluate their capacity to inhibitHSPA6 expression. Two siRNA sequences per cell line were estab-lished for HSPA6 inhibition with 50% to 60% of gene knockdown. For HeyA8 cell line, seq # 1 at 60.2 nmol/L and seq #2 at100 nmol/L were able to inhibitHSPA6. In the case of A2780cp20cells, seq #3 at 60.2 nmol/L and seq. #4 at 100 nmol/L (Supple-mentary Fig. S2A) were also successful in inhibiting HSPA6. Thein vitro siRNA effects were evaluated in each cell line (Fig. 2C and

Figure 1.

Geneexpression afterMFH in ovarian cancer cells.A,Cell viabilitywhenMFHwas applied to ovarian cancer cells at 41, 43, 45�C for 30 and60minutes; (n¼ 3; error SE).B, Pathway of upregulated genes from IPA after MFH was applied to HeyA8 cell lines for 30 minutes at 43�C. C, Top 20 upregulated genes after MFH wasapplied to HeyA8 cell lines for 30 minutes at 43�C. Analysis revealed a significant upregulation of HSP70-related genes (one-way ANOVA, n ¼ 3, P < 0.02).

Table 1. Biological functions after MFH treatment of HeyA8 cells (43�C and 30minutes)

Biological function Number of genes P

Response to unfolded proteins 14 9.99E�17Response to protein stimulus 15 8.43E�16Protein folding 14 1.88E�11






D). Combination treatment (HSPA6 siRNAþMFH) resulted in ahigher decrease in cell viability when compared toMFHorHSPA6siRNA applied independently.

Antitumor effects of HSP70 inhibition and MFH in vitroThe effects ofHSP70 inhibition using PES inhibitor were tested.

PES cytotoxicity was measured (Fig. 3A) and results presented areduction in cell viability as PES concentration was increased.Supplementary table S4 illustrates the IC50 values for PES whenexposed to ovarian cancer cell lines. A270cp20 cells showed lowerIC50 values to PES when compared to HeyA8 and SKOV3 cells.Previous work by Granato and colleagues, revealed PES affectedmembrane permeability since HSP70 supports lysosome mem-brane integrity, especially in cancer cells (33). To confirm suchmechanism in ovarian cancer cells lysosomal permeabilizationwas measured. Results indicated that lysosome permeabilization

increased as the concentration of PES increased as depicted by thehigher percentage of pale cells (Supplementary Fig. S3A). Lyso-some permeabilization was also investigated by imaging therelease of cathepsin B into the cytosol. Cathepsin B in controlcells was located in lysosomes as small red points (SupplementaryFig. S3B). In PES-treated cells a diffuse pattern was observed,indicating the relocation of cathepsin B into the cytosol confirm-ing lysosome permeabilization (Supplementary Fig. S3B).

The effect of in vitro combination treatment (MFH þ PES) wasassessed in ovarian cancer cells (Fig. 3B). PES concentrations wereselected as to provide viabilities higher than 90% when usedindependently (Fig. 3A and Supplementary Fig. S3E), 5 mmol/Lfor A2780cp20 and 10 mmol/L for HeyA8 and SKOV3. Two MFHtemperatures were selected, 41�C representative of mild hyper-thermia, and 43�C representative of hyperthermia. Combinationtreatment decreased cell viability to approximately 40% at 41�C

Figure 2.

HSP70 genes are upregulated afterMFH andHSPA6RNA interference andMFH in vitro.A, Levels of HSP70 geneswere confirmed by quantitative real-time PCR after MFH treatment for 30minutes, three temperatures 39�C,41�C, 43�C and up to 8 hours ofrecovery time. Higher expression wasobserved after 1-hour recovery;(� , P < 0.05; n ¼ 3; error, SD). B,Western blot analysis of HSP70protein level in A2780cp20 after30-minuteMFHheat treatment at 41�Cand 43�C, with recovery time of 1, 4,and 6 hours. C, MFH cell viability afterHSPA6 gene is knockdown withHSPA6 siRNA sequence 1 at 41�C and43�C and sequence 2 at 41�C in HeyA8cell line.D,A2780cp20 cell viability forsequence 3 and 4. cells treated withMFH at 41�C for 30 minutes(� , P < 0.05).

Court et al.

Mol Cancer Ther; 16(5) May 2017 Molecular Cancer Therapeutics970




compared with MFH alone (Fig. 3B). Results indicated that thecombination treatment generated a cytotoxic effect on cells,whichcould indicate synergy. For this purpose, combination index(CI; Fig. 3C) was calculated using the Chou–Talalay (27) andCompuSyn methods. Cell viabilities and dose response curvesfrom PES and MFH (Supplementary Fig. S3E) were used tocompute CI values. Themedian effect principle of themass-actionlaw was evaluated using parameters described in SupplementaryTable S5. The degree of synergismdefined byChou andMarinwasanalyzed (27). According to this analysis, A2780cp20 and HeyA8cells at 41�C showed slight synergism with PES treatment. Undersimilar conditions, SKOV3 cells also demonstrated synergism. Ata higher temperature (43�C), the effect of all cell lines wassynergistic. Further experiments were performed to evaluate syn-ergy at various PES concentrations and MFH exposure times. Forthis purpose, HeyA8 cells were exposed to MFH at 30, 45 or 60

min in combination with 5, 10, and 20 mmol/L. Results areillustrated in Supplementary Fig. S3D and the CIs shown inSupplementary Table S6 for each experimental condition. Datademonstrated synergism for PES concentrations above 10 mmol/Land all exposure times. These results confirmed that MFH appliedin combination with non-lethal doses of PES produced a syner-gistic effect.

HSPA6 silencing and MFH reduced tumor growth in vivoTo identify the in vivo effect of HSP70 inhibition during MFH,

HSPA6 siRNA was used to silence HSPA6 in a subcutaneousovarian tumor model. siRNA was delivered with DOPC nanoli-posomes. Mice were divided into four groups, control siRNA-DOPC, control siRNA-DOPC andMFH treatment,HSPA6 siRNA-DOPC and combinedHSPA6 siRNA-DOPCwithMFH treatment.The efficacy of siRNA delivery and gene silencing was confirmed

Figure 3.

PES exposure results in significant celldeath for ovarian cancer cell linesdue to lysosomal damage andcombination treatment is synergistic.A, Cytotoxicity for A2780cp20,HeyA8, and SKOV3 at 24 and48 hours.B, Combination treatment PES andMFH, HeyA8, A2780cp 20, and SKOV3cell were treated with PES and MFH at41�C (top) and 43�C (bottom) for 30minutes after 48 hours of recovery. C,Combination index of MFH and PES forovarian cancer cell lines.






(Supplementary Fig. S2B). Figure 4A presents the experimentalschedule. Research demonstrated that reduction in tumor growthcan be enhanced by the number of MFH treatments (34, 35). Forthis purpose, three MFH treatments were scheduled 48 hoursapart. CMDx-IO nanoparticles were injected intratumorally, 4hours before treatment, to allow them to distribute in the tumor.The siRNA-DOPC was administered according to previous pub-lications, two times per week and injected intraperitoneally (28).Tumor growth was slower in groups that were treated with MFHwhen compared with those not treated with MFH (Fig. 4B andSupplementary Fig. S4B). Supplementary Table S7 presents thestatistical values of the final tumor weight. By the end of thetreatment, relative tumor volume for the control groups and

control siRNA-DOPC with MFH presented a 3- to 4.4-foldincrease when compared to their initial value whereas HSPA6siRNA-DOPC was only a 1.4-fold. Final tumor weight for HSPA6siRNA-DOPC and MFH was 65% lower than control siRNA-DOPC and MFH (Fig. 4C). The temperature profile is exhibitedin Supplementary Fig. S4A. Tumor temperatures of 43�C werereached.

A second tumor model was evaluated using the A2780cp20cells under a similar treatment schedule (Fig. 4D and E). Tumorvolume and weight were measured. Tumor weight for the com-bination treatment, HSPA6 siRNA-DOPC and MFH, was approx-imately 65% smaller when compared with MFH and controlsiRNA-DOPC groups. For the control groups and control

Figure 4.

HSPA6 RNA interference and MFHdecreased subcutaneous HeyA8 andA2780cp20 tumor model growth. A,Timeline for MFH in vivo. B, Relativetumor volume in function of time ofHeyA8 tumor model. C, Ex vivo HeyA8tumorweight. Number ofmice 4–5 pergroup. D, Relative tumor volume ofA2780cp20 tumor model. E, Ex vivoA2780cp20 tumor weight. F, PrussianBlue staining to determine distributionof iron oxide nanoparticles fromcollected tumor from A2780cp20 andHeyA8; (� , P < 0.05; �� , P < 0.01).

Court et al.





siRNA-DOPC MFH, an increase in relative tumor volume wasobserved with a fold increase between 2 and 6.6. HSPA6 siRNA-DOPC and MFH presented a relative 0.7-fold change in volume.HSPA6 inhibition improved the outcome of MFH treatment insubcutaneous models. The biological effect of MFH was assessedfor apoptosis. Caspase-3 activity was observed in MFH-treatedtumors (Supplementary Fig. S4B). The distribution of CMDx-IOnanoparticles was evaluated in both tumors models using Prus-sian Blue (Fig. 4F). Iron oxide nanoparticles remained in thetumor tissue of both subcutaneous tumormodels until the tumorwas collected at the end of the experiment.

MFHwas also tested in theHeyA8 intraperitoneal tumormodelin combination with PES. Treatment started when PES wasinjected IP at day 12 after cells injection (Fig. 5A). PES dose wasselected from previous reports where it was administered every 5days (25, 36, 37). The following day, CMDx-IO nanoparticleswere injected intraperitoneally, the technique allows the inter-nalization of nanoparticles in intraperitoneal tumors as describedelsewhere (38). At day 14 mice were exposed to MFH for 30minutes. A total of three MFH treatment and PES injection wereperformed. Mice were sacrificed when controls appeared mori-bund. Tumor weight of PES and MFH treated mice (Fig. 5B), was65% smaller when compared with the control MFH group. Micewere monitored and no sign of sickness or pain were observeddays after MFH. Body weight and feeding habits remainedunchanged. Results agreed with in vitro outcomes of synergybetween PES and MFH.

DiscussionThe present work investigated the molecular and cellular

responses of ovarian cancer cells to MFH. We and others haveattempted to elucidate the cellular and molecular features ofMFH; however, there are still aspects at the molecular level thatneed to be understood (6–12). Results described herein indicatedthat there is overexpression of HSPs, in particular, Hsp70 as aresult ofMFH treatment. The resulting genomic analysis providedthe means to design targeted combination therapies using direct-

ed nanoscale heat delivery with magnetic nanoparticles Theinhibition of Hsp70 by RNA interference and the novel Hsp70inhibitor, PES, enhanced the effect of MFH both in vitro and invivo in various ovarian cancer cell lines and tumor models(subcutaneous and intraperitoneal). Furthermore, in vitro obser-vations with PES demonstrated, for the first time, that the com-bined treatment resulted in a synergistic effect at mild hyperther-mia temperatures. Findings of this work also indicated that theintraperitoneal injection of iron oxide nanoparticles is a feasiblealternative to nanoparticle delivery to peritoneal tumors and thatMFH treatment can be successfully administered without dam-aging healthy tissue.

To further understand such cellular responses, gene expressionand pathway analyses were conducted. An experimental temper-ature of 43�C was selected because an inflection point in cellviability is observed for conventional hyperthermia (1, 39). Suchbehavior was also observed when MFH was applied as a soletreatment in vitro for various ovarian cancer cell lines as describedherein.

Results are supported by prior work demonstrating that HSP-related genes, including Hsp70, Hsp40, Hsp105, Hsp110, Hsp47,and BAG3 are upregulated when conventional hyperthermia wasapplied to cancerous andnormal human cells (13, 15, 16, 40–44).However, such experiments have not been previously conductedfor MFH-treated ovarian cancer cells or any other cancer cell line.The genetic analysis of the response of ovarian cancer cellsexposed to MFH revealed that several HSPs were upregulated aswell as ubiquitinC (UBC). Thesefindings demonstrated thatMFHupregulated the expression of HSPs genes in ovarian cancer celllines similar to conventional hyperthermia. Interestingly, MFHalso overexpressed UBC, which has not been previously reportedas a top overexpressed gene in microarray for conventionalhyperthermia in treatments where the range in thermal dose wasbetween7.5and60cumulative equivalentminutes (CEM; refs. 15,40–42). At higher CEM (�300) ubiquitin genes expression havebeen reported (45, 46). In this particular work MFH treatmentswith a thermal dose of only 30 cumulative equivalent minutessignificantly upregulated UBC. These results may provide furtherevidence that the extent ofMFHdamage in the cell might bemoreextensive than conventional hyperthermia as reported by ourgroup and others (6, 7, 12).

After studying the gene expression resulting fromMFH at 43�C,Hsp70 was selected as it was the most upregulated gene of themicroarray, as confirmed by qPCR and Western blot analysis. Inaddition, Hsp70 is overexpressed in various cancer models, andhigh levels are associatedwith poor prognosis (31).Hsp70 plays asignificant role in cancer, suppressing apoptosis and participate intumor development. Under stress conditions, such as hyperther-mia, Hsp70 avoids accumulation of unfolded proteins and refoldaggregated proteins (31, 47). Several Hsp70 inhibitors have beendesigned with favorable results in vitro, but have failed efficacy inpre-clinical or clinical trials, with limitations as toxicity andreduced antitumor effect (11, 31, 48, 49). An alternative of twoapproacheswere investigated in this work, inhibition ofHsp70 byRNA interference and, inhibition of protein function using PES.

Previous reports have demonstrated that Hsp90 inhibition incombination with MFH therapy increased cell susceptibility tohyperthermia and in vivo tumor regression (50–52). Hsp90 inhi-bition is commonly used in hyperthermia because it plays acentral role in cancer signalingmolecules, but research establishedthat Hsp90 inhibition also upregulates Hsp70 production

Figure 5.

PES and MFH decrease orthotopic HeyA8 tumor model growth. Timelinefor MFH in vivo (A), ex vivo HeyA8 tumor weight (B). Number of mice3–5 per group.






(50, 51). The goal of this work was to demonstrate that onlyHsp70 inhibition will improve the antitumor effect of MFH.

Hsp70 knockdown with siRNA has been achieved in vitro inhuman colon cells, prostate and the ovarian cancer cell lineA2780 (37, 53, 54). The combination of Hsp70 RNA interfer-ence with MFH has not been previously reported in vitro or invivo. Results demonstrated thatHSPA6 inhibition with siRNA incombination with MFH enhanced treatment outcomes both invitro and in vivo. Data confirmed that Hsp70 inhibition andMFH appear as an effective selective alternative, resulting inenhanced MFH therapeutic effects. The second approach was toinhibit the Hsp70 protein function. An alternative to thisapproach was to employ PES because it has been identified asa novel Hsp70 inhibitor with direct specificity (25, 36). In thiswork, PES was able to reduce cell viability in various ovariancancer cells by similar mechanisms as those proposed in theliterature (55, 56). The combination of MFH and PES resultedin a synergistic effect in ovarian cancer cells. This is the first timethat synergy is demonstrated from the combination treatmentbetween PES and MFH. Sekihara and colleagues (37) demon-strated that when PES and conventional hyperthermia werecombined in prostate cancer cells the number of cells in earlyapoptosis and necrosis and late apoptosis were increased,concluding that the cell death mechanism of the combinationtreatment partially depended on the caspase pathway. Further-more, they demonstrated that the combination increased thenumber of cells in the G2–M phase and decreased the expres-sion of cell cycle related molecules such as c-Myc and cyclin D1causing cell growth arrest. These findings validated that Hsp70protein function inhibition is enough to enhance the thera-peutic outcome of MFH.

Hsp70 inhibition and MFH were further evaluated in intraper-itoneal tumor models with peritoneal administration of nanopar-ticles. The reason to select thismodel is that intratumoral injectionsare not practical in clinical settings. Besides, othermethods such asnanoparticle targeting for intravenous administration still facesignificant challenges achieving the required levels of nanoparticleaccumulation in tumors. Administration in the peritoneal area ofantineoplastic agents is currently an alternative for treating smalltumors located in the area. High drug concentrations and longerhalf-life can be achieved with intraperitoneal injections (57).Previous work has reported the intraperitoneal administration ofiron oxide nanoparticles for MFH treatment in a mouse ovariancancer model with peritoneal tumors but did not assess tumorgrowth, focusing solely on demonstrating cell death throughhistological analysis (38). In this work, we administered nanopar-ticles intraperitoneally to the HeyA8 intraperitoneal model andapplied MFH. Results demonstrated that iron oxide nanoparticleswhen injected intraperitoneal in combination with MFH on intra-peritoneal ovarian tumors showed an antitumor effect. We werealso able to enhanceMFH therapeutic outcomewith the inhibitionof HSP70 function using PES.

In summary, this work introduces the use of microarray geneexpression to rationally design targeted combination therapiesusingdirectednanoscale heat deliverywithmagneticnanoparticles.Results demonstrated that MFH gene expression in ovarian cancerwas dominated by HSPs and ubiquitin C. Hsp70 was found to bethemost upregulated gene and its combination enhanced the effectof MFH in vitro and in vivo. In vitro combination treatments dem-onstrated for the first time the synergistic behavior between MFHand PES. The intraperitoneal delivery of iron oxide nanoparticlesrevealed that tumorswere significantly affectedwhen the combinedtreatment of MFH and HSP70 inhibition was applied. Therefore,results presented herein suggested that iron oxide magnetic nano-particles under the influence of an alternating magnetic field incombinationwithheat response impairment drugs are a significantmilestone for the translation of nano-based adjuvant therapies forovarian cancer into the clinic.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.A. Court, S.Y. Wu, G. L�opez-Berestein, C. Rinaldi,E.J. Juan, A.K. Sood, M. Torres-LugoDevelopment of methodology: K.A. Court, S.Y. Wu, C. Rodríguez-Aguayo,G. L�opez-Berestein, C. Rinaldi, E.J. Juan, A.K. Sood, M. Torres-LugoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.A. Court, H. Hatakeyama, C. Rodríguez-Aguayo,L. Ju-Seog, E.J. Juan, A.K. SoodAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.A. Court, H. Hatakeyama, G. L�opez-Berestein,L. Ju-Seog, C. Rinaldi, E.J. Juan, A.K. Sood, M. Torres-LugoWriting, review, and/or revision of themanuscript:K.A. Court, H.Hatakeyama,S.Y. Wu, M.S. Lingegowda, G. L�opez-Berestein, C. Rinaldi, E.J. Juan, A.K. Sood,M. Torres-LugoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.A. Court, G. L�opez-Berestein, A.K. SoodStudy supervision: A.K. Sood, M. Torres-Lugo

AcknowledgmentsThe authors are grateful toDr. CamiloMora andDr.Marisel S�anchez for their

support in confocal imaging and flow cytometer.

Grant SupportThis work was supported by grants from the following: HHS (NIH;

CA 963000/CA 96297; to M. Torres-Lugo; P50CA083639 and CA016672,A.K. Sood), PR Institute for Functional Nanomaterials (EPS-100241; to Made-line Torres-Lugo), Nanotechnology Center for Biomedical, Environmental andSustainability Applications (HRD-1345156; to Madeline Torres-Lugo) OvarianCancer Research Fund (OCRF; RP101502/RP, to S.Y. Wu), and Cancer Preven-tion and Research Institute of Texas training grants (RP101489, to S.Y. Wu;RP110595 and RP120214, to A.K. Sood).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 10, 2016; revised September 6, 2016; accepted February 4,2017; published OnlineFirst February 21, 2017.

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Court et al.




2017;16:966-976. Published OnlineFirst February 21, 2017.Mol Cancer Ther Karem A. Court, Hiroto Hatakeyama, Sherry Y. Wu, et al. Fluid Hyperthermia in Ovarian Cancer

Inhibition Synergistically Enhances the Effects of MagneticHSP70

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