Bisphosphonates Inhibit Stellate Cell Activity and Enhance Antitumor Effects of Nanoparticle Albumin-Bound Paclitaxel in Pancreatic Ductal Adenocarcinoma

Bisphosphonates Inhibit Stellate Cell Activity and Enhance Antitumor Effects of Nanoparticle Albumin-Bound Paclitaxel in Pancreatic Ductal Adenocarcinoma
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   Published OnlineFirst September 5, 2014. Mol Cancer Ther Vianey Gonzalez-Villasana, Cristian Rodriguez-Aguayo, Thiruvengadam Arumugam, et al. in Pancreatic Ductal AdenocarcinomaBound Paclitaxel − Antitumor Effects of Nanoparticle Albumin Bisphosphonates Inhibit Stellate Cell Activity and Enhance   Updated version   10.1158/1535-7163.MCT-14-0028doi:Access the most recent version of this article at:   MaterialSupplementary the most recent supplemental material at:   E-mail alerts  related to this article or journal.Sign up to receive free email-alerts   SubscriptionsReprints and .pubs@aacr.orgDepartment atTo order reprints of this article or to subscribe to the journal, contact the AACR Publications   Permissions  .permissions@aacr.orgDepartment atTo request permission to re-use all or part of this article, contact the AACR Publications Research. on October 28, 2014. © 2014 American Association for Downloaded from  Published OnlineFirst September 5, 2014; DOI: 10.1158/1535-7163.MCT-14-0028 Research. on October 28, 2014. © 2014 American Association for Downloaded from  Published OnlineFirst September 5, 2014; DOI: 10.1158/1535-7163.MCT-14-0028  Small Molecule Therapeutics Bisphosphonates Inhibit Stellate Cell Activity and Enhance Antitumor Effects of Nanoparticle Albumin – BoundPaclitaxel in Pancreatic Ductal Adenocarcinoma  Vianey Gonzalez-Villasana 1 , Cristian Rodriguez-Aguayo 1 , Thiruvengadam Arumugam 2 ,Zobeida Cruz-Monserrate 2 , Enrique Fuentes-Mattei 3 , Defeng Deng 2 , Rosa F. Hwang 4 ,Huamin Wang 5 , Cristina Ivan 6 , Raul Joshua Garza 7 , Evan Cohen 7 , Hui Gao 7 ,Guillermo N. Armaiz-Pena 2,6 , Paloma del C. Monroig-Bosque 1,8 , Bincy Philip 2 ,Mohammed H. Rashed 1,9 , Burcu Aslan 1 , Mumin Alper Erdogan 1 , Yolanda Gutierrez-Puente 10 ,Bulent Ozpolat 1 , James M. Reuben 7 , Anil K. Sood 2,6,11 , Craig Logsdon 2 , and Gabriel Lopez-Berestein 1,11  Abstract Pancreatic stellate cells (PSC) have been recognized as the principal cells responsible for the production of fibrosisinpancreaticductaladenocarcinoma(PDAC).Recently,PSCshavebeennotedtosharecharacteristicswithcellsofmonocyte-macrophagelineage(MMLcells).Thus,wetestedwhetherPSCscouldbetargetedwiththe nitrogen-containing bisphosphonates (NBP; pamidronate or zoledronic acid), which are potent MML cellinhibitors. In addition, we tested NBPs treatment combination with nanoparticle albumin–bound paclitaxel(nab-paclitaxel) to enhance antitumor activity.  In vitro , we observed that PSCs possess  a -naphthyl butyrateesterase(ANBE)enzymeactivity,aspecificmarkerofMMLcells.Moreover,NBPsinhibitedPSCsproliferation,activation, release of macrophage chemoattractant protein-1 (MCP-1), and type I collagen expression. NBPsalso induced PSCs apoptosis and cell-cycle arrest in the G 1  phase.  In vivo , NBPs inactivated PSCs; reducedfibrosis;inhibitedtumorvolume,tumorweight,peritonealdissemination,angiogenesis,andcellproliferation;and increased apoptosis in an orthotopic murine model of PDAC. These  in vivo  antitumor effects wereenhanced when NBPs were combined with nab-paclitaxel but not gemcitabine. Our study suggests thattargeting PSCs and tumor cells with NBPs in combination with nab-paclitaxel may be a novel therapeuticapproach to PDAC.  Mol Cancer Ther; 13(11); 1–12.   2014 AACR. Introduction Pancreatic ductal adenocarcinoma (PDAC) is thefourth leading cause of cancer-related death and oneof the most lethal human cancers (1). The prognosis of patients with PDAC is extremely poor, with a mediansurvival time of 6 months and an overall 5-year survivalrate of less than 5% (1–3). Recently, the contribution of stromal elements to the initiation and progression of this disease has come to be appreciated (3–5). Targetingtumor microenvironment represents a potential strate-gy for improving the delivery and efficacy of chemo-therapeutic agents for PDAC and, hence, the prognosis.The activated stroma of PDAC comprises extracellularmatrix and several cell types, including stellate cells,endothelial cells, nerve cells, and immune cells (4). Theprincipal cells responsible for the production of fibrosisin PDAC are the pancreatic stellate cells (PSC; refs. 6, 7).In normal pancreas, PSCs are quiescent, but whenpancreatic injury or inflammation occurs, these cellsundergo morphologic and functional changes to become activated, expressing  a -smooth muscle actin( a -SMA) and secreting growth factors, a wide varietyof proinflammatory cytokines, such as macrophagechemoattractant protein-1 (MCP-1) and inteleukin-8(IL8), and excessive amounts of extracellular matrixproteins such as type I collagen (5, 6, 8). If the injuryorinflammationissustainedorrepeated,PSCsmaintainpermanentactivation,allowingthedevelopmentofpan-creatic fibrosis and tumor progression (7). 1 Department of Experimental Therapeutics, The University of Texas MD AndersonCancerCenter,Houston,Texas. 2 DepartmentofCancerBiology,The University of Texas MD Anderson Cancer Center, Houston, Texas. 3 Department of Molecular and Cellular Oncology, The University of TexasMD Anderson Cancer Center, Houston, Texas.  4 Department of SurgicalOncology, The University of Texas MD Anderson Cancer Center,Houston, Texas.  5 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.  6 Department of GynecologicOncology,TheUniversityofTexasMDAnderson CancerCenter,Houston,Texas.  7 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.  8 University of Puerto RicoSchool of Medicine, San Juan, Puerto Rico.  9  Al-Azhar University, Cairo,Egypt. 10 UniversidadAutonomadeNuevoLeon,SanNicolasdelosGarza,Nuevo Leon, Mexico.  11 Center for RNAi and Non-coding RNA, The Uni-versity of Texas MD Anderson Cancer Center, Houston, Texas. Note: SupplementarydataforthisarticleareavailableatMolecularCancer Therapeutics Online ( Corresponding Author:  Gabriel Lopez-Berestein, Department of Exper-imental Therapeutics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3508; 713-792-0362; E-mail: doi:  10.1158/1535-7163.MCT-14-0028  2014 American Association for Cancer Research. Molecular Cancer Therapeutics  OF1 Research. on October 28, 2014. © 2014 American Association for Downloaded from  Published OnlineFirst September 5, 2014; DOI: 10.1158/1535-7163.MCT-14-0028  TheoriginationofPSCsisunclear.However,ithasbeendemonstrated that PSCs display phagocytic activity (9)and express TLR2, TLR4, TLR3, and TLR5 (10), whichsuggests that these cells may play a macrophage-like roleinthepancreasthatiscomparablewiththeroleofKupffercells in liver. Highly phagocytic cells, such as macro-phagesandosteoclasts,whichsharethesamelineage,arethe major target of bisphosphonates (11, 12). Thompsonand colleagues (13) showed that bisphosphonates areinternalized by J774 murine macrophages by fluid-phaseendocytosis and vesicular acidification, suggesting thathighly phagocytic cells, such as macrophages, can inter-nalize bisphosphonates, which makes them ideal targetsforthesedrugs.Therearetwoclassesofbisphosphonates,those that contain nitrogen and those that do not (14).Nitrogen-containing bisphosphonates (NBP), such aspamidronate, alendronate, and ibandronate, have beenshown to inhibit cell proliferation, reduce cell viability,andcausetheapoptoticcelldeathofmacrophages(11).Inthis study, we explored the use of NBPs to reduce PSCs-mediated fibrosisasawaytoreducetheburdenofPDACand its use in combination with nanoparticle albumin– bound paclitaxel (nab-paclitaxel), which is known toenhance drug delivery in tumors. Materials and Methods Reagents Pamidronate(Pam)waspurchasedfromSigma-Aldrich,zoledronic acid (ZA) from Novartis Pharmaceuticals,Corporation, gemcitabine (Gem) from APP Pharmaceuti-cals, LLC, and nab-paclitaxel from Abraxis BioScience, Inc. Cell culture Human PSCs isolated by Dr. Rosa Hwang [The Uni-versity of Texas MD Anderson Cancer Center (MDACC),Houston, TX; ref. 15] were maintained in DMEM/F12(Invitrogen Corporation) supplemented with 10% FBS.Capan-2 human PDAC cells [American Type CultureCollection (ATCC)] were transfected with luciferase andmaintained in McCoy 5A medium (Invitrogen Corpora-tion)with10%FBSin5%CO 2 /95%airat37  C.Peripheral blood monocytes (PBM) isolated from buffy coats of healthyadultdonors(MDACC)andTHP-1humanmono-cytic leukemia cells (ATCC) were both maintained inRPMI-1640 medium (Invitrogen) with 10% FBS. TheATCC uses the Promega PowerPlex system to authenti-cate their cell lines. All cell lines were expanded, cryo-preserved, used within 6 months after resuscitation, andscreened for  Mycoplasma  using the MycoAlert Mycoplas-ma Detection Kit (Lonza Rockland, Inc.) as described bythe manufacturer. Isolation of PBMs by adherence PBMs were isolated from buffy coats of three humandonorsbyFicoll-Hypaquedensity-gradientcentrifugation.The mononuclear fraction was washed in PBS, counted,adjusted at 2.5    10 6 cells/mL in serum-free RPMI-1640medium, and incubated for 3 hours. We next removed themedium and washed the adherent cell layer twice withserum-free RPMI-1640 medium. Cells were then culturedwith 10% FBS RPMI-1640 medium for 6 days. Isolation of PBMs by CD14 microbeads PBMswereisolatedfrombuffycoatsofhumandonorsbyFicoll-Hypaque density-gradient centrifugation, and thenwerecountedandcentrifugedat300   g for10minutes.Thepellet was resuspended in running buffer (MACS MiltenyiBiotec), and then 20  m L of CD14 microbeads (MACSMiltenyi Biotec) per 1    10 7 total cells was added and thecells were incubated for 15 minutes at 4  C. Then, the cellswere washed with running buffer and centrifuged at300   g for10minutesat4  C,thesupernatantwasremoved,andthecellswereresuspendedinrunningbuffer.Magneticseparation was conducted using the MACS separator(MACS Miltenyi Biotec). The collected CD14-positive cellswere counted, adjusted at 12    10 6 cells in RPMI-1640medium with 10% FBS, and incubated at 37  C.  ANBE activity  PSCs, THP-1, and PBMs were harvested, counted, andadjusted at 5    10 4 cells in their respective medium.Samples were centrifuged in a Shandon CytoSpin 3 cen-trifugeat600rpmfor2minutesandthenfixedinacitrate–acetone–formaldehyde solution. Staining was performedwith an  a -naphthyl acetate esterase kit (Sigma-Aldrich)according to the manufacturer’s protocol (pH ofthe incu- batingmediumadjustmentto6.3).Cellswereviewedwithan inverted microscope (Nikon Corporation InstrumentsCompany) at   10 magnification.  Western blotting  Cells were lysed with ice-cold lysis buffer. Lysateswere centrifuged, supernatants were collected, and pro-teinconcentrationwasdeterminedusingtheDCProteinAssay (Bio-Rad Laboratories). Samples were electro-phoresed using 10% and 4% to 15% gradient polyacryl-amide gels (Bio-Rad) and then transferred to nitrocel-lulose membranes (Bio-Rad). Membranes were blocked,rinsed, and incubated with primary antibodies against a -SMA (Dako), unprenylated Rap1A (Santa Cruz Bio-technology, Inc.), transglutaminase 2 (TG2; Abcam),cleaved caspase-3 (eBioscience), cleaved PARP-1 (CellSignaling), fibronectin (Santa Cruz Biotechnology, Inc.),laminin (Thermo Fisher Scientific, Inc), caveolin-1 (BDBioscience), farnesyltransferase beta (FNTB; Abcam),and type I collagen (SouthernBiotech). After overnightincubation at 4  C, membranes were washed and incu- bated with their corresponding secondary antibodyconjugatedwithhorseradishperoxidase(HRP).Proteins bands were detected with an enhanced chemilumines-cence detection kit (GE Healthcare). Immunoblots werescanned by an Alpha Imager densitometer (Alpha Inno-tech Corp.) for quantification of protein expression. b -Actin (Sigma-Aldrich) and vinculin (Santa Cruz Bio-technology, Inc.) were used as a loading control. Gonzalez-Villasana et al.Mol Cancer Ther; 13(11) November 2014  Molecular Cancer Therapeutics OF2 Research. on October 28, 2014. © 2014 American Association for Downloaded from  Published OnlineFirst September 5, 2014; DOI: 10.1158/1535-7163.MCT-14-0028  Cell viability assay  Cells were seeded on 96-well plates at 1,250 cells perwell and incubated overnight. Cells were then treated for72 hours with pamidronate. The MTS cell proliferationassay(PromegaCorporation)wasperformedaccordingtothemanufacturer’sinstructions.Theresultsareexpressedintermsofpercentageofgrowthinhibitionwithrespecttountreated (control) cells.  Apoptosis Cell apoptosis was measured with an FITC apoptosisdetection kit (BD Biosciences), according to the manufac-turer’s instructions. Apoptotic cells were analyzed with aFACSCalibur flow cytometer (BD Biosciences). The Cell-Quest Pro software (BD Biosciences) was used to deter-mine the number of apoptotic cells. Apoptosis was alsoassessed using freshly cut frozen tissue and a DeadEndfluorometric TUNEL system kit (Promega) according tothe manufacturer’s instructions. The number of terminaldeoxynucleotidyltransferase–mediated dUTP nick endlabeling (TUNEL)-positive cells was counted in five ran-dom fields per slide (one slide per mouse, 10 slides/group) at   200 magnification. Cell-cycle analysis Cells were fixed with 70% cold ethanol for 24 hours,thenwerecollectedandresuspendedinPBScontaining50 m g/mL propidium iodide and 100 U/mL of RNAse A.Cells were incubated at 37  C for 30 minutes in the darkand then analyzed by a FACSCalibur flow cytometer (BDBiosciences).TheCellQuestProsoftware(BDBiosciences)wasusedtodeterminethenumberofcellsineachphaseof the cell cycle. Measurement of MCP-1 levels We measured the MCP-1 levels in the supernatant of PSCstreatedwithpamidronatebymultiplexbeadimmu-noassay using a Luminex kit (Millipore Corp.). Orthotopic tumor implantation and drug treatment  At week 0, 8-week-old female athymic nude mice pur-chased from Taconic were anesthetized with Nembutal[20 mg/kg body weight (BW)/i.p.], and Capan-2 cellstransfectedwithluciferasewereinjected(1  10 6 cells/100 m L) directly into tail of the pancreas. One week afterimplantation (week 1), bioluminescence was measuredwithIVISimagingtoconfirmthatthecellshadimplanted,and the mice were randomly assigned to one of fourtreatment groups. Starting at week 3.5, mice were treatedweekly with ( n  ¼  10 mice/group): (i) saline solution (200 m L i.p.), (ii) zoledronic acid alone (1 mg/kg BW/once perweek/i.p.), (iii) gemcitabine alone (100 mg/kg BW/onceperweek/i.p.), or (iv)acombination ofzoledronic acid (1mg/kg BW/once per week/i.p.) and gemcitabine (100mg/kgBW/onceperweek/i.p.).Anothersetofmice( n ¼ 10 mice/group) was treated with nab-paclitaxel (10mg/kg BW/once per week/i.v.) instead of gemcitabine2weeksaftercellimplantation(SupplementaryFig.S1A).All experiments wereconducted three times.In each one,10 mice were used per group.All drugs wereused in a clinically relevant doses, basedon the equivalent surface area dosage conversion factorsdescribed by Freireich and colleagues (16) and the DrugGuidances by U.S. Food and Drug Administration (17).And because bisphosphonates appear to be associatedwith nephrotoxicity (18), we also determined the levels of  bloodureanitrogen(BUN)inC57BL/6micewithouttreat-ment, treated with saline solution, 0.05, 0.5, and 1 mg/kgof zoledronic acid, and we did not observe toxicity at anydose of zoledronic acid as is shown in SupplementaryFig.S1B.Noovertdeleteriousclinicaleffectswereobserved.Tumor volume was monitored weekly with the biolu-minescence IVIS spectrum system (Caliper Life Sciences)using the Living Image software (Caliper Life Sciences).Mice were sacrificed the fifth week after treatment began.The complete pancreas was weighed to obtain tumorweight.Tumortissuewasfixedinformalinandembeddedinparaffinforimmunohistochemicalanalysisandstainingwith hematoxylin and eosin (H&E). The animal experi-ments were conducted in accordance with the AmericanAssociation for Laboratory Animal Science (AALAS) reg-ulations and the approval of the MD Anderson CancerCenter Institutional Animal Care and Use Committee. Immunohistochemical analysis Unstainedsectionsofmousetissuesweredeparaffinizedand rehydrated. Antigen retrieval was performed withDAKO antigen retrieval solution (Dako). Endogenous per-oxidase was blocked by hydrogen peroxide (3%). Forproteinblocking,cold-waterfishskingelatin40%(ElectronMicroscopy Sciences) in PBS was applied for 1 hour( a -SMA) or 5% normal horse serum and 1% normal goatserum inPBS(Ki67andCD31).Primaryantibodiesagainst a -SMA (ab5694; Abcam), Ki67 (Thermo/Lab Vision), andanti-CD31 (Pharmingen) were incubated overnight at 4  C.For  a -SMA, a biotinylated rabbit anti-goat secondary anti- body (Vector Labs) was incubated for 20 minutes. Slideswere then incubated with HRP streptavidin solution (Vec-torLabs)for30minutes.ForKi67,thegoatanti-rabbitHRPsecondary antibody diluted in blocking solution wasadded and incubated for 1 hour at room temperature. ForCD31, a goat anti-rat HRP-conjugated secondary antibody(Jackson ImmunoResearch Laboratories Inc.) diluted in blocking solution was added and incubated for 1 hour atroomtemperature.Finally,slidesweredevelopedwith3,3-diaminobenzidinesubstrate(DAB;VectorLabs)andcoun-terstained with Gill no. 3 hematoxylin. To quantify Ki67and CD31 expression, the number of positive (DAB-stained) cells was counted in five random fields per slide(one slide per mouse, 10 slides/group) at   200 magnifi-cation, and the percentage of cells that were Ki67- andCD31-positive was calculated for each group. Statistical analysis Statistical analyses were conducted using the Student t  test.  P  <  0.05 was considered statistically significant for Inhibition of PSCs by Bisphosphonates in Mol Cancer Ther; 13(11) November 2014  OF3 Research. on October 28, 2014. © 2014 American Association for Downloaded from  Published OnlineFirst September 5, 2014; DOI: 10.1158/1535-7163.MCT-14-0028  all assays. The nonparametric test Kruskal–Wallis wasapplied to assess the relationship between tumor volumeand treatment. A box-and-whisker plot [Box plot repre-sents first (lower bound) and third (upper bound) quar-tiles, whiskersrepresent1.5timestheinterquartilerange]was used to visualize the data.To examine the interaction index between gemcitabineandzoledronicacid,anisobologramanalysiswasusedasdescribed by Vivas-Mejia and colleagues (19) using thefollowing equation:Interaction index  ¼ ð Gem c = Gem a Þ þ ð ZA c = ZA a Þ where Gem and ZA are concentrations of gemcitabineand zoledronic acid, respectively, that produce somespecified effect when used alone ( a ) or in combination( c ). The interaction is considered synergistic when theinteraction index value is lower than 1.0 and antagonisticwhen the value is higher than 1.0. Results a -Naphthyl butyrate esterase activity in PSCs Recently, PSCs have been noted to share character-istics with cells of monocyte-macrophage lineage (MMLcells; refs. 7, 9). We first assessed whether PSCs display a -naphthyl butyrate esterase (ANBE) activity, which is aspecific enzyme present in MML cells. PSCs, undifferen-tiated PBMs, differentiated PBMs, and THP-1 cells allexpressed ANBE (Fig. 1A), suggesting that PSCs sharea key marker with MML cells. PSCs express  a -SMA and TG2 when treated with12-O-tetradecanoyl-phorbol-13-acetate 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is a tu-mor promoter and protein kinase C (PKC) activator thatinducesthedifferentiationofleukemiccellssuchasTHP-1tomonocyticlineage(20–22).TodeterminewhetherPSCscan be differentiated such as monocytes, PSCs, and THP-1, cells were treated with TPA at 50 and 100 ng/mL,respectively, for 72 hours, which was followed by immu-noblottingtodetermineTG2and a -SMAexpressionlevelsin the presence and absence of TPA treatment. TG2 is ahighly selective marker of monocyte differentiation (23,24),and a -SMAisthehallmarkmarkerofPSCsactivation.We found that TPA induced the expression of TG2 and a -SMA in PSCs, PBMs, and THP-1 cells (Fig. 1B). Theseresults indicate that PSCs follow a similar pattern of differentiation markers as MML cells do. PamidronateinhibitsPSCsproliferationandleadstoapoptosis and cell-cycle arrest  Our observations that PSCs behave in a similar fashionas MML cells suggested that they might be susceptible toMMLinhibitorssuchasNBPs(11,12).Forthisreason,wetargeted the PSCs with pamidronate. Seventy-two hoursoftreatmentwithpamidronate(0.1,1,10,and100 m mol/L)led to a dose-dependent reduction in cell proliferation(Fig. 2A). We examined the effect of pamidronate inproliferation at 24, 48, and 72 hours and because weobserved the best response at 72 hours, we decided touse these time point for the rest of the experiments. It isconceivable that the concentrations used maybe achievedin bone marrow but not in peripheral organs.We next determined whether the inhibition of PSCsproliferationbypamidronatewasthroughapoptosis.Themaximal induction of apoptosis was observed at 100 m mol/L pamidronate (89.7% of apoptotic cells; Fig. 2B).Our results were confirmed by Western blotting, whichshowed that pamidronate induced the expression of cleaved caspase-3 and cleaved PARP-1 in a dose-depen-dent manner; maximum induction of these apoptoticmarkers was at 100  m mol/L pamidronate (Fig. 2C).We also assessed the effect of pamidronate on thedistribution of PSCs in the cell cycle. Pamidronateresulted in G 1  arrest at a concentration of 100  m mol/L(Fig.2D).Westernblottingconfirmedthatcomparedwith Figure 1.  PSCs possess characteristics of MML cells. A, representative ANBE activity staining in undifferentiated PBMs, differentiated PBMs,THP-1 cells, and PSCs. Brown-red color of ANBE staining (pH 6.3)denotes enzyme activity. Magni fi cation,   10. B, immunoblotting for  a -SMA and TG2 expression in PSCs, PBMs, and THP-1 cells. Gonzalez-Villasana et al.Mol Cancer Ther; 13(11) November 2014  Molecular Cancer Therapeutics OF4 Research. on October 28, 2014. © 2014 American Association for Downloaded from  Published OnlineFirst September 5, 2014; DOI: 10.1158/1535-7163.MCT-14-0028
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