A Polymeric Nano Particle Formulation of Curcumin Inhibits Growth, Clonogenicity and Stem-like Fraction in Malignant Brain Tumors

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Cancer Biology & Therapy 11:5, 1-10; March 1 , 2011; © 2011 Landes Bioscience

RESEARCH PAPER RESEARCH PAPER

*Correspondence to: Charles G. Eberhart; Email: [email protected]: 10/12/10; Revised: 12/06/10; Accepted: 12/06/10Previously published online: www.landesbioscience.com/journals/cbt/article/14410DOI:

Introduction

Medulloblastoma and glioblastoma are the most common malig-nant cancers arising in the central nervous systems o childrenand adults, respectively.1 Current therapeutic strategies includesurgery, radiation therapy and chemotherapy, but these are asso-ciated with signi cant side e ects and only limited e cacy,particularly in patients with glioblastoma. More e ective, lessharm ul therapeutic agents are urgently needed. A number o preclinical and clinical studies suggest that natural compoundssuch as curcumin may represent use ul additions to therapeuticregimens o cancer patients.2,3

Turmeric has been historically used in Indian Ayurvedic med-icine to treat a variety o disorders.4 More recently, curcumin,

also known as di eruloyl methane, a polyphenolic compoundderived rom turmeric, has been shown to exert antitumor e ectsin many di erent cancer cell lines and animal models.5,6 Theclinical e ects o curcumin are currently being investigated inhuman clinical trials or a variety o conditions including pan-creatic cancer, colorectal cancer and multiple myeloma (www.clinicaltrials.gov/ct2/show/NCT00094445, www.clinicaltrials.

Curcumin is a polyphenolic compound derived rom the Indian spice turmeric. We used nanoparticle-encapsulated

curcumin to treat medulloblastoma and glioblastoma cells. This ormulation caused a dose-dependent decrease ingrowth o multiple brain tumor cell cultures, including the embryonal tumor derived lines DAOY and D283Med, andthe glioblastoma neurosphere lines HSR-GBM1 and JHH-GBM14. The reductions in viable cell mass observed wereassociated with a combination o G 2 /M arrest and apoptotic induction. Curcumin also signi cantly decreased anchorage-independent clonogenic growth and reduced the CD133-positive stem-like population. Downregulation o the insulin-like growth actor pathway in DAOY medulloblastoma cells was observed, providing one possible mechanism or thechanges. Levels o STAT3 were also attenuated. Hedgehog signaling was blocked in DAOY cells but Notch signaling wasnot inhibited. Our data suggest that curcumin nanoparticles can inhibit malignant brain tumor growth through themodulation o cell proli eration, survival and stem cell phenotype.

A polymeric nanoparticle formulationof curcumin inhibits growth, clonogenicity

and stem-like fraction in malignant brain tumorsKah Jing Lim,1,2 Savita Bisht, 2 Eli E. Bar,2 Anirban Maitra 2,3 and Charles G. Eberhart 2-4,*

1Graduate Program in Pathobiology; Departments o 2Pathology, 3Oncology and 4Ophthalmology; Johns Hopkins University School o Medicine; Baltimore, MD USA

Key words:curcumin, hedgehog, IGF, glioblastoma, medulloblastoma, nanocurcumin, nanoCurcTM

Abbreviations: Bcl2, B cell lymphoma 2 associated oncogene; IGF, insulin-like growth actor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gli, glioma-associated oncogene; MAPK, mitogen-activated protein kinase; NVA622,

N-isopropylacrylamide (NIPAAM), vinylpyrrolidone (VP) and acrylic acid (AA), in a molar ratio o 60:20:20; Ptch, patchedhomolog; PI3K, phosphoinositide-3-kinase; STAT, signal transducer and activator o transcription

gov/ct2/show/NCT00118989, www.clinicaltrials.gov/ct2/show/

NCT00113841).The mechanisms by which curcumin is thought to inhibittumorigenesis are diverse, and pro-apoptotic, anti-angiogenic,anti-infammatory, immunomodulatory and anti-mitogenice ects have been described in various systems.3,7 Some o cur-cumin’s potential molecular targets include insulin-like growth

actor (IGF), Akt, mitogen-activated protein kinase (MAPK),signal transducer and activator o transcription 3 (STAT3),Nuclear actor kappa B (NFκ B) and Notch.6,8 These pathwaysare all thought to be active in malignant brain tumors,9,10 rais-ing the possibility that curcumin could be e ective in treatingmedulloblastoma or glioblastoma.

Several groups have begun to examine the potential o cur-

cumin in neuro-oncology. Curcumin was rst ound to repressthe in vitro invasion o astrocytic glioma cells through inhibitinggene expression o matrix metalloproteinases.11 In another study,the ERK, JNK and MAPK/Elk-1/Egr-1 pathways were oundto be required or p53-independent transcriptional activation o p21 Wa /Cip in U87MG glioblastoma cells in response to curcumintreatment.12 In addition, there was evidence that curcumin



2 Cancer Biology & Therapy Volume 11 Issue 5

lower-passage cells generated rom a tumor recently resected aour institution (JHH-GBM14) as well as an established line cul-tured or several years (HSR-GBM1). Both were sensitive to thee ects o nanocurcumin, with 36 ± 0.8% to 53 ± 4.3% statisti-cally signi cant inhibition respectively (±SEM) in growth o thespheres over 6 days at higher doses (Fig. 1E and F, p < 0.001). Allexperiments were repeated at least two times with similar results.

To determine the mechanisms o growth inhibition, weexamined both apoptosis and cell cycle parameters in nanocur-cumin and vehicle treated cells. The percentage o apoptoticcells increased signi cantly rom 2.3–30.5% (13- old, p < 0.001 when DAOY cultures were treated or 1 day with 10μ M cur-cumin (Fig. 1G). The percentage o dead cells also dramaticallyincreased over this time period. It is not clear why the level oapoptotic induction was higher than the overall growth suppres-sion seen using MTS assay. A signi cant, dose-dependent induc-tion in the raction o apoptotic and dead cells was observed inthe medulloblastoma line D283Med (Fig. 1H) and in HSR-GBM1 (Fig. 1I) and JHH-GBM14 glioblastoma neurospheres(data not shown). Cell cycle arrest also contributed to the slowergrowth in cultures exposed to nanocurcumin. In medulloblas-toma (DAOY, D283Med) and glioblastoma (HSR-GBM1) celllines, the percentage o cells in G2/M increased by 74, 63 and25% respectively, with a roughly corresponding reduction in theG1 raction (Fig. 1J–L).

Curcumin inhibits clonogenicity and depletes stem-like cellsrom malignant brain tumor cultures. We next examined the

ability o brain tumor cell lines to orm anchorage-independentcolonies over several weeks when treated with nanocurcumin.Equal numbers o DAOY single cells were seeded in so t agar anthen grown in the presence o empty nanoparticles or increas-ing doses o nanocurcumin. Compared to vehicle-treated cells,

5 and 10μ

M curcumin signi cantly reduced the normalizedclonogenicity o DAOY cells rom 100 to 10% (p < 0.0001) and1.8% (p < 0.0001) respectively (Fig. 2A ). Similar statistically sig-ni cant reductions were observed in the D283Med and U87 celllines (Fig. 2B and C). In the glioblastoma neurosphere cell lineHSR-GBM1, statistically signi cant reductions were observedonly with 10 μ M curcumin (Fig 2D). The above ndings raisethe possibility that stem-like cells required or clonogenic tumorgrowth are depleted by nanocurcumin.

To more directly address the e ects o curcumin on stemlike cancer cells, we examined changes in the percentage ocells expressing the marker CD133. Glioblastoma cells express-ing this marker have been shown by some investigators to be

more clonogenic in vitro, and are more e ective in establishing xenogra ts in vivo.19-21 However, it has recently been shownthat CD133-negative cells can also be tumorigenic,22-24 andmay contain an alternative subpopulation o stem-like cells. Inour hands, CD133 expression in HSR-GBM1 neurospheres isassociated with an approximately 2- old increase in clonogenicpotential.25 Using fow cytometric analysis, we ound that theCD133-positive population in the JHH-GBM14 and HSR-GBM1 neurosphere lines decreased markedly over 2 days ollow-ing addition o nanocurcumin (Fig. 2E and F). In JHH-GBM14neurospheres, a step-wise 92% decrease rom 7.7-0.6% was seen

induces G2/M arrest and non-apoptotic autophagic cell death inboth U87MG and U373MG glioma cells.13

Most prior reports have ocused on glioblastoma, and rela-tively little is known about the potential o curcumin to treatother brain malignancies such as medulloblastoma. Also, whileseveral studies have suggested that curcumin can suppress clo-nogenicity,14,15 the direct e ects o curcumin on stem-like brain

tumor cells are not well understood. Past investigators have usedadherent glioblastoma cells grown in high serum rather than neu-rosphere lines, limiting investigations into stem cell e ects. Wethere ore investigated the e ects o curcumin on both medullo-blastoma and glioblastoma, and examined in particular changesin the stem-like tumor subpopulations, using neurosphere cul-tures when possible. NanoCurcTM , a recently described polymericnanoparticle ormulation o curcumin amenable to systemicdelivery,16 was used in this study in order to acilitate eventualclinical translation. We ound that medulloblastoma and glio-blastoma cells, including neurospheres derived rom malignantgliomas, had their growth and clonogenicity signi cantly inhib-ited, with a concomitant reduction in the percentage o stem-liketumor cells. A variety o signal transduction pathways possibly mediating these e ects were altered ollowing treatment. Thesepreliminary in vitro studies set the stage or the in vivo translationo NanoCurcTM (hence orth “nanocurcumin”) in preclinical ani-mal models o medulloblastoma and glioblastoma, respectively.

Results

Curcumin nanoparticles inhibit the growth o brain tumor celllines via programmed cell death and G2/M cell cycle arrest. Nanocurcumin treatment caused a dose-dependent decrease incell growth as measured by MTS assay in multiple malignant

brain tumor cell lines (Fig. 1). A ter 3 days o treatment using 10μ M curcumin, we noted a statistically signi cant 35% growthreduction in adherent DAOY medulloblastoma cells (Fig. 1A , p< 0.001). A second non-adherent line, D283Med, had growthinhibited by 87% over a somewhat longer period (Fig. 1B). Toinvestigate the speci city o the observed inhibitory e ects, wealso examined NIH-3T3 cells. Although ree curcumin concen-trations above 30μ M had been previously shown by Jiang et al.17 to be toxic in NIH-3T3 cells, the doses o nanocurcumin (up to20 μ M) used in this study which slowed or arrested brain tumorgrowth resulted instead in increased growth rate o NIH-3T3cells (Fig. 1C). This suggests that the e ects are at least somewhatselective, and do not inhibit cellular proli eration and growth in

all cells at these doses. We also investigated the inhibitory e ects o curcumin on thecommonly used adherent glioblastoma cell line U87, and oundthat the growth o these was 32 ± 3.9% slower with 20μ M cur-cumin treatment over 5 days (Fig. 1D). Finally, we examinedseveral glioblastoma neurosphere lines, which are thought torepresent genetically and pathologically superior models as they stably maintain the genomic changes o primary tumors with-out accumulating signi cant additional alterations, allow themaintenance o stem-like tumor cells, and more aith ully reca-pitulate the invasive behavior o glioblastoma.18 These include




observed decreases o 6 and 53% in the proportion o D283Medand DAOY medulloblastoma cultures expressing this stem cellmarker when treated with 20 μ M curcumin as compared tovehicle (Fig. 2F).

Curcumin inhibits IGF and STAT3 signaling. The IGF-1receptor pathway is aberrantly regulated in tumors o the nervoussystem, including both medulloblastoma and glioblastoma.27,28

with increasing doses o curcumin (Fig. 2E). In HSR-GBM1, weobserved an approximately 5, 23 and 49% reduction in the per-centage o CD133-positive stem-like cells ollowing 5, 10 and 20μ M doses o curcumin respectively (Fig. 2F). While the biologi-cal signi cance o CD133 expression in DAOY and D283Medis less clear, we have previously shown that CD133 marks cells with increased xenogra t initiating potential in these lines,26 and

Figure 1. Growth o brain tumor cell lines is selectively inhibited by nanocurcumin via programmed cell death and cell cycle arrest. MTS assays per-ormed on brain tumor cell lines DAOY (A), D283Med (B), U87 (D), JHH-GBM14 (E) and HSR-GBM1 (F) showed signi cant growth reduction a ter treat-

ment over the period o time indicated. This was due to both apoptotic induction (G–I) and G 2 /M cell cycle arrest (J–L). However, the non-neoplasticNIH-3T3 line instead showed greater growth a ter nanocurcumin treatment (C). Statistical signi cance was calculated using the nal time points in(A–F). *p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle.




Figure 2. Curcumin reduces clonogenicity o brain tumor cell lines DAOY (A), D283Med (B), U87 (C) and HSR-GBM1 (D) in a dose-dependent ashion.Equal number o cells were seeded and treated with the indicated nanocurcumin concentrations or void NVA622 nanoparticles (vehicle). Curcuminreduces the CD133-positive stem-like raction in glioblastoma cell lines JHH-GBM14 (E) and HSR-GBM1, as well as D283Med medulloblastoma cell line(F). JHH-GBM14 (E) and HSR-GBM1 (F) cells were treated with void NVA622 nanoparticle (vehicle), 5 μ M, 10 μ M or 20 μ M nanocurcumin or 2 days andcollected or fow cytometry analysis with CD133 antibodies. DAOY and D283Med (F) cells were treated with the same doses or only 1 day.*p < 0.05, **p < 0.01, ****p < 0.0001 compared to vehicle control.




phospho-Akt (Ser 473). Supplemental Figure 1 shows a reduc-tion o phospho-Akt levels a ter 6 hours o nanocurcumin treatment by 34%, but this had begun to normalize by 26 hours(normalized intensities are depicted as histograms in the bottomsub-panel). Total Akt levels were also reduced by curcumin treat-ment, and could account or at least a portion o the decrease inthe phosphorylated raction. This experiment was repeated twotimes with similar results. We also examined MAPK pathway activity a ter curcumin treatment. Most p42/p44 MAPK, p42/p44 phospho-MAPK, ERK1/2 and phospho-ERK1/2 proteinsappeared unchanged ollowing treatment, although some phos-phoproteins were present at levels too low to be detected (data notshown). We also examined the e ects o curcumin on STAT sig

naling, as this pathway has been reported to act downstream o IGF in some contexts,31,32 and is also known to promote a stem-like ate in brain tumors.33,34 STAT3 α proteins were expressedin both medulloblastoma (Fig. 3C) and glioblastoma cells (datanot shown). Immunoblotting with phospho-speci c antibodiescon rmed that STAT3 α is phosphorylated on both Tyr 705 andSer 727 residues, suggesting that the pathway is active. Treatmento the cells with dose-escalating nanocurcumin reduced totalSTAT3 α protein levels, and to a greater extent, phosphorylationo STAT3 at Tyr 705 in DAOY cells (Fig. 3C). There was also avery slight reduction o the same phospho-protein in HSR-GBM1

These tumors o ten overexpress IGF-1R and secrete both IGF-1 andIGF-2 ligands.27-30 We identi ed a remarkable 18- old down-regu-lation o IGF-1 expression ollowing curcumin treatment in a pre-liminary oligonucleotide microarray analysis experiment in DAOY cells (data not shown). We there ore examined the e ects o cur-cumin in this pathway using quantitative PCR and protein analysis.IGF-1, IGF-2 and IGF-1R levels were measured in RNA extracted

rom cells treated with nanocurcumin or void NVA622 nanopar-ticle (vehicle), revealing a dose-dependent reduction in expressiono both the IGF ligands and IGF-1R in DAOY cells which wasstatistically signi cant at 10μ M and above (Fig. 3A ). A signi cantreduction in IGF-1 mRNA levels, but not in IGF-2 or IGF-1R, wasalso observed in D283Med cells (data not shown). Western blot

analysis showed a modest reduction in overall IGF-1R β protein lev-els, as well as a more pronounced decrease in the phosphorylated,active orm o the receptor (p-IGF-1R β , Tyr 1135/1136) a ter cur-cumin treatment in DAOY cells (Fig. 3B). Normalized intensitiesare depicted as histograms in the right sub-panel.

IGF signaling activates two key downstream signal-trans-duction cascades, the lipase kinase PI3K/Akt pathway and theGTPase Ras-Ra -ERK/MAPK pathway. To determine i cur-cumin can modulate Akt signaling in medulloblastomas, wetreated DAOY cells with vehicle or 20μ M curcumin or 6 or26 hours and carried out western blot analysis or total and

Figure 3. The IGF-1R pathway is attenuated a ter nanocurcumin treatment in medulloblastoma cells. DAOY (A) cells treated with the indicated concen-trations o curcumin showed decreased transcript levels o IGF-1, IGF-2 and IGF-1R. *p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle control. Ly-sates rom DAOY (B) cells treated or 26 hours with vehicle or the indicated nanocurcumin concentrations show reduction in p-IGF-1R β (Tyr 1135/1136)and IGF-1Rβ protein levels. (C) p-STAT3 (Tyr 705) levels were reduced ollowing curcumin nanoparticle treatment in DAOY cells. Normalized intensitiesmeasured by densitometry o 2 independent experiments are depicted as bar graphs (B and C, right parts).




nanocurcumin did not inhibit Hh signaling in a second medul-loblastoma cell line (D283Med) or in glioblastoma neurospheres(Fig. 4B and C). Because we have previously shown that Hh cancontrol Bcl2 transcription in DAOY medulloblastoma cells andin primary tumors,39 we measured levels o this key antiapoptoticprotein, and ound reductions which corresponded to reductionsin Gli1 (Fig. 4D).

As Notch activity has been implicated in the propagation o the stem-like phenotype in glioblastoma,21 and has been pro-

posed as a potential target o curcumin,40,41

we also measured tar-gets o this pathway. However, expression levels o transcripts othe Notch target genes Hes1, Hes5 and Hey2 were not suppresseda ter nanocurcumin treatment in DAOY or HSR-GBM1 cells(Sup. Fig. 2), suggesting that curcumin does not block pathway activity in these cells.

Discussion

We investigated i nanocurcumin, a ormulation that has sig-ni cantly greater aqueous solubility and systemic bioavailability

(data not shown). In contrast, while in some experiments Ser 727p-STAT3 levels appear to be reduced when grown in both serum-

ree media (Fig. 3C, le t part) and 2% FBS (data not shown), inothers they remained relatively constant or increased slightly, asrefected in the combined serum- ree data depicted in the rightpart.

Inhibition o the hedgehog (Hh) pathway, but not notch,by curcumin in a subset o cell lines.Both Hh and Notch havepreviously been implicated in medulloblastoma and GBM biol-

ogy.21,35-37

Our microarray data revealed a 2.4- old downregu-lation o Gli1 expression a ter 20μ M curcumin treatment inDAOY cells. Gli1 is a key target and e ector o the Hedgehog(Hh) pathway, which has been associated in the initiation andgrowth o both medulloblastoma and glioblastoma.35,37,38 To vali-date our microarray results, we used quantitative real-time PCR to evaluate Gli1 transcripts and a second marker o Hh activ-ity, Ptch1B . These targets were ound to be reduced by 76 and55% respectively in DAOY cells a ter treatment with 20μ M cur-cumin (Fig. 4A ). In contrast, Gli2 transcript levels, which are notthought to refect pathway activity, did not decrease. However,

Figure 4. The Hh pathway is downregulated a ter curcumin treatment. Transcript levels o Hh pathway targets ( Gli1 and Ptch1B ) are reduced ollowing

treatment with nanocurcumin in DAOY (A) cells, but not in D283Med (B) or HSR-GBM1 (C). Protein levels o Bcl2 are downregulated ollowing nanocur-cumin treatment in DAOY cells (D). *p < 0.05, ***p < 0.001 compared to vehicle.




has been implicated in the survival and di erentiation o braintumors,50,51 and could also play a role in the modulation o tumorgrowth and clonogenicity by nanocurcumin.

Two other pathways known to play critical roles in the stemcell phenotype o brain tumors are Notch21,36 and Hh. 38,52 Bothhave also been previously implicated as targets o curcumin Wang et al.41 showed that curcumin could downregulate Notch1

in pancreatic cancer cells, but we did not nd any suppressiono Notch targets in our tumor lines ollowing curcumin treat-ment. Elamin et al.42 recently ound that curcumin had inhibi-tory e ects on the Hh pathway in medulloblastoma cells. Usingthe MED-5 cell line, they showed more than 5- old reductionsin Gli1 and 2- old reductions inPtch1, a ter treatment with 40μ M curcumin. Our ndings were similar, with 20μ M curcuminreducing expression o Ptch1B and Gli1 by 55 and 76% respec-tively in DAOY cells (Fig. 4A ). We have previously shown thatGli1 can regulate Bcl2 levels in this line,39 and this may there oreexplain the changes we observed in that anti-apoptotic protein(Fig. 4D). Also, reductions in Bcl2 protein levels may lead to theinduction o apoptosis observed inFigure 1G. However, we didnot nd pathway suppression in a second medulloblastoma cellline D283Med (Fig. 4B), indicating that Hh inhibition by cur-cumin is not universal or all medulloblastomas. We also did not

nd down-regulation o Gli1 and Ptch1B expression in treatedHSR-GBM1 cells.

In summary, we have ound that a nanoparticle ormulation ocurcumin NanoCurcTM can reduce the growth and clonogenicity o medulloblastoma and glioblastoma cell lines, and deplete thesubpopulation o cells expressing the stem cell marker CD133.Downstream signaling pathways a ected by curcumin in ourmodels included IGF, STAT3, Akt and Hh. These data provide

urther support or the development o curcumin as a new ther-

apy or brain tumors, and indicate that pathways a ecting bothsurvival and neoplastic stem cell phenotype can be modulatedby this natural compound. The availability o nanocurcumin, which is readily amenable to systemic delivery and biodistribu-tion16 should acilitate the in vivo application in preclinical ani-mal models o these tumors.

Materials and Methods

Materials. Ultra-pure curcumin (>99% di eruloylmethane) wasa kind gi t rom Sabinsa Corporation. Monomers or polymernanoparticle synthesis—speci cally N-isopropylacrylamide(NIPAAM, 415324), vinylpyrrolidone (VP, V3409) and acrylic

acid (AA, 147230)—were obtained rom Sigma Aldrich.Reagents or the polymerization step, including NN’-methylene-bis-acrylamide (MBA, 146072), ammonium persul ate (APS,248614) and errous sul ate (FeSO4, F8048) were also romSigma. For each experiment, nanocurcumin (NanoCurcTM)16 wasdissolved in sterile PBS to prepare a resh 1 mM stock solutionand diluted urther in cell culture media to the appropriate reecurcumin-equivalent concentrations.

Synthesis o nanocurcumin (NanoCurcTM ). The predistilledmonomers o NIPAAM, VP and AA are mixed together in a molarratio o 60:20:20, respectively, hence the acronym “NVA622”

than ree curcumin,16 can e ectively inhibit the proli eration andclonogenicity o medulloblastoma and glioblastoma cell lines.Nanocurcumin was highly e ective in blocking growth o theDAOY and D283Med medulloblastoma cultures, with a moremodest inhibition o glioblastoma neurospheres. Both apoptoticcell death and G2/M cell cycle arrest contributed to the antitu-mor e ects. While nothing was known about the e ects o cur-

cumin on medulloblastoma until recently, two other groups havenow reported growth inhibition and the induction o caspase-mediated cell death in medulloblastoma cells ollowing ree cur-cumin treatment.14,42

This curcumin ormulation also e ectively inhibited the clo-nogenic potential o both medulloblastoma and glioblastomalines, raising questions regarding its e ects on stem-like tumorinitiating cells. Recently, curcumin was ound to target thestem-like side population in the adherent rat C6 glioma cells.43 We used a di erent marker, CD133 and neurospheres grownin serum- ree conditions thought to help maintain stem cellpopulations or our glioma studies. In our tumor-derived neuro-spheres, we ound that 20μ M curcumin induced a remarkable49% decrease in the percentage o CD133 positive GBM cells. Italso reduced this population in the D283Med medulloblastomaline. Consistent with the notion that stem-like tumor cells weredepleted by nanocurcumin, so t agar clonogenic assays (Fig. 2)revealed much more pronounced e ects than short term growthassays (Fig. 1). It remains to be seen, however, whether curcuminmight also deplete non-neoplastic stem cells in the brain, which would have potentially signi cant side e ects.

I curcumin is to be most e ectively used therapeutically, it will be necessary to understand which signaling cascades it modu-lates. We there ore examined the molecular pathway(s) curcuminalters in brain tumors. Preliminary gene expression array analy-

sis suggested that curcumin downregulates the IGF pathway inmedulloblastoma via reduction o IGF-1 and 2 ligands, and we were able to con rm suppression o IGF-1R β receptor expres-sion and activity using phospho-speci c antibodies. Curcuminhas been previously shown to suppress IGF-1 expression in breastcancer cells,44 suggesting that this may be a common target inmultiple tumor types, although to our knowledge it has not beenpreviously identi ed in brain tumors. A number o prior studieshave also shown that IGF-1, IGF-2 and IGF-1R play an activerole in the ormation and growth o medulloblastoma and otherbrain tumors,45,46 supporting the biological relevance o theirdownregulation by curcumin.

In some contexts, the STAT pathway can be activated by IGF

signaling.31,32

STAT has also been implicated in modulating stemcell phenotype in non-neoplastic cells47,48 and in several types o cancer, including brain tumors.33,34 Given the suppression o IGFactivity and stem cell markers observed, we examined i STAT3 was also modulated by nanocurcumin. Indeed, the phosphoryla-tion o Tyr 705 residue on STAT3, which induces dimerization,nuclear translocation and DNA binding,49 was reduced in DAOY cells (Fig. 3C). This suggests that suppression o IGF and STAT3by curcumin could play a role in its e ects on growth and stemcell phenotype in brain tumors. We also observed less pronouncede ects o curcumin on Akt expression and phosphorylation. Akt




Clonogenic assay. We assessed anchorage-independent tumorgrowth potential with clonogenic assay using so t agar. Briefy,the base layer consists o serum-supplemented media in 1% agar(18300012, Invitrogen) containing void NVA622 nanoparticle,5 or 10 μ M nanocurcumin in each well o a 6-well plate. Thecell layer contains 1 x 104 cells mixed with serum-supplementedmedia in 0.5% agar on top o the base layer. A ter 3-4 weeks, the

cells were stained with p-Nitro blue tetrazolium chloride (19535,USB Corporation) and the numbers o colonies larger than 50μ M in three high-powered elds per well were determined by useo computer-assisted image analysis with the MCID Elite so t- ware. Each experiment was done at least twice in triplicates.

Flow cytometry analysis.Flow cytometric analysis o CD133 was done with antibodies rom Miltenyi Biotec according tomanu acturer’s instructions using a FACScan machine (BD). Inbrie , cells were treated or 1 (DAOY, D283Med) or 2 (HSR-GBM1, JHH-GBM14) days with void NVA622 nanoparticleor nanocurcumin and harvested. Cells were blocked with FcR blocking reagent (130-059-901) and incubated with CD133/1(AC133)-phycoerythrin antibody (130-080-801) in the dark at4°C. The cells were then washed and resuspended in PBS con-taining 0.5% BSA and 2 mM EDTA. Cells expressing levels oCD133 higher than those seen in unconjugated CD133 IgG con-trols (130-090-422) were considered positive.

Quantitative PCR analysis. RNA was extracted with Qiagen’sRNeasy kit. RNA levels were assayed by real-time PCR analy-sis per ormed in triplicate with SYBR Green reagent (4364346, Applied Biosystems) according to the manu acturer’s instructions on an I-Cycler IQ real-time detection system (Bio-Rad), with all reactions normalized to GAPDH. Primer sequences arein the Supplemental Materials and Methods.

Protein analysis. For IGF-1R western blot analysis, cells

were treated with nanocurcumin in serum- ree media or 26hr and stimulated with 50 ng/ml IGF-1 (I3769, Sigma) or 5minutes be ore extracting proteins. In other western analyses,cells were cultured in serum- ree media during nanocurcumintreatment. Proteins were extracted in a bu er containing 50mM Tris, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1mM PMSF, 10 mM NaF, 2 mM Na 3VO4 and complete prote-ase inhibitors (11697498001, Roche). Western blots containingat least 20 μ g total protein per lane on a NuPAGE 4–10% Bis-Tris gel (NP0321BOX) were electrophoresed in 1x NuPAGEMOPS SDS running bu er (NP0001) on XCell SureLock minigel apparatus (all rom Invitrogen). Proteins were then trans-

erred to Biorad’s Immun-blot PVDF membrane (162-0177) in

1x NuPAGE trans er bu er in X Cell II Blot module (Invitrogen)Membranes were blocked or 1 hour at room temperature in 5%non- at milk or 5% BSA ( or phospho-antibodies) and incubatedat 4°C overnight in primary antibodies containing 5% non- atmilk or 5% BSA ( or phospho-antibodies). Mouse anti-rabbitGAPDH antibodies were purchased rom Research Diagnostics(RDI-TRK5G4-6C5). Akt (9272), STAT3 (9132), Phospho-Akt(Ser 473) (4060), phospho-IGF-IR β (Tyr 1135/1136)(3024),Phospho-STAT3 (Tyr 705 and Ser 727) antibodies were allpurchased rom Cell Signaling Technology. IGF-IR β antibod-ies were purchased rom Santa Cruz (sc-713). Bcl2 antibodies

or the resulting polymer. Polymerization was per ormed or 24hours at 30°C under an inert (nitrogen) atmosphere, using APSand FeSO4 as initiator and activator, respectively. A ter com-plete polymerization, the total aqueous solution o polymer waspuri ed using dialysis, and then lyophilized or post loading o curcumin, as described in re erence 53. Typically, a 10 ml stock solution o polymeric nanoparticles (100 mg) was slowly mixed

with 150 μ l o curcumin solution in chloro orm (10 mg/ml), andgently stirred or 15–20 minutes on low heating, in order to loadcurcumin and evaporate chloro orm simultaneously. The result-ing solution, corresponding to 1.5% (w/w) loading o curcuminin nanoparticles, was then snap rozen on a dry ice/acetone bathand lyophilized. The lyophilized nanocurcumin powder is storedat 4°C until urther use.

Cell lines and cultures. The DAOY and D283Med cell lines were obtained rom the American Type Culture Collection(HTB-186 TM and HTB-185 TM), cultured in MEM and ImprovedMEM Zn ++ Option (Richter’s Modi cation) (10373017,Invitrogen) respectively. The ATCC-obtained adherent GBMcell line U87 was cultured in MEM media. Cell culture media were all supplemented with 10% FBS unless otherwise noted.The glioblastoma-derived neurosphere line HSR-GBM1 waspropagated in serum- ree media as previously described in re -erence 54. The glioblastoma-derived low passage neurospheres JHH-GBM14 originated rom a tumor resected at JohnsHopkins Hospital, and was propagated the same way.

Cell proli eration assay.Approximately 2 x 103 viable cells were seeded in each well o a 96-well plate and allowed to attachovernight in their respective culturing media. Wells were washed with PBS and replaced with media containing 2% FBS ( orDAOY cells) and various concentrations o nanocurcumin. Onthe day o assay, resh media was replaced in each assayed well.

Cell mass was measured using CellTiter (MTS) assays accord-ing to manu acturer’s instructions (G3580, Promega). For neu-rospheres, the overnight attachment step and replacement with

resh media be ore each reading step were omitted. Apoptosis assay. Cells were plated in 12-well plates at 6 x

104 cells per well (and allowed to attach overnight or adher-ent cells) and treated with void NVA622 nanoparticle or nano-curcumin or 24 hours unless otherwise stated. Apoptotic cellinduction was evaluated using the Guava Annexin V assay kit(4500-0455, Millipore) according to the manu acturer’s pro-tocol. Quanti cation o apoptotic and dead cells were doneusing the GUAVA-PCA fow cytometry system. Each experi-ment represents a minimum o 2 x 103 gated events rom each o

three wells. Apoptotic ractions were assigned using the Guavaso tware.Cell cycle analysis.Cells were plated in six-well plates at 1.5

x 105 cells per well to attach overnight and treated with voidNVA622 nanoparticle or nanocurcumin or 24 hours unlessotherwise stated. Cells were then stained with the cell cyclereagent (4500-0220, Millipore) and assessed on the GuavaPCA ollowing the manu acturer’s protocol. Each experimentrepresents a minimum o 5 x 103 gated events rom each o three wells. Cell cycle ractions were assigned using the Guavaso tware.




Acknowledgements

We thank Karisa Schreck or help ul discussions, and AngelVescovi and Francesco DiMeco or their kind gi t o HSR-GBM1 cells. NanoCurcTM development and synthesis supportedby R01CA113669 (A.M.); R01CA13767 (A.M.); P01CA134292(A.M.); Flight Attendants Medical Research Institute (FAMRI)(A.M.); and SignPath Pharmaceuticals Inc., (A.M.). Brain tumor

assays are supported by R21 AT003893 (C.G.E.).

Conficts o Interest

NanoCurcTM is a registered trademark o SignPathPharmaceuticals Inc., Quakerstown, Pennsylvania. Dr. Maitra isa member o the scienti c advisory board o SignPath PharmaInc., and any conficts o interest under this arrangement arehandled in accordance with the Johns Hopkins University O ceo Policy Coordination (OPC) guidelines.

Note

Supplemental materials can be ound at: www.landesbioscience.com/supplement/LimCBT11-5-Sup.pd

were rom Calbiochem (OP60). Secondary antibodies romKPL (peroxidase-conjugated goat anti-mouse (074-1806) or rab-bit IgG (074-1506) were diluted 1:5,000 in blocking solution.Blots were developed with enhanced chemiluminescence reagent(NEL103001EA, PerkinElmer).

Statistics. Cell proli eration, apoptosis, cell cycle, clonogenicand quantitative PCR assays were per ormed in triplicate, and

repeated at least twice unless otherwise noted. As JHH-GBM14is a low passage line, experiments with these cells were done onetime in triplicate (other than the MTS assay, which was per ormedtwice). The so tware GraphPad Prism was used or all statisti-cal analyses. Mean values ± SEM or representative experimentsare shown. Statistical di erences were determined by Student’stwo-tailed t-test. A p value o < 0.05 was considered signi cant.Bands visualized via western blot were subjected to band den-sitometry analysis with ImageJ so tware (National Institutes o Health). The amount o speci c signal or each protein was cor-rected or sample loading and represented as a value comparedto vehicle. Normalized intensities rom two experiments aredepicted as histograms.

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A Polymeric Nano Particle Formulation of Curcumin Inhibits Growth, Clonogenicity and Stem-like Fraction in Malignant Brain Tumors