Liposome-encapsulated curcumin suppresses neuroblastoma growth through nuclear factor-kappa...

Liposome-Encapsulated Curcumin Suppresses NeuroblastomaGrowth Through Nuclear Factor-κB Inhibition

W. Shannon Orr, MDa,b, Jason W. Denbo, MDa,b, Karim R. Saab, MDa, Adrianne L. Myers,MDa,b, Catherine Y. Ng, MSa, Junfang Zhou, MDa, Christopher L. Morton, BSa, Lawrence M.Pfeffer, Ph.Dc, and Andrew M. Davidoff, MDa,b

aDepartment of Surgery, St. Jude Children’s Research Hospital, Memphis, TennesseebDepartment of Surgery, University of Tennessee Health Science Center, Memphis, TennesseecDepartment of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee

AbstractBackground—Nuclear factor-κB (NF-κB) has been implicated in tumor cell proliferation andsurvival, and tumor angiogenesis. We sought to evaluate the effects of curcumin, an inhibitor ofNF-κB, on a xenograft model on disseminated neuroblastoma.

Methods—For in vitro studies, neuroblastoma cell lines, NB1691, CHLA-20, and SK-N-AS,were treated with varying doses of liposomal curcumin. Disseminated neuroblastoma wasestablished in vivo by tail vein injection of NB1691-luc cells into SCID mice which were thentreated with 50mg/kg/day of liposomal curcumin 5 days/week intraperitoneal.

Results—Curcumin suppressed NF-κB activation and proliferation of all neuroblastoma celllines in vitro. In vivo, curcumin treatment resulted in a significant decrease in disseminated tumorburden. Curcumin treated tumors had decreased NF-κB activity and an associated significantdecrease in tumor cell proliferation and an increase in tumor cell apoptosis, as well as a decreasein tumor VEGF levels and microvessel density.

Conclusions—Liposomal curcumin suppressed neuroblastoma growth, with treated tumorsshowing a decrease in NF-kB activity. Our results suggest that liposomal curcumin maybe a viableoption for the treatment of neuroblastoma that works via inhibiting the NF-κB pathway.

BackgroundNeuroblastoma is an aggressive malignancy of the sympathetic nervous system and is themost common solid extracranial tumor of childhood.1 Children with high-riskneuroblastoma have a very poor prognosis, with the 5-year disease free survival rate beingbetween 25%-35%, despite aggressive multi-modality therapy.1 This highlights the need fornew therapeutic strategies.

The nuclear factor-kappaB (NF-κB) family of transcription factors regulates expression ofgenes that affect multiple biological processes, including immune and inflammatory

© 2011 Mosby, Inc. All rights reserved.Address correspondence to: Andrew M. Davidoff, MD, Department of Surgery, St Jude Children’s Research Hospital, 262 DannyThomas Place, Memphis, Tennessee 38105. Phone: 901-595-4060; Fax: 901-595-6621; [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptSurgery. Author manuscript; available in PMC 2013 May 01.

Published in final edited form as:Surgery. 2012 May ; 151(5): 736–744. doi:10.1016/j.surg.2011.12.014.

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responses, developmental processes, cellular growth, and apoptosis.2 NF-κB is tightlyregulated by interactions with inhibitory IκB proteins.3 In most cells, NF- κB is bound to theIκB-complex in the cytoplasm and is inactive.3 The canonical and non-canonical pathwaysto NF-κB activation have a common upstream regulatory step which involves activation ofthe IκB kinase (IKK) complex. This activation results in IKK-mediated, phosphorylation-induced degradation of the IκB inhibitor.3 Degradation of the IκB inhibitor allows NF-κBdimers to translocate into the nucleus and activate specific target genes.3 Underphysiological conditions, the NF- κB pathway is generally inactive in normal cells, exceptneurons, B cells, and thymocytes4, but has been shown to be constitutively active in manycancer cell lines including human neuroblastoma.5

Curcumin (diferuloylmethane), a polyphenol and the active component of turmeric, amedicinal compound first used for its anti-inflammatory properties, has been shown tosuppress NF-κB activity and down regulate expression of NF-κB regulated gene productsknown to regulate cell proliferation, invasion, angiogenesis, metastasis and apoptosis.6-9 Inthis current study, we evaluated the in vitro and in vivo antitumor activity of liposomalcurcumin against human neuroblastoma. We demonstrated that curcumin inhibitsneuroblastoma NF-κB activity in vitro and in a murine model of disseminatedneuroblastoma. The inhibition of NF-κB activity in the animal model was associated withsuppression of tumor growth, inhibition of angiogenesis, and increased tumor cell apoptosis.

Materials and MethodsCell lines

The NB1691 (P. Houghton, Columbus, Ohio), SK-N-AS (American Type CultureCollection, Manassas, Virginia), and CHLA-20 (C.P. Reynolds, Lubbock, Texas) humanneuroblastoma cells were used. These cells were engineered to constitutively express fireflyluciferase as previously described.10

Curcumin preparation1, 2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1, 2-dimyristoylsn-glycero-3-[phosphor-rac- (1-gylcerol)] (DMPG) were obtained as dry powder from Avanti PolarLipids (Alabaster, Alabama). Curcumin (ACROS, Morris Plains, New Jersey),dimethylsulfoxide (DMSO), and tert-butanol were obtained from Sigma Chemical Company(St. Louis, Missouri). The lyophilization process involved several steps. A 10:1 total lipid tocurcumin ratio (weight/weight) was used.11 Curcumin was dissolved in DMSO at 50mg/mland the lipid was dissolved 9:1 (DMPC:DMPG) in 20mg/ml tert-butanol. Aliquots of thissolution were placed in lyophilization vials frozen at −20°C and then lyophilized to removeall DMSO and tert-butanol. The lyophilized powder was stored at −20°C.

Alamar blue cell viability assayThe effects of increasing doses of curcumin on cell viability were determined by AlamarBlue Viability Assay using the manufacturer’s instructions (Invitrogen, Carlsbad,California). Alamar blue solution was added to treated cells and after 4 hours the absorbancewas read on a Synergy 2 Multi-Mode Microplate Reader (Biotek, Winooski, Vermont). Theexperiment was repeated three times.

Annexin V and cell cycle analysisTo analyze the effects of curcumin on cell cycle and apoptosis in these cell lines, cells weretreated with 10 mol of liposomal curcumin for 48 hours, after which time cells wereanalyzed by flow cytometry for DNA content and Annexin V staining.

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Nuclear factor KappaB reporter assayNB1691 cells were transduced to stably express a NF-kB luciferase reporter. A 1.9kbfragment of the plasmid pGL4.32 luc2 NF-κB (Promega, Madison, Wisconsin) containingthe NF-κB response elements was excised using Eco53KI and EagI and ligated onto theCL20c-MSCV plasmid backbone. These cells were plated at a density of 5,000 cells/well ina 96 well plate and then treated with varying concentrations of lyophilized curcumin andafter 3 hours of incubation, a set of plates was stimulated with 10ng/mL TNFα (Promega)for 30 minutes. Following this, the media was then removed and replaced with 1:100 15mg/mL luciferin containing media. The plate was allowed to incubate for 5 minutes and thenread on a Synergy 2 Multi-Microplate Reader.

Nuclear protein extractionFor in vitro analysis of NF-κB activity, neuroblastoma cells were treated with 5 molliposomal curcumin for 3, 24, 48 and 72 hours. After various time points, a set of plates wasstimulated with 10ng/mL TNFα for 30 minutes. Cell nuclei were isolated using AffymetrixNuclear Extraction Kit (Affymetrix, Santa Clara, California). Nuclear extracts and allworking reagents were prepared per manufacturer’s protocol. For tumors, nuclear extractionwas again done using the Affymetrix Nuclear Extraction Kit, starting with 0.5 grams oftissue.

Nuclear protein concentration was quantified using Pierce BCA Protein Assay Kit (ThermoScientific, Rockford, Illinois) following manufacturer’s guidelines. Aliquots of each nuclearextract were stored at −80°C until use.

Electrophoretic mobility shift assayTo determine the effect of curcumin on NF-κB activation, an electrophoretic mobility shiftassay (EMSA) was performed on nuclear extracts after liposomal curcumin treatmentfollowing the protocol by Promega Gel Shift Assay Systems (Promega). Briefly, nuclearextracts were incubated with a 32P-end-labed double-stranded NF-κB oligonucleotidecontaining a tandem repeat consensus sequence of 5′-AGT TGA GGG GAC TTT CCCAGG C-3′. Subsequently, all samples were electrophoresed through a 4% Acrylamide gel.Dried gels were visualized, and radioactive bands were quantified by a PhosphorImager(Molecular Dynamics, Sunnyvale, California) using ImageQuant 5.2 software (MolecularDynamics)

Animal modelAll murine experiments were performed in accordance with a protocol approved by theInstitutional Animal Care and Use Committee at St. Jude Children’s Research Hospital. Adisseminated model of neuroblastoma was established by injecting 2 × 106 NB1691-luc cellsvia tail vein into 4- to 6-week old male CB-17 SCID mice (Taconic, Hudson, New York).Two weeks after tail vein injection, animals were imaged using IVIS Imaging System 100Series (Xenogen Corporation, Alameda, California) as previously described10. Living ImageSoftware (Xenogen) was used to analyze tumor bioluminescence. Animals were divided into2 groups (n=10 per group) with equivalent tumor burden (≈4.90 × 107 photons/second). Thetreatment group received 50mg/kg of liposomal curcumin intraperitoneal 5 days/week andthe control group received empty liposome intraperitoneal 5 days/week. For real-time invivo assessment of tumor burden, bioluminescence imaging was performed biweekly. At theend of 3 weeks, all animals were sacrificed.

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Real time polymerase chain reaction analysisAssay-on-demand gene expression primer and probe sets for human vascular endothelialgrowth factor and glyceraldehyde-3-phosphate dehydrogenase (Hs00173626_m1, andHs99999905_m1); Applied Biosystems, Foster City, California) were used for real-timePCR as previously described10.

Tumor immunohistochemistryFormalin-fixed, paraffin-embedded, 4 m thick tumor sections were stained with ratantimouse CD34 (RAM 34; PharMingen, San Diego, California) antibodies as previouslydescribed12. Apoptosis in tumors was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling using a commercially available in situ apoptosis detectionkit (Serologicals, Billerica, Massachusetts) as previously described13. Tumor cellproliferation was analyzed by staining formalin-fixed, paraffin-embedded sections (4 μm)with anti-Ki67 (rabbit monoclonal clone SP6; Neomarkers, Fremont, California). Resultswere expressed as percentage of Ki-67 positive cells ± SE per 400x magnification.

Statistical AnalysisContinuous variables are reported as mean ± SEM and were compared using an unpairedStudent’s t test. A p value of less than .05 was considered significant. Data was analyzed andgraphed using Sigma Plot (Version 9; SPSS Inc, Chicago, Illinois).

ResultsLiposomal curcumin inhibits the proliferation and survival of neuroblastoma cell lines invitro

The effect of curcumin on proliferation was assessed on the human neuroblastoma cell linesNB1691, CHLA-20 and SK-N-AS. Exposure to liposomal curcumin inhibitedneuroblastoma cell proliferation in all 3 cell lines in a concentration and time -dependentmanner. 96 hour IC50 concentrations varied from 10.57 mol for CHLA-20 to 17.97 mol forSK-N-AS (Figure 1). The anti-proliferative effects of liposomal curcumin were equivalent tofree curcumin, confirming that there was no loss in curcumin’s potency during liposomalformulation. The neuroblastoma cell lines were also treated with empty liposome; no anti-proliferative effects were seen (Figure 1).

The effect of curcumin on cell cycle distribution and apoptosis was next assessed. Platedcells were treated for 48 hours with 10 mol of curcumin prior to FACS cell cycle andAnnexin V analysis. All three neuroblastoma cell lines treated showed an increase in thepercentage of cells in the G2/M phase of cell cycle. Curcumin treatment resulted in an 8.7%,11.3%, and 13.7% increase in the number of cells in the G2/M phase of cell cycle (p =0.007, p = 0.002, and p = 0.008) in the NB1691, SK-N-AS, and CHLA-20, respectively.Curcumin treatment resulted in 6.7, 4.83 and 5.87% increase in the percentage of AnnexinV-positive apoptotic cells (p = 0.01, p= 0.02, and p = 0.03) in the NB1691, SK-N-AS andCHLA-20 cell lines respectively (Figure 2). Thus curcumin treatment appeared to inhibittumor cell proliferation by causing G2/M phase cell cycle arrest and inducing apoptosis.

Liposomal curcumin inhibits NF-κB activation in neuroblastoma cell linesTo determine the effect on NF-κB activity by liposomal curcumin, an NF-κB gene reporterassay was assessed using NB1691 cells modified to express a dual-luciferase NF-κBreporter (luc). Untreated NB1691 NF-κB-luc cells had a relative luciferase unit (RLU) of5,796 per well. Curcumin decreased NF-κB activation in a dose dependent manner. (p =0.001) (Figure 3). NF-κB activity was also determined by EMSA which showed a decrease

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in activity in a time dependent manner (Figure 3). After 72 hours of curcumin treatment,there was an 85.4%, 80%, and 74.5% decrease in NF-κB activity compared to control cellsin the NB1691, CHLA-20, and SK-N-AS cell lines, respectively.

Cellular stresses such as irradiation and chemotherapy have been shown to increase NF-κBactivity. Therefore, we next sought to examine the effects of curcumin together with TNFα,a known activator of NF-κB, to determine whether curcumin pretreatment could prevent NF-κB activation by adjuvant therapies. This was again done using both NB1691 NF-κB (luc)cells and by EMSA. NB1691 NF-κB (luc) cells stimulated with TNFα had a 438% increasein RLU per well (23,962 RLU/well), while cells that were pretreated with 10 μmol curcuminbefore the addition of TNFα showed a significant decrease in RLU (p < 0.0001). Cells thatwere treated with 25 mol curcumin 3 hours before the addition of TNFα showed noinduction of NF-κB activity (98% of control). Cells that were pretreated with 100 mol ofcurcumin before TNFα stimulation had a RLU of 80% of control (Figure 3). Next, weexamined the effects of the combination of curcumin and TNFα by EMSA. TNFα induced a33.3% increase in NF-κB activity relative to control cells based on band quantification.Curcumin inhibited the TNFα-induced activation of NF- κB when added 3 hours beforeTNFα; there was no increase in NF-κB activity when curcumin was added 24 hours beforeTNFα. We performed supershift with antibodies directed to specific NF-κB subunits todetermine which components of the NF-κB complex were involved in its activation. Thesupershift assay showed that TNFα increases neuroblastoma NF-κB activity through thecanonical pathway, specifically through increases in the p50 and p65 subunits; curcuminblocked the p50 and p65 subunit activation as seen by antibody shift (Figure 3).

Liposomal curcumin inhibits neuroblastoma tumor xenograft growth in a disseminatedmurine model

We next evaluated the effects of curcumin in a model of disseminated neuroblastoma.Fourteen days after NB-1691 tumor cell inoculation, animals were divided into a controlgroup (n=10) and a treatment group (n=10), with equal tumor burden based onbioluminescence imaging. (4.89 ± 0.89 × 107 vs 4.91 ± 0.78 × 107 photons per second, p=0.98). Mice in the treatment group received 50mg/kg of curcumin via intraperitonealinjection 5 days a week and the control group received empty liposome via intraperitonealinjection 5 days a week. After 3 weeks of therapy, control animals appeared ill and cachetic(animal weight 21.95 ± 0.5 grams) compared to those in the treatment group (animal weight25.99 ± 0.86 grams, p= 0.0002). Real time assessment of tumor burden demonstratedsignificantly less disease in curcumin treated mice (7.43 ± 1.0 × 109 photons/second)compared to control mice (3.0 ± 0.4 × 1010 photons/second, p=0.00003) (Figure 4). Due tothe extent of disease in the control group, animals in both groups were sacrificed for furtherevaluation and comparison of disease burden on day 35 after tumor cell injection. Uponnecropsy, mice in the control group were found to have bulky tumor replacement of theliver, kidney, and lungs (Figure 4) compared to those in the treatment group (liver weight6.15 ±.83 grams vs 2.58 ±.23, p=0.00002; kidney weight .233 ± .025grams vs .166 ± .016,p= .0005, lung weight .158 ±.01 grams vs .111 ± .024grams, p=0.0004). Hematoxylin andeosin staining of these organs further demonstrated the tumor growth restriction within micetreated with curcumin (Figure 5). Thus, curcumin was able to cause a significant decrease intumor burden compared to the control group in a disseminated neuroblastoma model.

Liposomal curcumin inhibits NF-κB activity in neuroblastoma murine tumorsEMSA was used to evaluate the effect of curcumin on NF-κB activity in treated tumors. NF-κB activity was inhibited in curcumin treated tumors compared to control tumors, (Figure 5)being diminished by 26.2%, thus, confirming that lyophilized curcumin treatment iseffective in decreasing NF-κB activity in vivo.

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Liposomal curcumin inhibits tumor cell proliferation and increases tumor cell apoptosisThe effect of curcumin on tumor cell proliferation and apoptosis was assessed by Ki67 andTUNEL staining. The percentage of proliferating tumor cells was significantly decreased intumors treated with curcumin (68.1 ± 3.4%) compared to control tumors (78.5% ± 2.82%,p=0.006) (Figure 6). There was also a significant increase in tumor cell apoptosis asassessed by TUNEL staining in the curcumin-treated group (15.87 ± 4.9 per 1000 cells)compared to the control group (6.09 ± 2.6 per 1000 cells, p=0.0003) (Figure 7). Thus,curcumin-treated tumors have a decrease in cell proliferation and an increase in tumor cellapoptosis, which are both known to be regulated by NF-κB.

Liposomal curcumin also inhibits angiogenesis in vivoNF- κB has been to shown to play a key role in regulating the expression of vascularendothelial growth factor (VEGF). We examined the effects of curcumin-mediated NF-κBinhibition on VEGF expression by RT-PCR analysis and found that curcumin treated tumorsshowed a 29.7% decrease in VEGF gene expression as compared to control tumors(p=0.005). We further examined the effect of curcumin-mediated NF-κB inhibition on thetumor vasculature by CD34 immunohistochemistry staining. Curcumin treated tumors had asignificant decrease in tumor vascular endothelial cells (12,988 ± 4,173 CD34 pixels/hpf)compared to control (38,045 ± 16,182 CD34 pixels/hpf, p=0.0004) (Figure 7). These datasuggest that there is a loss of the more immature vessels through VEGF inhibition incurcumin treated tumors.

DiscussionNeuroblastoma frequently presents with metastatic disease, with 5 year disease free survivalrates ranging between 25%-35%. Despite advances in multi-modality therapy, manychildren with advanced neuroblastoma face a poor prognosis and so new treatment strategiesare needed.

The data presented here demonstrate that curcumin has significant anti-tumor activityagainst human neuroblastoma. In vitro all three cell lines tested demonstrated a dose-dependent and time-dependent decrease in cell proliferation after being treated withcurcumin. There was also a time-dependent decrease in tumor cell NF-κB activity whencells were treated with curcumin doses less than their IC50.

In addition to the effects in vitro, curcumin had significant activity against neuroblastoma invivo, significantly restricting tumor growth in a murine disseminated disease model. Inaddition to diminished bioluminescence signal, the curcumin-treated mice had significantlyless tumor burden at necropsy than did the control group. There was bulky tumorreplacement in the liver, kidney and lungs of the control group compared to the treatedgroup.

NF-κB regulates the expression of genes that induce cell proliferation, invasion,angiogenesis, metastasis as well as suppress of apoptosis.14,1 The curcumin treated tumorshad a decrease in NF-κB activity as compared to control tumors demonstrated by NF-κBDNA binding activity. Immunohistochemical analysis confirmed inhibition of cellproliferation and induction of apoptosis in curcumin treated tumors compared to the controlgroup. Immunohistochemistry also confirmed inhibition of angiogenesis as seen by thedecrease in the endothelial cell count in the treatment group as compared to the controlgroup.

Based on the role that NF-κB plays in cell proliferation, promoting tumorigenesis, andinhibiting apoptosis, it is an attractive clinical therapeutic agent. Radiation therapy and

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several chemotherapeutic agents including doxorubicin, daunomycin, vincristine, andvinblastine have been reported to induce NF-κB activation.16-18 Based on curcumin’s abilityto block the TNFα-mediated increase NF-κB activity, further investigation is needed inusing curcumin as an adjuvant therapy with radiation therapy and chemotherapeutic agents.

In conclusion, curcumin has significant activity against human neuroblastoma in vitro aswell as in clinically relevant murine model of disseminated disease. The data here suggestthat targeting the inhibition of the NF-κB pathway is a promising new way to treat a difficultdisease and that further research and clinical trials are warranted.

AcknowledgmentsWe would like to extend special thanks to Dorothy Bush of St. Jude Children’s Research Hospital for her assistancewith all immunohistochemistry.

This work was supported by the Assisi Foundation of Memphis, the US Public Health Service Childhood SolidTumor Program Project Grant No. CA23099, the Cancer Center Support Grant No. 21766 from the National CancerInstitute, Grant No. CA133322 from National Cancer Institute, and by the American Lebanese Syrian AssociatedCharities (ALSAC).

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Figure 1. Effects of curcumin on tumor cell viability(A) Dose-dependent decrease in tumor cell viability in all 3 neuroblastoma cell lines tested(NB-1691, CHLA-20, SK-N-AS). The IC50s varied from 10.57 mol for CHLA-20 to 17.97mol for SK-N-AS. (B) Treatment of all three cell lines with empty liposome had no effect.(C) No significant differences were found in cell viability when NB1691 cells were treatedwith liposomal curcumin versus curcumin dissolved in DMSO.

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Figure 2. Effects of curcumin on cell cycle and apoptosis(A) Curcumin treated cells showed an increased percentage of cells in the G2/M phase ofcell cycle compared to control cells. (B) Curcumin treatment resulted in an increase inAnnexin V-positive apoptotic cells.

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Figure 3. Effects of curcumin on NF-κB in vitro(A) NB1691 NF-kB luc cells showed a dose dependent decrease in NF-kB activity after 3hours of treatment. (B) Pretreating NB1691 NF-κB luc cells with curcumin before beingstimulated with TNFα prevented NF-κB activation. (C-E) All three cell lines (NB1691,CHLA-20, SK-N-AS, respectively) were treated with 5 mol of curcumin for 3 hours, 24hours, 48 hours and 72 hours. EMSA showed time dependent inhibition of NF-κB activity inall three cell lines. (F) NB1691 cells stimulated with TNFα showed an increase in NF-κBactivity, that was abrogated when curcumin was added 24 hours before TNFα. Antibodysupershift shows that TNFα induced NF-κB complexes contains the p50 and p65 subunits.

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Figure 4. Effect of curcumin on tumor burden in a murine model of disseminated neuroblastomaBioluminescence imaging showed a reduction in tumor burden in the mice withdisseminated neuroblastoma after 3 weeks of curcumin therapy. *p value = 0.00003. Alsoshown are representative photographs of bioluminescence in treat and control mice.

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Figure 5. Effects of curcumin treatment on disseminated tumor burden(A) After 3 weeks of treatment, control mice appeared ill and cachetic compared totreatment group. At necropsy, the control group was found to have bulky tumor replacementof the liver compared to treatment group. *p value = 0.00003, **p value = 0.00002 (B) Thecontrol group also had significant tumor replacement in the kidney and the lung compared totreatment group *p value = 0.0005, **p value = 0.0004 (C) Hematoxylin and eosin stainingof representative kidney and lung sections. Magnification 200x (D) EMSA showsdiminished activity in curcumin treated tumors

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Figure 6. Curcumin treatment decreased tumor cell proliferation and increased tumor cellapoptosis(A) Quanitative assessment of tumor cell proliferation (Ki-67 staining) and apoptosis(TUNEL staining). *p value = 0.006 **p value = 0.0003 (B-C) Representative images ofKi67 staining of control and treated tumor, respectively, (D-E) Representative images ofTUNEL staining of control and treatment tumor, respectively. Magnification 400x.

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Figure 7. Curcumin inhibits tumor angiogenesisA) VEGF was assessed by RT-PCR. Curcumin treated tumors showed a significant decreasein VEGF levels. *p value = 0.005. (B) Tumor endothelial cell density was assessed by CD34immunohistochemistry. Curcumin treated tumors showed a signficant decrease tumorvascular endothelial cells. *p value = 0.0003. (C-D) Representative images of CD34immunohistochemistry of control and treated tumors, respectively. Magnification 400x.

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