Therapeutics Curcumin Enhances the Effect of Cisplatin in ...mct.aacrjournals.org/content/molcanther/9/10/2665.full.pdf · with curcumin, which will allow effective suppression of

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cumin Enhances the Effect of Cisplatin in Suppression ofd and Neck Squamous Cell Carcinoma via Inhibition of

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M. Duarte1,2, Eugene Han1, Mysore S. Veena1, Amanda Salvado1,2, Jeffrey D. Suh1,
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ious experiments have shown that curcumin or cisplatin treatment suppresses growth of head andquamous cell carcinoma (HNSCC). To study the potential cooperative effect of both agents, twoC cell lines were treated with curcumin or cisplatin alone or in combination. In vivo studies consistedavenous tail vein injection of liposomal curcumin, with intraperitoneal cisplatin, into nude mice grow-nograft HNSCC tumors. Introduction of curcumin and suboptimal concentrations of cisplatin showed acant suppressive effect compared with treatment with either agent alone. Reduced expression of cyclinBα, phospho-IκBα, and IKKβ occurred in cisplatin- and curcumin-treated cell lines. Confocal micros-howed expression of IKKβ in the nucleus of the cell lines. Chromatin immunoprecipitation assay onisolated from IKKβ immunoprecipitated samples showed PCR amplification of interleukin-8 promoterces, a binding site of NFκB, indicating an interaction between IKKβ and NFκB. Curcumin inhibitedin the cytoplasm and nucleus, leading to reduced NFκB activity, with no effect on phospho-AKT. In vivos showed significant growth inhibition of xenograft tumors treated with a combination of liposomalin and cisplatin. The suppressive effect of curcumin was mediated through inhibition of cytoplasmic

uclear IKKβ, resulting in inhibition of NFκB activity. Cisplatin treatment led to cellular senescence, in-g an effect mediated by p53 activation. The mechanisms of the two agents through different growthing pathways suggest potential for the clinical use of subtherapeutic doses of cisplatin in combination
signal
with curcumin, which will allow effective suppression of tumor growth while minimizing the toxic sideeffects of cisplatin. Mol Cancer Ther; 9(10); 2665–75. ©2010 AACR.

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d and neck squamous cell carcinoma (HNSCC) isgressive cancer with a poor prognosis for advanceds. There were 40,490 new cases of HNSCC in thed States in 2006, accounting for ∼3% of adult malig-s (1). The worldwide incidence exceeds half a mil-ases annually. The significant morbidity associated

eatment modalities, which include disfig-chemotherapy, and radiation, has led to

nationmanyple ofin thecancethe mcisplaresistalular usis, anthat cp53-ddepenbonecisplausefu

ns: 1Department of Surgery, VA Greater Los Angeles, West Los Angeles, California and 2Division of Heady, 3Department of Medicine, and 4Pasarow Massoratory, Neuropsychiatric-Semel Institute, UCLAol of Medicine, Los Angeles, California

ary material for this article is available at Molecularcs Online (http://mct.aacrjournals.org/).

. Han contributed equally to this work.

thor: Marilene B. Wang, Division of Head and Neckvid Geffen School of Medicine, 200 UCLA Medicalos Angeles, CA 90095. Phone: 310-268-3407; Fax:ail: [email protected]

7163.MCT-10-0064

ssociation for Cancer Research.

ls.org

on November 7, 2020. © 20mct.aacrjournals.org from

uing investigation of potential alternative and lesstherapies.rent treatment regimens for HNSCC include use ofum-based chemotherapy such as cisplatin (2).atin is not effective when used as a single agent,s efficacy is significantly enhanced when used withion therapy and/or in combination with other che-rapeutic drugs. Thus, cisplatin/radiation or combi-chemotherapy is the cornerstone of treatment ofcancers, including head and neck cancer. An exam-this is the combination of cisplatin and cetuximabtreatment of metastatic/recurrent head and neck

r (3). Initial platinum responsiveness is high, butajority of cancer patients eventually relapse withtin-resistant disease. Many mechanisms of cisplatinnce have been proposed, including changes in cel-ptake and efflux of the drug, inhibition of apopto-d increased DNA repair. We have hypothesizedisplatin induces cell cycle arrest through a p16/ependent pathway (4). Cisplatin has several dose-dent side effects, including renal, otologic, andmarrow suppression. Thus, a suboptimal level of
tin in combination with nontoxic agents would bel for effective treatment of HNSCC.
2665

10 American Association for Cancer Research.

http://mct.aacrjournals.org/

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cumin (diferuloylmethane) is the major componentspice turmeric and is derived from the rhizome ofst Indian plant Curcuma longa (Fig. 1A). It has beenmed as a dietary supplement for centuries and haseen shown to prevent tumor initiation, prolifera-nd metastasis in breast, colon, oral, and other hu-cancers (5). Curcumin is soluble only in organicts, but liposomal formulations have been studiedtreatment of pancreatic cancer (6).ivery of curcumin is limited by its poor bioavail-and insolubility in saline. Previous studies from

aboratory have shown that curcumin treatmentesses growth of HNSCC cell lines in vitro and re-tumor volume in vivo via topical application (7).recently, our laboratory has shown the use ofme-encapsulated curcumin for intravenous ad-tration of curcumin, and bioavailability studiesmed the presence of curcumin in circulatingand liver (8). Its growth-inhibitory effect was
ted through the inhibition of transcription factor tained
064). In comparison with the untreated control, the combination treatment of cuto that seen with 20 μmol/L cisplatin (P = 0.0011).

ancer Ther; 9(10) October 2010

on November 7, 2020. © 20mct.aacrjournals.org wnloaded from

yclooxygenase-2 (COX-2), interleukin-6 (IL-6), andukin-8 (IL-8) genes.he present study, we investigated whether curcu-ould enhance the suppressive effect of cisplatin inC. Two HNSCC cell lines, CAL27 and UM-SCC1,treated with curcumin and cisplatin individuallycombination. Nude mice with subcutaneous xeno-CAL27 tumors were administered with cisplatinor a combination of cisplatin and liposomal curcu-In addition, the mechanism of action of curcumin,iting transactivation of NFκB, mediated throughlasmic and nuclear IKKβ, was studied.

rials and Methods

inesSCC cell lines CAL27 and UM-SCC1, representingancers, were used. CAL27 was obtained from theican Type Culture Collection, and UM-SCC1 was ob-
from Dr. Thomas E. Carey (University of Michigan,
, resulting in the downregulation of IκBα, cyclin Ann Arbor, MI). CAL27 cells were characterized by the

1. Growth inhibition of HNSCC in vitro with cisplatin and curcumin. A, molecular structure of curcumin. Cell viability by MTT assay was done on(B) and UM-SCC1 (C) cells with cisplatin or curcumin alone or in combination. We compared the time trends (or slopes) among different groups, andimated slopes from the lowest to the highest in CAL27 are as follows: untreated control (0), empty liposomes (−13.89), cisplatin alone (−15.61),al curcumin (−20.80), and liposomal curcumin + cisplatin (−23.35). Estimated slope values in UM-SCC1 cells are as follows: untreated control (0),liposomes (−4.90), curcumin (−11.46), 10 μmol/L cisplatin (−15.52), curcumin + cisplatin (−22.83), and 20 μmol/L cisplatin (−26.79). The calculationsat empty liposomes themselves have a significant growth inhibition in CAL27 cells over time (i.e., slope of growth inhibition) compared with theed control (P = 0.0015). However, the estimated slope of growth inhibition is greater with liposomal curcumin treatment in comparison with theed control (P < 0.0001). There is a significantly greater effect in liposomal curcumin–treated cells in combination with cisplatin compared with cellswith cisplatin alone (P = 0.0152). Statistical significance was not observed for combination treatment of liposomal curcumin and cisplatin versuses and cisplatin. In the UM-SCC1 cells, liposomal effect is minimal and a significant growth inhibition over time is seen in the presence of curcumin

rcumin with 10 μmol/L cisplatin shows an effect (P = 0.0016)

Molecular Cancer Therapeutics



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ican Type Culture Collection using polyphasictypic and phenotypic) testing methods. UM-SCC1ere not characterizedbyour laboratory after receivinghe University of Michigan. Cell lines were grown inM containing high glucose (4,500 μg/mL) andol/L glutamine, 100 IU/mL penicillin, and 10% fetale serum (Sigma).

omal curcumin preparation:1 ratio of lipids 1,2-dimyristoyl-sn-glycero-3-phocholine and 1,2-dimyristoyl-sn-glycero-3-ho-rac-(1-glycerol) (Sigma-Aldrich) was dissolvedt-butanol at a concentration of 10 mg/mL. Sterile(1/20 volume) was added and 1 part curcuminy, 97%; Cayman Chemical) was added for a finalcurcumin ratio of 10:1. The solution was steriled, frozen in dry ice and acetone, and lyophilizedight. Liposomal curcumin was suspended in ster-% NaCl at 65°C to yield a 100 mmol/L stockon.

min and cisplatin treatment of HNSCCnesls were plated in 24-well plates (30,000 per well)llowed to grow for 24 hours. After serum starvationhours, cells were treated with cisplatin [10 μmol/Lμg/mL) or 20 μmol/L (i.e., 6 μg/mL)] for 5 hoursth liposomal curcumin (100 μmol/L) for 8 hours.ination treatment consisted of addition of cisplatinrs after the addition of liposomal curcumin. Un-d cells or those treated with empty liposomes(100 μmol/L) were used as controls. Cells wereallowed to incubate in serum-containing mediumC, and cell viability was measured at 24 hours.th assays were done at least twice and each timelicates.

iability assaywth medium was aspirated out of wells and 1.0 mLTT solution (1 mg/mL in complete medium;-Aldrich) was added to each well. Cells were thenated at 37°C for 4 hours. The MTT solution wasted out of the wells, air dried for 5 minutes,issolved in isopropanol, and absorbance valuesread in an ELISA microplate reader at 570 nmscribed (4).

ern blot analysisteins were extracted from cells or xenograft tumorsradioimmunoprecipitation assay lysis buffer con-g a complete protease inhibitor cocktail (Roche Di-tics). Western blotting was done using 20 to 30 μgatured proteins on 4% to 20% SDS acrylamide gelsrogen, Inc.). Proteins transferred onto nitrocellulosehybridized following the established protocol (7) totibodies (cyclin D1, phospho-IκBα, IκBα, histone
nd histone H3; Santa Cruz Biotechnology). Allrn blot analyses were done at least twice.
sis wtomet

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nofluorescencels were grown overnight on coverslips to semicon-e (70–80%) and treated with liposomal curcuminminutes, 1 hour, or 4 hours. Cells were then trea-ith tumor necrosis factor α (TNFα) for 15 minutes.ated cells and cells treated with empty liposomeshours were included as controls. Immunofluores-was done using the IKKβ, NFκB, phospho-AKT,hospho-IκBα antibodies (Santa Cruz Biotechnolo-llowing the established protocol (7). Confocal mi-py was done following established protocol usings microscope.

matin immunoprecipitation assay-SCC1 cells grown to 80% confluency were treatedTNFα for 1 hour and cross-linked with 37% form-yde for 10 minutes at 37°C. Chromatin immuno-pitation (ChIP) assays were done using ChIPkit (Millipore) following the manufacturer's pro-Immunoprecipitations were carried out using anti-s to IgG (Santa Cruz Biotechnology), IKKβ (Cellling), and NFκB (Calbiochem). DNA isolated fromput as well as the immunoprecipitated samplesamplified using IL-8 and β-actin primers (9). Theing primers were used in the PCR: IL-8, 5′-CCATCAGTTGCAAATC-3 ′ ( forward) andCCTTCCGGTGGTTTCTTC-3′ (reverse); β-actin,ACGCCAAAACTCTCCCTC-3′ (forward) and 5′-GCAAAGGCGAGGCTCTG-3′ (reverse). DNAsdenatured at 95°C for 1 minute, annealed atfor 1 minute, and extended at 72°C for 1 minutecycles. The PCR products were separated on

polyacrylamide gels and stained with ethidiumide, and images were captured using the Kodakocumentation system.

cence assayL27 cells grown to semiconfluence in six-wells were treated with l iposomal curcuminmol/L) for 8 hours or cisplatin (20 μmol/L) fors.Growthmediumwas replacedand cellsweregrownays. Cells were then stained with β-galactosidase,lue senescence colonies were visualized undericroscope. Photographs were taken using acamera.

min binding studiesL27 cells grown to semiconfluence in 100-mmdishestreated with liposomal curcumin (100 μmol/L) forrs. After medium replacement, cells were grownhours and proteins were extracted from total cell ly-Immunoprecipitation was done with IKKβ or phos-6K antibody, and the precipitate was washed fivewith the cell lysate buffer. Curcumin was extractedethyl acetate/methanol mixture and vacuum

, and tandem mass spectrometry (MS/MS) analy-
as carried out on a triple-quadrupole spectropho-er as described (8). Curcumin peak identification
Mol Cancer Ther; 9(10) October 2010 2667



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ntensity measurement was determined using aard curcumin run as a control. Untreated cellsells treated with liposome alone were used asive controls.

C xenograft tumors in micee-week-old female athymic nude mice (nu/nu;n) were used for in vivo experiments. Animals wered with 2 × 106 cells in the right flank to form xeno-tumors. Animals were housed in sterile rodent mi-lator caging, with filtered cage top. Two to fourls were housed in each cage, with animals restingly on bedding. They were given free access to sterileand food. All cages, covers, and bedding were ster-weekly. All animal procedures were approved bystitutional Animal Care and Use Committee ofest Los Angeles Veterans Affairs Medical Center,ordance with the USPHS Policy on Humane Carese of Laboratory Animals.

ment of nude mice with liposomal curcuminisplatine subcutaneous nodules were visible at the inocu-site (days 7–10 after inoculation), liposomal curcu-50 mg/kg, 1 mg for the 20-g mouse in a maximale of 100 μL) or equal amount of empty liposomesdministered via tail vein injection three times afor 4 weeks. In the fourth week, cisplatin (100 μLμg/mL) was administered through intraperitonealon. There were four groups in the study: mice withatment (nine), mice treated with cisplatin (seven),treated with empty liposomes and cisplatin (five),ice treated with liposomal curcumin and cisplatinTumor size was measured weekly using Vernier ca-, and tumor volume was calculated using the for-V = 4/3pW2L, where W is half of the shorter axister and L is half of the longer axis diameter as de-d (10). At the end of the fourth week, mice wereced and tumors were removed.

tical analysisthe MTT absorbance, cell viability was set to beat baseline, and the percent of viable cells [(viableount in the experiment group/mean viable cell) × 100] per replicate was calculated at each of theeatment days. To examine the differences in thetion of cell viability over time among groups,VA modeling was used with the following covari-roup, time (measured in days), and a group-by-timection term. The model also allows heterogeneousce across groups.e trends in tumor volume among experimentals were examined using the piecewise regressionl, which allows for the trend after the injectionplatin at week 3 to be different from the trende injection of cisplatin. Although mice were
ed over time, their IDs were not recorded duehnical difficulties. Thus, the common repeated-
curcuand p



res model cannot be used in this situation. In-, the piecewise regression model was used, withssumption of heterogeneous variance acrosss to analyze tumor volume growth. All statisticalses were carried out with the SAS System forows (version 9.2)

lts

th inhibition of HNSCC cell lines in vitro withmal curcumin and cisplatin

o HNSCC cell lines, CAL27 and UM-SCC1, repre-g aggressive oral cancers were tested for growthtion with cisplatin or liposomal curcumin alone orbination. All experiments were done in triplicate

well plates, and viable cell counts were measured48, 72, and 96 hours after treatment. Empty lipo-, curcumin alone, and cisplatin alone had a sig-ntly greater growth inhibition in CAL27 cellstime (i.e., slope of growth inhibition) comparedthe untreated control (P = 0.0015, <0.0001, and01, respectively; Fig. 1B and C). There was signif-y greater effect in liposomal curcumin–treatedin combination with cisplatin compared with cis-alone–treated cells (P = 0.0152). Statistical signif-was not observed for combination treatment ofmal curcumin and cisplatin versus liposomesisplatin. In UM-SCC1 cells, liposomes alone didow an inhibitory effect. Significant growth inhibi-ver time was seen in the presence of cisplatin0.0486 and 0.0011, for 10 and 20 μmol/L, respec-), curcumin alone (P = 0.0064), curcumin in com-on with 10 μmol/L cisplatin (P = 0.0016), andmes in combination with 10 μmol/L cisplatin0 .0247 ) compared wi th cont ro l . We havebserved a significantly greater effect over time inmin-treated cells compared with cells treated with li-es (P = 0.0011). The combination treatment, liposo-

urcumin and 10 μmol/L cisplatin, showed an effectr to that seen in cells treatedwith 20μmol/L cisplatin.

ced expression of NFκB-activated genes inmin and cisplatin-treated cellshave previously shown that curcumin and cisplatinregulate the expression of genes involved in cell cy-d apoptosis and also that cisplatin induces cellularcence through the upregulation of p16 and p53 tu-uppressor genes (4, 7). Our studies have also shownhe effect of curcumin is mediated by the inhibitionβ kinase, resulting in reduced phosphorylation of

(inhibitor of NFκB) and retention of NFκB in the cy-m (11). Thus, cisplatin and curcumin seem to sup-cell growth through independent mechanisms.verify that the two agents act via different mech-s, we did expression analysis of proteins collectedthe drug-treated cell lines. Both cisplatin and
min showed reduced expression of cyclin D1hospho-IκBα in CAL27 and UM-SCC1 cell lines



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Figurewith cisanalyzeBecausthe expcisplatitherapywith cisphosphin phossenescence-mediated cell death in cisplatin-treated CAL27 cells. Magnification, ×100.


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2A). Because our previous studies have shown-level expression of these two proteins in CAL27in the present experiments, the cells wereated with TNFα for 15 minutes before the addi-of drugs. A higher concentration of cisplatinol/L) showed greater reduction in the expression

clin D1 and phospho-IκBα in comparison withent with 10 μmol/L cisplatin. In addition, com-treatment with 10 μmol/L cisplatin and liposomalin resulted in greater inhibition, equaling that of
ent with 20 μmol/L cisplatin. Empty liposomesot affect the expression of these two proteins. West-
withempty

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lot analysis showing reduced expression of IκBαKKβ in cisplatin (20 μmol/L)– and liposomal cur-(100 μmol/L)–treated CAL27 cells (Fig. 2B) fur-

ndicated a possible direct effect on the expressionse two proteins by cisplatin and curcumin.confirm further that curcumin inhibited phospho-expression, immunofluorescence was done

AL27 cells treated with liposomes or liposomalmin. Phospho-IκBα expression was mostly seencytoplasm, and the expression level increased

2. Inhibition of NFκB-regulated genes by cisplatin and curcumin. A, total protein lysate extracted from CAL27 and UM-SCC1 cell lines treatedplatin (10 or 20 μmol/L) or liposomal curcumin (100 μmol/L) or a combination of cisplatin (10 μmol/L) and liposomal curcumin (100 μmol/L) wasd by PAGE. Untreated cells and those treated with empty liposomes (100 μmol/L) or empty liposomes and cisplatin (10 μmol/L) served as controls.e CAL27 cells expressed lower level of cyclin D1, cells were also pretreated with TNFα. Empty liposomes do not show any inhibitory effect onression of cyclin D1 or phospho-IκBα. However, reduced protein expression is observed with cisplatin or curcumin. The inhibitory effect ofn-curcumin combination reaches that of 20 μmol/L cisplatin, indicating usefulness of the nontoxic curcumin as a chemotherapeutic drug for adjuvant. B, hybridization of CAL27 total protein lysates to IκBα and IKKβ antibodies shows decreased expression of these two proteins in cells treatedplatin (10 μmol/L) or liposomal curcumin (100 μmol/L). C, immunofluorescence analysis of CAL27 cells with TNFα shows increased expression ofo-IκBα, indicating enhanced phosphorylation through IKKβ. This expression is not affected by the addition of liposomes. However, a decreasepho-IκBα expression level is visualized with the addition of curcumin. D, β-galactosidase assay shows increased blue staining, indicating

the addition of TNFα (Fig. 2C). Treatment withliposomes resulted in increased localization of




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rotein to the perinuclear region. However, curcu-reatment led to reduced protein expression in thelasm and in the perinucleus of the cells, confirminghibitory activity of curcumin on IKK kinase, result-decreased phosphorylation of IκBα.

show that cisplatin inhibits cell growth through aence pathway, CAL27 cells treated with liposomalmin or cisplatin were stained for the expression of β-osidase, a marker for cellular senescence. Untreatedontained a low-level background blue staining duedeath from confluent cell culture in 4 days of studycol (Fig. 2D). Although there was cell killing inmin-treated cells, therewas no observable blue stain,ting a senescence-independent cell death by liposo-rcumin. As expected, cisplatin-treated cells showed-level expression of β-galactosidase, correlating toence-mediated cell death of HNSCC cells.

min binds to IKKβ protein subunit of thecomplexause we have previously shown that curcumin in-d IKK kinase activity (11), we did MS/MS analysisermine whether there was a direct binding of cur-to IKKβ protein. Cell lysates of CAL27 treated

curcumin were immunoprecipitated with IKKβ ortrol phospho-S6K antibody, and the antibody-products were extracted with organic solvents.

sis by MS showed the presence of curcumin peakcell lysates. Intensity measurements using a curcu-tandard showed a 5-fold increased binding of cur-n to IKKβ in comparison with phospho-S6Klementary Table S1), indicating an interaction be-curcumin and IKKβ.

ar expression of IKKβ protein in HNSCCneserify further whether curcumin binding resulted inhibition of IKKβ expression as was seen in Westerng, confocal microscopy was done. We observed ex-on of IKKβ both in the cytoplasm and in the nucleusL27 and UM-SCC1 cell lines (Fig. 3A). Although ther presence of the IKKα protein has been shown be-2), there is only one recent report indicating the ex-on of IKKβ protein in the nucleus of normal andastic B lymphocytes (9). Thus, for the first time, weeen IKKβ expression in the nucleus of HNSCC cellTo confirm this finding, we used two different IKKβodies and HeLa cells, a cervical cancer cell linen before to contain cytoplasmic IKKβ expression.ssion of IKKβ in HeLa cells was seen in the cyto-as well as in the nucleus of the cells (Fig. 3A). Cost-with 4′,6-diamidino-2-phenylindole (DAPI; left

n), a nuclear-specific stain, showed magenta stain-the nucleus (last column in Fig. 3A), confirming nu-xpression of IKKβ in HeLa and HNSCC cell lines.eas treatment with liposomes did not have an effect
expression of IKKβ, liposomal curcumin treatmented in an inhibitory effect both in the cytoplasmic
hibiteHNSC



the nuclear expression of this protein (last row inA). As a confirmation to cytoplasmic and nuclearzation of IKKβ, we did confocal microscopy forar-specific proteins histones H1 and H3. The re-showed complete localization of histone H1 toucleus (Fig. 3B). Expression of histone H3 wass speckles inside the nucleus as wells in the peri-ar/cytoplasmic region (Fig. 3B). Western blot hy-ation studies of the cytoplasmic and nuclearts of CAL27 and HeLa cells further confirmedresence of IKKβ in the nucleus of these two cell(Fig. 3C). Immunoprecipitation with nuclear IKKβCAL27 cells and hybridization to histone H3 an-showed binding of histone H3, indicating a role

uclear IKKβ in chromatin remodeling (Fig. 3C).was also hybridization to NFκB in these immu-ts, indicating an interaction between NFκB andproteins in the cytoplasm as well as in the nucle-ese interactions were confirmed by the hybridiza-f IKKβ antibody to the immunoprecipitates oflasmic and nuclear NFκB (Fig. 3C). Finally, ChIPshowed amplification of IL-8 promoter sequencesbinding site) in the DNA isolated from the IKKβdy immunoprecipitated samples, indicating an in-ion between IKKβ and NFκB (Fig. 3D). IL-8 pro-r amplification was not seen with the IgGdy precipitated samples used as a control. Thereo amplification of β-actin promoter sequences inf the immunoprecipitated samples again used as al in the ChIP assay.verify the effect of curcumin on nuclear IKKβ andFκB, liposome- and liposomal curcumin–treated7 cells were studied by immunofluorescence. Ex-ion was also measured with the addition of TNFα,B upregulating molecule. IKKβ expression wasin the nucleus, and there was a minimal effectthe addition of TNFα (Fig. 4A). Treatment withme did not alter the expression. However, re-expression was observed in as little as 30 min-fter curcumin treatment. The presence of IKKβcleosome-like particles again showed a possiblef this protein in chromatin remodeling. Expres-f NFκB was seen in the cytoplasm, and increasedar transport occurred with the addition of TNFα4B). NFκB expression in liposome-treated cells re-led that of control cells. Reduction in the expres-f NFκB could be seen in curcumin-treated cells.ver, the inhibitory effect was pronounced withockage of NFκB transport in the presence of TNFα,ming the inhibitory effect of curcumin on the tran-ional activation of NFκB. Here, the results clearlythe effect of curcumin on cytoplasmic IKKβ, lead-the inhibition of IKKβ kinase activity. Finally,

d not see an inhibitory effect on phospho-AKT ex-ion in the presence or absence of TNFα (Fig. 4C),rming our previous finding that curcumin in-
d NFκB by an AKT-independent mechanism inC cells.



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nced growth inhibition of in vivo xenograftrs with combination (liposomal curcuminisplatin) therapy

w the PCR product, confirming the specificity of the ChIP assay.

L27 xenograft tumors were grown in nude mice,umor dimensions were measured weekly with

timesalso r

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rs. Once it was evident that xenograft tumorsforming (7 days), liposomes and liposomal cur-n in saline were injected via the tail vein three

3. Nuclear expression of IKKβ in HNSCC cell lines. A, confocal microscopy with a Zeiss microscopic system done on HeLa, CAL27, and UM-SCC1ows cytoplasmic and nuclear localization of IKKβ in all three cell lines. However, nuclear expression is more prominent in HeLa and UM-SCC1s. Costaining with DAPI shows magenta staining of the nuclei, confirming nuclear localization of IKKβ in all three cell lines. Cytoplasmic stainingvisualized in these photographs. Whereas treatment with liposomes does not affect the expression of IKKβ in CAL27 cells, treatment with liposomalin results in reduced expression of the protein in the nucleus and cytoplasm (last row). Arrows point to nuclear magenta staining indicatinging of DAPI and IKKβ. Magnification, ×63. B, confocal microscopy on HeLa cells shows nuclear expression of histones H1 and H3. Costaining withdicates the presence of a small fraction of histone H3 in the perinuclear/cytoplasmic region. Speckle-like staining of histone H3 could indicatetion to nucleosomes involved in chromatin remodeling. C, Western blot analysis of cytoplasmic and nuclear fractions of CAL27 and HeLa cells withows the presence of IKKβ in both the cell compartments. Whereas histones H1 and H3 and lamin A/C used as controls represent proteins ofleus, β-tubulin is present both in the cytoplasm and in the nucleus. Immunoprecipitation with IKKβ antibody followed by Western blot analysis showsinant binding of nuclear histone H3 to IKKβ, indicating a role for IKKβ in chromatin remodeling. Minor hybridization seen in the cytoplasmiccould represent hybridization to the cytoplasmic H3 protein. Binding of nuclear IKKβ to NFκB is also seen in the immunoblots. The cytoplasmic IKKβo NFκB and IκBα, confirming its role in the phosphorylation and ubiquitination of IκBα. The interaction between NFκB and IKKβ in the nucleusoplasm is confirmed by the immunoprecipitation of proteins with NFκB antibody followed by hybridization to IKKβ. D, ChIP assay done on DNAfrom IKKβ and NFκB immunoprecipitated samples shows a PCR product of 182 bp for IL-8 promoter sequences. PCR product is not seen in the

a week for 4 weeks. In the fourth week, miceeceived cisplatin by intraperitoneal injection. A




slowetreatefindinmodea greslopecombcisplaof gro0.109depicnumbuppeoverright)

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r tumor growth rate of liposomal curcumin–d mice for the first 3 weeks confirmed our earliergs (8). Results from the piecewise regressionl indicated that in comparison with the control,ater inhibitory effect over time (i.e., estimatedof growth) was seen with the curcumin-cisplatinination treatment before and after receivingtin. However, the estimated difference in slopeswth did not reach statistical significance (P =

8). A box plot is a convenient way of graphicallyting groups of numerical data through their five-er summaries (minimum, lower quartile, median,r quartile, and maximum). The tumor volume

α. C, the absence of an effect on phospho-AKT points to an AKT-indepenexpression. Magnification, ×100.

the first 3 weeks for cisplatin alone (Fig. 5B, topand curcumin-cisplatin (Fig. 5B, bottom right)

tumoment



ot go beyond 100 mm (Fig. 5B, dashed lines),t one observation at week 3 in the cisplatin alonep. In addition, a smaller variation in tumore is seen 1 week after the injection of cisplatin

k 4) for these two groups. This analysis agained growth inhibition of xenograft tumors in theination treatment in comparison with cisplatinor the controls (Fig. 5B). The tumor size shown. 5C showed growth inhibition with cisplatinand an enhanced growth reduction with the in-n of curcumin in the combination treatment.rn blot analysis showed a marginal inhibitory ef-n the expression of cyclin D1 in cisplatin-treated

ownregulation of NFκB by curcumin. Arrows indicate cells with

4. Confirmation of IKKβ protein expression in the nucleus. A, immunofluorescence studies show inhibition of nuclear IKKβ with a 30-minin treatment and the inhibition is greater for longer treatments. The speckle-like particles possibly represent nucleosomes involved in chromatinling. B, NFκB is mostly localized to the cytoplasm and is transported to the nucleus with the addition of TNFα. Whereas there is no difference inxpression in liposome-treated cells, curcumin-treated cells show inhibition of nuclear transport, and this inhibition is clearly visible after the addition

rs (Fig. 5D). However, liposomal curcumin treat-in combination with cisplatin resulted in a




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ed decrease in cyclin D1 expression correlating tohibitory effect on tumor growth.

ssion

mechanism of action of cisplatin includes cell cycleand initiation of apoptosis (13). We and others havethat cisplatin induces cellular senescence through

tion of p53 and p16 proteins, and there is strong ev-that p53 plays a role in cisplatin sensitivity. It alsothat cisplatin-induced growth arrest in human can-lls has characteristics of senescence in addition toosis (14). In the present investigation, we usedC cell lines containing mutated p53 and lackingpression. The senescence-associated β-galactosidasey was seen in the CAL27 cells following cisplatin ex-e, suggesting that growth of cancer cells containing
ed p53 function can also be inhibited by cisplatin (8, 16
1 expression in cisplatin-treated tumors in comparison with the untreated contrin shows significant reduction in the expression of cyclin D1, correlating to tumo

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ts further suggest that functional expression ofugments the effect of cisplatin in cell killing. Be-p16 and p53 are functionally active in the nucleus,kely that cisplatin plays an essential role in the nu-transport and stabilization of these proteins for cellarrest and apoptosis. We have shown here that inesence of cisplatin, there is reduced expression of-regulated proteins cyclin D1 and phospho-IκBα,by indicating control of NFκB transactivation bytin through the nuclear p53 protein.or cells evade apoptosis by downregulation of

totic proteins and upregulation of antiapoptoticns. Expression of growth signaling pathways givessurvival advantage and allows them to resist therapy-ed apoptosis (15). We and others have shown thatmin-mediated head and neck cancer cell growthtion occurs through the inhibition of NFκB activity
–18). Recently, we have reported that the inhibitory
clear transportation of p53 protein (4). Previous effect of curcumin on NFκB is by the inhibition of IKK

5. Inhibition of CAL27 mouse xenograft tumors with cisplatin or cisplatin-curcumin combination. Mice were treated with empty liposomes oral curcumin for 3 wk after the appearance of tumor nodules. Intraperitoneal injection of cisplatin was administered on the fourth week, and a weekmors were excised. A, tumor volume was calculated using the method described in Materials and Methods. Compared with control, the resultsmor growth inhibition with cisplatin treatment. A greater inhibitory effect was seen with the curcumin-cisplatin combination treatment before andceiving the cisplatin. However, the estimated difference in slopes of growth between the curcumin-cisplatin combination and control did nottatistical significance (P = 0.1098). B, a box plot is a convenient way of graphically depicting groups of numerical data through their five-numberries (minimum, lower quartile, median, upper quartile, and maximum). The analysis shows reduced growth of the xenograft tumors in the combinationnt in comparison with other groups. C, representative tumors show reduced growth with cisplatin treatment and greater tumor growth inhibitioncisplatin-curcumin combination treatment. D, Western blot analysis of proteins isolated from the xenograft tumors shows a marginal reduction in

ols. However, treatment with the combination of cisplatin andr size reduction in the combination treatment.




kinasthat thtweenhavefunctiof NFincludproteiTra

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Duarte et al.

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e (11). In the present investigation, we have shownis inhibition could be due to direct interaction be-curcumin and IKKβ. Further, for the first time, we

shown the presence of IKKβ in the nucleus whoseon seems to be related to transcriptional activationκB by chromatin remodeling. Additional studiesing ChIP assay will be required to determine thens recruited by IKKβ for this NFκB transactivation.nscription factor NFκB and the serine/threoninee AKT play critical roles in cancer cell survivalave been shown to be activated in various malig-s (19, 20). Some have shown that curcumin acts viaT-dependent pathway in cell growth regulation (21,evious studies in our laboratory revealed that liposo-rcumin treatment suppressed the activation of NFκBut affecting the expression of phospho-AKT or itsstream target phospho-S6 kinase (8). Here, we havemedusing immunofluorescence that curcumin affectsexpression via an AKT-independent mechanism.ent investigative trials include gene-targeted therapiesSCC, including cetuximab, bevacizumab, and erloti-argeted against epidermal growth factor receptor; refs. 23–27).Althoughanti-EGFR therapies are activeepatients, eventuallydisease innearly all patientswille refractory to therapy (28). Mutations in the EGFRor seem to play a significant role in the resistance to

6. Mechanism of action of curcumin and cisplatin. The available data sugge. Inhibition of NFκB seems to occur through the downregulation of IKKβ by c

therapy. Thus, efforts are under way to identify alter-erapies, including the use of curcumin in combination

We tZhao fo



adiation and/or chemotherapeutic drugs in hepaticoma, ovarian carcinoma, and HNSCC (29–31).onclusion, we have used the combination of curcu-nd cisplatin to show enhanced growth suppressionSCC. We believe that this occurs through two dif-t molecular pathways. Curcumin affects NFκBctivation by inhibiting IKK kinase activity both intoplasm and in the nucleus, possibly through an in-ion with IKKβ (Fig. 6). We hypothesize that nuclearin combination with histone H3 is involved in chro-remodeling of the NFκB transcription binding sites.tion of IKKβ by curcumin therefore results in reduc-NFκB-occupied sites in chromatin, leading to a de-

e in NFκB-mediated transcription. Cisplatin, inst, downregulates NFκB through a p53-mediateday. We therefore believe that the therapeutic poten-cisplatin will be enhanced with the addition of cur-, with lower, less toxic doses of cisplatin requiredcytotoxic effect.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

B activation to be inhibited through a p53-directed mechanism byin. Histone H3 (H3) bound to IKKβ, forming a nucleosomal complex.

hank Dr. Yasutada Akiba for confocal microscopy and Dr. Ke Weir help in acquiring photographic images.




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diated NF-κB activation and proinflammatory gene expression byibiting inhibitory factor I-κB kinase activity. J Immunol 1999;163:4–83.

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costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedement in accordance with 18 U.S.C. Section 1734 solely to indicatet.

ived 01/27/2010; revised 08/04/2010; accepted 08/08/2010;
21 CA116826-01 (M.B. Wang); gton, DC) Merit grant (E.S. Srivatsan). published OnlineFirst 10/11/2010.
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2010;9:2665-2675. Mol Cancer Ther Victor M. Duarte, Eugene Han, Mysore S. Veena, et al.

B Pathwayκ Protein of the NFβHead and Neck Squamous Cell Carcinoma via Inhibition of IKKCurcumin Enhances the Effect of Cisplatin in Suppression of

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