Silibinin Prevents Lung Tumorigenesis in Wild-Type but not in … · Cancer Therapy: Preclinical...

Cancer Therapy: Preclinical

Silibinin Prevents Lung Tumorigenesis in Wild-Type but not iniNOS�/� Mice: Potential of Real-Time Micro-CT in Lung CancerChemoprevention Studies

Kumaraguruparan Ramasamy1, Lori D. Dwyer-Nield1, Natalie J. Serkova2, Kendra M. Hasebroock2,Alpna Tyagi1, Komal Raina1, Rana P. Singh1,3, Alvin M. Malkinson1, and Rajesh Agarwal1

AbstractPurpose: Sustained nitric oxide (NO) generation positively correlates with lung cancer development

and progression. Herein, we genetically confirmed this role of iNOS and evaluated the chemopreventive

efficacy of silibinin in carcinogen-treated B6/129 wild-type (WT) and iNOS�/� mice.

Experimental Design: Male B6/129-Nos2tm1Lau (iNOS�/�) and B6/129PF2 WT mice were injected i.p.

with 1 mg/g body weight urethane once weekly for 7 consecutive weeks, followed by silibinin gavage

(742 mg/kg body weight) for 5 d/wk for 18 weeks.

Results: Quantification of micro-CT data in real-time showed that silibinin significantly decreases

urethane-induced tumor number and size inWTmice, consistent with measurements made ex vivo at study

termination. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 87% (P < 0.001)

compared to WT mice. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in WT mice, but did

not show any such considerable effect in iNOS�/� mice. Tumors from WT mice expressed more iNOS

(P < 0.01) but almost similar eNOS and nNOS than those in silibinin-treated mice. In these tumors,

silibinin moderately (P < 0.01) inhibited cell proliferation but strongly (P < 0.01) reduced the number of

newly formed nestin-positive microvessels. Silibinin decreased VEGFR2 level, and STAT3 and NF-kBactivation in tumors.

Conclusions: The lack of effect of silibinin in iNOS�/� mice suggests that silibinin exerts most of its

chemopreventive and angiopreventive effects through its inhibition of iNOS expression in lung tumors.

Our results support iNOS as a potential target for controlling lung cancer, and demonstrate the value of

real-time noninvasive micro-CT imaging modality for evaluating the efficacy of lung cancer chemopre-

ventive agents. Clin Cancer Res; 17(4); 753–61. �2010 AACR.

Introduction

Lung cancer is the leading cause of cancer-related deathsin the United States. Lung cancer patients are usuallydiagnosed at a late stage when prognosis for most patientsremains poor, with a 5-year survival rate of less than 16%(1). There has been little improvement in the efficacy oflung cancer treatments in recent decades, indicating theneed for alternative strategies to help control this disease(2, 3). One way to reduce the risk of this disease is bychemoprevention using phytochemicals administration tohigh-risk populations. A deeper molecular understanding

of the events regulating tumor progression in model organ-isms should enhance development of novel chemopreven-tive and therapeutic approaches to this problem.

The flavolignan, silibinin, is a major constituent in sily-marin, an extract of milk thistle (Silybum marianum).Recently, silibinin received significant attention for itsstrong chemopreventive and anticancer efficacy. Silibinininhibited growth in cancer models of skin (4, 5), prostate(6–9), bladder (10), and colon (11). Silibinin inhibitsmultiple cytokine-induced signaling pathways that regulateinducible nitric oxide synthase (iNOS) expression in A549cells, and inhibited the in vivo growth of A549 xenografts,and reduced the systemic toxicity of doxorubicin in thesestudies (12, 13). Dietary silibinin decreased cell prolifera-tion and angiogenesis by targeting iNOS in a urethane-induced lung tumorigenesis model (14). It has also shownchemotherapeutic efficacy in urethane-induced, establishedlung adenocarcinomas where it also inhibited angiogenesis(15). Silibinin also reduced proliferation of human non–small cell lung carcinoma (NSCLC) H1299, H460, andH322 cells by targeting cell cycle associated proteins (16).

Silibinin antiangiogenic efficacy may be mediatedthrough nitric oxide (NO) signaling, which plays a major

Authors' Affiliations: Departments of 1Pharmaceutical Sciences and2Anesthesiology and Radiology, University of Colorado Cancer Center,University of Colorado Denver, Aurora, Colorado; and 3Cancer BiologyLaboratory, School of Life Sciences, Jawaharlal Nehru University, NewDelhi, India

Corresponding Author: Rajesh Agarwal, Department of PharmaceuticalSciences, University of Colorado Cancer Center, University of ColoradoDenver, Aurora, CO. Phone: 303-724-4055, Fax: 303-724-7266; E-mail:[email protected].

doi: 10.1158/1078-0432.CCR-10-2290

�2010 American Association for Cancer Research.

ClinicalCancer

Research

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role in tumor angiogenesis. NO is a free radical thatregulates diverse physiologic and pathologic events. Sus-tained and/or excess NO generation occurs during lungcancer development and progression (17). NO is synthe-sized by three major isoforms of NO synthase (NOS),constitutively expressed endothelial (eNOS), neuronal(nNOS) NOS and inducible NOS (iNOS). Of these iso-zymes, iNOS produces the most NO (18). More iNOS isexpressed in lung tumors than in surrounding normaltissue, and iNOS levels are high in alveolar and tumor-associated macrophages, pulmonary endothelium, airwayepithelium, and human AC (19). iNOS expression/activitycorrelates with angiogenic status and metastatic potentialin a wide range of tumors, and iNOS inhibition by naturaland synthetic compounds has been efficacious in cancerchemoprevention studies (20, 21). Previously we hypothe-sized that dietary silibinin mediates its antiangiogeniceffects by targeting iNOS in urethane-induced A/J mouselung tumorigenesis (14).

Herein, we investigate the chemopreventive efficacy ofsilibinin on urethane-induced lung tumorigenesis in termsof tumor growth and progression in B6/129 wild-type(WT) mice and their iNOS�/� counterparts. We hypothe-sized that if lung cancer development is regulated by NOproduction, silibinin should, at least in part, exert itschemopreventive effects through iNOS inhibition. Thefindings in this study supported this hypothesis, and alsohighlighted, for the first time, the usefulness of microcom-puted tomography (micro-CT), a noninvasive imagingtechnique, tomonitor real-time progression of lung tumorsin evaluating the efficacy of silibinin.

Materials and Methods

AnimalsMale B6/129-Nos2tm1Lau (iNOS�/�) and B6/129PF2 WT

mice (5–6 weeks of age), from Jackson Laboratory wereinjected i.p. with 1 mg/g bodyweight urethane onceweeklyfor 7 consecutive weeks (22). Eight weeks after the initialurethane injection, mice were randomly divided into twogroups and gavaged with either 0.2 mL vehicle [0.5% (w/v)carboxy methyl cellulose and 0.025% Tween 20 in distilledwater] or silibinin (742 mg/kg bodyweight) in vehicle for 5d/wk for 18 weeks. This dose of silibinin corresponds to 1%(w/w) of silibinin in diet (14) and has been used in ourprevious study (15). Animal care and experiments wereconducted in accordance with an IACUC-approved proto-col at University of Colorado Denver. Twenty-five weeksafter the first urethane injection, mice were euthanized,tumors enumerated, and tumor diameters measured usingdigital calipers. Lungs from 4 mice/group were perfused,formalin-fixed, and paraffin-embedded for histologic andimmunohistochemical analyses.

Noninvasive micro-CT imagingAfter the final urethane injection, a subset (n¼ 6) ofmice

from control and silibinin-treated groups underwent4 monthly CT scans (at 0, 4, 8, and 12 weeks of silibinintreatment) for longitudinal assessment of number anddiameter of lung lesions. Mice were anesthetized with80 mg/kg ketamine and 12 mg/kg xylazine mixture(i.p.), placed on a warming pad, and inserted into a Sie-mens Inveon micro-CT scanner (Siemens Preclinical Solu-tions). A single 3-dimensional (3D) micro-CT image setwas acquired for each mouse using following parameters:270� rotation; 240 rotation steps; CCD read-out of 2,304/2,048; 4 binnings for matrix size reduction; exposure timeof 30mswith 80 kV voltage and 450 mA current; with a fieldof view (FOV) of 34.68 � 30.83 mm. The 6-minuteacquisition with middle-to-high magnification resultedin effective isotropic resolution of 60 mm (after Shepp-Logan reconstruction algorithm). Animals were monitoredduring recovery from the anesthesia and returned to theircages. CT image reading was performed by an imagespecialist (NJS) who was blinded to animal group assign-ments. All postprocessing analysis was performed using ahigh-speed CT reconstruction system (COBRA, SiemensPreclinical Solutions) and AsiPro image software for totallesion counts. Maximal diameters of each lesion were alsodetermined for each anatomical direction (transaxial, cor-onal, and saggital) andmean diameter determined. Motionartifacts were reduced by restraining anesthetized mice.

Immunohistochemical analysis and quantification ofprotein expression

Serial tissue sections (5 mm) were sliced from paraffin-embedded formalin-fixed lungs and immunohistochem-ical staining performed as described previously (15).Briefly, tissue sections were deparaffinized, hydrated, and

Translational Relevance

Overexpression of inducible nitric oxide synthase(iNOS) and sustained nitric oxide (NO) generationpositively correlate with lung cancer developmentand progression. This study genetically confirmed thisrole of iNOS using urethane-induced lung tumorigen-esis in B6/129-Nos2tm1Lau (iNOS�/�) and B6/129PF2WTmice. This study also showed that silibinin, a cancerchemopreventive agent, decreases lung tumor multi-plicity by 71% (P < 0.01) in WT mice, but failed tohave such efficacy in iNOS�/� mice, suggesting iNOS asa potential chemopreventive target during lung carci-nogenesis. The findings also showed that silibinin exertsits chemopreventive and angiopreventive effects mostlythrough its inhibition of iNOS expression involvingdownregulation of STAT3 and NF-kB signaling, whichotherwise are up-regulated in lung tumors. Overall,these results support iNOS as a potential target forcontrolling lung cancer by silibinin, and demonstratethe application of real-time noninvasive micro-CT ima-ging for evaluation of the efficacy of lung cancer che-mopreventive agents.

Ramasamy et al.

Clin Cancer Res; 17(4) February 15, 2011 Clinical Cancer Research754




stained using specific primary antibodies, biotin-conju-gated secondary antibodies, and horse radish peroxidase(HRP)-conjugated avidin. Specific antibody interactionswere detected with the HRP substrate 3, 30-diaminobenzi-dine (DAB). Primary antibodies used were mouse mono-clonal anti-PCNA (1:250, Dako), nestin (1:100 dilution;Abcam) rabbit polyclonal anti-eNOS (1:100 dilution;Abcam) nNOS (1:100 dilution; Abcam), iNOS (1:100dilution; Abcam), pSTAT3 (Ser727; 1:200 dilution;Abcam), and p65NF-kB (Ser276; 1:100 dilution; Abcam)followed by appropriate secondary antibodies (VectorLaboratories). PCNA, pSTAT3, and p65/NF-kB-positivecells were quantified by counting the number of brown-stained nuclei/total number of cells in 5 randomly selected400� magnified fields. eNOS, nNOS, and iNOS werequantified by immunoreactivity (represented by intensityof brown staining) and scored as 0 (no staining), þ1 (veryweak), þ2 (weak), þ3 (moderate), and þ4 (strong) at5 randomly selected 400�magnified fields. Newly formednestin-positive microvessels were quantified as mean num-ber of positive vessels in 5 randomly selected 400� mag-nified fields per tumor. All microscopic, histologic, andIHC analyses were performed with a Zeiss Axioskop2 microscope (Carl Zeiss, Inc.), and photomicrographswere captured with a Carl Zeiss AxioCam MRC5 camera.

Immunoblot analysisRandomly selected frozen tumor samples from four

individual mice from WT urethane and silibinin-treatedgroups were homogenized and lysates prepared as reportedpreviously (15). Equal protein per lysate was denaturedwith 2� sample buffer and resolved on Tris-glycine gels,transferred onto nitrocellulose membrane, and blocked for1 hour with 5% nonfat dry milk. Membranes were incu-bated with specific primary antibodies including anti-iNOS(BD BioSciences), anti-nestin and anti-VEGFR2 (SantaCruz Biotechnology), followed by peroxidase-conjugatedappropriate secondary antibody. These antibodies forimmunoblot analyses provide better results. Finally, pro-teins were visualized by enhanced chemiluminescencedetection. To confirm equal protein loading, membraneswere stripped and reprobed with mouse monoclonal anti–b-actin primary antibody (Sigma) in each case.

Statistical analysisStatistical analyses were performed using SigmaStat soft-

ware version 3.5 (Jandel Scientific). Quantitative data arepresented as mean � SEM. Statistical significance of differ-ences between control and silibinin-treated groups wasdetermined by an unpaired Student’s t-test, with P < 0.05considered statistically significant.

Results

Imaging analysis of lung tumorsImaging technology has been used to monitor lung

tumor progression and evaluate the efficacy of preventive/therapeutic approaches. Micro-CT provides 3D represen-

tations of high-density tissue structures needed to moni-tor therapeutic effects over time. This optimizes treatmentintervals and reduces the number of animals needed toget reliable results (23). Micro-CT is a preferred imagingmodality of choice for lung lesions. Magnetic resonanceimaging (MRI) exhibits low-signal intensity and suscept-ibility artifacts in lung (24). We monitored growth inhi-bitory effects of silibinin on urethane-induced lungtumorigenesis by subjecting control and silibinin-treated,tumor-bearing WT mice to micro-CT scanning at 4-weekintervals. Micro-CT images clearly distinguished lungtumors from surrounding tissue even without any con-trast agent, and the reconstructed 3D pulmonary imageseasily differentiated tumors from blood vessels (Fig. 1A).WT animals developed an average of 5, 7, and 9 tumors at4, 8, and 12 weeks, respectively, whereas silibinin-treatedanimals developed only 2, 3, and 3 tumors, respectively,at the same time points (Fig. 1B). Average tumor diameterincreased from 0.5 to 2 mm in control animals graduallyover 12 weeks. Silibinin treatment significantly reducedtumor diameter by 43% (P < 0.02) and 72% (P < 0.005)at the 8th and 12th weeks compared to correspondingcontrols, respectively (Fig. 1C). These results directlycorrelate with our ex vivo tumor data measured at theend of the experiment (Fig. 2B), and confirm the applic-ability of micro-CT imaging as a noninvasive and quan-titative, real-time monitoring tool of lung tumorprogression. The calculation of lung lesions for eachmouse in each CT session was performed in triplicateby an imaging scientist who was ignorant to the groupassignment and histologic findings. The interday varia-bility for CT reads on number of lesions was 8% and onlesion diameters 14%.

Silibinin inhibits urethane-induced lungtumorigenesis in WT but not iNOS�/� mice

Tumors were extracted from lungs, and tumor numberand diameters compared between WT and iNOS�/� mice.WT mice developed an average of 15 lung tumors/mouse,whereas iNOS�/� animals developed only 2 lung tumors/mouse, an 87% (P < 0.001) reduction in tumor multi-plicity (Fig. 2A) in agreement with our earlier studies(25). Silibinin treatment significantly reduced lung tumormultiplicity by 71% (P < 0.01) in WT mice, but there wasno statistically significant change in tumor number incontrol versus silibinin-treated iNOS�/� mice (Fig. 2A).WT mice developed lung tumors with diameters varyingfrom 0.5 to 4.5 mm, whereas all tumors from iNOS�/�

mice were <1 mm in diameter, an 82% reduction. Silibininretarded tumor progression of the <1 mm lesions by 67%(P < 0.01) and of the 1.0 to 1.5 mm lesions by 62%, andcompletely suppressed the progression to1.5 to 2.5 mm(P < 0.05) and >2.5 mm diameter lesions in WT mice.However, silibinin did not further reduce tumor size ornumber in iNOS�/� mice (Fig. 2B). Because of small sizeand number of iNOS�/� tumors, we were unable to usethem for molecular analysis.

Silibinin Targets iNOS in Lung Cancer Chemoprevention

www.aacrjournals.org Clin Cancer Res; 17(4) February 15, 2011 755




Histopathologic characteristics of lung tissue andtumors

Histopathologic examination of lung tissue and tumorsin B6/129mice 16 weeks after the last of 7 weekly urethaneinjections yielded adenomas that exhibited uniformcellular organization, mild nuclear dysmorphology, and

a normal nuclear/cytoplasmic ratio. Airways and alveolarspaces immediately adjacent to the tumors were com-pressed slightly, and the tumors were surrounded by asmall number of macrophages (data not shown). Therewere no significant differences in histopathology betweenlesions from silibinin-fed and control groups.

A

B C

Figure 1. Micro-CT images and quantitative end-points for lung tumor growth and chemopreventive effect of silibinin. A, axial micro-CT images of tumorgrowth in urethane and urethane þ silibinin treated WT mice at the indicated weeks after silibinin treatment. Tumors observed in the lungs are circled.Quantitative analysis of tumor number (B) and diameter (C) by micro-CT imaging. Each tumor scanned by micro-CT was reconstructed to three-dimensionalimages (axial, sagittal, and coronal), and diameters measured periodically as indicated. Data represent mean � SEM value of 6 animals in each group. Sb,silibinin.

Ramasamy et al.





Silibinin selectively reduces the level of iNOSexpression in urethane-induced lung tumorsBecause in iNOS�/� mice silibinin did not show any

considerable effect on urethane-induced lung tumorigen-esis, we anticipated that antitumor effect of silibinincould be mostly mediated via iNOS in WT mice. There-fore, we analyzed the levels of all the three forms of NOSin tumors. Immunohistochemical examination of iNOS,eNOS, and nNOS levels in control and silibinin-treatedtumor-bearing WT mice showed that silibinin decreasediNOS levels in tumors by 57% (P < 0.001), which wasalso confirmed by immunoblot analysis (Fig. 3A). Silibi-nin did not show any considerable effects on eNOS andnNOS expression levels (Fig. 3B and C). These resultsprovided evidence that silibinin targets iNOS for itsantitumor activity in urethane-induced lung tumorigen-esis of mouse model.

Silibinin inhibits cell proliferation andneoangiogenesis in urethane-induced lung tumors

Because we observed silibinin-mediated decreases innumber and sizes of lung tumors, we next analysedwhether silibinin affected cell proliferation and angiogen-esis in these tumors. Silibinin decreased expression of theproliferative marker, PCNA, by 18% (P < 0.01) comparedto controls (Fig. 4A).Microscopic examination of nestin, anangiogenic marker for newly formed microvessels, showednumerous nestin-positive microvessels and increasedimmunoreactivity in tumors of control mice. Silibinindecreased the number of nestin-positive tumor vessels by61% (P < 0.01), which was also confirmed by immunoblotanalysis (Fig. 4B). We also observed a significant decrease(55%; P < 0.01) in the intensity of nestin immunostainingin silibinin-treated versus control tumor-bearing WT mice(data not shown).

Figure 2. Silibinin preventsurethane-induced lung tumorgrowth and progression in WT butnot in iNOS�/� mice. A, lungtumors were dissected from allgroups and tumor multiplicity/mouse determined. B, tumordiameters were measured withdigital calipers under a dissectingmicroscope and grouped bydiameter: <1.0 mm, 1.0 to 1.5 mm,>1.5 to 2.5 mm, and >2.5 mm.Data represent mean � SEMvalue from tumors of four animalsin each group. Sb, silibinin; iNOSKO, iNOS knock out.

A

B






The growth of new blood vessels in tumors depends onsignaling pathways regulated by vascular endothelialgrowth factor (VEGF) and its major receptor, VEGFR2(26). VEGFR2 expression analysis by immunoblottingrevealed a significant decrease (P < 0.01) in the silibinin-treated group compared to controls (Fig. 4C). These resultssuggest that silibinin-caused downregulation of VEGFR2could mediate its anti-neoangiogenesis in lung tumors.

Silibinin inhibits STAT3 and NF-kB activationin urethane-induced lung tumors

The transcription factors, STAT3 and NF-kB, play vitalroles in tumor angiogenesis, and the promoter region ofiNOS has binding sites for both STAT3 and NF-kB (27). Wedetermined STAT3 and NF-kB activities by examiningpSTAT3 (Ser727) and p65NF-kB (Ser276) expressionand localization in lung tumors by IHC. Silibinin treatmentsignificantly decreased nuclear pSTAT3 (Ser727)-positivecells by 38% (P < 0.001) compared to the control group oftumors (Fig. 5A). Similarly, silibinin also decreased nuclearp65NF-kB (Ser276) positive cells by 31% (P < 0.001) ascompared to the control group of tumors (Fig. 5B). Theseresults indicate the inhibitory effect of silibinin on STAT3and NF-kB signaling in lung tumors, which could havemediated its chemopreventive and angiopreventive effectsmost likely through iNOS regulation.

Discussion

The focus of this study was to assess the chemopreven-tive potential of oral silibinin on multiple injections ofurethane-induced lung tumors in B6/129 wild type andiNOS�/� mice. Previous studies from our laboratoryshowed that silibinin inhibits tumor formation and thegrowth of advanced lung tumors in the A/J mouse model(14, 15). We previously observed that silibinin inhibitediNOS and COX-2 expression, the two enzymes whichpromote lung tumor growth and progression in thismodel (14). Herein, we demonstrated the usefulness ofmicro-CT for evaluating the chemopreventive efficacy ofsilibinin on urethane-induced lung tumorigenesis. Micro-CT images revealed that the number of tumors increasedover time after carcinogen exposure, and that silibinintreatment inhibited this growth. We determined that thetotal number of tumors detected by CT (extrapolated for18 weeks) was slightly lower than that detected by macro-scopic study at 18 weeks, suggesting that micro-CT couldnot detect all tumors in this study. This may be explainedby the differences in spatial resolution of a macroscopicimaging technique in the living animal versus micro-scopic histologic examination of tissues. However, thetrends correlated well and further improvement of themicro-CT imaging protocol is under development. Ourresults also support recent findings that micro-CT candetect lung cancer lesions as small as 1-mm diameter withhigh resolution (28–31). Haines et al. (30) longitudinallydemonstrated that micro-CT could be used to study lungcancer progression and treatment response to erlotinib inmice genetically engineered to produce mutant KRASdriven lung tumors. Fushiki et al. (29) described astrong correlation between bioluminescence and micro-CT imaging in early lung lesion detection and quantita-tively assessed cancer response to cisplatin and gemcita-bine therapy in an orthotopic model. Wang andcolleagues (31) showed that both MRI and micro-CTcan differentiate squamous cell carcinoma from adeno-carcionma induced by N-nitroso-trischloroethylurea. We

A

B

C

Figure 3. Silibinin selectively decreases iNOS expression in lung tumors.Lung tumors from WT mice were processed for IHC staining andquantification for: A, iNOS; B, eNOS; and C, nNOS. IHC quantitative datarepresent mean � SEM of 4 animals in each group. A (lower), iNOS levelswere also analyzed by Western blot in four randomly selected tumorsamples from 4 individual animals in control and silibinin-treated WTmice.Sb, silibinin.

Ramasamy et al.





quantitatively assessed silibinin efficacy in carcinogen-treated, genetically modified mice and demonstrated astrong correlation between histopathologic endpointsand real-time quantitative micro-CT.

To determine whether silibinin chemopreventive activitywas working through iNOS, mice lacking the iNOS genewere given a multiple urethane carcinogenesis regimen. Wehave previously determined that this regimen induces ahigh multiplicity and incidence of lung tumors in themoderately resistant B6/129 strain (25). This study demon-strates that silibinin is an effective chemopreventive agentfor lung tumor formation in strains other than A/J injectedonce with urethane as compared with a more aggressivecarcinogenesis protocol employing multiple injections ofurethane. Eighteen weeks of silibinin administrationreduced lung tumor multiplicity as well as size, and thiswas accompanied by significant antiangiogenic effects. B6resistance alleles present in these chimeric mice did notaffect silibinin efficacy, suggesting that silibinin exerts itschemopreventive efficacy on lung tumorigenesis irrespec-tive of genetic background. The 87% decrease in tumornumber in iNOS�/� mice confirms that iNOS is an impor-tant mediator of lung tumorigenesis in this model. Becausesilibinin did not considerably affect either tumor numberor size in iNOS�/� mice, these mice suggest that silibininexerts its chemopreventive effects mostly through inhibit-ing iNOS expression in lung tumors.

A

B

C

Figure 4. Silibinin inhibits proliferation and neoangiogenesis in lungtumors. Lung tumors were immunohistochemically analyzed for: A, PCNAand B, nestin, and quantified for PCNA-positive cells and nestin-positivemicrovessels (arrows, nestin-positive vessels), respectively, as detailed insection Materials and Methods. Four randomly selected tumor samplesfrom 4 individual animals in control and silibinin-treated WT mice wereanalyzed by Western blot for: B (lower), Nestin and C, VEGFR2 expressionlevels. IHC quantitative data for PCNA and nestin represent the mean �SEM value of four animals; quantitative densitometric data for VEGFR2represent the mean� SEM value of four bands in each group. Sb, silibinin.

A

B

Figure 5. Silibinin decreases the activation of STAT3 and p65NF-kB inlung tumors. Lung tumors from WT mice were processed for IHC stainingand quantified for: A, pSTAT3 (Ser727) and B, p65NF-kB (Ser276)using phospho-specific primary antibodies as detailed in section Materialsand Methods. IHC quantitative data in each case represent themean � SEM of 4 animals in each group. Sb, silibinin.






To further explore the mechanisms of lung cancer che-moprevention by silibinin, we evaluated the effect ofsilibinin on cancer cell proliferation. Cell proliferationwas assessed by PCNA expression, a cofactor of DNApolymerase required for DNA replication and nucleotideexcision repair (32). We observed that lung tumors fromsilibinin-treated mice had significantly lower PCNA expres-sion than their urethane-treated controls, suggesting that atleast part of silibinin’s mechanism of chemoprevention isthrough inhibition of tumor cell proliferation.

Neoangiogenesis facilitates tumor growth, progression,and metastasis. Newly formed microvessels were observedin urethane-induced lung tumors inWTmice, and silibinintreatment significantly decreased the number of thesevessels. Because VEGF and its receptors are highly expressedin many tumors (33, 34), iNOS expression is associatedwith increased VEGF expression, and VEGF expression isdecreased in the lung tumors of iNOS-deficient mice (25),we analyzed the expression of angiogenic factors in tumorsfrom control and silibinin-treated mice. VEGFR2 expres-sion level and nestin immunoreactivity were decreased insilibinin-treated mice, implying that angiopreventivemechanisms of silibinin are related to iNOS-dependentsuppression of vascular growth in lung tumors.

Next we evaluated the activation state of STAT3 and NF-kB, transcription factors which play major roles in tumor-igenesis of a wide variety of cancers including lung cancer(35, 36). STAT3 nuclear translocation and activation isknown to increase expression of genes, which influencecell cycle progression (such as cyclin D1) and angiogen-esis (such as VEGF) (35). Other studies have demon-strated the importance of NF-kB signaling in chemicallyinduced and genetic models of lung cancer (37, 38). Inthis study, silibinin strongly inhibited the activation ofSTAT3 and NF-kB in multiple urethane-induced lungtumors of WT mice. This is further supported by ourearlier observations where silibinin inhibited macro-phage infiltration as well as the activation of STAT3and NF-kB in tumors from urethane-treated A/J mice,and has also inhibited doxorubicin-induced NF-kB acti-vation in A549 cells (12, 15).

Nitric oxide production by iNOS is involved in thepathogenesis of many cancers. When we analyzed iNOSexpression by immunohistochemistry and immunoblot-

ting in WT mice, we found that silibinin inhibited iNOSexpression without affecting that of eNOS and nNOS. HighiNOS levels are shown in lung large cell carcinoma andlung adenocarcinoma but not in squamous cell carcinomaof human non–small cell lung cancer (39). iNOS/NOlevels are higher in lung tumors from smokers than non-smokers, and in human lung tumors compared to sur-rounding tissue (17, 40). Inhibition of iNOS activityreduces aberrant crypt foci formation and colon tumorformation (41). Silibinin is shown to inhibit iNOS expres-sion and cytokine-induced activation of STAT andNF-kB innon–small cell lung cancer A549 cells (12). Because theiNOS gene promoter region has binding sites for STAT3and NF-kB, the inhibition of the activation of these tran-scription factors by silibinin may account for the observeddecrease in iNOS expression (27).

In summary, we demonstrate the chemopreventive effi-cacy of silibinin in the B6/129 WT lung tumorigenesismodel. The key findings are: (a) iNOS�/� mice developedfew tumors, indicating the importance of iNOS in lungtumorigenesis; (b) silibinin decreased the number and sizeof tumors in B6/129WT but not in iNOS�/�mice; (c) thesedecreases in tumor number and size correlate with silibi-nin-mediated inhibition of cell proliferation and angiogen-esis inWTmice, most likely through the inhibition of iNOSexpression involving down-regulation of STAT and NF-kBsignaling; and (d) micro-CT analysis represents a sensitiveand reliable imaging modality to longitudinally assessthe inhibition of growth of lung tumors by chemopreven-tive agents including silibinin, and therefore could also beuseful in other lung cancer intervention studies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

NCI RO1 grants CA113876, CA33497, CA96310, and P30 CA046934.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 25, 2010; revised October 22, 2010; accepted November5, 2010; published OnlineFirst December 10, 2010.

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2011;17:753-761. Published OnlineFirst December 10, 2010.Clin Cancer Res Kumaraguruparan Ramasamy, Lori D. Dwyer-Nield, Natalie J. Serkova, et al. Cancer Chemoprevention Studies

Mice: Potential of Real-Time Micro-CT in Lung−/−in iNOS Silibinin Prevents Lung Tumorigenesis in Wild-Type but not

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