Growth Inhibition and Regression of Lung Tumors by Silibinin: Modulation of Angiogenesis by...

Growth Inhibition and Regression of Lung Tumors by Silibinin:Modulation of Angiogenesis by Macrophage-associatedCytokines, and NF-κB and STAT3

Alpna Tyagi1, Rana P. Singh1,3, Kumaraguruparan Ramasamy1, Komal Raina1, Elizabeth F.Rodente1, Lori D. Dwyer-Nield1, Richard A. Radcliffe1, Alvin M. Malkinson1,2, and RajeshAgarwal1,21Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver,Denver, Colorado, USA2University of Colorado Cancer Center, University of Colorado Denver, Denver, Colorado, USA3Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

AbstractThe latency period for lung tumor progression offers a window of opportunity for therapeuticintervention. Herein, we studied the effect of oral silibinin (742 mg/kg body wt, 5 days/wk for 10wks) on the growth and progression of established lung adenocarcinomas in A/J mice. Silibininstrongly decreased both tumor number and tumor size, an antitumor effect that correlates withreduced antiangiogenic activity. Silibinin reduced microvessel size (50%, p<0.01) with no changein the number of tumor microvessels and reduced (by 30%, p<0.05) formation of nestin-positive newmicrovessels in tumors. Analysis of several proteins involved in new blood vessel formation showedthat silibinin decreased tumor expression of IL-13 (47%) and TNF-α (47%), and increased TIMP-1(2-fold) and TIMP-2 (7-fold) expression, without significant changes in VEGF levels. HIF-1αexpression and nuclear localization were also decreased by silibinin treatment. Cytokines secretedby tumor cells and tumor associated macrophages (TAMs) regulate angiogenesis by activating NF-κB and STAT. Silibinin decreased phosphorylation of p65NF-κB (ser276) (38%, p<0.01) and STAT3(ser727) (16%, p<0.01) in tumor cells and decreased the lung macrophage population.Angiopoietin-2 (Ang-2) and Ang-receptor tyrosine kinase (Tie-2) expression were increased bysilibinin. Therapeutic efficacy of silibinin in lung tumor growth inhibition and regression byantiangiogenic mechanisms appears to be mediated by decreased TAMs and cytokines, inhibition ofHIF-1α, NF-κB and STAT3 activation, and up-regulation of the angiogenic inhibitors, Ang-2- andTie-2.

KeywordsSilibinin; lung tumor; angiogenesis; macrophages; cytokines; NF-κB; STAT3

IntroductionLung cancer is the leading cause of cancer death in both men and women in the United States,with an estimated 213,380 new lung cancer cases and 160,390 associated deaths in 2007 (1).The 5-year survival rate of 14% has shown little improvement over the last 30 years, even with

To whom correspondence should be addressed, E-mail: [email protected]; Phone: 303-315-1228; Fax: 303-315-6281.

NIH Public AccessAuthor ManuscriptCancer Prev Res (Phila). Author manuscript; available in PMC 2010 January 1.

Published in final edited form as:Cancer Prev Res (Phila). 2009 January ; 2(1): 74–83. doi:10.1158/1940-6207.CAPR-08-0095.

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the development of molecularly targeted therapies such as epidermal growth factor receptor(EGFR) inhibitors. Tobacco exposure has been implicated in 90% of lung carcinomas;compared to never smokers, smokers have a 20-fold greater risk of developing lung cancer(2). Because smoking is the major risk factor for developing lung cancer and most smokershave small pulmonary nodules, strategies for inducing nodule regression or preventing theirfurther growth should decrease the number of patients diagnosed with advanced malignantdisease.

Efforts are being made towards identifying dietary supplements to prevent and treat lungcancer. One such agent, silibinin, inhibits the growth of various cancer cell lines and primarytumors in several chemically-induced rodent models, including mouse lung (3-7). Silibinin isa flavonolignan, a major component in the silymarin complex of flavonolignans andpolyphenols present in milk thistle (Silybum marianum) seeds. Silymarin has been extensivelyused in patients with liver disease for decades (8). Silibinin has shown strong anticancerefficacy against SHP-77 and A549 lung cancer cells, where it inhibits cell growth and inducescell cycle arrest (9). It also inhibits the invasion of lung cancer cells via down-regulating PI3K-Akt and MAPK signaling pathways and decreased production of urokinase-plasminogenactivator and matrix metalloproteinase-2 (10,11). Silibinin inhibits in vivo growth of A549xenografts in nude mice, reduces systemic toxicity of doxorubicin, and reduces doxorubicin-induced chemo-resistance by inhibiting NF-κB signaling (12).

Angiogenesis, the formation of new blood vessels, is required early on in tumor development.It is essential for tumor expansion, progression, and metastasis (13). Angiogenesis is a complexprocess that involves degradation of the basement membrane and invasion of the stroma byendothelial cells, which then proliferate, migrate and become organized into capillarystructures (14). Tumor associated macrophages (TAMs) constitute an important interfacebetween tumor cells and the immune system, and influence neoplastic growth and progressionin several ways (15). Macrophage infiltration affects angiogenesis by influencing theproduction of angiogenic cytokines and growth factors including interleukins, TNF-α and IFN-γ (16). Therefore, modulation of TAMs may be critical in nascent vessel formation in tumors,and could be targeted to inhibit tumor angiogenesis.

Recently, we showed that dietary silibinin markedly reduced the growth and progression ofurethane-induced primary lung tumors in A/J mice. In a previous study, silibinin was fed tomice 2 weeks after carcinogen administration to model a chemopreventive treatment regimen(7). We now examine the chemotherapeutic effect of silibinin on established advanced lungtumors in this animal model. Tumors at this stage are adenocarcinomas with malignantcharacteristics such as invasiveness, nuclear dysmorphology, and cellular heterogeneity. Ourfindings are novel and significant because this chemopreventive agent that is known to inhibitcell proliferation and induce apoptosis did not effect these biological activities in a therapeuticsetting. Instead, silibinin targeted vessel size and nascent tumor microvessel formation via itseffects on TAMs, known modulators of angiogenesis.

Materials and MethodsChemicals

Urethane (ethyl carbamate) and silibinin were purchased from Sigma (St. Louis, MO). Purity(>98%) of silibinin was confirmed by high-performance liquid chromatography, as previouslydescribed (17). Mice were fed AIN-76A rodent diet pellets (Dyets Inc, Bethlehem, PA).

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Urethane-induced lung tumorigenesis experimental designA/J male mice (4-6 wks of age) were purchased from the Jackson Laboratory (Bar Harbor,ME) and housed under standard laboratory conditions in the Center of Laboratory Animal Careat the University of Colorado Denver. Animal care was performed in accordance withinstitutional guidelines, and all animal treatments performed under an institutionally approvedanimal protocol. Mice (n=25, 8 wks of age) were given a single i.p. injection of 1 mg/g bodywt of urethane in saline, as previously described (7). Urethane-induced lung tumors harbor amutation in codon 61 of the Kras proto-oncogene (18). Thirty two wks after urethane injection,5 mice were sacrificed and their lung tumors dissected, counted, and diameters measured withdigital calipers, and pooled tumors weighed to determine total tumor burden/mouse. Theremaining 20 mice were randomly divided into 2 groups (n=10 mice per group) and gavagedwith normal saline (control group) or silibinin 742 mg/kg body wt 5 days/wk for 10 wks. Thisdose of silibinin was extrapolated from the dietary consumption of A/J mice exposed to 1%silibinin (w/w) and fed AIN-76A diet in our previous study (7). At 43 wks, mice were sacrificedby i.p. pentobarbital injection, lung tumors harvested, and the parameters mentioned abovewere recorded from 5 mice in each group. Lungs from the remaining 5 mice from each groupwere perfused, formalin fixed, and paraffin embedded for histological andimmunohistochemical (IHC) analyses. Plasma samples from all mice were collected by cardiacpuncture into heparinized tubes, and stored at -80°C.

Immunohistochemistry (IHC)Fixed lung samples were sectioned (5 μm), deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0) for 30 min at 90°C. Nonspecific bindingsites were blocked with serum-free blocking reagent (DAKO), and antibodies againstproliferating cell nuclear antigen (PCNA, 1:400, mouse monoclonal, Dako, Carpinteria, CA),CD31 (platelet-derived endothelial cell adhesion molecule, 1:200, goat polyclonal, Santa CruzBiotechnology, Santa Cruz, CA), nestin (1:200, mouse monoclonal, Abcam, Cambridge, MA),pSTAT3 (ser727) (1:200, rabbit polyclonal, Abcam), inducible nitric oxide synthase (iNOS;1:200, BD-Transduction Laboratory, San Diego, CA), cyclooxygenase-2 (COX-2; 1:2000,goat polyclonal, Santa Cruz Biotechnology), p65NF-κB (ser276) (1:200 rabbit polyclonal, CellSignalling, Beverly, MA), HIF-1α (1:200, mouse monoclonal, Novus, Littleton, CO) and themacrophage specific marker, F4/80 (1:100, Caltag laboratories, Burlingame, CA) were appliedovernight at 4°C followed by appropriate secondary antibodies. Proteins were visualized using3, 3′-diaminobenzidine as previously described (7). Sections were counterstained withhematoxylin. Apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining using Dead End Colorometric TUNELsystem (Promega Corp).

The PCNA, TUNEL, p65NF-κB (ser276) and pSTAT (ser727) -positive cells (brown) werequantified by counting the numbers of stained cells compared to the total number of cells infive randomly selected tumor fields, at 400X magnification. HIF-1α nuclear staining wasvisualized at 1000X using an oil immersion lens. Blood vessels (CD31-positive) and newlyformed vessels (nestin-positive) were quantified as the mean number of positive vessels per400X field in five randomly selected tumor fields. CD-31 positive blood vessel diameter ofcircle-shaped vessel cross sections was measured using Spot Advanced software on anOlympus BX-41 micrscope equipped with an Olympus U-TVX-1 camera. Intensity of COX-2and iNOS immunoreactivities were scored as 0 (no staining), 1+ (weak staining), 2+ (moderatestaining), 3+ (strong staining) or 4+ (very strong staining) as recently published (7). Since mostepithelial cells express these proteins basally, intensity of staining described increases inprotein expression more clearly than did ratios of expressing cells to non-expressing cells.

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Immunoblot analysisLung tumor lysates from 3-4 individual mice from each group, were prepared and analyzed byimmunoblotting, as previously described (7). Primary antibodies against PCNA, cyclin D1,VEGF, TIMP-1 and Tie-2 (Santa Cruz Biotechnology); p65NF-κB (ser276), total p65NF-κB(ser536), pSTAT3 (ser727), total STAT3 and phospho-Tie-2 receptor (Tyr992) (CellSignalling, Beverly, MA); HIF-1α (Novus Biologicals, Inc); nestin, angiopoietin 2 and TNFα(Abcam, Cambridge, MA); TIMP-2 (Chemicon, Temecula, CA), and IL-13 (Sigma-Aldrich)and secondary antibodies against anti-rabbit IgG (Cell Signalling) or anti-mouse IgG (GEHealthcare, Piscataway, NJ) were used. Equal protein loading was determined by stripping andreprobing membranes with anti-β actin primary antibody (Sigma Aldrich). Protein bands werevisualized by enhanced chemiluminescence detection.

Mouse angiogenesis antibody arrayTwo randomly selected lung tumor lysates from each group were applied to a MouseAngiogenesis Antibody Array (RayBiotech Inc, Norcross, GA) to analyze the expression ofangiogenesis-related molecules according to the manufacturer's protocol. Expression of eachprotein was represented in duplicate on the membrane. Duplicate dots identifying each proteinwere scanned using Adobe Photoshop software and quantified by ScionImage Program. Themean intensity of these dots (arbitrary units) was determined for intergroup comparisons.

ELISA assay for IL-13Plasma samples from control and silibinin-fed group were applied to a mouse IL-13 ELISAkit, following vendor's protocol (RayBiotec Inc, Norcross, GA) to quantify secreted IL-13. Theassay is based on quantitative sandwich enzyme immunoassay using an antibody specific formouse IL-13 coated onto a microplate for solid phase ELISA. Briefly, 100 μl plasma samples(collected from 4-5 individual mice/group) were assayed, and absorbance of the developingcolor was determined by microplate reader at 450 nm wavelength. IL-13 concentration wasextrapolated from a recombinant mouse IL-13 standard curve.

Statistical and microscopic analysesStatistical analyses were carried out by using Sigma Stat software version 2.03 (JandelScientific, San Rafael, CA). All statistical tests were two-sided, and p<0.05 was consideredstatistically significant. The differences between controls (urethane treated group) andsilibinin-fed groups were analyzed by unpaired two-tailed Student's t-test and one-wayANOVA followed by a Bonferroni t-test for pair-wise multiple comparisons. Densitometricanalysis of immunoblots was performed using the Scion Image program (NIH, Bethesda, MD).IHC staining was visualized with a Zeiss Axioscope 2 microscope (Carl Zeiss, Inc, Jena,Germany), and photographs were captured with a Carl Zeiss AxioCam MrC5 camera at 400Xor 1000X magnification.

ResultsSilibinin inhibits urethane-induced lung tumor progression in A/J mice

A/J mice given a single 1 mg/g body wt i.p. injection of urethane develop microadenomas (<0.3mm) within 3 wks, macroscopic adenomas (tumors <1.5 mm with uniform nuclei that aren'tinvasive) within 16 wks, and adenocarcinomas (invasive tumors with nuclear dysmorphologyand cellular heterogeneity) within 30 wks (19,20). Metastasis is rare in this model as the micedie from oxygen deprivation before tumors progress to that stage. To determine whethersilibinin could act as a chemotherapeutic agent in this model, we examined the effect of oralsilibinin (742 mg/kg body wt, 5 days/wk for 10 wks, a dose determined to be equivalent to the1% wt/wt dietary consumption of silibinin) treatment on mice with 32-wk established lung

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tumors; also, a group of mice were sacrificed 32 wks after urethane injection as a referencecontrol (Fig. 1A). Mice receiving silibinin had 33% (p<0.05) fewer tumors than age-matched43-wk controls; this control group developed slightly more tumors than a 32-wk control group(Fig. 1B). From 32 - 43-wks, a high tumor growth rate (p<0.001) was observed. Silibinindecreased tumor burden (pooled tumor wt/mouse) by 37% (p<0.05) compared to micereceiving vehicle (Fig. 1C). When tumors were categorized according to size, smaller tumors(<1.5 mm) progressed to a larger size (≥1.5 mm) from 32 wks to 43 wks (p<0.05-0.001) (Fig.1D). Silibinin treatment significantly decreased the number of larger tumors (>2.5 mm) by37% (p<0.01), and caused a 50% (p<0.01) decrease in the number of tumors between of 1.5and-2.5 mm diameter compared to the 43-wk control group (Fig. 1D). The decreased numberof tumors in the silibinin group indicates regression, whereas the decreased number of largertumors in the silibinin group indicates its growth inhibitory effects. For molecular analysis, weused samples from the 43-wk control and silibinin-treated groups.

Histopathological characteristics of lung tissue and tumorsHistopathological examination of lung tissue and tumors in A/J mice at 32 and 43 wks aftertreatment with a single urethane injection showed well-vascularized adenocarcinomas thatexhibited invasiveness, nuclear dysmorphology, decreased cytoplasm/nuclear ratio, andundifferentiated cellular orgainization (data not shown). In the 43 wk groups, most tumors werelarge (>1.5-2.5 mm in size), and the surrounding alveolar spaces contained numerous largefoamy macrophages. Airways and alveolar spaces immediately adjacent to the tumors werecompressed. Bronchus-associated lymphatic tissue (BALT) was present adjacent to largetumors. When sections from untreated tumor bearing mice were compared to those from micegiven silibinin for 10 wks prior to harvest, few differences besides the reduction in tumor sizewere evident, but large alveoli with characteristic ‘emphysematous’ hooked alveolar wallsappeared in the silibinin-treated samples. Since silibinin decreased tumor number, these largeopen spaces may indicate areas of tumor regression. To determine whether tumor regressionleft fibrotic deposits, as described in other tumor models, pentachrome stains were performedon lung sections from control and silibinin treated mice. However, no differences in stainingfor collagen I, mucin, ground substance, muscle, or elastic fibers were observed between thetwo groups (data not shown).

Effect of silibinin on lung tumor cell proliferation and apoptosisSince we observed tumor growth inhibition by silibinin, tumors from both control and silibinin-treated groups were analyzed for PCNA and TUNEL IHC staining. No significant differencein PCNA immunoreactivity (Supplementary Fig. 1A) was observed in the silibinin-fed group(39% ± 1.97) as compared to control (43%±2.34). To confirm our IHC results, tumor lysatesfrom control and silibinin groups were analyzed by western immunoblotting for PCNA andcyclin D1. Little change in PCNA and cyclin D1 expression was observed in silibinin-fed groupof tumors as compared to control tumors (Supplementary Fig. 1B). TUNEL staining, performedto assess the apoptotic effect of silibinin in tumors, showed similar numbers of TUNEL-positivecells in both silibinin and control groups (Supplementary Fig. 1C). Additionally, we did notobserve any considerable effect of silibinin on ERK1/2 and Akt phosphorylation in lung tumors(data not shown). These results suggest that silibinin does not considerably affect cellproliferation and apoptosis in established lung tumors although a trend toward lowerproliferative and higher apoptotic rates was observed. Since small differences in tumorproliferative rates could amount to big differences in tumor size over 10 wks, the significanceof these slight changes cannot be accurately assessed.

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Silibinin inhibits angiogenesis in urethane-induced lung tumorsSince significant antiproliferative and apoptotic effects of silibinin on lung tumors were notdetected, we assessed the effect of silibinin on tumor angiogenesis using CD-31 staining, amarker for endothelial cells (both established and nascent). Image analysis followed byquantitative counting of CD-31 positive vessels did not reveal significant differences in tumormicrovessel density between control and silibinin-fed groups (data not shown). However,vessel cross-sectional area in the silibinin-fed decreased by 50% (p<0.01; Fig. 2A). Nestin, astem cell marker, is expressed on newly formed microvessels in advanced tumors but is lostupon endothelial cell maturation (21). Tumors in mice gavaged with silibinin showed fewernestin-positive microvessels (30% decrease, p<0.05) as compared to control tumors (Fig. 2B).Western immunoblotting for nestin in tumor lysates confirmed that less nestin was expressedin mice treated with silibinin (Fig. 2C). Overall, silibinin had little significant effect on tumorcell proliferation and apoptosis but may suppress tumor angiogenesis by inhibiting increasedmicrovessel size and the formation of new microvessels.

Silibinin modulates angiogenesis-related cytokines in tumors and decreases tumor-associated macrophages

Mechanisms of inhibition of angiogenesis by silibinin in lung tumors were explored byperforming protein array analysis for biomolecules that regulate angiogenesis in tumor lysates.Silibinin treatment decreased levels of several interleukins and cytokines including IL-1α(34%), -6 (44%), -9 (29%), -13 (47%) and -16 (44%), as well as IFN-γ (16%) and TNF-α(47%) in tumors compared to controls (Fig. 3A). Expression levels of TIMP-1 and TIMP-2were increased 2- and 7-fold, respectively, by silibinin (Fig. 3A). Results for the most stronglyeffected proteins, IL-13, TNF-α, TIMP-1 and TIMP-2, were confirmed by western blotanalysis, which showed changes similar to those observed in the antibody arrays (Fig. 3B;densitometry confirmed decreases of 14% and 31% in IL-13 and TNF-α, respectively andincreases of 1.5 and 3-fold in TIMP-1 and TIMP-2, respectively.

IL-13 plays an important role in regulating angiogenesis, and circulating IL-13 couldfunctionally affect tumor angiogenesis. To test this, we measured levels of IL-13 in mouseplasma by ELISA, and observed that the silibinin treated group had less circulating IL-13 thancontrols (317.8 ± 43.4 pg/ml in control group versus 137.5 ± 10.6 pg/ml in silibinin group;57% decrease; p<0.01; Fig. 3C). Silibinin treatment did not influence VEGF or Fas ligandexpression either in the protein array or western blot analysis (Fig. 3B). Since macrophagescritically modulate cytokine secretion (22), and several cytokines decreased upon silibinintreatment, we used F4/80 staining of macrophages to examine whether silibinin affected thenumber of macrophages in tumor bearing lungs. In sections from control mice, macrophageswere more populous around tumors compared to sections from the silibinin-treated group.Quantification of these macrophages showed a 38% (p<0.05) decrease in TAM numberfollowing silibinin feeding (Fig. 3D). Overall, these results suggest that silibinin targets TAMsto suppress the angiogenic tumor microenvironment.

Effect of silibinin on HIF-1α, iNOS and COX-2 expression in lung tumorsHIF-1α, iNOS and COX-2 can promote tumor angiogenesis (23,24). To examine whether theantiangiogenic effects of silibinin were mediated at least in part by HIF-1α, iNOS and COX-2,we performed IHC analysis of these proteins in lung tumors. Lung tumors from mice in thecontrol group displayed more cells containing HIF-1α positive nuclei (8% ± 1.5 in the controlgroup compared to 2% ± 0.5 (p<0.01) in the silibinin-treated group; Fig. 4A). The effect ofsilibinin on HIF-1α expression was also analyzed by immunoblotting tumor lysates, whichexhibited less 25% HIF-1α protein in the silibinin-treated group of tumors (Fig. 4A). Ourprevious studies showed that urethane-induced mouse lung tumors usually express high levelsof iNOS and COX-2 enzymes (25,26), but no effects of silibinin on iNOS and COX-2

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immunoreactivity by either IHC or immunoblot analysis were noted in these samples (data notshown). These results suggest a selective effect of silibinin on HIF-1α rather than on iNOS orCOX-2 in these advanced tumors.

Silibinin inhibits the activation of p65NF-κB in urethane-induced lung tumorsIn addition to HIF-1α, the NF-κB transcription factor also regulates expression of many genesthat control angiogenesis (27,28). High levels of NF-κB expression have been reported in non-small cell lung cancer cell lines compared to normal bronchial epithelial cell lines (29). In orderto examine the status of NF-κB activity in urethane-induced lung tumors, IHC staining forphospho-p65NF-κB (ser276) was performed. Thirty-eight percent less nuclearimmunoreactivity was observed in tumors from silibinin-treated mice (Fig. 4B). Whenphospho-p65NF-κB (ser276 and ser536) levels were examined by western blot in tumorslysates, the silibinin group had lower expression of both p65(ser276, 15%) and p65(ser536,40%) than controls, with no effect on total p65 in tumors (Fig. 4B). These results indicatesilibinin inhibits the activity of the angiogenic cytokine-NF-κB loop in urethane-induced lungtumors.

Silibinin inhibits the activation of STAT3 in urethane-induced lung tumorsSince we observed a down-regulation of cytokine expression by silibinin treatment in urethane-induced tumors, inhibited STAT signaling was anticipated. STAT3 was originally discoveredas a mediator of cytokine signaling pathways that play an active role in oncogenesis,inflammation and angiogenesis (30). Accordingly, we analyzed STAT3 activity in lung tumorsby assessing phospho-STAT3 (ser727) by IHC. Fifty eight percent of the tumor cells hadnuclear staining of phospho-STAT3 in the silibinin-treated group as compared to 70% (p<0.01)in control group (Fig. 4C). Western immunoblot analysis of phospho-STAT3 (ser727) and totalSTAT3 in tumor lysates showed that, consistent with the IHC data, control tumor samples hadmore phospho-STAT3 (ser727) than the silibinin-treated group with no change in total STAT3protein levels (Fig. 4C). These results revealed inhibition by silibinin of STAT3 (ser727)phosphorylation reduced its nuclear translocation and, thus, its transcriptional activity in lungtumors.

Silibinin enhances Angiopoietin-2 and Tie-2 levels in urethane-induced lung tumorsAngiopoietin-2 (Ang-2) is the ligand for the Tie-2 (tyrosine kinase) receptor in endothelial aswell as immune cells. Ang-2 can act as both an antagonist and agonist after binding to the Tie-2receptor (31). Transgenic overexpression of Ang-2 impairs angiogenesis as well asvasculogenesis (31). In the present study, lung tumors from silibinin-treated mice expressedhigher levels of Tie-2 (1.5 fold increase, p<0.05) protein, compared to control tumors (Fig. 5).We do not observe any considerable change in phospho-Tie-2 (tyr992) levels, adjusted withtotal Tie-2 level, by silibinin treatment (Fig. 5). Consistent with immunoblot data, we alsoobserved increased Tie-2 protein in lung tumors lysate in silibinin-treated mice using a Kinexusassay, a global protein kinase antibody-based assay system (data not shown). Concurrently,we observed enhanced Ang-2 expression (1.6 fold increase, p<0.01) in lung tumors fromsilibinin-treated mice as compared to the controls (Fig. 5). These results suggest that silibininincreased Ang-2 and Tie-2 levels as well as enhancing Ang-2 activated Tie-2 receptorsignaling, which may in part be responsible for destabilizing and regressing tumor vasculature.

DiscussionThe focus of the present study was to assess the chemotherapeutic potential and relatedmechanisms of oral silibinin on urethane-induced advanced lung tumors in A/J mice, a modelof human adenocarcinoma. Previously we showed that silibinin was a potent chemopreventiveagent in preventing tumor formation and adenoma to adenocarcinoma prevention in this model

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(7). Our novel findings are that silibinin reduced tumor number, tumor burden, and progressionof adenocarcinomas without adverse effects on mice. Tumor angiogenesis, as opposed to tumorcell proliferation or survival was targeted by silibinin. Silibinin did not exert any significanteffect on tumor microvessel density, but rather inhibited increases in vessel size and inhibitednew microvessel growth. This antiangiogenic effect of silibinin was accompanied by a decreasein the number of TAMs as well as reduced levels of cytokines known to promote inflammationand angiogenesis (Fig. 6). Enzymes important for inhibiting metalloproteinases such asTIMP-1 and TIMP-2 were up-regulated by silibinin treatment of mice bearing advanced lungtumors. Activation of the transcription factors, HIF-1α, NF-κB and STAT3, was also inhibitedby silibinin. Finally, a higher content of Ang-2 and Tie-2 proteins were observed; sustainedlevels of these proteins impair microvessel development (31).

In this study, A/J mice were given urethane, and after 33 wks, treated with oral silibinin for 10wks. Lung tumors at this stage were mainly adenocarcinomas i.e., invasive tumors with nucleardysmorphology and cellular heterogeneity. Silibinin feeding for 10 wks reduced lungadenocarcinoma multiplicity as well as tumor burden, and this was accompanied by significantantiangiogenic effects. In a recently completed chemoprevention study in this urethane-induced lung tumorigenesis model we observed that in early lung lesions, silibinin inhibitstumor growth and progression by strongly inhibiting tumor microvessel density and tumor cellproliferation (7). However, in the present therapeutic experimental design, in which tumorswere adenocarcinomas with established tumor vasculature prior to silibinin treatment, silibininsuppressed the growth of existing tumor vasculature as well as microvessel size. Slight but notsignificant differences were seen in tumor cell proliferation and apoptosis. We also did notobserve any considerable effect of silibinin on ERK1/2 and Akt phosphorylation. It appearsthat at later stage of lung tumor progression, silibinin has no effect on mitogenic and survivalpathways. Increased cellular heterogeneity and further mutations in key growth and cell deathregulatory pathway components may render cells in these advanced tumors resistant to theeffects of silibinin seen in our previous chemoprevention study. These observations suggestthat silibinin can target tumors at different stages of progression through its antiangiogeniceffects.

Another novel finding is that silibinin inhibited infiltration of macrophages into tumor bearinglungs; macrophages were sparse around the tumors from silibinin-treated mice as compared tothose in untreated controls. This observation suggested that silibinin may target TAMs as partof its mechanism in blocking angiogenesis. Numerous studies showed macrophageinvolvement in promoting tumor angiogenesis (15). Other agents can also influence thesemacrophages to down-modulate angiogenesis, such as linomide and thiol containingcompounds (32,33). Activated macrophages create a proangiogenic microenvironment bysecreting high levels of cytokines, but tumor cells at advanced stages of neoplasia also expresshigh levels of angiogenic cytokines possibly in response to TAMs (16,34). Silibinin treatmentdecreased tumor expression of many inflammatory and angiogenic cytokines, includingIL-1α, IL-6, IL-9, IL-13, IL-16, IFN-γ and TNF-α which may represent a combined effect ofsilibinin on macrophages as well as tumor cells. Metalloproteinase activity is required forpaving a path for growing microvessels, which can be regulated by TIMPs (35). Silibinintreatment increased expression of inhibitors of metalloproteinases TIMP-1 and TIMP-2,providing support for another mechanistic explanation of the inhibition of tumor microvesselgrowth and size by silibinin.

Angiogenesis is a highly orchestrated process involving sprouting of new capillary-likestructures from the existing vasculature that mature into new blood vessels (36). It can betriggered and modified by many factors, including cytokines, growth factors and theirreceptors, chemokines, and extracellular matrix macromolecules (36). These factors regulatemany transcription factors, including HIF-1α, NF-κB and STAT (23,24). Hypoxia within

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tumors stimulates production of HIF-1α, among other pro-angiogenic molecules. HIF-1α islow in normal cells, but reaches high intracellular concentrations in many cancers, and theseconcentrations strongly correlate with poor prognosis and resistance to therapy (37). Silibininsignificantly decreased the number of HIF-1α-positive nuclei, indicating inhibition of itstranscriptional activity. Therefore, it is likely that down-regulation of HIF-1α, in part plays arole in the antiangiogenic effect of silibinin on advanced lung tumors.

Recently, activated macrophages have been linked to NF-κB activation in urethane-inducedlung carcinogenesis (34). To our knowledge, our finding of inhibition of STAT3 activation isthe first report showing STAT3 signaling in urethane-induced lung tumors in mice. Pulmonarymacrophages can produce various cytokines, such as TNFα, interleukins, and IFN-γ (asobserved in the present study), which in turn activate NF-κB and STAT3 signaling in tumors.As anticipated, we observed activation of both NF-κB and STAT3 in tumors, as indicated byincreased levels of their phosphorylated forms, namely, p65 NF-κB (ser276 and ser536) andpSTAT3 (ser727). Silibinin treatment inhibited macrophage infiltration in tumor bearing lungsand inhibited activation of both NF-κB and STAT3 in tumors, which correlates with thediminished size of microvessels as well as the decrease in nestin-positive newly formedmicrovessels.

We were surprised to note that even after inhibition of HIF-1α, NF-κB and STAT3 signalingby silibinin in tumors, we observed no effect on expression of VEGF, iNOS and COX-2,potential targets of these transcription factors that usually play important roles in tumorangiogenesis. This is relevant because in our previous chemoprevention study, silibinindecreased expression of all three of these proteins (VEGF, iNOS and COX-2) as well asangiogenesis in developing lung tumors (7). This suggests that lung tumor progression in thisanimal model recruits different angiogenic mediators in early stages of tumor development(microadenoma to adenoma) than late adenocarcinoma stages. Further, we observed thatsilibinin enhances expression of Ang-2 and Tie-2 receptor tyrosine kinase factors whichregulate vessel stabilization and angiogenesis (31). Ang-1, the ligand for the Tie-2 receptor,promotes angiogenesis and recruits pericytes to stabilize vessels (38). However, Ang-2 is aconditional antagonist and agonist for Tie-2 receptor, whose systemic over expression leadsto tumor vessel regression without concomitant inhibition of VEGF (31). Similar results wereobserved with silibinin treatment; we saw no change in VEGF levels but observed more Ang-2and Tie-2 levels. This could be a potential antiangiogenic mechanism of silibinin on establishedlung adenocarcinoma.

In summary, oral silibinin showed antitumor effects in urethane-induced and established lungadenocarcinomas most likely by decreasing microvessel size and inhibiting newly formedmicrovessel growth in tumors. The decrease in TAM infiltration into lungs as well as lowerlevels of angiogenic cytokines, and greater TIMP-1 and TIMP-2 concentrations, along withthe inhibition of HIF-1α, NF-κB and STAT3 activation, could account for the antiangiogeniceffects of silibinin. Additionally, elevating levels of Ang-2 and Tie-2 without changing VEGFamounts could have led to microvessel regression in tumors by silibinin. Overall, our findingshere, together with our earlier studies (7), suggest that silibinin is a promising agent forintervention in human lung cancer oncogenesis.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsGrant support: This work was supported by RO1 grant CA 113876 and CA 33497 from the National Cancer Institute

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Abbreviations

IL interleukin

TNFα tumor necrosis factor α

IFNγ interferon γ

TIMP tissue inhibitor of metalloproteinase

VEGF vascular endothelial growth factor

HIF-1α hypoxia inducing factor 1α

NF-κB nuclear factor kappa B

STAT signal transducer and activator of transcription

Ang-2 angiopoietin-2

TAM tumor associated macrophage

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Figure 1.Silibinin inhibits urethane-induced lung tumorigenesis in A/J mice. A, Eight wk old male A/Jmice (n=25) were given a single i.p. injection of 1 mg urethane/g body weight. Thirty-two wkslater, 5 mice were sacrificed and the remaining 20 mice randomly divided into 2 groups (n=10mice per group), and gavaged with saline (control group) or silibinin 742 mg/kg body weightfor 5 days/wk for 10 wks and sacrificed. B, lung tumors dissected from all groups and lungtumor multiplicity/mouse determined. C, tumors were pooled together for each mouse andweighed to determine burden. D, tumor diameters were measured with digital calipers undera dissecting microscope, and tumors were grouped by diameter: <1.5 mm, 1.5-2.5 mm and

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>2.5 mm. Columns, mean; error bars, SEM for each group. $, p<0.05; #, p<0.01; *, p<0.001versus 43 wks for tumor size; SB, silibinin

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Figure 2.Silibinin inhibits angiogenesis in urethane-induced lung tumors. Tumor-bearing lungsharvested from A/J mice after 43 wks of urethane (n = 5 mice/group) injection were analyzedby IHC staining for CD31 and nestin, as described in Materials and Methods. A, IHC of CD31-positive (brown) endothelial cells from urethane (control) and urethane + silibinin treatedgroups (left). Microvessel diameter was quantified in 5 randomly selected fields in tumors fromeach of 5 different mice in both groups. Columns, mean; error bars, SEM for each group(right). B, nestin-positive (brown), newly formed endothelial cells from urethane (control) andurethane + silibinin treated groups of lung tumors (left). Microvessel numbers were quantifiedby measuring the nestin-positive cells in 5 randomly selected fields at 400X magnification intumors from each of 5 different mice in each group (right). Columns, mean; error bars, SEMfor each group (right). C, lung tumor lysates from three individual randomly chosen mice fromeach group were analyzed for nestin protein levels by immunoblotting. Membranes werestripped and reprobed with β-actin as loading control. SB, silibinin. Magnification 400X.

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Figure 3.Silibinin modulates angiogenesis-related cytokines in lung tumors and decreases tumor-associated macrophages. A, lung tumor lysates from control and silibinin-treated mice at 43wks post urethane injection were analyzed for angiogenesis related protein expression with anangiogenesis antibody array kit, as described in Material and Methods. Reactive protein spotswere visualized by enhanced chemiluminescence detection. Densitometric analysis ofangiogenesis related protein dot intensity was adjusted with positive control, and the datashown as percent or fold change by silibinin treatment. B, three lung tumor samples from eachgroup were analyzed for IL-13, TNFα, TIMP-1, TIMP-2 and VEGF protein levels byimmunoblotting. Membranes were stripped and reprobed with β-actin as a loading control.

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C, Elisa for mouse IL-13 determined plasma IL-13 levels from control and silibinin treatedgroups using recombinant mouse IL-13 as a standard. Columns, mean of four samples; errorbars, SEM for each group. D, the presence of TAMs were assayed by F4/80 IHC (brownstaining) of tumor-bearing lungs harvested from control and silibinin-treated A/J mice after 43wks (n = 5 mice/group). Macrophage numbers were quantified by counting brown stainingcells in the pulmonary stroma in 5 randomly selected fields at 400X magnification from eachof 5 different samples in both groups. Columns, mean; error bars, SEM for each group(right). SB, silibinin; IL, interleukin; IFN, interferon; TNF, tumor necrosis factor; TIMP, tissueinhibitor of metalloproteinase; VEGF, vascular endothelial growth factor.

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Figure 4.Silibinin inhibits activation of HIF-1α, p65NF-κB and STAT3 in urethane-induced lung tumorcell nuclei. Tumor-bearing lungs harvested from A/J mice after 43 wks of urethane (n = 5 mice/group) and analyzed by using IHC for HIF-1α (A), p65NF-κB (ser276) (B) and pSTAT3(ser727) (C), as detailed in Materials and Methods (left). HIF-1α, p65NF-κB (ser276) andpSTAT3 (ser727) immunoreactivities in tumors were quantified by counting (brown) positivecells in 5 randomly selected fields at 400X magnification from each of 5 different samples inboth groups. The percent positive cells was determined as the number of positive-stained cellsX 100/total number of cells counted. Columns, mean; error bars, SEM for each group(right). Final magnification 1000X for HIF-1α and 400X for phospho-p65NF-κB and phospho-STAT3. (A-C, right), three lung tumor samples were randomly taken from each group and

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analyzed for HIF-1α, p65NF-κB (ser276), p65NF-κB (ser536), p65NF-κB, pSTAT3 (ser727)and STAT3 protein levels by immunoblotting total cell lysates. Bands were visualized byenhanced chemiluminescence detection. Membranes were stripped and reprobed with β-actinas loading control. SB, silibinin; NF-κB, nuclear factor kappa B; STAT, signal transducer andactivator of transcription factor.

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Figure 5.Silibinin enhances angiopoietin-2 and Tie-2 levels in urethane-induced lung tumors. Threelung tumor samples were randomly taken from each group and analyzed for pTie-2(Tyr992),Tie-2 and angiopoietin-2 protein levels by immunoblotting. Bands were visualized byenhanced chemiluminescence detection. Membranes were stripped and reprobed with β-actinas a loading control. Densitometric analysis of band intensity for each protein was adjustedwith β-actin. Columns, mean intensity of three bands; error bars, SEM for each group. SB,silibinin; Ang-2, angiopoietin-2.

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Figure 6.Potential anti-angiogenic mechanism of silibinin in advanced lung tumor cells in A/J mice.Silibinin inhibits the production and secretion of cytokines and interleukins via inhibition oftumor associated macrophages. Further, silibinin inhibits the activation of transcription factors(NF-κB/STAT/HIF-1α) but induces the expression of Ang-2/Tie2 to inhibit the angiogenesisin urethane induced-advanced lung tumors in A/J mice. The green color scheme follows theevents involved in driving angiogenesis, and all red color schemes show the effect of silibinin.

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