Silibinin Preferentially Radiosensitizes Prostate Cancer ...Silibinin Preferentially Radiosensitizes...

Small Molecule Therapeutics

Silibinin Preferentially Radiosensitizes ProstateCancer by Inhibiting DNA Repair SignalingDhanya K. Nambiar1,2, Paulraj Rajamani2, Gagan Deep3, Anil K. Jain3,Rajesh Agarwal3, and Rana P. Singh1,4

Abstract

Radiotherapy, a frequent mode of cancer treatment, is oftenrestricted by dose-related toxicity and development of thera-peutic resistance. To develop a novel and selective radiosensi-tizer, we studied the radiosensitizing effects and associatedmechanisms of silibinin in prostate cancer. The radiosensitizingeffect of silibinin with ionizing radiation (IR) was assessed onradioresistant prostate cancer cell lines by clonogenic, cell cycle,cell death, and DNA repair assays. Tumor xenograft growth,immunohistochemical (IHC) analysis of tumor tissues, andtoxicity-related parameters were measured in vivo. Silibinin (25mmol/L) enhanced IR (2.5–10 Gy)-caused inhibition (up to96%, P < 0.001) of colony formation selectively in prostatecancer cells, and prolonged and enhanced IR-caused G2–Marrest, apoptosis, and ROS production. Mechanistically, silibi-nin inhibited IR-induced DNA repair (ATM and Chk1/2) and

EGFR signaling and attenuated the levels of antiapoptoticproteins. Specifically, silibinin suppressed IR-induced nucleartranslocation of EGFR and DNA-PK, an important mediator ofDSB repair, leading to an increased number of g-H2AX (ser139)foci suggesting lesser DNA repair. In vivo, silibinin stronglyradiosensitized DU145 tumor xenograft inhibition (84%, P <0.01) with higher apoptotic response (10-fold, P < 0.01) andreduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibininenhanced the radiotherapeutic response via suppressing IR-induced prosurvival signaling and DSB repair by inhibitingnuclear translocation of EGFR and DNA-PK. Because silibinin isalready in phase II clinical trial for prostate cancer patients, thepresent finding has translational relevance for radioresistantprostate cancer. Mol Cancer Ther; 14(12); 2722–34. �2015 AACR.

IntroductionRadiotherapy is a frontline treatment option in prostate cancer,

next only to radical prostectomy, with 25% of men ages 18 to 65years and 42% of men ages 65 to 74 years undergoing radiother-apy (1). However, the positive facets of radiotherapy are moder-ated by its detrimental consequences onnormal dividing cells andemergence of therapeutic resistance (2). Hence, development ofnovel radiosensitizing agents with a capacity to improve thera-peutic index mandates urgent attention.

The major mechanisms responsible for radiotherapeutic resis-tance are the activationof prosurvival andDNA repair pathways inresponse to IR (3, 4). In general, IR-induced cell death ismediatedby induction of double-stranded breaks (DSB) in DNA, whichleads to faulty cell division and death by mitotic catastrophe.However, in response to IR, the damaged cells also activate their

DNA repair machinery, including ATM and DNA-PK, whichreduces the extent of radiation-induced damage and resultantdeath (5). IR also activates mitogenic signaling especially EGFR(6), and IR-induced EGFR nuclear translocation, which activatesDNA repair pathways (7, 8). Understandably, these mechanismsare unfavorable to the use of radiation in cancer treatment, andsuggest that agents that could inhibit EGFR-mediated signalingand/or DNA repair following radiotherapy could be successfulradiosensitizers.

Here, we have evaluated the radiosensitizing properties of aplant flavonoid silibinin in prostate cancer, a malignancy withlate-stage radioresistance. Silibinin possesses strong anticanceractivity against prostate cancer in preclinical studies and is cur-rently in phase II clinical trial against prostate cancer (9, 10).Subsequently, we rationalized that silibinin could enhance radio-response andmayhave additional translational potential. Indeed,our results suggested that silibinin preferentially sensitizes pros-tate cancer cells to IR, reduces IR-induced systemic injury andtissue toxicity and in mice, and enhances radiotherapeutic indexfor prostate cancer.

Materials and MethodsCell lines and reagents

Human prostate carcinoma DU145, PC-3, and 22RV1 cells,mouse keratinocyte JB6 cells, human lung cancer A549 cells werefrom the ATCC. DU145, 22RV1, and A549 cell lines were char-acterized by short tandem repeat analysis in August 2013, and PC-3 in July 2015. JB6 cells were procured in January 2010. HEK-293cells were purchased from National Centre for Cell Science inAugust 2010. Initially, cells were grown and frozen in liquid

1CancerBiologyLaboratory, School of Life Sciences, Jawaharlal NehruUniversity,NewDelhi, India. 2School of Environmental Sciences, Jawa-harlal Nehru University, NewDelhi, India. 3Department of Pharmaceu-tical Sciences, Skaggs School of Pharmacy and PharmaceuticalSciences, University of Colorado Denver, Aurora, Colorado. 4Schoolof Life Sciences, Central University of Gujarat, Gandhinagar, India.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Rana P. Singh, Jawaharlal Nehru University, New Delhi,India 110067. Phone: 91-11-2670-4503; Fax: 91-11-26742558. E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0348

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 14(12) December 20152722

on March 7, 2021. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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nitrogen. Cells grown from a vial were always monitored for theirmorphology and used for the experiments within 5 months.Other cell culture materials were from Himedia. Silibinin (MW¼ 482.4), RPMI media, propidium iodide (PI), DCFH-DA, anti-bodies to Cdc25C, DNA-PK, b-actin were obtained from Sigma-Aldrich Chemical Co. Antibodies for Cyclin B1, Cdc-2, PCNA andsurvivin, phospho-EGFR, ATM, phospho-Chk1, phospho-Chk2,Chk1, Chk2 and anti-rabbit peroxidase–conjugated secondaryantibody were from Cell Signaling Technology. Anti-tubulin andanti-mouse peroxidase–conjugated secondary, goat anti-rabbitIgG were from Santa Cruz Biotechnology. ECL detection systemwas from Millipore.

Cell culture and treatmentsDU145, PC-3, A549, and 22RV1 cells were cultured in RPMI-

1640mediumwith 10%FBS andpenicillin–streptomycin at 37�Cin 5% CO2 incubator. HEK-293 cells were grown in DMEM with10% FBS and JB6 cells in MEMmedium with 5% FBS. Cells weretreated with varying doses of radiation (2.5–10 Gy) and/orsilibinin (25–100 mmol/L), which was dissolved in DMSO. Thetreatment time varied from 3 to 72 hours depending upon theexperiment. An equal volume ofDMSO (0.1% v/v) was present ineach treatment.

Irradiation protocolThe cells were irradiated in 60Co gamma chamber (Model

4000A, Bhabha Atomic Research Centre) at a dose rate of 4.6Gy/min. For the animal experiment,micewere irradiated using RS2000 Biological Irradiator (Rad Source Technologies) housed atAnschutzMedical campus. The dose rate of the irradiator was 1.34Gy/min. The silibinin and IR treatments were started whenthe tumor volume reached 200 mm3. Silibinin was given as200 mg/kg body weight per day in 0.5% CMC by oral gavageand immediately followed with the first dose of IR (2.5 Gy/fraction) on day 0. Silibinin treatment continued as 5 days perweek and each IR fractionwas separated by 2days. The cumulativedose of 15 Gy (six doses each with 2.5 Gy) was given to theanimals. After last dosing of silibinin and IR on day 15, theexperiment was terminated on day 16.

Colony formation assayCells were seeded in 6-well plates (500 cells/plate). After 24

hours of plating, cells were treated with respective doses ofradiation and/or silibinin (co-treatment) and eventually culturedfor 10 days; the colonies formed were fixed with ice-cold meth-anol: glacial acetic acid (3:1) for 10minutes; and stained with 1%crystal violet. Plating efficiency was calculated by dividing theaverage number of colonies perwell by the amount of cells plated.Survival fractions were calculated by normalization to the platingefficiency of appropriate control groups. Dose-enhancement ratio(DER) was calculated as the ratio of radiation dose withoutsilibinin to dose of radiation with silibinin required to achievethe same amount of cell kill. If DER is >1, the agent is consideredto be radiosensitizing, whereas a value <1, the agent is consideredto be radioprotective.

Cell-cycle analysis by flow cytometryCell-cycle distribution was analyzed by flow cytometry using

BD FACS Calibur by BD Biosciences and data were analyzed withModFit LT software, as described in earlier studies (11).

Reverse transcriptase PCRCells were seeded and grown in 100-mm culture plates to 70%

confluency under regular growth conditions andwere treatedwithsilibinin (25 mmol/L) and/or radiation (5 Gy) in 10% serumsupplemented RPMI-1640medium. Total RNAwas isolated usingTRizol reagent and cDNA was synthesized as described earlier.This was followed by standard PCR reactions using gene-specificforward and reverse primers (Supplementary Methods). PCRproducts were analyzed by running on 1% agarose gel stainedwith ethidium bromide and photographed under low intensityUV in GelDoc system (Applied Biosystems).

BrdUrd incorporation assayBriefly, the cells were cultured in 96-well plates at a density of

5,000 cells/100 mL/well in complete growthmedia. After 48 hoursof respective treatments, the cells were labeled with bromodeox-yuridine (BrdUrd) using the Cell Proliferation ELISA, BrdUrd(colorimetric) Kit (Roche Applied Science), and the percentBrdUrd incorporation was measured in each treatment, as perthe manufacturer's protocol.

Analysis of ROS levelsCells were treated with DMSO or 25 mmol/L silibinin and/or 5

Gy for 12, 24, 48, and 72 hours. Cells were incubated with 20mmol/L DCF-DA during the last 30 minutes of treatment at 37�C.The probe waswashed off with PBS and the cells were trypsinized,resuspended in PBS, and analyzed for DCF-positive cells by flowcytometry.

DNA fragmentation assayAt the end of the treatment time, cells were gently scraped and

centrifuged at 1,000� g for 5 minutes at 4�C following which thecells were lysed by adding 500mL of the lysis buffer containing 1%NP-40, 20 mmol/L EDTA, 5 mmol/L Tris-HCl-pH, 8.0. The lysedsample was then centrifuge at 12,000 � g at 4�C for 20 minutesand the supernatant was collected and treated with RNase at 37�Cfor 1hour. The fragmentedDNAwas then extractedby thephenol-chloroformmethod. FragmentedDNAwasprecipitated overnightat �20�C after adding 30 mL of 5 mol/L NaCl to a final concen-tration of 300mmol/L and 2.5 volume of ice-cold 100% ethanol.The pellet obtained after centrifugation, was washed with 70%ethanol, air dried and resuspended in TEbuffer, and runona1.2%agarose gel containing 0.5 mg/mL ethidium bromide.

AO-EtBr apoptosis assayAfter the completion of desired treatments, total cells were

collected by centrifugation and added with 50 mL of stainingsolution containing the acridine orange (AO) and EtBr mix (100mg/mL each) in PBS. Then cells were put on a slide and visualizedunder 100� field of a fluorescent microscope. Aminimumof 250cells were scored and the percentage of apoptotic cells weredetermined. Early apoptotic cells showed green nuclei with con-densed chromatin whereas late apoptotic cells showed orangenuclei with condensed chromatin.

Immunoblot analysisProstate cancer cells were grown in regular serum conditions

to 70% confluency and treated with the desired doses ofsilibinin and/or radiation. At the end of the treatment timeperiods, whole-cell lysates were prepared in nondenaturinglysis buffer as published recently (12). Cytoplasmic and nuclear

Silibinin Preferentially Radiosensitizes Prostate Cancer

www.aacrjournals.org Mol Cancer Ther; 14(12) December 2015 2723




extracts were prepared as described earlier (12). Protein con-centrations were determined by Bradford assay and 60 to 80 mgprotein lysates were resolved on 8% to 16% SDS-PAGE. Pro-teins were blotted onto nitrocellulose membranes and probedwith specific primary antibodies followed by detection withhorseradish peroxidase–conjugated appropriate secondaryantibodies using the ECL detection system.

Immunofluorescence staining assayCells were grown on coverslips in 24-well plate, washed with

PBS,fixed in 4% formaldehyde for 15minutes, andpermeabilizedwith 0.3% TritonX-100 for 15 minutes. Cells were blocked with[10% (v/v) FBS, 0.3% (w/v) TritonX-100] for 1 hour, incubatedwith a primary rabbit anti-pEGFR (1:200) or DNA-PKcs (1:250)overnight at 4�C, and then incubatedwith the secondary antibodyanti-rabbit IgG (1:500) for 1 hour, andfinally counterstainedwith300 nmol/L DAPI as published earlier (12). Cells were examinedusing a confocal microscope at our Central Instrument Facility inthe school.

In vivo tumor xenograft studyAthymic (nu/nu) male nude mice (NCI, Frederick, MD)

approved by the Institutional Animal Care and Use Committeeof the University of Colorado Denver were s.c. injected in theright flank with 6� 106 DU145 cells mixed with Matrigel. Fromthe day following xenograft implantation, mice were moni-tored regularly for tumor growth and once the tumors reachedapproximately around 200 mm3, the mice were randomlydivided into four groups and respective treatments were given.For the radiation alone group, IR treatment (2.5 Gy/dose) wasgiven at an interval of 2 days, until a cumulative dose of 15 Gywas achieved (day 15). Mice were anesthetized with ketamine/xylazine before radiation and positioned under a lead shieldsuch that only the tumor-bearing flank was exposed. Silibinin(200 mg/kg) was given in 0.5% CMC (w/v) to animals as oralgavage just before the first dose of IR and continued for 5 daysper week. Group I (vehicle control): 200 mL of 0.5% CMC (w/v)in saline; Group II: mice treated with IR alone, group III: micetreated with IR and silibinin (SB); group IV: mice treated withsilibinin. Tumor sizes were measured twice weekly using digitalcaliper and tumor volume was calculated by the formula:0.5236 L1 (L2)2, where L1 is long diameter and L2 is shortdiameter. Mice were sacrificed on day 16 (Fig. 5A).

Immunohistochemical analysis of tumorsAfter 24 hours following the final irradiation and silibinin

treatment, the mice were euthanized and tumors were dissect-ed out, weighed, fixed in formalin, and further processed andembedded in paraffin. Paraffin-embedded tissue sections weredeparaffinized and stained using specific primary antibodyfollowed by 3,30-diaminobenzidine (DAB) staining, as previ-ously described. Biotinylated secondary antibodies used wererabbit anti-mouse IgG (1:200; Dako) and goat anti-rabbit IgG(1:200; Santa Cruz Biotechnology). Apoptotic cells were iden-tified by TUNEL staining using Dead End Colorimetric TUNELSystem (Promega Corp.) as published (13). The percentage ofKi-67, TUNEL, pChk2, pH2A.X-positive cells were quantifiedby counting brown-stained cells within total number of cells atfive arbitrarily selected fields from each tumor at 400�magnification.

Densitometric and statistical analysesBands on X-ray films were scanned and their mean density was

analyzed by ImageJ (NIH, Bethesda, MD). Densitometry data,which is represented below the bands, are the "fold change" ascompared with respective DMSO control, after normalizationwith respective loading controls (b-actin). The data were statisti-cally analyzed using the Jandel Scientific Sigma Stat 3.5 software.The Student t test was used for statistical significance (P < 0.05).The paired Student t test was used for tumor volumes.

ResultsSilibinin preferentially radiosensitizes prostate cancer cells

Effect of silibinin in sensitizing prostate cancer cells to IR wasassessed using two radioresistant human prostate cancer cell linesDU145 and PC-3, using clonogenic survival assays. PC-3 weremore sensitive to IR treatment than DU145, showing 47%decrease in colony formation compared with 32% in DU145 at5 Gy dose (Fig. 1A and B). IR (2.5–10 Gy) inhibited colonyformation by 17% to 68%, which increased to 29% to 92% (P <0.001) in combination with 25 mmol/L silibinin in DU145 cells(Fig. 1A). Similar results were observed for PC-3 cells (Fig. 1C).The DER at 50% inhibition in colony formation was 1.67 forDU145 and 1.4 for PC-3 cells. Silibinin also radiosensitized othertype of cancer cells, for example, human lung carcinoma A549cells, with a DER of 1.6 (Fig. 1D). More importantly, in non-neoplastic human embryonic kidney cells (HEK-293), similartreatment with IR and/or silibinin did not radiosensitize the cells;in fact, it resulted in radioprotection with a DER of 0.83 at 50%inhibition (Fig. 1E). In radioresponsive 22RV1 cells, combinationtreatment did not show any significant increase in radiosensitivity(Supplementary Fig. S1).

Silibinin enhanced and prolonged IR-induced G2–M arrest ofprostate cancer cells

IR alone increased G2–M cell population from 20% in controlto 37%, which was further increased to 59% (P < 0.001) whencombined with 25 mmol/L silibinin at 24 hours, and in thecombination treatment, the effect was prolonged (P < 0.01) evenuntil 48 hours (Fig. 2A, left and Supplementary Fig. S2). A similartrend in cell-cycle effects was observed in PC-3 cells with combi-nation treatment (Fig. 2A, right). Concurrently, the expressionlevels of Cyclin B1 and Cdc2 decreased as early as 6 hoursfollowing the combination treatment (Fig. 2B). This effect wassustained until 48 hours, where we also observed IR-inducedincrease in Cdc25C, which was strongly decreased in the IR plussilibinin treatment, supporting prolonged G2–M block in thecombination treatment (Fig. 2B, right and Supplementary Fig.S3A). In HEK-293 cells, combining SB with radiation did notshow significant difference in gene expression of cell-cycle reg-ulators as compared with radiation treatment alone, which againsuggests that silibinin shows a differential response (Supplemen-tary Fig. S3A).

Silibinin strongly inhibited cancer cell proliferation followingIR exposure and downregulated IR-induced expression ofprosurvival molecules

Following irradiation, a fraction of cells that are lethally dam-aged undergo apoptosis, but the remaining cells that are sublethally irradiated try to evade apoptosis, by activating a prosur-vival response. After 48 hours of treatment, BrdUrd incorporation

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Mol Cancer Ther; 14(12) December 2015 Molecular Cancer Therapeutics2724




decreased by 36% (P < 0.001) in silibinin with IR as comparedwith IR alone (16%) in DU145 cells, whereas in PC-3 cells,combination treatment resulted in 39% (P < 0.01) inhibitionversus 30% in IR alone (Fig. 2C). This inhibition was aided viadecreased expression of both PCNA and survivin, which did notchange with IR alone in DU145 cells (Fig. 2D). In PC-3 cells, IRappeared to posttranscriptionally modify and increase proteinlevels of PCNA and survivin, which were decreased by the com-bination treatment (Fig. 2D and Supplementary Fig. S3B).

Silibinin enhanced IR-induced ROS production and led toprolonged oxidative stress

Although, silibinin has antioxidant activity, it is now welldocumented that many polyphenols, including silibinin, alsobehave as pro-oxidants under certain conditions (14, 15). Wefound that IR showed an established distribution pattern of ROS

production during 12 to 72 hours of treatments with a peak at 24hours, whereas silibinin showed peak of ROS production atapproximately 48 hours, with 27% positive cells (Fig. 2E, left).In the combination treatment, therewas a dramatic increase in theROS-positive cellswith apeak at 24hours (61%,P<0.001; Fig. 2E;Supplementary Fig. S4A and S4B). Combination of silibinin withIR also led to reduction in the mRNA expression of antioxidantenzymes including SOD1, SOD2, Catalase, and GST, supportingthe data showing enhanced oxidative stress in these cells (Sup-plementary Fig. S4C).

Because instead of radiosensitization, we had observedradioprotection of HEK-293 cells by silibinin, we examinedwhether this differential effect could be facilitated via themodulation of redox status. Surprisingly, presence of silibininwith IR showed an inverse effect on HEK-293 cells to that ofcancer cells. Silibinin treatment alone showed 7% ROS-positive

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Figure 1.Silibinin selectively radiosensitizesprostate cancer cells. Cells wereplated at 600 cells per well in a 6-wellplate and after 24 hours treated withthe indicated dose of IR and/or 25mmol/L of silibinin (SB). Cells werethen maintained for another 10 days.The colonies were fixed and stained.Number of colonies containing >50cells were counted and thepercentage of colony formation wasdetermined for each cell line withrespect to the nontreated controls.Survival curves for advanced humanprostate carcinoma DU145 (A) withrepresentative picture for stainedcolonies of DU145 cells (B), PC-3 cells(C), A549 cells (D), and transformednonneoplastic human embryonickidney HEK-293 (E) cells are shown;P < 0.01 (#), P < 0.001 (�) comparedwith respective control.






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Figure 2.Silibinin potentiates IR-inducedG2–Marrest and inhibition in cell proliferation, and augments oxidative stress selectively in prostate cancer cells. DU145 andPC-3 cellswere exposed to IR with or without silibinin (SB). After treatment time points, cells were processed for cell-cycle analysis using saponin-PI staining. A,quantitative data showing cell-cycle distribution in DU145 (left) and PC-3 cells (right) after treatment with IR (5 Gy) and/or SB (25 mmol/L). B, Western blotsfor G2–M cell-cycle–related proteins at 3, 6, and 48. Cell proliferation rate in cells was assessed by BrdUrd incorporation assay. C, the percentage of BrdUrdincorporation was calculated with respect to control after 48 hours treatment in both DU145 and PC-3 cells. D, RT-PCR and immunoblotting analysis of PCNA andsurvivin proteins after 48 hours treatment. E, for oxidative stress analysis, cells were analyzed for DCF fluorescence by flow cytometry, after treatmentwith IR (5 Gy)and/or SB (25 mmol/L). The percentage of positive cellswas thosewith a fluorescent intensity >102 on the histogram. Graph showing change in theDCF-positive cellsin different groups after 12 to 72 hours of treatment of DU145 cells. Bar diagram showing DCF-positive cells for DU145 and HEK-293 cells after 24 hours of treatment.P < 0.001 (�) and P < 0.01 (#) compared with respective control or indicated treatment.

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Figure 3.Silibinin potentiates radiation-induced apoptosis and attenuates DNA repair activation signaling in prostate cancer cells. A, representative pictures (left) andquantitative data (right) showing DNA laddering in DU145 and PC-3 cells treated with IR (5 Gy) and/or silibinin (25 mmol/L) for 48 hours. B, graphical datadepicting the percentage of cells positive for apoptosis after AO–EtBr staining. C, Western blot analysis of Bcl-2 and cleaved caspase-3 in DU145 andPC-3 cells after 48 hours of treatment. D, immunoblotting for damage signaling molecules activated in response to IR (5 Gy) and/or SB (25 mmol/L). E,RT-PCR for cell-cycle check-point regulators Chk1 and Chk2; and F, phospho/total Chk1 (threonine 345 and serine 296) and Chk2 levels (threonine 68) inDU145 cells. P < 0.001 (�) and P < 0.05 ($) compared with respective control or indicated treatment.






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Figure 4.Silibinin inhibits nuclear translocation of EGFR andDNA-PK in cancer cells and reduces repair of pH2A.X (Ser 139) foci in prostate cancer cells. A, confocalmicroscopyshowing distribution of EGFR (red) in DU145 cells in response to IR (5 Gy) and/or SB (25 mmol/L). B, immunoblotting for EGFR in cytoplasmic and nuclearfractions after 3 hours of treatments; tubulin and laminwere used as loading control for cytoplasmic and nuclear compartments, respectively. C, confocalmicroscopyshowing distribution of DNA-PK (red), nucleus (Sytox-green) in DU145 cells in response to IR (5 Gy) and/or SB (25 mmol/L) at 3 hours; and D, representativeimages of pH2A.X foci (red) in the nucleus at 12 hours and quantitation of number of pH2A.X foci after 6 and 12 hours of treatments in DU145 cells.P < 0.001 (�) and P < 0.05 (#) compared with respective control or indicated treatment.

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Figure 5.Silibinin treatment enhances radiation-induced tumor growth inhibition of human prostate cancer DU145 xenograft in athymic nude mice. A, diagrammaticrepresentation of the time line followed for the tumor study. Mice were s.c. injected with DU145 cells (6 � 106) mixed with Matrigel (1:1) and monitored for tumorgrowth until the tumor size reached approximately 200 mm3. Then mice were treated with IR (2.5 Gy) with a gap of 2 days between two IR fractions, withor without SB (200 mg/kg), which was given 5 days per week. Control and IR-alone group of mice were gavaged with 0.5% CMC in saline. The treatment wascontinued until the cumulative irradiation dose reached 15 Gy. Twenty-four hours after the final fraction of IR (day 16), the tumors were excised and processedfurther for immune-histochemical staining. B, tumor volume/mouse as a function of cumulative radiation dose; C, tumor weight/mouse at the end of study;D, mean body weight per mouse; and E, average diet consumption per mouse per day were analyzed as detailed in Materials and Methods. Data, mean� SE from 8mice in each group. Effect of IR and/or silibinin was also checked on the hematopoietic system at the end of the experiment, meanWBC count per mouse (F), meanlymphocyte count/mouse (G), and represents mean platelet count/mouse (H); P < 0.01 (#) and P < 0.001 (�) compared with respective control.






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cells, whereas in IR alone, there were 25% ROS-positive cells at48 hours (Fig. 2E, right and Supplementary Fig. S4D). However,combining silibinin with IR significantly reduced ROS-positivecells to 7.6% (P < 0.001).

Silibinin enhanced IR-induced apoptosisBecause prosurvival molecules were suppressed and ROS level

was enhanced in combination treatment, we assessed whether itled an increase in radiation-induced apoptosis. Compared witheither silibinin or IR, an intense DNA laddering (>2-fold) incombination treatment was observed in both DU145 and PC-3cells (Fig. 3A). AO–EtBr assay also showed an increase in apo-ptosis from 25% to 27% in IR alone to 44% to 46% (P < 0.001) incombination after 48 hours in both prostate cancer cells (Fig. 3B).Bcl-2 overexpression, amajor player in the development of radio-resistant phenotype (16), was upregulated by IR in DU145 cells,whereas combining it with silibinin led to a profound decrease inBcl-2 expression in DU145 cells with a moderate effect on PC-3cells (Fig. 3C).

Silibinin augmented the therapeutic efficacy of radiation byinhibiting DNA repair

One of the major mechanisms for acquired radioresistance incancer cells is the DNA repair, DNA being the principle target ofradiation-induced damage (17). Our results revealed that IRenhances ATM expression as early as 3 hours but in combinationtreatment, especially at 6 hours, it downregulated the expressionofATM inDU145 cells (Fig. 3D). Also, thephosphorylated level ofEGFR (Y1068) was enhanced with IR, which was reduced bysilibinin treatment. Chk1 and Chk2, the downstream effectors ofATM involved in activation ofDNA repair (18), were also inducedby IR showing an increase inmRNA levels (Fig. 3E) and enhancedphosphorylation of Chk1 (S345 and S269) and Chk2 (T68).These IR-induced levels were downregulated in the combinationtreatment (Fig. 3F).

Silibinin inhibited IR-induced nuclear translocation of EGFRThe role of nuclear EGFR in development of radioresistance by

acting as a mediator for DNA repair is gaining grounds (6, 8).Furthermore, IR-induced EGFR activation is a prominent contrib-utor to radioresistance. Because we observed inhibition of IR-activated EGFR,we further analyzedwhether silibinin can alter IR-induced nuclear translocation of EGFR in prostate cancer cells. IRexposure ofDU145 cells resulted in nuclear translocation of EGFR(red) at 3 hours that was almost completely inhibited by silibininand EGFR localization was limited to the cytosol (Fig. 4A). Thiswas further confirmed by measuring EGFR protein levels incytosolic and nuclear fractions (Fig. 4B). We also checked theeffect of these treatments onnuclear translocationof EGFR innon-neoplastic JB6 mouse keratinocyte cells (Supplementary Fig. S5).Compared with prostate cancer cells, we did not observe consid-erable reduction in the nuclear localization of EGFR in JB6 cells,

suggesting that this effect of silibinin may be selective to neo-plastic cells.

Silibinin inhibited IR-induced nuclear translocation of DNA-PK and prolonged the presence of pH2A.X foci

Confocal microscopy showed that after IR treatment, most ofDNA-PKwas localized into the nucleus.However, when cells weretreated with silibinin and IR, like EGFR, DNA-PK too remainedexcluded out of the nucleus, thereby blocking it from carrying outits DNA repair function (Fig. 4C). To further support that silibinininhibits IR-induced DNA repair signaling, pH2A.X foci wereassessed as indicator of DNA damage. We observed that IRexposure increased pH2A.X foci at 6 hours, which was reducedby 12 hours (59% decrease, P < 0.001), whereas in presence ofsilibinin, it was increased by 38% (Fig. 4D). Furthermore, by 12hours in the presence of silibinin, the number of pH2A.X foci wasmore than 2.5-fold (P < 0.001) from that of IR alone (Fig. 4D).This persistence of pH2A.X foci levels in combination as com-pared with IR alone suggests that silibinin-mediated radiosensi-tization involves an inhibition of repair of IR-induced DNAdamage.

Silibinin enhanced radiation-induced tumor growth inhibitionand protected the normal tissue from radiation injury

After establishing the radiosensitizing properties of silibininin vitro, we substantiated these findings in the DU145 xenograftmodel. Once the tumors reached approximately 200 mm3,mice were treated with IR and/or silibinin, as detailed in theMaterials and Methods (Fig. 5A). Silibinin and IR inhibitedtumor growth (volume) by 56% (P < 0.01) and 61% (P < 0.01)from control, respectively; however, their combination led to84% (P < 0.001) growth inhibition when compared withcontrol (Fig. 5B). Similarly, tumor weight was decreased by46% (P < 0.01), 43% (P < 0.01), and 82% (P < 0.001) insilibinin, IR and IR with silibinin-treated groups from control,respectively (Fig. 5C). The tumor volume and weight weredecreased by 61% (P < 0.01) and 69% (P < 0.01) in combi-nation when compared with IR alone, respectively.

IR treatment alone led 13% and 30% decrease in body weightand diet consumption, respectively, at the end of treatment;however, silibinin treatment reversed these losses by 8% and19% (P < 0.01 for both), respectively (Fig. 5D and E). IRleverages heavy toxicity to the hematopoietic system (18). Weobserved that total WBC, neutrophil, monocyte, and plateletcounts were reduced by 33% to 50% by IR; however, treatmentwith silibinin completely blocked (P < 0.01–0.001) theseadverse effects of IR on hematopoietic system (Fig. 5F–H andSupplementary Fig. S6B–S6E). We also observed a lesser dam-age to genitourinary tract (GUT; Supplementary Fig. S6A).These results indicate that the combination treatment was nottoxic to normal tissues and in fact, silibinin showed radiopro-tective response in normal tissues.

Figure 6.Combination of silibinin with IR leads to reduced expression of cell proliferation and DNA repair markers and enhances apoptosis in DU145 xenograft. Tumorxenograft tissue samples were immunohistochemically analyzed for Ki67, pChk-2 (T68), pH2A.X, and TUNEL-positive cells as detailed in Materials andMethods. A, the representative pictograph (�400, magnifications) for positive brown-stained cells (dark color) for each of the markers are shown from control, IR,SB, and IRþSB groups. Quantitative data for Ki67 (B), pChk-2(T68) (C), pH2A.X(S139) (D), and apoptotic cells (E) from 5 to 6 mice in each group; P < 0.01 (#) andP < 0.001 (�) compared with respective control. F, a model summarizing the radiosensitizing action of silibinin in prostate cancer (PCa) cells. Black dotted arrowsrepresent pathways activated by IR in prostate cancer cells, bold arrows represent the action of silibinin (SB), and crosses represent IR-activated responsesblocked by silibinin.






Silibinin-mediated radiosensitization of prostate tumorinvolved inhibition of DNA repair and enhanced apoptosis

The immunohistochemical analysis of tumor samples showedthat IR and/or silibinin reduced the immunostaining for Ki67(Fig. 6A). IR or silibinin alone decreased Ki67-positive cells by25% and 21%, respectively; however, their combination resultedin 54% (P < 0.001) decrease versus control, and 33% (P < 0.01)versus IR alone treatment (Fig. 6B), which supported the corre-sponding decrease in tumor burden. For the translational rele-vance of the in vitro observations of DNA repair signaling, tumorswere analyzed for the pChk2 (Fig. 6A andC), whichwas increasedby approximately 7-fold (P < 0.001) by IR treatment andthat was reduced by the silibinin treatment to approximately 2-fold (P < 0.001) when compared with control (Fig. 6C). Next, theDNA DSBs were analyzed by immunostaining of pH2A.X (S139)(Fig. 6A and B), which was increased to 4% as compared with 1%in control, whereas in combination with silibinin, as anticipated,it increased to 13% (P < 0.001), suggesting that the mechanismof silibinin-mediated radiosensitization involved reduced DSBrepair signaling (Fig. 6D). By TUNEL staining of tumor tissue(Fig. 6A), no significant apoptosis induction was observedwith radiation or silibinin alone whereas their combinationincreased apoptotic cells by 10-fold (P < 0.001) from controland 5-fold (P < 0.001) from IR treatment (Fig. 6E).

DiscussionRadiotherapy is one of the principal and affordable treatment

choices for locally or regionally advanced prostate cancer (19).However, development of radioresistance in these cells delimitsits effectiveness in patients. With an aim to strengthen therapeuticoutcomes, radiotherapy is often used in combination with drugsthat are either cytotoxic or can radiosensitize or both (20, 21).These agents help in achieving the required remission at a muchlower dose of radiation, thereby reducing the damage to normaltissues, which is a very germane issue in cancer treatment. How-ever, currently, there is barely any radiosensitizer that has beensuccessful in clinics.

In this study, we demonstrated the radiosensitizing effects ofsilibinin in prostate cancer cells. Silibinin enhanced the efficacy ofradiotherapy in prostate cancer via (i) enhancing and prolongingthe G2–M cell-cycle arrest induced by IR, (ii) augmenting ROSlevels and sustaining high level of oxidative stress, (iii) inhibitingIR-induced prosurvival signaling and antiapoptotic pathways,(iv) inhibiting IR-inducedDNA repair signaling in prostate cancercells and tumors. These mechanisms eventually contributed todecreased cell growth, clonogenicity, and increased cell death;subsequently improving radiotherapeutic response. In addition,silibinin also helped in countering IR-induced toxicity in normaltissues.

One of the mechanisms for radiosensitizing effect of a drugcould be its ability to affect cell-cycle progression especially byblocking it in G2–M phase of the cell cycle (22). Our study foundthat silibinin could enhance IR-induced G2–M arrest and alsoprolongs the duration of the arrest. This is of high significance infractionated radiotherapy, G2–M phase being the most radiosen-sitive phase in the cell cycle, arresting a maximal population ofcells in this phasewould subsequently sensitize them to next cycleof radiation and enhance cell killing (23).

Other than cell-cycle perturbations, radiation-induced damageis essentially orchestrated via the production of ROS, which

targets macromolecules, causing severe damage leading to celldeath (24). Radiation-induced ROS levels peak withinminutes ofexposure and after the peak, it maintains a medium level of ROSlasting for days after irradiation (25). Unlike in normal cells, thismoderate level of ROS is well tolerated by cancer cells, asthese cells manipulate their redox system and generally havehigh levels of antioxidant enzymes to counter these conditions(26, 27). It has also been shown that in cancer stem cells,persistent low levels of ROS could eventually help indevelopmentof radioresistance (28). We demonstrated that addition of silibi-nin along with IR, can dramatically increase the level of ROSwhich, when retained for a longer duration, overpowers therobust antioxidant defense in cancer cells and drives the cell todeath. Thus, silibinin enhances the ionizing radiation (IR)–induced oxidative stress to a level where it does not contributeto development of resistance, instead maintains it high andpersistent enough to induce cell death. Another significantfindingwas that silibinin showed this pro-oxidant behavior exclusively incancer cells, but not in normal cells. This biased behavior inmodulating the redox status of cancer cells is a valuable asset for aradiosensitizer. This finding could also explain the disparity inresponse observed in clonogenic assay with HEK-293 cells whencompared with other cancer cells.

Studies done in the past looking at the mechanisms of radio-resistance in prostate cancer have pointed out that the upregula-tionof prosurvival signaling and the tipping of the balance towardantiapoptosis, compromises with the therapeutic efficacy of IR.The overexpression of Bcl-2 enhances radiation resistance inprostate cancer andother cancer cells (29, 30) and the suppressionof which could overcome resistance (16). We demonstrate thatsilibinin could downregulate IR-induced survival signaling. Sili-binin downregulated IR-induced Bcl-2 and survivin expression inDU145 and PC-3 cells, thereby maneuvering the cells towardapoptosis. Most cancer cells boast of a robust DNA repair system,which also contributes to acquiring a radioresistant phenotype.Thus, DNA repair proteins are now regarded as key targets forradiosensitization (17, 31). Our study found that silibinin coulddownregulate the repair process by inhibiting the expressionof ATM as well as other downstream effectors, including Chk1and Chk2.

One of the major players, which is involved in both DNArepair and upregulation of prosurvival signaling, is EGFR (7,8). Most of the earlier studies linking EGFR and radioresis-tance focused on the receptor signaling induced by EGFR afterligand-independent activation in response to IR (32). Butrecent literature suggests that in response to IR, EGFR couldcontribute directly to development of radioresistance by itsrole in the nuclear compartment where it regulates DNA repairalong with DNA-PK, which is a key regulator of NHEJ (7,8, 33, 34). We found that silibinin treatment blocked nucleartranslocation of EGFR. Silibinin also modulated the distribu-tion of DNA-PK, restraining it from entering the nucleus tocarry out its function, in concurrence with EGFR. The exclu-sion of EGFR and DNA-PK from the nucleus prevented therepair of DNA lesions, as shown by significantly enhancednumber of g-H2A.X foci. This is the first report of a phyto-chemical-modulating DNA repair by blocking the nucleartranslocation of EGFR.

Many radiosensitizers though seem effective in vitro, but donot work under in vivo conditions and also have problemsassociated with toxicity. IR and silibinin combination strongly


Nambiar et al.




decreased tumor burden and also reduced Ki67-positive cells inthese tumors. We also observed intense staining for g-H2A.X andinhibition in Chk2 phosphorylation, suggesting inhibition ofDNA repair signaling induced by IR. From our previous prostatecancer xenograft study (11),we know that the selected oral dose ofsilibinin used for the combination with IR is nontoxic. Weobserved significant reduction in the body weight and diet con-sumption in the IR alone group; however, the combinationshowed substantial improvement in these parameters. We alsofound that systemic toxicity of IR, mainly on the hematopoieticsystem was greatly reduced by silibinin treatment.

In conclusion, we, for the first time, report that silibinin func-tions as a potent radiosensitizer in human prostate cancer cells,and more importantly it offers substantial protection to thenormal tissues from unwarranted IR toxicity. Silibinin targetsmultiple pathways, including DNA repair signaling involvingnuclear translocation of EGFR, which are implicated in develop-ment of radioresistance (Fig. 6F). Earlier studies done with sili-binin showed that a concentration of up to 100 mmol/L could beachieved in blood plasma in mouse (35) as well as in humans(36), which signifies that the dose used in study (25 mmol/L)could be realized in patients undergoing radiotherapy for prostatecancer, and thus underlines the translational significance of thisstudy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D.K. Nambiar, R. Agarwal, R.P. SinghDevelopment of methodology: D.K. Nambiar, G. Deep, A.K. JainAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Deep, R. Agarwal, R.P. SinghAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.K. Nambiar, G. Deep, R. Agarwal, R.P. SinghWriting, review, and/or revision of the manuscript: D.K. Nambiar, G. Deep,R. Agarwal, R.P. SinghAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Agarwal, R.P. SinghStudy supervision: P. Rajamani, R. Agarwal, R.P. SinghOther (immunohistochemistry analysis): A.K. Jain

AcknowledgmentsD.K. Nambiar is supported by fellowships from CSIR, India and Fulbright,

USA.

Grant SupportThe study was supported in part, by grants from CSIR (37/1391-09/EMR-II)

and Department of Science and Technology (DST), University Grant Commis-sion (UGC) Resource Networking, DST-PURSE, and UGC-University of Poten-tial Excellence, India (to R.P. Singh), and NCI RO1 grant CA102514 (toR. Agarwal).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 29, 2015; revised August 21, 2015; accepted September 23,2015; published OnlineFirst October 29, 2015.

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2015;14:2722-2734. Published OnlineFirst October 29, 2015.Mol Cancer Ther Dhanya K. Nambiar, Paulraj Rajamani, Gagan Deep, et al. Inhibiting DNA Repair SignalingSilibinin Preferentially Radiosensitizes Prostate Cancer by

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