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Small Molecule Therapeutics

Role of P53-Senescence Induction inSuppression of LNCaP Prostate Cancer Growthby Cardiotonic Compound BufalinYong Zhang1, Yinhui Dong1,2, Michael W. Melkus1, Shutao Yin1,2, Su-Ni Tang1,Peixin Jiang1, Kartick Pramanik1,3,Wei Wu1,3, Sangyub Kim3, Min Ye4, Hongbo Hu2,Junxuan Lu1,3,5, and Cheng Jiang1,3

Abstract

Bufalin is a major cardiotonic compound in the tradi-tional Chinese medicine, Chansu, prepared from toad skinsecretions. Cell culture studies have suggested an anticancerpotential involving multiple cellular processes, includingdifferentiation, apoptosis, senescence, and angiogenesis. Inprostate cancer cell models, P53-dependent and indepen-dent caspase-mediated apoptosis and androgen receptor(AR) antagonism have been described for bufalin at micro-molar concentrations. Because a human pharmacokineticstudy indicated that single nanomolar bufalin was safelyachievable in the peripheral circulation, we evaluated itscellular activity within range with the AR-positive and P53wild-type human LNCaP prostate cancer cells in vitro. Ourdata show that bufalin induced caspase-mediated apoptosisat 20 nmol/L or higher concentration with concomitantsuppression of AR protein and its best-known target, PSAand steroid receptor coactivator 1 and 3 (SRC-1, SRC-3).

Bufalin exposure induced protein abundance of P53 (notmRNA) and P21CIP1 (CDKN1A), G2 arrest, and increasedsenescence-like phenotype (SA-galactosidase). Small RNAiknocking down of P53 attenuated bufalin-induced senes-cence, whereas knocking down of P21CIP1 exacerbatedbufalin-induced caspase-mediated apoptosis. In vivo, dailyintraperitoneal injection of bufalin (1.5 mg/kg body weight)for 9 weeks delayed LNCaP subcutaneous xenograft tumorgrowth in NSG SCID mice with a 67% decrease of finalweight without affecting body weight. Tumors from bufalin-treated mice exhibited increased phospho-P53 and SA-galactosidase without detectable caspase-mediated apopto-sis or suppression of AR and PSA. Our data suggest potentialapplications of bufalin in therapy of prostate cancer inpatients or chemo-interception of prostate precancerouslesions, engaging a selective activation of P53 senescence.Mol Cancer Ther; 17(11); 2341–52. �2018 AACR.

IntroductionProstate cancer is the second leading cause of cancer-related

death in American men. In spite of significant advances in hor-monal- (1, 2), and taxane-based chemotherapeutic (3, 4) modal-

ities that have improved patient survival measured by months,there is currently no cure for castration-resistant prostate cancer(CRPC), following standard-of-care androgen deprivation thera-py (ADT). Unfortunately, the heavily treated CRPC does notrespond to checkpoint inhibitor immunotherapies that exertdurable remission in some "high mutation load" malignanciessuch as melanoma and non–small cell lung cancer. New drugswith mechanisms distinct from current modalities will likely,therefore, help to improve patient survival to prevent or delayrecurrence at different stages of a patient's therapeutic course.

Chansu, a traditional Chinesemedicine prepared from the skinand auricular gland secretions of giant toads, has been used totreat various cardiac diseases, infections, and pains for millen-niums (5). Chansu-containing prescriptions have been used forcancer treatment in China and are being evaluated clinicallyelsewhere (6). Bufalin (Fig. 1A) is a major cardiotonic steroid-like compound isolated from Chansu (5, 7). In cell culturemodels, bufalin exerts growth inhibitory and/or cytotoxic activityon leukemia (8–10) and solid cancers of many organ sites,including prostate (7, 11, 12) and breast (13–15). Reportedcellular responses include differentiation, apoptosis, cell-cyclearrests, autophagy, antiangiogenesis, and antimetastasis (5).Some of these reports suggest a preferential growth inhibitionof malignant cells over "normal" noncancer cells. Most studiesemployed bufalin exposure concentrations in sub- tomicromolarrange, except the few early studies with leukemia differentiation

1Department of Biomedical Sciences, Texas Tech University Health SciencesCenter School of Pharmacy, Amarillo, Texas. 2Department of Nutrition andHealth, College of Food Science and Nutritional Engineering, China AgriculturalUniversity, Beijing, China. 3Department of Pharmacology, Penn State College ofMedicine, Hershey, Pennsylvania. 4The State Key Laboratory of Natural andBiomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beij-ing, China. 5Penn State Cancer Institute, Pennsylvania State University, Hershey,Pennsylvania.

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

Current address for K. Pramanik: Kentucky College of Osteopathic Medicine(KYCOM), University of Pikeville, Pikeville, Kentucky; and current address forW.Wu, Department of Chemistry and Pharmacology, Zhuhai College of JilinUniversity, Zhuhai, Guangdong, China.

Corresponding Authors:Cheng Jiang, Penn State College of Medicine, Hershey,PA 17033. Phone: 717-531-8964; Fax 717- 531- 5013; E-mail:[email protected]; and Junxuan Lu,[email protected]

doi: 10.1158/1535-7163.MCT-17-1296

�2018 American Association for Cancer Research.

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Figure 1.

Effects of bufalin on apoptosis in LNCaP, DU145, and PC-3 prostate cancer cells and on AR, P53, and DDR signaling in relationship to apoptosis in LNCaP cells.A, Chemical structure of bufalin. B, Concentration-dependent reduction of number of adherent LNCaP cells exposed to bufalin for 48 hours. Thedata were shown as Mean � SEM of crystal violet stain optical density, n ¼ 3 wells. C, Western blot analysis detection of caspase-driven apoptotic marker(c-PARP) and P53 protein in LNCaP (wild-type P53), DU145 (mutant P53), and PC-3 (P53 null) cells after bufalin treatment for 48 hours. D, Westernblot analysis detection of c-PARP, AR, PSA, SRC-1, and SRC-3 in LNCaP cells exposed to bufalin for 48 hours. E, Growth suppression by bufalin on PC-3 cellsestimated by crystal violet stain optical density, n ¼ 3 wells, Mean � SEM. F, Western blot analysis detection of P53, p-P53 (ser15), P21CIP1, andkey DDR proteins in LNCaP cells exposed to bufalin for 48 hours. G, Western blot analysis detection of LC3 lipid modification as a marker of autophagy inLNCaP cells exposed to bufalin for 24 hours and 72 hours.

Zhang et al.

Mol Cancer Ther; 17(11) November 2018 Molecular Cancer Therapeutics2342




induction (5, 9, 16). For example, prostate cancer cell culturestudies reported concentrations up to 15 mmol/L (7, 11, 12).Within such ranges, bufalin was shown to induce caspase-mediated apoptosis in association with changes of BAX/BCL2ratio, cytochrome release, and cellular Caþþ level. It is note-worthy that one study reported the mediator role of P53 (TP53)tumor suppressor protein in micromolar bufalin–inducedcaspase activation and apoptosis of LNCaP cells (11).

In light of the potent cardiotonic indication of bufalin, whichalso represents a major adverse effect of overdosing, the thera-peutic window is likely tight (5). A human pharmacokinetic studywith Huachansu (a commercial Chansu preparation) at up to8-fold higher doses than typical use showed that bufalin infusioncould reach a plasma concentration of approximately 9 nmol/L,without apparent side effects (6). Therefore, the relevance ofmechanisms studied in cell culture models should be validatedwith in vivo efficacy and safety data to facilitate meaningfultranslation into clinical benefits. Given the crucial role of andro-gen receptor (AR) signaling for prostate cancer genesis and pro-gression (17), the steroid-like backbone of bufalin and itsreported AR antagonism (18, 19) and inhibition of select steroidreceptor coactivator SRCs (SRC1 and SRC3, but not SRC2) inmultiple cancer cell types (15), we evaluated the effects of bufalinin the AR-positive and P53 wild-type human LNCaP prostatecancer cells to explore the relationship of AR and P53 signalingpathways with cell-cycle arrest and apoptosis induced by phar-macokinetically relevant concentrations of bufalin. Owing to themultifaceted roles of P53 in regulating cell fates and survival suchas apoptosis, autophagy, and senescence (20) and the reportedP53 activation by bufalin in the LNCaP model (11), we investi-gated the cross-talk of thesemolecular and cellular pathways withrespect to bufalin action. Furthermore, we assessed the in vivoanticancer efficacy of amaximumtolerated dose (MTD)of bufalinusing LNCaP xenograft model in NSG SCID mice and examinedthe tumor tissues for key pathway molecules studied in vitro forconcordance. Our data indicate P53-mediated cancer cell senes-cence as a primary cellular mechanism for the in vivo growthinhibitory efficacy of bufalin, with application implication for theadjuvant therapy of prostate cancer in patients or for the chemo-prevention of precancerous lesion progression.

Materials and MethodsChemicals and cell culture

Bufalin (99.5% purity) was isolated as described previously(21). siRNA for nonspecific sequence (scramble) or targetingTP53 and CDKN1A (P21CIP1) were obtained from Santa CruzBiotechnology. Human LNCaP proate cancer cells were pur-chased from ATCC and used within 25 passages of thawingfrom cryopreservation. Authentication of the LNCaP cell linewas accomplished by next-generation DNA whole-genomesequencing (Macrogen Clinical Laboratory) and RNA sequenc-ing (RNA-seq; Penn State Hershey Genome Sciences Facility).At the genotype level, the cells display XY gender trait and thecharacteristic T877A mutation in the AR gene (22). At themRNA level, these cells express AR, KLK3 (PSA), TP53 (P53),and CDKN1A (P21CIP1) mRNA. Cells were maintained inRPMI-1640 medium (ATCC) supplemented with 10% FBS(Atlanta Biologicals) without antibiotics and incubated instandard 37�C and 5% CO2 humidified environment, exceptwhen indicated otherwise.

The DU145 and PC-3 prostate cancer cells were also obtainedfrom the ATCC and used within 25 passages of thawing fromcryopreservation. In terms of P53 functional status, DU145 cellscontain nonfunctional mutant protein and PC3 cells are null (23,24). DU145 cells were cultured in EMEM essential medium(ATCC) supplemented with 10% FBS without antibiotics. PC-3cells were cultured in F-12Kmedium (ATCC) supplemented with10% FBS without antibiotics. Mycoplasma testing was performedon LNCaP cells and was found negative.

Flow cytometry analysesThe LNCaP cells were harvested with trypsinization for flow

cytometry as previously described (25). Phospho-histone H3(Ser10) was used as a marker of mitotic cells to distinguish thecell cycle phase between G2 and M. Cells were isolated and fixed(4% formaldehyde þ 0.1% Triton X-100 in PBS) at room tem-perature for 10 minutes. The cells were washed with PBS andresuspened in 75 mL of 2% FBS in PBS. The cells were thenincubated with anti-phospho-histone H3 (Ser10; Cell SignalingTechnology) antibody together with propidium iodide/RNAse Asolution at room temperature in the dark for 60minutes. The cellswere rinsed two times and resuspended in 500 mL of 2% FBS inPBS and analyzed by flow cytometry with BD FACSverse (BDBiosciences).

Cell growth assaysThe LNCaP and other prostate cancer cells were exposed to the

indicated treatment and duration, and then the adherent cellswere quantized by crystal violet staining as described previously(26). For long-term growth evaluation, we pretreated LNCaP cellswith vehicle or 20 nmol/L bufalin for 48 hours and reseeded thesame number of trypsin-recovered live cells in fresh completegrowth medium for the indicated days in 6 cell plates. Theadherent cells were stained by crystal violet and photographed.

ImmunoblottingThe whole-cell lysates were prepared for immunoblotting as

described previously (27). Antibodies for P53, phospho-P53(Ser15), P21CIP1, cleaved-PARP (c-PARP), cleaved caspase-3(c-caspase-3), SRC-1, SRC-3, HK2, phospho-ATM (Ser1981), andphospho-H2A.X (Ser139) were obtained from Cell SignalingTechnology. Antibody for LC3 was purchased from MBL. Anti-body for AR was obtained from Millipore and PSA antibody wasobtained from DAKO. Antibody for b-actin was obtained fromSigma-Aldrich. Positive and negative controls were included inimmunoblot analyses whenever possible.

Assessment of senescence-associated b-galactosidase activityTwo methods were performed to assess the senescence-

associated b-galactosidase (SA-b-gal) activity at pH 6.0 in LNCaPcells or frozen tumor sections. For in situ detection, we used5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside (x-gal) asa substrate to detect the SA-b-gal activity with a staining kit fromCell Signaling Technology. Cultured cells grown on 6-wells platesor frozen tumor tissues (10mmthickness)mounted on slideswerefixed with the fixative solution and stained with the stainingworking solution provided by the kit. The stained cells and tissueswere viewed and photographed under a microscope. For thesecond method, we used 5-dodecanoylaminofluorescein di-b-D-galactopyranoside (C12FDG), obtained from Sigma-Aldrichas a substrate to detect SA-b-gal activity (28, 29). Briefly, cells

Bufalin and Prostate Cancer Cell Senescence

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grown on 6-well plates were pretreated with 100 nmol/L bafilo-mycin A1 (Sigma-Aldrich) for 1 hour at 37�C and 5% CO2

humidified environment, and then were incubated with 33mmol/L C12FDG for another 2 hours. The SA-b-gal activity waseither visualized under fluorescence microscopy or with a BDFACSVerse Flow Cytometer (BD Biosciences) following trypsini-zation. Nuclei were stained with 40,6-diamidino-2-phenylindole,dihydrochloride (DAPI; Thermo Fisher Scientific) for visualiza-tion under fluorescence microscopy. The flow cytometry data wasanalyzed with FlowJo software (FLOWJO).

Microarray analysis of RNA transcriptomeThe cells were treated with 0, 10, and 20 nmol/L of bufalin for

24 hours for concentration-dependent changes. Total RNA wasextracted using RNeasy Mini Kit (QIAGEN). The quality andquantity of the total RNA samples were determined using Nano-Drop 1000 Spectrophotometer (Thermo Fisher Scientific). TheRNA expression profiles of the indicated samples were performedon Illumina Human HT-12 Bead Chips (Illumina Inc.). All RNAlabeling and hybridization was performed at the BioMedicalGenomics Center of the University of Minnesota according tothe protocols specified by the manufacturers. Raw data werenormalized by rank invariant normalization with Illumina Bead-Studio 3.0 software. Data filter was performed on detectionP value (P � 0.05) in at least 2/3 of the samples, signal intensity(�100.0), and cut-off fold-change�1.1 and�0.9 between vehicleand bufalin-treated groups. Formultiple probes of the same gene,the ratio of each probe signal intensity to vehicle was averaged.Thereafter, the normalized and filtered data consisted of 13,758genes. Differentially expressed gene sets were subjected to bioin-formatic analysis using Ingenuity Pathway Analyses (IPA) soft-ware to corroborate affected signaling networks. Microarray datahave been deposited with accession GSE118261.

Animal experimentThe animal study was approved by the Institutional Animal

Care andUseCommittee of Texas TechUniversityHealth SciencesCenter and carried out in the Amarillo campus facility. A total of40maleNSG SCIDmice at 8weeks of agewere obtained fromTheJackson Laboratory. One week after acclimation, the mice wererandomly divided into 2 groups and subcutaneously inoculatedwith 1 � 106 LNCaP cells suspended in 100 mL of inoculumcontaining serum free 50% RPMI-1640 medium and 50%Matri-gel (BD Biosciences) in the lower right flank. One day afterinoculation, the mice started to receive once daily solvent vehicle(5% alcohol in saline) or bufalin (1.5 mg/kg) treatments viaintraperitoneal injection (7 days per week), respectively. Thebufalin dosage was chosen based on published data (30). Xeno-graft tumors were calipered once per week. The tumor volumeswere calculatedwith a formula as L�W2� 0.526. After 9weeks oftreatments, the mice were sacrificed 2 hours after the last dose;tumors, blood (plasma), and tissues were collected.

Immunohistochemical (IHC) staining stainingIHC staining was performed as described previously (31). After

antigen retrieval, the sections were blocked with 6% horse serumand incubated with a primary antibody for c-caspase-3 (CellSignaling Technology), phospho-P53 (Ser15; R&D Systems), AR(Millipore), PSA (KLK3; DAKO). Biotinylated secondary anti-body–horseradish peroxidase conjugate (Vector Labs) was usedat 1:200 dilutions. The sections were developed with 3.30-diami-

nobenzidine and counterstained with hematoxylin (Sigma-Aldrich). After dehydration and mounting in Permount, imageswere captured under light microscopy.

Statistical analysisWhen more than two groups are compared, ANOVA was used

to analyze the statistical significance with appropriate post hoccomparison among the specific groups. For comparison involvingonly 2 groups, Student t test was used with a¼ 0.05 for statisticalsignificance.

ResultsBufalin-induced suppression of AR and PSAprotein abundancewas not dissociable from apoptosis

Exposure of LNCaP cells to bufalin led to a strongconcentration-dependent decrease of the number of adherentcells, estimated with crystal violet staining, with an IC50 value ofapproximately 25 nmol/L, when assessed at 48 hours (Fig. 1B)and approximately 7 nmol/L by 96 hours (SupplementaryFig. S1A). Under a phase contrast microscope, detached floaterstypical of apoptotic response were easily observed in 50 nmol/Lbufalin exposed cells (Supplementary Fig. S1B). To determine thecontribution of apoptosis to the observed cell number suppres-sion, we immunoblotted c-PARP as a molecular indicator ofcaspase-mediated death execution. Bufalin exposure for 48 hoursincreased c-PARP readout in concentration-dependent mannerwith a threshold of 20 nmol/L for apoptosis (Fig. 1C and D;Supplementary Fig. S2A). Bufalin exposure for 24 hours showed asimilar pattern, albeit less intense (Supplementary Fig. S2B).

The P53-mutant DU145 cells exhibited similar caspase-medi-ated c-PARP readouts as the P53 wild-type LNCaP cells (Fig. 1C).On the other hand, the P53-null PC-3 cells did not show anydetectable c-PARP with as high as 100 nmol/L bufalin exposure(Fig. 1C). Nevertheless, bufalin induced cytostasis of PC-3 cells at20 nmol/L or higher, as reflected by the time-independent steadycell number estimation (Fig. 1E). LNCaP and PC-3 cell linesexpress high level of constitutive AKT survival activity due todefective PTEN, whereas DU145 cell line is low for this survivalpathway due to wild-type PTEN (32). Therefore, the apoptosissensitivity of DU145 cells to bufalin was more likely due to theirlow basal survival signaling than the non-functional mutant P53protein (23, 24).

The protein level of AR and its best characterized transcriptionaltarget, PSA, was decreased by bufalin at 20 nmol/L or higher,mirroring the reciprocal increase of c-PARP (Fig. 1D; Supplemen-tary Fig. S2). Because bufalin in the range of 1–10 nmol/L hadbeen reported to downregulate the abundance of two steroidreceptor coactivator (SRC1 and SRC3, but not SRC2) in breastcancer and other cancer types (15), we immunoblotted SRC1 andSRC3 in bufalin-treated LNCaP cells to assess whether they wereaffected in our cellular model. As shown in Fig. 1D, these ARcoactivators were suppressed by bufalin in a manner highlysimilar to AR and PSA suppression and not dissociable fromapoptosis.

Microarray detection of mRNA of AR, KLK3 (PSA), and itsfamily member KLK2 (Table 1) indicated little to modest effect of24-hour bufalin exposure on their steady state levels, but themRNA level of another AR target gene,NKX3-1,was decreased byas much as four folds. Whereas the lack of probes for SRC-1 andSRC-3 mRNA in our microarray precluded us from examining

Zhang et al.





their responses to bufalin exposure, a related gene steroid recep-tor, RNA activator 1 (SRA-1), was not affected by 24-hour bufalinexposure (Table 1). Taken together, in LNCaP cells, the suppres-sing effects of bufalin on abundance of AR, PSA, and SRC-1, -3proteins were not separable from its apoptotic action and werenot tightly linked to their steady state mRNA abundance, andtherefore, were likely through posttranscriptional mechanism ofprotein stability and/or protein synthesis.

Bufalin-induced P53/P21CIP1 protein abundance, cellularstress, and DNA damage response and senescence genes inpreference over apoptosis and autophagy genes

Because P53 was found to mediate apoptosis induced bybufalin exposure in the micromolar range (11) and P53 regulatesnot only apoptosis, but also senescence, autophagy, DNAdamageresponse (DDR), and cellular oxidative stresses (20), we immu-noblotted the protein extracts of LNCaP cells treated with increas-ing concentrations of bufalin for total P53 (Fig. 1C and F) and itsbest known transcriptional target, P21CIP1 (CDKN1A; Fig. 1F).Bufalin treatment for 48 hours led to an increased total P53protein abundance at 10–20 nmol/L as well as that of P21CIP1

in a concentration-dependent manner. We detected bufalin-induced increased phosphorylation of ATM kinase (p-ATM)(Ser1981) and an increased DNA double-strand break marker,phospho-H2A.X (Ser139), which were concomitant with c-PARP(Fig. 1F). For autophagymarker LC-3 lipidmodification,we failedto detect any bufalin-induced increase of the fast-moving form,LC-3-II at 24 or 72 hours of exposure (Fig. 1G).

Microarray transcriptomic analyses of bufalin-inducedchanges at the mRNA level in LNCaP cells after 24-hourexposure showed no change of TP53 mRNA (Table 1), consis-tent with well-known posttranscriptional (e.g., increased pro-tein stability) and posttranslational activation (e.g., phosphor-ylation) of P53 in cells undergoing DDR. Bufalin exposureinduced classical P53 target genes such as CDKN1A (P21CIP1)and GADD45A involved in typical DDR, some classical andnonclassical senescence genes (e.g., CCN1/CYR61, CCN2/CTGF, and GADD34), a number of cellular, oxidative stress,and endoplasmic reticulum (ER) stress genes (e.g., GADD45B,HMOX1, DUSP1, and DUSP5) and some unique tumor sup-pressors (e.g., GDF15/NAG-1; Table 1, showing top 30 genesand Supplementary Table S1). It is apparent that many of these

Table 1. Effect of bufalin on TP53, AR and selected AR-target genes, and top 30 upregulated genes (24-hour exposure) in LNCaP cells

Symbol 10 nmol/L bufalin 20 nmol/L bufalin Major functions PMID

GAPDH 0.94 0.86 Housekeeping gene, glycolysisACTB 1.8 2.0 b-actin, Housekeeping geneACTG1 1.2 1.6 g-actin1ACTA2 1.6 1.4 Smooth muscle actinAR 1.1 0.75 ARKLK3 (PSA) 0.99 0.73 AR target geneKLK2 0.68 0.58 KLK family memberKLK4 1.4 0.49 KLK family memberNKX3-1 0.57 0.21 AR target geneSRA-1 0.91 0.79 Steroid receptor RNA activator 1TP53 1.2 1.0 P53 tumor suppressor geneCYR61/CCNI 10.6 25.2 Senescence, DDR, P53, ROS, P16 activator 22566095CTGF/CCN2 3.7 13.1 Senescence, TGFb target, cancer catabolism 22684333PPP1R15A/GADD34 3.8 10.0 Senescence, DDR, P53 activator 14635196BCL6 6.0 7.4 Senescence, P53 target, Apoptosis suppressor 18524763CDKN1A (P21Cip1) 7.2 7.2 Senescence, P53 target, DDR, cycle arrest 8242752HMOX1 2.7 14.3 Cellular stress, ROS, P53 target 17933770DUSP1 7.5 11.2 Cellular stress, ROS, P53 target 18403641DUSP5 3.1 5.3 Cellular stress, ROS, P53 target 12944906TXNIP 5.7 5.5 Cellular stress, ROS, P53 activator 23823478IFRD1/PC4/Tis7 4.1 5.7 Cellular stress, P53 activator 17785449SDC4 3.9 7.5 Cellular stress, hypoxia 15141079TRIB1 3.8 5.9 Cellular stress, ER cheperone, p53 inactivator 25832642ATF3 5.8 12.7 Cellular stress sensor, P53 target 21927023TSC22D3 7.6 8.0 Cellular stress sensor, osmotic 17147695SIPA1L2 3.9 6.6 Cellular stress response 22178795IER5 3.2 7.3 Cellular stress, chaperone, checkpoint 23273978RNF103/Kf-1 3.2 5.4 Cellular ER stress, ubiquitin ligase 18675248NUPR1/Com1/p8 3.3 5.3 Cellular stress, P53 interactor, P21 activator 18690848GADD45B 7.2 13.6 Growth arrest, p53 target 23948959GADD45A 6.4 12.1 DDR, p53 target 10779360BTG2 4.3 5.4 DDR, P53 target, cycle arrest 8944033GDF15/NAG-1 9.0 7.6 Tumor suppressor, P53 target 23220538EFNA1 3.9 7.4 Tumor suppressor, antiproliferation 17949899ERRFI1 3.9 7.2 Tumor suppressor, antiproliferation 17351343TNFRSF12A 5.1 9.1 AR-repressed metastasis gene 24970477ELF3 6.2 8.4 AR repressor 23435425GABARAPL1 3.5 5.5 Autophagy, maturation 20404487ISG20L1 2.1 5.6 Autophagy, P53 target 20429933SLC38A2 5.2 6.6 Solute transport 21622135KLHL28 3.3 6.2 Ubiquitination regulator 21145461

NOTE: Expressed as fold over control set as unity.






top 30 upregulated genes were related to P53 in the capacity ofdirect transcriptional targets, activators, or interactors.

Of proapoptotic P53 target genes, NOXA was moderatelyincreased (4.2–4.5�; Supplementary Table S1), whereas BAX(1.5–1.8�) and PERP (1.5–1.8�) were within the fluctuation ofthe 3 actin genes (Table 1). Two nonclassical autophagy genes,GABARAPL1 and ISG20L1, were within the top 30 bufalin-induced genes (Table 1), and P53-target autophagy gene,DRAM1, was modestly increased (3.0–4.8�), but many otherclassical autophagy genes regardless of P53 dependence (e.g.,ATG9A and ATG16L1) were within the fluctuation of the actingenes (Table 1; Supplementary Table S1). A couple of AR-repressor/repressed genes, such as TNFRSF12A, ELF3, were alsoamong the top 30 upregulated genes, perhaps a reflection of theandrogen-sensitive nature of LNCaP cells. Bufalin exposure for24 hours decreased the gene expression of many proliferativeoncogene-like entities, mitochondrial function, DNA replica-tion and chromatin, and cyclins (Supplementary Tables S2 andS3). Prompted by these changes and those indicating cellularsenescence signaling (Table 1), we analyzed cell-cycle arrest(s)and senescence next.

Bufalin exposure increased cellular senescenceCell cycle distribution analyses showed increased sub G0/1

apoptotic fraction at bufalin exposure of 20 nmol/L (Fig. 2A),consistent with the c-PARP pattern in Fig. 1C and D. However,the overall "apoptotic" fraction remained rather minor (�6%point above control). There was no enrichment of G1 or S phasecells upon bufalin exposure, but the "G2–M" fraction wasmoderately enriched (Fig. 2A). Staining for phospho-HistoneH3 (Ser10) as a marker of mitotic cells showed decreasedpercentage of mitotic cells in spite of increased bufalin-enriched "G2–M" % (Fig. 2A), revealing a G2 phase arrest ratherthan M phase arrest. Transcriptomic profiling of changes ofcyclin genes (CCN) and minichromosome maintenance(MCM) genes showed greater effect on S–G2 CCNA2 as wellas S phase helicase MCM 4, 6 core (Supplementary Table S3),consistent with G2 arrest.

Cellular senescence is an irreversible terminal arrest, playing anespecially important role in suppressing Pten deficiency–drivenprostate carcinogenesis (33). We examined cellular senescencephenotype in bufalin-exposed LNCaP (PTEN-negative, high AKTactivity) cells by evaluating SA-b-gal activity as a cytochemicalmarker (Fig. 2B) and observed increased proportion of cells withblue staining and flattened morphology. Using a fluorogenicsubstrate, we detected SA-b-gal activity with stronger senescencesignal in the cytoplasm of bufalin-treated cells compared with thecontrol cells under a fluorescence microscope (Fig. 2C). Whenquantified by flow cytometry (Fig. 2D), bufalin at 20 nmol/Linduced peak SA-b-gal signal. Higher concentrations of bufalinexposure compromised senescence detection due to apoptosis. AtthemRNAexpression level, bufalin exposure for 24hours induceda number of genes related to senescence, including the topupregulated gene, CYR61/CCNI, and its related family member,CTGF/CCN2, and P53 target gene,CDKN1A (P21CIP1) (Table 1).

As apoptosis eliminates cancer cells and senescence commitsthem to an irreversible terminal arrest fate, we tested whetherbufalin pretreatment at 20 nmol/L for 48 hours would lead to apersistent growth inhibitory action on LNCaP cells in a reseed-ing experiment. With the same initial number of trypsin-harvested "live" bufalin-pretreated cells versus control cells in

fresh culture medium, the bufalin-pretreated cells almostcompletely lost their growth capability, whereas the controlcells grew exponentially, forming large visible colonies by 14days (Fig. 2E).

P53-dependent upregulation of P21CIP1 by bufalin-promotedsenescence and attenuated apoptosis

To further probe the role of P53-P21CIP1 in bufalin inducedapoptosis and senescence in LNCaP cells, we used siRNAapproach to knockdown (KD) expression of each. As shownin Fig. 3A, TP53 silencing by siRNA (lane 4) was effective atdecreasingbasal (lane1) and scramble siRNA-transfection elicited(lane 2) P21CIP1 expression and blocked the induction ofP21CIP1 after bufalin treatment (lane 8 vs. 5 & 6), indicatingthat both transfection- and bufalin-induced P21CIP1 expressionwere P53-dependent in LNCaP cells. However, knocking downCDKN1A (P21CIP1) did not decrease transfection-elicited (lane 3vs. 2) and bufalin-induced P53 expression (lane 7 vs. 6), asexpected of P53 role as a classic upstream regulator of P21CIP1.Notably, CDKN1A (P21CIP1)-silencing increased transfection-elicited apoptotic cleavage of PARP (lane 3 vs. 2) and bufalin-induced cleavage activation of caspase-3 and PARP (lane 7 vs. 6),supporting a prosurvival role of P21CIP1 in LNCaP cells. Becauseof the increased cell death in P21CIP1-KD cells, it was not possibleto assess their senescence status. Knocking down P53 did notaffect bufalin-induced caspase-driven c-PARP (lane 8 vs. 6). Oneinterpretation was that siRNA-silencing of P53 disabled not onlythe P21CIP1-mediated protection against bufalin-induced apo-ptosis, but also decreased the other P53 transcriptional deathtargets, therefore the magnitude of overall death by bufalin wasless than in P21CIP1-KD cells.

To test the role of P53 induction by bufalin in senescence, wetransfected LNCaP cells with TP53 siRNA for 24 hours (Fig. 3B)and replated them to attach for 48hours, then treatedwithbufalinfor 5 additional days before subjecting the cells to SA-b-gal flowcytometric detection. By the time SA-b-gal was assessed, trans-fected TP53 siRNA had lost considerable potency (Fig. 3B). Inspite of the partial recovery of P53 and P21CIP1, we observed asubstantial attenuation of bufalin-induced SA-b-gal (Fig. 3C).Therefore, P53/P21CIP1 signaling induced by bufalin exposurelikely promoted LNCaP cell fate, switching from apoptosis tosenescence.

Bufalin inhibited LNCaP xenograft tumor growth in NSG SCIDmice

We randomized male NSG SCID mice inoculated with LNCaPcells into two groups (N ¼ 20 per group) and administeredsolvent vehicle (5% ethanol in saline) or 1.5 mg/kg of bufalin,respectively, by once daily intraperitoneal injection starting theday after inoculation. We chose this bufalin dosage based on areported toxicity study (30). After 9 weeks of treatments, thetumor take rate of the vehicle group was 80 % (16 of 20),consistent with the reported tumor take rates of LNCaP xenograftin immunodeficientmice. The take rate in bufalin groupwas 75%(15 of 20). Compared with vehicle alone, bufalin significantlydelayed LNCaP xenograft tumor growth (Fig. 4B and C) withoutaffecting animal bodyweights (P > 0.05; Fig. 4A). At necropsy, themean tumor weight of bufalin-treated mice showed a decrease ofapproximately 67% (P < 0.05) compared with that of the vehicle-treated mice (Fig. 4D).

Zhang et al.





Figure 2.

Effects of bufalin on cell cycle distribution and senescence-like phenotype in LNCaP cells. A, Flow cytometry analyses of cell cycle distribution inLNCaP cells exposed to bufalin for 48 hours and of mitotic-specific protein marker, Histone H3 (Ser 10) phosphorylation. B, Representative light-microscopicimage of SA-b-gal activity using X-gal as substrate in LNCaP cells exposed to 20 nmol/L of bufalin treatment for 3 days. C, Representative fluorescentmicroscopic image of SA-b-gal activity using C12FDG as substrate in LNCaP cells exposed to 10 nmol/L of bufalin for 9 days. D, Flow cytometry detection ofSA-b-gal activity using C12FDG as substrate in LNCaP cells exposed to 20 nmol/L of bufalin treatments for 4 days. Bar graph shows the summary ofSA-b-gal activity enhancement by bufalin. The experiments were done in triplicate. The data were shown as Mean � SEM. � , P < 0.05. E, Durable growthinhibition of LNCaP cells by 48-hour bufalin exposure in a reseeding experiment. After 48 hours of pretreatment with ethanol or 20 nmol/L of bufalin, the cellswere rinsed with PBS and harvested by trypsinization, then the same number of "live" cells were replated into new 6-well plates with fresh complete medium.Adherent cells/colonies were visualized by crystal violet staining over 2 to 14 days.






Increased senescence in bufalin-treated LNCaP tumors withoutdetectable increase of caspase apoptosis

We selected the 3rd to 8th ranked tumors from each group andanalyzed them by hematoxylin and eosin (H&E) staining (Fig.5A), IHC (Fig. 5B), and immunoblot (Fig. 5C). In vehicle-treatedtumors, H&E staining showed dark hematoxylin-stained regionsthatweredensely packedwith cells (Fig. 5A, left panel). Therewere

variable and large eosinophilic necrotic areas (without nuclei,middle panel marked N) within these tumors (5 of 5 vehiclecontrol tumors >40% area). The control tumors displayed pro-found acute hemorrhage (profuse presence of erythrocytes onH&E section) such that their paraffin blocks appeared dark versusthe translucent appearance of the bufalin-treated tumor blocks(Supplementary Fig. S3). The bufalin-treated tumors showed lessnecrotic proportion and their acute hemorrhage status rangedfrom scant to moderate. H&E staining revealed loosely packedcells in the bufalin-treated tumors (Fig. 5A, right panel). Therewasno evidence of increased in situ apoptotic cleavage of caspase-3 inthe bufalin-treated tumors with IHC detection (Fig. 5B). Immu-noblot analyses did not detect c-caspase-3 or c-PARP in eithergroup (Fig. 5C).

The "proliferation biomarker" Ki67 IHC staining revealed largepositive nuclei in bufalin-treated group and the proportion ofpositive cells were same as vehicle group, if not higher (Fig. 5B).The strong Ki67-stained large nuclei in the bufalin-treated tumorswere therefore consistent with the in vitro observation that bufalininduced G2 arrest (Fig. 2A). The proportion of p-P53 (Ser15)–positive large nuclei was increased in bufalin-treated tumors (Fig.5B), afinding consistentwith immunoblot analyses (Fig. 5C). Yet,there was no detectable effect of bufalin on AR (Fig. 5B nuclearstaining, 5C) and PSA (Fig. 5C). The abundance of p-AKT or HK2,the two crucial kinases for prostate cancer survival and growth(34), was not altered between the two tumor groups (Supple-mentary Fig. S4).

Compared with the vehicle group, in situ SA-b-gal activity offrozen xenograft tumor sections of bufalin-treated mice washigher (blue staining in Fig. 5D, top right panel). In contrast, theprostate epithelium of the NSG SCID mice from vehicle group orbufalin group did not show a difference of the basal SA-b-galreadout (Fig. 5D, bottompanels). Taken together, the data suggestP53-mediated cellular senescence was a likely cancer-selective,primary cellularmechanism for bufalin to inhibit LNCaP prostatecancer tumor growth in vivo.

DiscussionWith ahumanpharmacokinetic studyoutcome inmind, that is,

intravenous infusion leading to a maximum level of approxi-mately 9 nmol/L at the end of the 2-hour infusion (6), weinvestigated the cellular effects of relevant concentration rangeof bufalin in LNCaP cell culture model and discovered theinduction of G2 arrest and increased senescence-like cellularphenotype as the primary cellular processes to inhibit LNCaPcancer cell proliferation (Fig. 2), involving P53-P21CIP1 as acrucialmolecular signaling axis (Fig. 3).Whereas P21CIP1 knock-down significantly increased the apoptosis in response to bufalin(Fig. 3A), P53 knockdown attenuated the extent of senescenceinduction (Fig. 3C), supporting P53-P21CIP1 in our cellmodel asa cell fate determinant from apoptosis to senescence (See Sup-plementary Fig. S5).

Whereas bufalin was found to suppress AR and PSA abundancein cell culture exposuremodel, thesemolecular readouts were notseparable from the apoptotic action; nor was the reduction ofSRC-1 and SRC-3 proteins (Fig. 1D). SRC1 and SRC3 are 2universal coactivators of steroid receptors including AR (15).Transcriptomic profiling of 24-hour bufalin-exposed cells showeddecreased AR target, NKX3-1, by as much as 4 folds at 20 nmol/Lbufalin, whereas the PSA (KLK3) gene was not much affected

Figure 3.

Impact of knocking down P53/P21CIP1 axis on bufalin induction of apoptosis andsenescence in LNCaP cells.A,Western blot analysis detection of P53 and P21CIP1knock down by siRNA and caspase-mediated PARP cleavage by bufalinafter 24-hour exposure.B,Western blot analysis detectionof P53 knockingdownby siRNA 24 hours after transfection and 5 days after initiation of bufalintreatment. C, Flow cytometry detection of SA-b-gal activity using C12FDG assubstrate for scramble-RNA transfected versus si-P53-KD cells exposed tobufalin for 5 days.

Zhang et al.





within this time frame (Table 1). Therefore, bufalin might exertdifferential posttranscriptional as well as transcriptional controlsover these AR targets. Given structure–activity relationship (SAR)investigation demonstrated bufalin as a poor ligand-bindingantagonist of AR (18, 19), the cell culture data suggest thatbufalin-induced reduction of AR and its coactivators was causedby apoptosis andmight contribute little to senescence signaling atthe sub-apoptotic range of bufalin exposure.

We demonstrated that daily injection of a MTD of bufalinsignificantly delayed LNCaP xenograft growth in vivo (Fig. 4), inassociation with a significant induction of SA-b-gal senescence(Fig. 5D), but without apparent changes of caspase-mediatedapoptosis biomarkers or a suppression of AR and PSA (Fig. 5Band C). However, we could not rule out the possibility thatapoptosis-susceptible cancer cells had already been eliminatedfrom the xenograft tumors at the time of necropsy, thus enrichingthe long-lived senescent cancer cells in the bufalin-treated mice.Given such caveats, it is imperative that we interpret the in vivobiomarker changes and mechanism of action with considerablereservation.

The mRNA profiling of 24-hour bufalin-exposed LNCaP cellsrevealed gene signatures of P53-driven DDR, oxidative cellularstress and ER stresses, as well as senescence in top 30 upregulatedgenes (Table 1). AnchoredbyATM/ATR-activated P53,DDRelicitsmultiple cellular responses ranging from transient responses (cell-cycle arrest and DNA repair) to terminal fates (death or senes-cence; refs. 35–39). In vivo restoration of P53 function in P53-dysfuntional sarcomas or liver tumors led to significant tumorregression (40, 41) and surprisingly, through P53-mediatedsenescence and innate immune cells, instead of apoptosis (40).Recent studies further reveal P53-mediatedmitosis skip in cellularsenescence (42, 43). Activation of P53 by DNA damage promotesG2 arrest, leading to cell-cycle exit and senescence without enter-

ingmitosis (42, 43). Themitosis skip is executed byP53 inductionand upregulated P21CIP1 abundance to bind and inhibit themitosis-promoting activity of nuclear cyclin B1with its CDK (42).Even transient activation of P53 without DNA damage wassufficient to lead to mitosis skip and senescence (40, 42). Ourin vivo bufalin-treated cancers showed large Ki67 positive nuclei(Fig. 5B), and increased SA-b-gal senescence staining (Fig. 5D).We speculate that the daily bufalin exposure in vivo (bufalin sharpspikes) promoted DDR and the P53-P21CIP1–mediated mitoticskip and senescence cascade, culminating to an enrichment oflong-lived senescent cells (Supplementary Fig. S5).

The wild-type P53 inmost primary prostate cancer (44) wouldbe advantageous for bufalin-induction of senescence as a meansto intercept prostate cancer progression. Whereas our NSG SCIDmice lacked key lymphocytes that had largely precluded a con-tribution of bufalin-induced immune responses, it is prudent tospeculate possible ramifications of bufalin-activation of P53senesence in immune competent hosts for prostate cancer therapyor chemoprevention. As noted above, Tp53 gene restoration inliver tumors led to senescent cells producing various inflamma-tory cytokines and chemokines (known as senescence-associatedsecretory phenotype, SASP) that activated innate immune cellsincluding natural killer (NK) cells, macrophages, and neutrophils(40). The innate immune response was indispensable becauseblocking these immune cells significantly abolished preventiveaction of Tp53 gene reactivation (40). A recent study shows thatP53-expressing senescent cells secrete factors that promote mac-rophage activation towards tumor-inhibiting M1-state, whereasP53-deficient cells secrete those that skew macrophage activationtowards tumor-promoting M2-state (45). Our top-upregulatedgene, CYR61/CCN1, by bufalin (Table 1) encodes a matricellularprotein that is secreted by tumor cells and stimulates macrophageactivation towards tumor-inhibiting M1-state (46), suggesting a

Figure 4.

Effect of once daily intraperitonealinjection of bufalin on the growth ofsubcutaneously inoculated LNCaPxenograft tumors in NSG SCID mice.A, Mouse body weight. B, Growthcurve of LNCaP xenograft tumors bytumor volumes, nontumor-bearingmice were excluded (effectiveN ¼ 15,16). C, Ranking order of tumorweight of vehicle- and bufalin-treatedmice. D, Mean tumor weight atnecropsy. The data were shown asMean � SEM. vehicle-treated(N ¼ 16); bufalin-treated. (N ¼ 15).� , P < 0.05.






Figure 5.

Biomarkers analyses of bufalin-treated LNCaP xenograft tumors. A, H&E-stained 3rd to 8th ranked LNCaP xenograft tumors from ethanol vehicle- and bufalin-treated groups, respectively. Large eosinophilic areas without nuclei in vehicle group represent necrotic regions (marked N in middle panel). Scale bars, 50 mm.B, Representative IHC images of Ki-67 proliferation marker and apoptosis marker (c-caspase 3), p-P53 (Ser15) and AR in tumors from vehicle- or bufalin-treated mice.Black scale bars, 200 mm; Yellow scale bars, 100 mm. C,Western blot analysis detection of c-PARP, c-caspase-3, p-P53 (Ser15), AR, and PSA in tumors from vehicle- orbufalin-treatedmice. LNCaPcells exposed to 1mmol/Ldoxorubicin (aDNA-damagingdrug) treatment for 24hourswereusedasapositivecontrol for apoptosis (LNþRx).PC3 extract and LNCaP extract (LN) were used as negative and positive control for AR and PSA, respectively. D, Representative images of SA-b-gal staining ofcryosections of LNCaP xenograft tumors and the normal prostate of NSG SCID mice treated with vehicle or bufalin.

Zhang et al.





potential for macrophage activation towards M1-state by bufalin.Given that bufalin induces a P53-mediated senescence program, itis likely that bufalin may lead to even greater tumor suppressionin immune competent hosts through coordination of cellularsenescence (growth arrest) and innate immune responses (tumorclearance). Such hypothesis should be tested in immune-competent animal cancer models.

A recent paper addressed the poor aqueous solubility andcardiotoxicity of bufalin with chemical modification throughphosphorylation of its 3-OH (14). The authors showed that3-phospho-bufalin was a water soluble prodrug suitable for in vivoadministration and significantly improved the safetymargin whileeffectively inhibiting tumor growth in anorthotopic triple-negativebreast cancer xenograft model. The development of 3-phospho-bufalin broadens the potential application of bufalin in cancerchemointerception and therapy for prostate and other organs.

In conclusion, our data suggest a probable primary in vivomechanism of prostate cancer suppression by bufalin throughinduction of P53-mediated cellular senescence. With the avail-ability of safer 3-phospho-bufalin as prodrug to deliver bufalin(14) or possible future-targeted delivery through novel bufalinformulations, it may be practical to apply bufalin in adjuvanttherapy of prostate cancer recurrence in patients or the chemoin-terception of precancerous prostate lesions.

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

Authors' ContributionsConception and design: Y. Zhang, M. Ye, J. Lu, C. JiangDevelopment of methodology: Y. Zhang, Y. Dong, M.W. Melkus, S. Tang,W. Wu

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Zhang, Y. Dong, M.W. Melkus, S. Yin, S. Tang,P. Jiang, W. Wu, M. YeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):Y. Zhang,M.W.Melkus, S. Tang, P. Jiang, K. Pramanik,S. Kim, J. Lu, C. JiangWriting, review, and/or revision of the manuscript: Y. Zhang, M.W. Melkus,S. Tang, P. Jiang, S. Kim, H. Hu, J. Lu, C. JiangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Zhang, M.W. Melkus, S. Kim, J. Lu, C. JiangStudy supervision: J. Lu, C. Jiang

AcknowledgmentsThe work was supported by institutional start-up funds at Texas Tech

University Health Sciences Center School of Pharmacy (to J. Lu) andPenn State Hershey College of Medicine (to J. Lu) and, in part, byNational Institutes of Health National Cancer Institute grant R01CA172169 (to J. Lu). The authors thank Jinhui Zhang, PhD, forguidance and assistance with microarray analyses and DeepkamalKarelia, PhD, for assistance with immunoblot analyses of DU145 andPC-3 cells. They thank Yuka Imamura, PhD, and the Penn State CancerInstitute Genomic Sciences Core facility for next-generation sequencingauthentication of cell line and veterinarian pathologist Timothy Coo-per, DVM, PhD, of Department of Comparative Medicine at Penn StateUniversity Hershey College of Medicine for evaluating xenograft tumorhistology sections.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 28, 2017; revised April 13, 2018; accepted August 22,2018; published first August 30, 2018.

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2018;17:2341-2352. Published OnlineFirst August 30, 2018.Mol Cancer Ther Yong Zhang, Yinhui Dong, Michael W. Melkus, et al. Prostate Cancer Growth by Cardiotonic Compound BufalinRole of P53-Senescence Induction in Suppression of LNCaP

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