natura-alpha targets forkhead box m1 and inhibits androgen ... · experimental design: the growth...

Cancer Therapy: Preclinical

Natura-Alpha Targets Forkhead Box M1 and Inhibits Androgen-Dependentand -Independent Prostate Cancer Growth and Invasion

Yirong Li1, Martin Ligr1, James P. McCarron5, Garrett Daniels1, David Zhang6, Xin Zhao6,Fei Ye6, Jinhua Wang3, Xiaomei Liu3, Iman Osman2,3, Simon K. Mencher7,Hebert Lepor2, Long G. Wang3,7, and Peng Lee1,2,3,4

AbstractPurpose: The development of new effective therapeutic agents with minimal side effects for prostate

cancer (PC) treatment is much needed. Indirubin, an active molecule identified in the traditional Chinese

herbal medicine—Qing Dai (Indigo naturalis), has been used to treat leukemia for decades. However, the

anticancer properties of Natura-alpha, an indirubin derivative, are not well studied in solid tumors,

particularly in PC.

Experimental Design: The growth kinetics and invasion ability of on human PC cell lines with or

without Natura-alpha treatment were measured by cell proliferation and invasion assays. The antitumor

effects of Natura-alpha were examined in nude mice tumor xenograft models, and in a patient with

advanced hormone-refractory metastatic PC. Signal network proteins targeted by Natura-alpha were

analyzed by using proteomic pathway array analysis (PPAA) on xenografts.

Results: Natura-alpha inhibited the growth of both androgen-dependent (LNCaP) and androgen-

independent (LNCaP-AI, PC-3, andDU145) PC cells with IC50 between 4 to 10mmol/L, and also inhibited

invasion of androgen-independent PC cells. Its antitumor effects were further evident in in vivo tumor

reduction in androgen-dependent and androgen-independent nude mice tumor xenograft models and

reduced tumor volume in the patient with hormone refractory metastatic PC. PPAA revealed that

antiproliferative and antiinvasive activities of Natura-alpha on PC might primarily be through its down-

regulation of Forkhead box M1 (FOXM1) protein. Forced overexpression of FOXM1 largely reversed the

inhibition of growth and invasion by Natura-alpha.

Conclusion: Natura-alpha could serve as a novel and effective therapeutic agent for treatment of both

hormone-sensitive and hormone-refractory PC with minimal side effects. Clin Cancer Res; 17(13);

4414–24. �2011 AACR.

Introduction

Prostate cancer (PC) is the most common cancer in menin the United States andwas expected to cause 217,730 newcases and 32,050 deaths in 2010 (1). Androgen ablation is

the most common therapy for advanced PC. The treatmentfailure of PC lies in the fact that, after androgen ablationtherapy, the disease inevitably progresses from androgen-dependence to androgen-independence. For patients whoare not cured by local treatment with ensuing metastases,neither androgen ablation nor chemotherapy can extendtheir survival time. Thus, the development of new effectivetherapeutic agents with minimal side effects is highlywarranted.

Cancer is increasingly being viewed as a cell-cycledisease because deregulation in the cell-cycle machinerycan be found in most cancers (2–4). Major componentsin the cell-cycle machinery are cyclin-dependent kinases(cdk) and their interacting partners, the cyclins and theendogenous inhibitors (e.g., cdki). Defects have beendescribed in the components of the cell-cycle machineryitself, or the checkpoint components that ensure orderlyadvancement through the cell-cycle phases, or inupstream signaling that triggers cell-cycle events (5–6).Strategies have been developed and intensified in the lastfew years by directly or indirectly targeting cdks and thesehave been reviewed extensively (3, 7–9). The first two cdk

Authors' Affiliations: Departments of 1Pathology and 2Urology, and3NYU Cancer Institute, New York University School of Medicine; 4NewYork Harbor Healthcare System; 5Department of Urology, Presbyterian-Cornell Medical Center; 6Department of Pathology, Mount Sinai School ofMedicine; and 7Natrogen Therapeutics, New York, New York

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Y. Li and M. Ligr equally contributed to this work.

Corresponding Author: Long G. Wang, Natrogen Therapeutics Inter-national, Inc., 315 East 68th Street, New York, NY 10065. E-mail:[email protected] or Peng Lee, Department of Pathologyand Urology, New York University School of Medicine, New YorkHarbor Healthcare System, 423 E. 23rd Street, Room 6139N, New York,NY 10010. Phone: 212-951-3418; Fax: 212-951-6341; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-11-0431

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(13) July 1, 20114414

Cancer Research. on September 24, 2020. © 2011 American Association forclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 23, 2011; DOI: 10.1158/1078-0432.CCR-11-0431





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inhibitors, Flavopiridol and UCN-01, have been in clin-ical trials either alone, or in combination with otherchemotherapeutic agents and have shown promisingresults with evidence of antitumor activity (10–12).Indirubin, an active molecule identified in the tradi-

tional Chinese herbal medicine—Qing Dai (Indigo natur-alis), has been used to treat leukemia for decades (13–14).In recent years, there has been a dramatic renewal of theinterest in indirubin due to the discovery of its greatpharmacologic potential (15). Increasing evidences showthat indirubin, and its derivatives and analogs, targetvarious important signal pathways involved in cancer,including inhibition of cdks (16). Natura-alpha (NTI-Onco2008-1), an indirubin derivative, shows an abilityto arrest leukemia cells at G1 phase, inhibit expression ofthe oncogene c-Myb, and induce cell differentiation andmaturation at low concentration, in which cell growthis completely inhibited without decrease in cell viability.At higher concentration, this agent blocks tumor cells atM–G2 phases.To further evaluate its potential clinical application and

to explore mechanisms of its anticancer action, in thisstudy, we examined therapeutic activities of Natura-alphaon androgen-dependent and androgen-independentprostate cancer in vitro and in vivo, and in a patient withadvanced hormone-refractory metastatic PC. Natura-alpha caused strong inhibition of cell proliferation andinvasion in various human PC cell lines and tumorgrowth in nude mouse xenografts. Most importantly,the patient with hormone-refractory metastatic prostatecancer achieved stable disease in response to Natura-alpha with his liver metastatic tumors reduced byapproximately 26% by using guidelines of ResponseEvaluation Criteria in Solid Tumors (RECIST; ref. 17).Further study indicated that the suppression of Forkheadbox M1 (FOXM1) was the primary target of inhibition ofproliferation and invasion by Natura-alpha.

Materials and Methods

ReagentsThe chemical name of Natura-alpha is N-methyl-D3,30-

dihydroindole-2,20-diketone. Natura-alpha was providedby Natrogen Therapeutics International, Inc. It was synthe-sized under cGMP conditions, and structure confirmed byIR, NMR, andmass spectrometry with a purity of 98.00%ormore.

Cell culture and cell proliferation assaysLNCaP and DU145 cells were maintained in RPMI 1640

(Gibco and PC3 cells were cultured in 50% RPMI 1640 and50% F2 Gibco) with 10% heat-inactivated FBS. The andro-gen-independent LNCaP-AI cells, a derivative of LNCaP(18–20), were maintained in RPMI 1640 medium contain-ing 10% charcoal-stripped, heat-inactivated FBS (CSFBS;Hyclone Laboratories, Inc.) and 5 mg/mL of insulin, asdescribed previously (18). Cell proliferation was deter-mined by MTT as described previously (21–22). Ancho-rage-independent cell growth in soft agar was done intriplicate with cells (1 � 104) suspended in 2 mL ofmedium containing 0.35% agar (Becton Dickinson) spreadon top of 5mL of 0.7% solidified agar (23). Colony volumewas calculated from the average radius of representativecolonies.

Matrigel invasion assaysEffect of Natura-alpha on invasive activity of LNCaP and

LNCaP-AI cells was determined via BD Matrigel invasionassay (Growth Factor ReducedMatrigel Invasion Chamber)as described (24). After rehydration of the insert withmedium for 2 hours, LNCaP and LNCaP-AI cells at theirexponential growth phases were added to the upper cham-ber at the density of 1 � 104 cells in 500 mL medium in thepresence or absence of indicated concentration of Natura-alpha and incubated at 37�C for 48 hours. Data wereadjusted by growth condition and expressed as mean ofmigrating cells in 3 fields � SD.

Western blot analysisWhole cell or cell fraction extracts were subjected to SDS-

PAGE and transferred to a nitrocellulose membrane forWestern blot analysis. Blots were incubated with primaryantibodies including FOXM1, cyclin D1, cyclin B, cyclin E,and b-actin (Cell Signaling Technology, Inc.) for 2 hoursat room temperature, washed with TBS-T, and incubatedfor 1.5 hours with horseradish peroxidase-conjugated sec-ondary antibody (1:5,000; Amersham Biosciences). Theprotein bands were detected by an enhanced chemilumi-nesence kit (Amersham Biosciences).

Nude mice xenograftsAndrogen-dependent LNCaP and androgen-indepen-

dent LNCaP-AI PC cells, mixed with Matrigel (BectonDickinson) at a ratio of 1:1 were inoculated into thebilateral flanks of 4- to 5-week-old male Nu/Nu BALB/cathymic nude mice by s.c. injection. The tumor growth and

Translational Relevance

Prostate cancer (PC) is the most common cancer inmen in the United States. The development of neweffective therapeutic agents with minimal side effectsfor PC treatment is much needed. In this study, we showanti-PC properties of Natura-alpha in vitro, in vivo byusing nudemice xenograft models, and in a patient withadvanced hormone refractory metastatic PC. Our find-ings revealed that antiproliferation and antiinvasionactivities of Natura-alpha on PC might primarily bethrough its downregulation of Forkhead box M1 pro-tein. This study provides comprehensive evidence tosupport that Natura-alpha could serve as a novel andeffective therapeutic agent for treatment of both hor-mone-sensitive and hormone-refractory PC in nearfuture with minimal side effects.

Natura-Alpha Inhibiting Prostate Cancer

www.aacrjournals.org Clin Cancer Res; 17(13) July 1, 2011 4415




volume were monitored every 3 days. When the tumorgrew to a diameter of 4 to 8 mm (4–5 weeks), animals wererandomly divided into 2 groups, 10mice each, according totumor size. One group of animals was treated with drugvehicle only as control, and another group was treated withNatura-alpha at dose of 100 mg/kg by gavage, once a day,5 days a week until the diameter of tumors in control groupreached approximately 15 mm. The tumor growth wasmonitored daily and tumor size recorded every 3 days.The tumor volume was calculated as l � d � h � 0.52 (25).

Proteomic pathway array analysisTotal cellular proteins were extracted from xenograft

tumors (3 mm3) by using a lysis buffer containing 20mmol/L Tris-HCl (pH 7.5), 20 mmol/L sodium pyropho-sphate, 40 mmol/L B-glycerophosphate, 30 mmol/Lsodium fluoride, 2 mmol/L EGTA, 10 mmol/L NaCl,and 0.5% NP-40. The lysate was sonicated 3 times for15 seconds each time, and then centrifuged (14,000rpm, 30 minutes, and 4�C). The tubes were kept on icethroughout the process. The protein concentration wasdetermined with the BCA Protein Assay Kit (PIERCE).Isolated proteins were separated by SDS-PAGE (10% acry-lamide). Three hundred micrograms of protein extractswere loaded in a well across the entire width of gel forSDS-PAGE, followed by electrotransferring to a nitrocellu-lose membrane. The membrane was then blocked for 1hour with 5% milk or 3% bovine serum albumin andclamped on to a Mini-PROTEAN II Multiscreen apparatusthat isolates 20 channels across the membrane (Bio-Rad).Two or 3 antibodies were added to each channel andincubated overnight at 4�C. Different sets of antibodieswere used for each membrane after stripping the previousset of antibodies. Antibodies were purchased either fromCell Signaling Technology, Inc., or from Santa Cruz Bio-technology, Inc. (Supplementary Table S1). The pathwayarray analysis was run in duplicate for each sample in eachset of antibodies and protein levels were normalized byusing b-actin and glyceraldehyde-3-phosphate dehydro-genase as standards. Chemiluminescence signals were cap-tured by using the ChemiDoc XRS System. Differences inprotein levels were determined by densitometric scanningand normalized to internal standards.

FDA- and IRB-approved single patient clinical trialAn 86-year-old patient with advanced androgen-inde-

pendent metastatic PC was consented for the Natura-alphatrial therapy for his disease with approval from the Foodand Drug Administration (FDA; IND# 104191) and Insti-tutional Review Board (IRB; IntegReview NTI-2007-1-PC).Natura-alpha was administered orally with increasingdoses from 40, 80, 160 to 200 mg per day every 2 weeks,and 200 mg later on for 3 months.

Statistical analysisThe growth inhibition (1 � T/C, Effect), median effect

dose (Dm), and combination indexes (CI) were calculatedand analyzed by using the computer program, CalcuSyn, of

Biosoft edited by T.C. Chou, Memorial Sloan-KetteringCancer Center, New York, and M.P. Hayball (Biosoft; refs.(21, 26). The CI was used to evaluate the results of thecombinations. A CI greater than 1 indicates the combina-tion is antagonistic, CI equal to 1 indicates the combina-tion is additive, and CI smaller than 1 indicates that thecombination is synergistic (26).

Results

Effects of Natura-alpha on PC growth and invasionin vitro

A response of different human PC cells to the treatmentof Natura-alpha was obtained by using colorimetric MTTand/or SRB methods after 3 days of exposure. Table 1shows the IC50 of Natura-alpha on hormone-dependentand hormone-independent human PC cell lines rangedbetween 3.96 to 9.39 mmol/L. IC50 data pertain to theconcentration of the tested drug that inhibits 50% cellgrowth in in vitro growth kinetic studies. The data of Table 1are mean � SD from 3 independent experiments.

To explore if Natura-alpha is able to enhance activity ofclinically available chemotherapeutic drugs used for PC,the commonly used antimicrotubule agent, paclitaxel(Taxol), was combined with Natura-alpha in 3 differentsequential exposures. In the first combination, LNCaP-AIcells were treated with Natura-alpha and Taxol simulta-neously for 6 days. In the second combination, the cellswere treated with Natura-alpha for the first 3 days followedby treatment with Taxol for additional 3 days (NTI !Taxol). In the third regimen, the cells were treated withTaxol for the first 3 days followed by treatment withNatura-alpha for additional 3 days (Taxol ! NTI). Expo-sure of the cells to either Natura-alpha or Taxol aloneserved as controls. After treatment, cell growth was deter-mined by MTT assays with the growth inhibition (1 � T/C,Effect), Dm, and CI calculated and analyzed as described inthe Materials and Methods.

The concurrent combination of Natura-alpha with theantimicrotubule agent, Taxol, achieved a strong synergisticeffect on LNCaP-AI PC growth. For example, the calculatedDm of Natura-alpha and Taxol alone against LNCaP-AIcells was found to be approximately 7.54 mmol/L and 41.57nmol/L, respectively. However, when the 2 drugs wereapplied to the cancer cells simultaneously at the ratio of

Table 1. IC50 of Natura in hormone-dependentand hormone-independent human cancer celllines

Cell lines IC50

LNCaP 4.54 � 0.73LNCaP-AI 6.89 � 0.57DU145 9.39 � 0.23PC-3 3.96 � 0.58

Li et al.

Clin Cancer Res; 17(13) July 1, 2011 Clinical Cancer Research4416




1,000:1 (Natura-alpha:Taxol), Dm was significantlyreduced to 0.78 mmol/L and 0.78 nmol/L, respectively(Supplementary Table S2) and CI was well below 1(Fig. 1A and B). One-way ANOVA test showed P < 0.001indicating that there is statistically significant differencebetween the combination and the 2 individual agents.Interestingly, the effects of the combination are highly

dependent on the sequence of the drug exposure indicated

by the CI (Fig. 1) and Dm values (SupplementaryTable S2). A strong synergistic growth inhibitory effect ofLNCaP-AI cells was achieved when the cancer cells wereexposed to Natura-alpha and Taxol concurrently (Fig. 1Aand B), in which case CI at each concentration points werewell below 1, whereas only a moderate synergism wasobserved when the cells were treated with Natura-alphafirst for 3 days followed by Taxol treatment for additional 3

Figure 1. Effects of Natura-alpha(NTI) and Taxol on cell growth ofandrogen-independent PC(LNCaP-AI) with differenttreatment regimens. A, C, and Eare dose–effect curves, and B, D,and F are CI–effect curves. Dashline in B, D, and F (CI ¼ 1) is thedivider of outcomes of 2-drugcombination. CI value above thedash line indicates an antagonisticeffect, below the line is asynergistic effect, and on the lineimplies an additive effect. Aregression analysis of CI valuesversus effects in B, D, and F weredone by Sigma plot 8, and the linedirection indicates trend of acombination. Androgen-independent LNCaP-AI cells weretreated simultaneously with NTIand Taxol (A and B); C and D(combination 2, NTI ! Taxol): thecells were exposed to NTI first for3 days followed by Taxoltreatment for additional 3 days;E and F (combination 3, Taxol !NTI): the cells were treated withTaxol first for 3 days followed byNTI treatment for additional 3days.

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days. Notably, the trend of the combination became antag-onistic when the cancer cells were exposed to Taxol for thefirst 3 days followed by exposure to Natura-alpha for anadditional 3 days (Fig. 1E and F). Similar results were alsoobtained in LNCaP cells (data not shown).

Growth inhibition of Natura-alpha on PC cells wasfurther supported by anchorage-independent assay.Although both LNCaP and LNCaP-AI cells could easilyform colonies in soft agar in the absence of Natura-alpha(Fig. 2A), LNCaP-AI cells showed stronger capacity ofcolony formation. However, the colony formation of bothLNCaP and LNCaP-AI cells was significantly inhibited byNatura-alpha (final concentration 2.5 mmol/L) as reflectedby remarkable decrease in numbers and size of coloniesunder the same experimental conditions (Fig. 2B).

To examine whether Natura-alpha inhibits the invasivepotential of PC cells, invasive activity of LNCaP andLNCaP-AI cells was determined via the BD Matrigel inva-sion assay. Results showed that invasive capacity of LNCaPcells was highly limited. Only a few cells migrated (data notshown). In contrast, LNCaP-AI cells showed strong invasivepotential. More than 4,000 cells invaded per hpf during 48hours culture in the presence of androgen. Interestingly, theinvasive capacity of LNCaP-AI cells was strongly blocked byNatura-alpha in a concentration-dependent manner. Inhi-bition of invasive LNCaP-AI cells reached more than 87%and 99% at concentrations of 2.5 and 5.0 mmol/L ofNatura-alpha, respectively (Fig. 2C).

Inhibition of prostate tumor growth in vivo byNatura-alpha

In an androgen-dependent (LNCaP) xenograft model,PC cells were injected s.c. into the flank region of malenude mice. When the prostate tumor grew for 4 to 5 weeks(20 to 30 mm3), animals were randomly divided into 2groups, 10 animals each, according to tumor size. A sus-

pension of Natura-alpha was given at dose of 100mg/kg bygavages once a day for 5 days a week. Mice fed with equalvolume of solution of 0.05% Tween 20 in water (a solutionused in preparing Natura-alpha suspension) served asvehicle controls. The tumor size was measured every 3days, and tumor growth curves (tumor size vs. time) wereplotted. As shown in Figure 3A and B, treating with Natura-alpha, starting at week 5, slowed tumor growth comparedwith the control group. By week 6, tumor growth in theNatura-alpha–treated group almost completely halted,whereas tumors in the vehicle-treated group continuedto grow. Continued feeding with Natura-alpha not onlycompletely halted tumor growth but significantly reducedthe tumor volume. For example, on day 78, the averagevolume of tumors in the Natura-alpha–treated group wasreduced by 53% (P ¼ 0.035). In addition, after dissection,tumor weight from the Natura-alpha–treated group wasreduced about 6-fold as compared with the control group(P ¼ 0.001) and HR is 0.168 (Fig. 3C).

To determine the effects of Natura-alpha on androgen-independent PC, we developed a xenograft model by usingandrogen-independent LNCaP-AI cells, with castration orsham castration. After 4 weeks of prostate tumor growth(20–30 mm3 in size), animals were castrated or shamcastrated and randomly divided into 4 groups, 10 animalseach, on the basis of tumor size. Groups A and B consist ofcastrated mice fed with Natura-alpha or with equal volumeof vehicle as control, respectively. Groups C and D consistof sham-castratedmice fed with Natura-alpha or with equalvolume of vehicle as control, respectively. A suspension ofNatura-alpha or equal volume of vehicle was given at doseof 100 mg/kg by gavage, once a day and 5 days a weekstarting on day 28.

As shown in Figure 3D–I, tumor volumes of castrated orsham-castrated mice from both vehicle groups showedconstant growth. In contrast, the growth of tumors in

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Figure 2. Inhibition of PC anchorage-independent growth and invasion. A and B, anchorage-independent cell growth in soft agar was done in triplicatewith cells (1 � 104) suspended in 2 mL of medium containing 0.35% agar (Becton Dickinson) spread on top of 5 mL of 0.7% solidified agar. Colony volumewas calculated from the average radius of representative colonies. C, LNCaP-AI cells at their exponential growth phases were added to the upper chamberat density of 1 � 104 cells in 500 mL medium in the presence or absence of indicated concentrations of Natura-alpha and incubated at 37�C for 48 hours,and the invading cells adherent at the bottom of the membrane were fixed, stained, and counted by tallying the number of cells in 3 random fieldsunder the microscope. Data were adjusted by growth condition and expressed as mean of migrating cells in 3 fields � SD.

Li et al.





the Natura-alpha–treated group was much slower. Thereduction of tumor volume between Natura-alpha andthe vehicle-treated group was found to be statisticallysignificant (Fig. 3E, P ¼ 0.019 and Fig. 3H, P ¼ 0.026)starting at week 7. The tumor weight from the Natura-alpha–treated group was reduced approximately 2.33-fold(castrated group; Fig. 3F) and 2.6-fold (sham-castratedgroup; Fig. 3I) as compared with the control group. TheHRs are 0.429 (castrated group) and 0.385 (sham-castratedgroup), respectively.In an effort to determine whether Natura-alpha would

prevent tumor growth, we fed mice with Natura-alpha2 weeks before LNCaP-AI cell injection. After tumor cellinjection, the mice were fed continuously with Natura-

alpha until sacrifice. As shown in Supplementary Figure S1,tumor growth from the prefeeding group mice stopped byweek 3 and did not grow any further. Tumor volume fromthe prefeeding group was reduced more than 3.5-fold ascompared with that of the vehicle control group (P <0.001). In addition, prefeeding reduced tumor volumealmost 2-fold when compared with that of mice fed withNatura-alpha beginning at week 5 postinjection (P <0.001).

Natura-alpha reduces tumor burden in a patient withhormone-refractory metastatic PC

An 86-year-old patient with advanced hormone refrac-tory and metastatic PC (liver metastasis confirmed by

Figure 3. Natura-alpha inhibits PCgrowth in vivo. PC cells (LNCaP orLNCaP-AI) were injected s.c. intothe flank region ofmale nudemice.After the prostate tumor grew for 4to 5 weeks (20–30 mm3), animalswere randomly divided into 2groups according to tumor size,10 animals each. A suspension ofNatura-alpha (or equal volume ofthe vehicle) was given at a doseof 100 mg/kg by gavage once perday for 5 days a week. The tumorsize was measured every 3 days,and tumor growth curves (tumorsize vs. time) were plotted. A–C,LNCaP tumors in mice withoutcastration. D–F, LNCaP-AI tumorsin castrated mice. G–I, LNCaP-AItumors in sham-castrated mice.

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biopsy) who had failed previous chemotherapy, was put onNatura-alpha therapy for his disease with permission fromthe FDAwith 3 treatment cycles (4 weeks per cycle). Duringthe 3 treatment cycles allowed by IRB, laboratory tests andimaging examinations have been done at the end of eachtreatment cycle (Supplementary Table S3).

Biological response. The value of alkaline phosphatase(APL) generally decreased during treatment period. Forexample, on December 28, 2008, it was 377 U/L, and itdecreased to 123 U/L on March 30, 2009. The decrease ofAPL may reflect improvement of liver and bone metastases.There was, however, no significant improvement in hisserum PSA after Natura-alpha treatment. Serum PSA initi-ally was at 270 ng/mL on January 2, 2009, decreased to 160ng/mL on January 20, 2009 and elevated to 294 ng/mL onMarch 20, 2009.

Assessment of target response. Target response wasevaluated by anterior and posterior whole body images(bone scan) and a CT scan of the chest, abdomen, andpelvis at the end of each cycle. These studies showed thatoverall tumor burden was reduced. Using the "Guide-lines to evaluate the RECIST" (17), 5 liver metastatictumors at end of third cycle were compared with theirbaseline before Natura-alpha treatment. Multiple meta-static lesions within the liver were unchanged in numberbut decreased in size. As shown in Supplementary FigureS2, a 26% decrease in a sum of the longest diameters of 5tumors was achieved, indicating Natura-alpha treatmentstabilized the disease condition. A bone scan at the endof each cycle showed mostly unchanged as comparedwith the baseline before the study except for the follow-ing lesions in which the radiotracer uptake was slightlydecreased; anterior left second rib, upper thoracic spine,and posterior upper ribs. Unfortunately, the patientexpired 10 months after 3-cycle Natura-alpha treatment.

Signal network proteins targeted by Natura-alpha bypathway array analysis on xenograft tumors

To further explore themechanism of tumor inhibition byNatura-alpha, we conducted proteomic pathway array ana-lysis (PPAA) by using tumor samples from androgen-dependent LNCaP and androgen-independent LNCaP-AIxenografts with or without treatments of Natura-alpha.

PPAA showed that Natura-alpha significantly affectsmolecules involved in regulating cell proliferation andmigration/invasion, or metastasis. Natura-alpha signifi-cantly (at least >2-fold) inhibited expression and activa-tions of cdk, such as cdk2, cdk6, p-cdc2Tyr15, and pRBSer780,which confirmed our previous observations in vitro (16). Asan inhibitor of cdks, it seems that the inhibition of cdkactivity of Natura-alpha (i.e., phosphorylation) was stron-ger than its reduction of protein expression. For example,only 2- to 3-fold decreases in levels of cdk2 and cdk6were achieved, whereas almost complete inhibition ofp-cdc2Tyr15 was obtained by the compound. Natura-alphashowed little effects on expression of cyclin D1 and E.Another key cell-cycle regulator, FOXM1, however, was alsosignificantly inhibited by Natura-alpha (Fig. 4).

Natura-alpha also significantly affected the expression of2 important molecules, E-cadherin and Mesothelin, inLNCaP xenografts (Supplementary Fig. S3). These proteinsare involved in adhesion, migration, and invasion/metas-tasis. Natura-alpha strongly upregulated expression ofE-cadherin (<10-fold) while it considerably inhibitedexpression of Mesothelin (>2-fold) in LNCaP xenografttumors.

In addition, PPAA study also showed that Natura-alphasignificantly (>2.5-fold) inhibited activations of variousprotein kinases, including p-PKCa, p-PKCd, p-ERK, andp-p38. Because overactivation of these protein kinases havebeen shown to be involved in prostate tumor growth,progression, and drug resistance (27–29), inhibition ofNatura-alpha on these protein kinases may also play animportant role in suppressing tumor growth and metasta-sis. Moreover, p-ERK and p-p38 are also involved in lipo-polysaccharide-mediated inflammatory signaling (30),suggesting inhibition of activation of p-ERK and p-p38may also play a role in the antiinflammatory activities ofNatura-alpha.

FOXM1 mediates inhibition of PC growth andinvasion by Natura-alpha

As mentioned above, the PPAA revealed that Natura-alpha significantly inhibited expression of cell-cycle regu-lator FOXM1. As shown in Figure 4A and B, expression ofFOXM1 was reduced more than 3-fold by Natura-alpha intumor samples from androgen-dependent LNCaP xeno-grafts. Similarly, Natura-alpha also repressed expression ofFOXM1 approximately 3-fold in tumor samples fromandrogen-independent LNCaP-AI xenografts (Fig. 4C andD). The PPAA results suggested that Natura-alpha could bean effective inhibitor of FOXM1 expression, resulted inrepressing the FOXM1 pathway-mediated the tumorgrowth promotion.

Because repression of FOXM1 was seen in vivo fromLNCaP and LNCaP-AI xenografts by Natura-alpha, weinvestigated in vitro expression of FOXM1 in LNCaP andLNCaP-AI cells. As shown inFigure 5A, endogenous FOXM1was expressed in both LNCaP and LNCaP-AI cells, however,about 2-fold higher expression was seen in LNCaP-AI cellscompared with LNCaP cells. Next, we examined the effectsof Natura-alpha on FOXM1 expression in both LNCaP andLNCaP-AI cells by incubating these cells in media contain-ing 5mmol/LNatura-alpha for 24 hours. FOXM1expressionwas reducedmore than3-fold inbothLNCaPandLNCaP-AIcells treated with Natura-alpha as compared with the con-trol group (Fig. 5B panel 1 and 2). RT-PCR also revealed thatNatura-alpha repressed FOXM1 expression at the transcrip-tional level (Fig. 5B panel 4 and 5).

To examine whether FOXM1 governs cell-cycle progres-sion in both LNCaP and LNCaP-AI cells, we conductedFOXM1 knockdown by using siRNA and observed that cellcyclewas arrested upon FOXM1knockdown in both LNCaPand LNCaP-AI cells (Supplementary Fig. S4). This observa-tion indicated that FOXM1 plays a key role in cell-cycleprogression which is consistent with previous report (31).

Li et al.





To further explore whether Natura-alpha mediatedrepression of FOXM1 would cause cell-cycle arrest, stablytransfected cell lines of LNCaP and LNCaP-AI with over-expression of FOXM1 were established by retrovirus system(Fig. 5C and E) and their proliferation rates were measured.Forced expression of FOXM1 was found to promote cellproliferation in both LNCaP and LNCaP-AI cell lines.Moreover, the overexpressed FOXM1 in both cell lineslargely reversed the growth inhibition by Natura-alpha,indicating that repression of FOXM1 mediated by Nat-ura-alpha was a primary cause of cell-cycle arrest by thecompound (Fig. 5D and F).Because invasion of LNCaP-AI cells was inhibited by

Natura-alpha (see Fig. 1), we examined whether overex-pression of FOXM1 played a role in the invasion of LNCaP-AI cells. As shown in Figure 5G and H, the overexpressionof FOXM1 resulted in more than 2-fold increase in theinvasion capability of LNCaP-AI cells as compared with thecontrol (Fig. 5G column 1 and 3). Although Natura-alphainhibited the invasion in both LNCaP-AI cells and FOXM1-overexpressed LNCaP-AI cells, the inhibitory effect of thecompound on invasion, however, was diminished to someextent by the overexpression of FOXM1 (Fig. 5G column 2and 4).FOXM1 promotes cell-cycle progression at both G1/S

and G2/M transitions, through regulating its direct targetgenes (cyclin B1, cyclin D1, cyclin E, and cdc23B) andindirectly regulated genes (cyclin A, cyclin F, cdc20, etc.;ref. 31). To further explore the mechanisms of Natura-alpha on inhibition of cell proliferation and invasion, we

investigated expression of several downstream genes ofFOXM1 in response to Natura-alpha treatment. We foundthat Natura-alpha slightly decreased the expression ofcyclin D1 and cyclin E which is consistent with our PPAAresults. Interestingly, Natura-alpha dramatically inhibitedexpression of FOXM1 direct targeted gene cyclin B1, indi-cating that Natura-alpha probably blocks cell-cycle pro-gression through FOXM1-mediated downregulation ofcyclin B1 (Fig. 5I).

Discussion

The development of new therapeutic drugs that simul-taneously target both cell proliferation and inflammationwould be ideal because it may not only suppress cancergrowth but also prevent tumor metastasis. Natura-alphahas antiproliferative and antiinflammatory activities bothin vitro and in vivo via inhibition of cdks and proinflam-matory cytokines (16, 32). In this study, our data showedthat Natura-alpha inhibited growth of PC cells LNCaP,LNCaP-AI, PC3, and DU145 by MTT and anchorage-inde-pendent assay. Matrigel assays also showed that Natura-alpha potentially inhibited invasive capability of cancercells in vitro. In particular, a significant synergistic effect wasalso seen when Natura-alpha was concurrently combinedwith the antimicrotubule agent paclitaxel at a ratio of1,000:1 (NTI-Onco2008-1:Paclitaxel) in androgen-depen-dent LNCaP and androgen-independent LNCaP-AI, indi-cating that Natura-alpha was able to enhance activity of aclinically available chemotherapeutic drug for PC.

Figure 4. PPAA of xenografttumors treated with Natura-alpha.A and B, expression of FOXM1 insamples from LNCaP xenografttumors. C and D, expression ofFOXM1 in samples fromLNCaP-AI xenograft tumors.

FoxM1 FoxM1

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Mechanisms by which different sequential combina-tions of Natura-alpha and Taxol produce different out-comes are currently not clear. It may depend on thetiming of cell-cycle progression affected by the 2 drugs.Natura-alpha and taxol target 2 different cell proliferativepathways, the former inhibiting molecules in cell-cycleregulation (it may arrest cells at G1, S, and G2 þM phase)and the latter interfering with microtubule stability andmostly arresting cells at G2 þ M phase. Thus, differentsequential combinations may lead to different timing ofcell-cycle progression dynamics by the 2 drugs, whichmay affect their ability to reduce growth. For example, ifthe cells are quickly arrested at G1, then the numbers of

cells entering G2þM phase will be reduced, and thereforethe G2 þ M phase target drug, the activity of Taxol willbe reduced (less cells it can attack), and so, a less additiveeffect will occur. However, if cells in G1 and G2 þ Mare simultaneously attacked, theoretically, at least an addi-tive effect or synergism will occur. Thus, our in vitro dataclearly showed the activity of Natura-alpha against PC.

The above in vitro results are substantiated by in vivoexperiments by using mouse models with xenografttumors from androgen-dependent (LNCaP) and andro-gen-independent (LNCaP-AI) PC cells. Continuedtreatment with the compound completely halted tumorgrowth from LNCaP cells and tumor volume was

LNCaP-AILNCaP

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Figure 5. Effects of Natura-alpha on FOXM1 expression in vitro. A, endogenous expression of FOXM1 in both LNCaP and LNCaP-AI cells. B, analysisof FOXM1 expression by RT-PCR and Western blot in LNCaP and LNCaP-AI. Lanes 1, 4: LNCaP, Lanes 2, 5: LNCaP-AI, Lanes 3, 6: b-actin and 18S RNAloading controls. C–F, expression of FOXM1 and cell proliferation in LNCaP and LNCaP-AI cells transfected with FOXM1 or control plasmid in response toNatura-alpha treatment. G and H, expression of FOXM1 and invasion in cells transfected with FOXM1 or control plasmid in response to Natura-alphatreatment; I, effects of Natura-alpha on the expression of cyclin B1, D1, and E in LNCaP-AD and LNCaP-AI cells.

Li et al.





significantly reduced as compared with the control group.Most encouragingly, with the permission of FDA, 1patient with advanced metastatic PC achieved stable dis-ease condition after treatment with Natura-alpha for 3months. All of his liver metastatic tumors reduced in sizeby approximately 26% as determined by using RECIST(17). Of note, although this patient showed response inliver metastases, PSA value remained the same. Thesephenomena need to be further studied. In general, theamount of PSA produced increases with the size of thetumor, thus PSA is widely used as a biomarker for PC anda biomarker for treatment response. However, there is apoor correlation between the PSA level and the actual sizeof the tumor (33). In addition, PC progression may occurin the presence of undetectable or low serum prostate-specific antigen level (34). A proof of concept clinical trialof Natura-alpha in treating patients with advanced meta-static PC is currently under preparation.PPAA showed that 2 categories of molecules involved in

regulating cell proliferation, and migration/invasion ormetastasis were found to be significantly affected by Nat-ura-alpha. Natura-alpha inhibited expression and activa-tion of cdk, such as cdk2, cdk6, p-cdc2Tyr15, and p-RBSer780,as well as FOXM1, which confirmed some of our previousobservations in vitro (16). E-cadherin and Mesothelin, 2critical molecules involved in cell adhesion, migration,invasion and metastasis were also significantly impactedby Natura-alpha treatment, which provided strong mole-cular basis for Natura-alpha in suppression of tumor pro-liferation and metastasis observed both in vitro and in vivo,as well as in one human case. As tumors become moreaggressive with the ability to metastasize, E-cadherin iscommonly lost, as in PC (35–38). There is evidence thatmesothelin may have potential as a new cancer diagnosticmarker and a novel molecular target for gene therapy (39–41). Changes of these molecules in response to Natura-alpha treatments may not only help us to understandmechanisms of its activities on PC invasion/metastasis,but may also make E-cadherin and mesothelin valuablebiological markers to monitor therapeutic activities ofNatura clinically.Our most important finding in this study is that Nat-

ura-alpha significantly inhibited FOXM1 in vivo as

revealed by PPAA study showing that significant repres-sion of FOXM1 by Natura-alpha may be the primary causeof cell-cycle arrest and inhibition of invasion by over-expression or siRNA to knockdown expression of FOXM1in both LNCaP and LNCaP-AI cells. FOXM1 is a humanproto-oncogene (42) and shown to regulate expression ofa large array of G2/M-specific genes, such as Plk1, cyclinB2, Nek2, and CENPF and plays an important role inmaintenance of chromosomal segregation and genomicstability (43). In addition, FOXM1 interacts with the cell-cycle–inhibitory pocket protein pRb and the cdk-activat-ing phosphatase cdc25B in G1 and in G1/S, respectively(44). Because FOXM1 is essential for cancer cell viability,targeting FOXM1 has become a new strategy for devel-oping novel anticancer drugs (45–46). Recent studyshowed that Iressa (Gefitinib) represses FOXM1 expres-sion in breast cancer (47). Repression of FOXM1 byNatura-alpha primarily resulted in cell-cycle arrest andinhibition of invasion, thus, FOXM1 may be one ofcritical targets of Natura-alpha against PC. Our datatogether strongly suggest that Natura-alpha could becomea novel and effective therapeutic agent in treating hor-mone-dependent and hormone-refractory PC with mini-mal side effects.

Disclosure of Potential Conflicts of Interest

L.G. Wang and S. Mencher are employees of Natrogen Therapeutics.

Acknowledgments

We thank Ms. X. Zou for technical assistance.

Grant Support

The study is supported by NYUSOM Urologic Center of Excellence andCTSI (1UL1RR029893) funds to P. Lee; NYU CTSI TL1 fellowship(1UL1RR029893) to G. Daniels; Natrogen Therapeutics International,Inc.; and James McCarron Jr. Medical Foundation.

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 February 16, 2011; revised April 20, 2011; accepted May 11,2011; published OnlineFirst May 23, 2011.

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Li et al.





Correction

Correction: Natura-Alpha Targets ForkheadBox M1 and Inhibits Androgen-Dependentand -Independent Prostate Cancer Growthand Invasion

In this article (Clin Cancer Res 2011;17:4414–24), which was published in theJuly 1, 2011, issue of Clinical Cancer Research (1), the author list should appear asnoted below. The authors regret this error.

Yirong Li, Martin Ligr, James P. McCarron, Garrett Daniels, David Zhang, XinZhao, Fei Ye, JinhuaWang, Xiaomei Liu, ImanOsman, Simon K.Mencher, HebertLepor, Long G. Wang*, Anna Ferrari, and Peng Lee*

*Co-corresponding authors.

Reference1. Li Y, Ligr M, McCarron JP, Daniels G, Zhang D, Zhao X, et al. Natura-alpha targets Forkhead box M1

and inhibits androgen-dependent and -independent prostate cancer growth and invasion. ClinCancer Res 2011;17:4414–24.

Published OnlineFirst August 9, 2011.�2011 American Association for Cancer Research.doi: 10.1158/1078-0432.CCR-11-1927

ClinicalCancer

Research

Clin Cancer Res; 17(17) September 1, 20115838

2011;17:4414-4424. Published OnlineFirst May 23, 2011.Clin Cancer Res Yirong Li, Martin Ligr, James P. McCarron, et al. Growth and InvasionAndrogen-Dependent and -Independent Prostate Cancer Natura-Alpha Targets Forkhead Box M1 and Inhibits

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