The Synthetic Compound CC-5079 Is a Potent Inhibitor of ... · dynamics of microtubule polymerization, leading to mitosis arrest, and consequently resulting in apoptosis of cancer

The Synthetic Compound CC-5079 Is a Potent Inhibitor of

Tubulin Polymerization and Tumor Necrosis Factor-A

Production with Antitumor Activity

Ling-Hua Zhang,1Lei Wu,

1Heather K. Raymon,

2Roger S. Chen,

1Laura Corral,

2Michael A. Shirley,

2

Rama Krishna Narla,2Jim Gamez,

2George W. Muller,

1David I. Stirling,

1J. Blake Bartlett,

1

Peter H. Schafer,1and Faribourz Payvandi

1

Divisions of 1Immunotherapeutics and 2Signal Research, Celgene Corp., Summit, New Jersey

Abstract

We have found that the synthetic compound CC-5079 potentlyinhibits cancer cell growth in vitro and in vivo by a novelcombination of molecular mechanisms. CC-5079 inhibitsproliferation of cancer cell lines from various organs andtissues at nanomolar concentrations. Its IC50 value rangesfrom 4.1 to 50 nmol/L. The effect of CC-5079 on cell growth isassociated with cell cycle arrest in G2-M phase, increasedphosphorylation of G2-M checkpoint proteins, and apoptosis.CC-5079 prevents polymerization of purified tubulin in aconcentration-dependent manner in vitro and depolymerizesmicrotubules in cultured cancer cells. In competitive bindingassays, CC-5079 competes with [3H]colchicine for binding totubulin; however, it does not compete with [3H]paclitaxel(Taxol) or [3H]vinblastine. Our data indicate that CC-5079inhibits cancer cell growth with a mechanism of actionsimilar to that of other tubulin inhibitors. However, CC-5079remains active against multidrug-resistant cancer cells unlikeother tubulin-interacting drugs, such as Taxol and colchicine.Interestingly, CC-5079 also inhibits tumor necrosis factor-A(TNF-A) secretion from lipopolysaccharide-stimulated humanperipheral blood mononuclear cells (IC50, 270 nmol/L). Thisinhibitory effect on TNF-A production is related to its inhi-bition of phosphodiesterase type 4 enzymatic activity. More-over, in a mouse xenograft model using HCT-116 humancolorectal tumor cells, CC-5079 significantly inhibits tumorgrowth in vivo . In conclusion, our data indicate that CC-5079represents a new chemotype with novel mechanisms of actionand that it has the potential to be developed for neoplasticand inflammatory disease therapy. (Cancer Res 2006; 66(2): 951-9)

Introduction

Microtubules, a major type of cytoskeletal filament in cells, areformed from tubulin subunits, including a-tubulin and h-tubulin.Microtubules are the target of a large and diverse group ofanticancer drugs derived from natural products. Given the successof this class of drugs in cancer treatment, microtubules can beenconsidered one of the best cancer target identified to date (1).Increasingly, anticancer drugs targeting microtubules are beingdiscovered from small molecules derived from either terrestrialplants or marine organisms. These small molecules can alter thedynamics of microtubule polymerization, leading to mitosis arrest,

and consequently resulting in apoptosis of cancer cells (2). Theycan be separated into two groups according to their effect onmicrotubules: inhibitors of microtubule polymerization, such asvincristine, vinblastine, colchicines, cryptophycins, and combretas-tatins, and microtubule stabilizers, such as paclitaxel (Taxol),docetaxel (Taxotere), discodermolide, epothilones, laulimalide, andeleutherobins (3). These small molecules bind to a variety of siteson tubulin and have diverse effects on microtubule dynamics. Inspite of the initial clinical success of some of these agents, themajority of cancer types are either inherently resistant to theseagents or eventually develop resistance. In addition, there areproblems of high toxicity (especially neurotoxicity), marginal oralbioavailability, poor solubility, difficulty of synthesis or isolationfrom natural sources, and, most importantly, development of drugresistance (4). Therefore, new synthetic compounds with oralbioavailability and an improved therapeutic index for first-line andsecond-line therapy are highly attractive therapeutics.Tumor necrosis factor-a (TNF-a) is a cytokine produced in vivo

mainly by activated macrophages/monocytes, with multiple effectson normal andmalignant cells. Although initial TNF-a expression inresponse to infection or injury is considered beneficial, excessiveproduction typically by activated monocytes and macrophages canproduce significant pathologic changes. Recent research has indi-cated that elevated levels of TNF-a may contribute to the patho-genesis of cancers, possibly promoting cell growth and inhibitingapoptosis (5, 6). In addition, elevated levels of TNF-a may allowmalignant cells to escape immune surveillance (7). Therefore, TNF-amay be a promising drug target for cancer. Recently, small-moleculeTNF-a inhibitors, such as thalidomide and its analogues, haveshown significant activity in the treatment of cancer (8, 9).In the course of our drug screening efforts to discover novel

anticancer and immunomodulatory agents from synthetic com-pounds, a novel class of cytokine inhibitory drugs has beendeveloped with distinct and multiple activities (i.e., inhibition ofboth mitosis and TNF-a synthesis). We report here that CC-5079, asa representative of this class of compound, binds tubulin anddestabilizes microtubules, leading to mitotic arrest and cell death.CC-5079 inhibits TNF-a production from lipopolysaccharide(LPS)–stimulated human peripheral blood mononuclear cells(PBMC) by inhibiting the enzymatic activity of phosphodiesterasetype 4 (PDE4), an essential cyclic AMP (cAMP)–metabolizingenzyme in immune and inflammatory cells for endotoxin-activatedTNF-a responses (10). CC-5079 displayed broad anticancer activityagainst a variety of cancer cell lines independent of multidrugresistance (MDR) phenotype status. Finally, CC-5079 exhibitedsignificant in vivo antitumor activity in a severe combined immuno-deficient (SCID) mouse xenograft model of human colorectalcancer.

Requests for reprints: Ling-Hua Zhang, Celgene Corp., 86 Morris Avenue, Summit,NJ 07901. Phone: 908-673-9000; Fax: 908-673-9001; E-mail: [email protected].

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-2083

www.aacrjournals.org 951 Cancer Res 2006; 66: (2). January 15, 2006

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Materials and Methods

Chemicals and biologicals. CC-5079 (Fig. 1A) was synthesized at

Celgene Corp. (Summit, NJ). General chemicals as well as tubulininhibitors Taxol, vinblastine, and colchicine were purchased from Sigma

(St. Louis, MO). All compounds were dissolved in 100% DMSO before

further dilution in cell culture medium. Final DMSO concentrations

were kept at a constant 0.1% for all samples, including controls, unlessotherwise stated. Streptavidin-coated yttrium scintillation proximity

assay (SPA) beads were obtained from Amersham Pharmacia Biotech

(Piscataway, NJ). [3H]Colchicine was from New England Nuclear(Boston, MA); [3H]Taxol and [3H]vinblastine were from Morevek

Biochemicals (Brea, CA). Purified tubulin and biotinylated microtubule-

associated protein-free bovine brain tubulin were from Cytoskeleton, Inc.(Denver, CO).

Cancer cell lines and primary cells. The human tumor cell lines

HT29 (colon adenocarcinoma, HTB-38), HT-144 (melanoma, HTB-63),

HCT-116 (colorectal carcinoma, CCL-247), A549 (non–small cell lung

cancer, CCL-185), NIH:OVCAR-3 (ovary adenocarcinoma, HTB-161), PC-3

(prostate adenocarcinoma, CRL-1435), HCT-15 (colorectal adenocar-

cinoma, CCL-225), MCF-7 (breast adenocarcinoma, HTB-22), MES-SA

(uterine sarcoma, CRL-1976), MES-SA/MX2 (CRL-2274), and MES-SA/

Dx5 (CRL-1977) were purchased from American Type Culture Collection

(Manassas, VA). MCF-7/Adr (also known as NCI/Adr-Res) was kindly

provided by the Signal Research Division of Celgene. All cell lines were

cultivated at 37jC, 5% CO2 in medium as published or as stated by

American Type Culture Collection. The detailed characteristics of

human parental MCF-7, MES-SA cell lines, and multidrug-resistant,

P-glycoprotein (P-gp) 170–overexpressing MCF-7/Adr, MES-SA/MX2, MES-

SA/Dx5, and HCT-15 cell lines have been reported (11, 12). Human umbilical

vein endothelial cells (HUVEC) were kindly provided by Celgene Cellular

Therapeutics (Cedar Knolls, NJ). PBMCs from normal donors were obtained

by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density centrifugation.

Cell proliferation assay. Cell proliferation was assessed in cancer celllines, HUVECs, and human PBMCs by [3H]thymidine incorporation assay.

Briefly, cells were seeded on 96-well microtiter plates 24 hours before

addition of compound to allow them to adhere to plates. Each compound

was tested at serial dilutions in triplicate. Following compound treatment,the cells were incubated at 37jC for additional 72 hours. [3H]Thymidine

(1 ACi in 20 AL medium) was added to each well for the last 6 hours of

incubation time. The cells were then harvested for detection of tritium

incorporation with a TopCount Microplate Scintillation Counter (PackardInstrument Co., Meriden, CT). IC50s were calculated using a nonlinear

regression analysis using GraphPad Prism program (San Diego, CA).

Flow cytometric analysis. For cell cycle analysis, cells were harvested

following treatment with test agents for 24 hours and stained withpropidium iodide (PI) according to the instruction of Cycle Test Plus DNA

Reagent kits from Becton Dickinson (San Jose, CA). Samples were examined

using FACSCalibur instrument (Becton Dickinson). Cell cycle distributionwas analyzed with CellQuest version 3.1 acquisition software and the

ModFit version 2.0 program.

For apoptosis analysis, cells were treated with test agents for 48 hours

and then harvested. Double staining for FITC-Annexin V binding and forDNA using PI was done as described previously (13).

Tubulin polymerization assay. The polymerization of purified tubulin

was monitored using the CytoDYNAMIX Screen (Cytoskeleton). Tubulin

polymerization was monitored spectrophotometrically by the change inabsorbance at 340 nm. The absorbance was measured at 1-minute intervals

for 60 minutes using a PowerWave HT microplate reader (Bio-Tek

Instruments, Highland Park, VT).Immunofluorescence microscopy. Detection of a-tubulin in A549

cells by immunofluorescence was done as described previously (14). Briefly,

cells were treated with test compounds for 24 hours and washed with

PBS. Cells were then fixed and permeabilized with warm PBS buffercontaining 3.7% formaldehyde and 1% Triton X-100 for 30 minutes. After

washing cells twice with PBS and saturation with 1% mouse blocking serum

in PBS for 30 minutes, staining was done with an anti-h-tubulin-FITCantibody (Sigma) alone or in the presence of 100 Ag/mL PI. Cells wereobserved under an epifluorescence microscope (Nikon Instruments,

Melville, NY) and imaged with a CCD camera using Image-Pro (Media

Cybernetics, Silver Spring, MD).Tubulin competitive binding SPA assay. The tubulin-binding assay

was done as reported previously (15) using biotin-labeled tubulin,

streptavidin-coated yttrium SPA beads, and 3H-labeled ligands ([3H]colchi-

cine, [3H]Taxol, or [3H]vinblastine). Briefly, the binding mixture includes0.08 Amol/L 3H-labeled ligand, 1 mmol/L GTP, and 0.5 Ag biotinylated

tubulin in 100 AL assay buffer containing 80 mmol/L PIPES (pH 6.9),

1 mmol/L MgCl2, 1 mmol/L EGTA, and 5% glycerol. The test compound and3H-labeled ligand were added before tubulin. After incubation at 37jC for2 hours, 20 AL SPA beads (80 Ag in the assay buffer) were added. After

Figure 1. Effect of CC-5079 on proliferation of HUVEC and human PBMC.A, chemical structure of CC-5079. B, HUVECs were grown in the presence orabsence of CC-5079 for 3 days, and the growth was assessed by [3H]thymidineincorporation. Representative of three independent experiments. C, PBMCswere cultured in the presence and absence of CC-5079 for 1, 2, or 3 days, andthe growth was assessed by [3H]thymidine incorporation.

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Cancer Res 2006; 66: (2). January 15, 2006 952 www.aacrjournals.org



further incubation for 30 minutes under agitation at room temperature, theSPA beads were allowed to settle down for 45 minutes, and scintillation

counting was done on the TopCount Microplate Scintillation Counter.

Caspase assay. Caspase activity was determined according to the

instructions from the assay kit supplier (R&D Systems, Minneapolis, MN).The results were expressed as fold change in caspase activity of drug-treated

cells over the vehicle control cells.

Immunoblot analysis of cell cycle regulatory proteins. Cancer cellswere treated with test compounds or 0.1% DMSO for 24 hours. Cells were

trypsinized, spun down for 6 seconds in a microfuge, and immediately lysed

in 0.1 mL lysis buffer containing 10 mmol/L Tris-HCl (pH 8.0), 10 mmol/L

EDTA, 150 mmol/L NaCl, 1% NP40, 0.5% SDS, 1 mmol/L DTT, 1 mmol/L

Na3VO4 plus Complete protease inhibitor cocktail (Roche Applied Science,

Indianapolis, IN) and then spun through a Qiashredder (Qiagen, Valencia,

CA) for 1 minute and frozen on dry ice. Samples were diluted with 3� SDS

sample buffer (New England Biolabs, Beverly, MA) and boiled 5 minutes.

The mixture (f30 AL) was loaded per lane on Tris-glycine polyacrylamide

gels (Invitrogen, Carlsbad, CA), electrophoresed, and transferred to

polyvinylidene difluoride (PVDF) membranes (Invitrogen). PVDF mem-

branes were blocked for 1 hour at room temperature in PBS containing

0.05% Tween 20 and 5% nonfat milk powder and then blotted overnight

at 4jC with antibodies against either MPM-2 (Upstate Biotechnology,

Lake Placid, NY), bcl-2, cyclin B1, p53, p21, Cdc25C, or a-tubulin (Santa

Cruz Biotechnology, Santa Cruz, CA). Membranes were washed and

incubated with horseradish peroxidase–conjugated anti-rabbit or anti-

mouse IgG (Santa Cruz Biotechnology; 1:1,000 dilution) for 60 minutes

at room temperature, washed thrice, and then developed using the

Enhanced Chemiluminescence Plus Detection System (Amersham Bio-

sciences, Piscataway, NJ).

PBMC culture and ELISA for TNF-A. PBMCs were prepared by densitycentrifugation on Ficoll-Hypaque. PBMCs, resuspended at 1 � 106/mL in

complete RPMI 1640/10% FCS, were stimulated with LPS (1 Ag/mL;

Escherichia coli serotype 0127:B8; Sigma) in 24-well plates by incubation at37jC in 5% CO2 for 24 hours with or without compounds (0.1-100 Amol/L).

Cell-free supernatants were collected and stored in aliquots at �70jC until

assayed by ELISA. Supernatants were assayed for TNF-a using an assay

procedure and reagents provided by R&D Systems.PDE4 assay. PDE4 purification from human histiocytic lymphoma U937

cells was done as described previously (16). Briefly, cells (1 � 109) were

washed in PBS and lysed in cold homogenization buffer [20 mmol/L Tris-

HCl (pH 7.1), 3 mmol/L 2-mercaptoethanol, 1 mmol/L MgCl2, 0.1 mmol/LEGTA, 1 Amol/L phenylmethylsulfonyl fluoride, and 1 Ag/mL leupeptin].

Following homogenization, the supernatant was collected by centrifugation

and loaded onto a Sephacryl S-200 column equilibrated in homogenizationbuffer. Phosphodiesterase was eluted in homogenization buffer and

rolipram-sensitive fractions were pooled and stored in aliquots. Phospho-

diesterase activity was assayed by a procedure described by DiSanto and

Heaslip (17) and in the presence of varying concentrations of compounds.IC50 was determined from dose-response curves derived from three

independent experiments.

Human tumor xenograft model. Female CB17 SCID mice (6-8 weeks

old) were obtained from the Charles River Laboratory (Wilmington, MA)and maintained in microisolator cages under sterile conditions. The right

hind legs of the mice were inoculated s.c. with HCT-116 cells suspended in

sterile PBS (2 � 106 cells per mouse). On day 6, tumors of all mice weremeasured with a digital caliper and volumes were calculated with a

formula of W2 � L / 2, where W is width (short axis) and L is length (long

axis). Mice bearing tumor size ranging between 75 and 125 mm3 were

pooled together and randomly distributed into cages. The mice were thenear tagged and cages were randomly assigned to treatment groups. On day

7, the tumors were measured and considered as starting volumes, and the

mice were then given i.p. with either vehicle (N-methyl-2-pyrrolidone/

polyethylene glycol 400/saline, 1:9:10), CC-5079 (5 and 25 mg/kg), orpositive control Camptosar (10 mg/kg; Pfizer, Inc., New York, NY). Both

doses of CC-5079 were initiated as once daily administrations for the first

5 days (days 7-11); the dosing of CC-5079 at 25 mg/kg was then switched to

q3d � 4 (days 14, 17, 20, and 23) due to potential toxicity of the compound.

Camptosar was given as q4d � 5 (days 7, 11, 15, 19, and 23). Mice weremonitored daily for health status as well as tumor growth. Tumors were

measured twice weekly.

Results

Effects of CC-5079 on various cancer cells, HUVECs, andPBMCs. To explore the effect of CC-5079 on cancer cell proli-feration, we treated human cancer cell lines from colon, prostate,breast, lung, ovary, uterus, and skin with different concentrations ofcompounds using Taxol and colchicine as positive controls. Cellproliferation was measured by [3H]thymidine incorporation assay.Proliferation of all cell lines was inhibited by CC-5079 in aconcentration-dependent manner. The growth inhibition constants(IC50) of the different tumor cell lines ranged from 4.1 to 50 nmol/Lfor CC-5079.One major mechanism of MDR of anticancer drugs is mediated

by the overexpression of the multidrug transporter P-gp (18, 19).The antitumoral efficacy of CC-5079 was compared with Taxol andcolchicine in a cytotoxicity assay using four MDR cell lines with highlevels of P-gp: HCT-15, MCF-7/Adr, MES-SA/MX2, and MES-SA/Dx5.These cell lines were markedly resistant to Taxol and colchicine,whereas no significant resistance to CC-5079 was observed. Asjudged from the resistance factors (the ratio of the IC50 of theresistant cell line relative to the IC50 of its parental cell line), MDRcells were >2,000- and >300-fold resistant to Taxol and colchicine,respectively. In contrast, the cytotoxic efficacy of CC-5079 againsttumor cells was unaltered by the MDR phenotype (Table 1).Because CC-5079 shows significant antiproliferative effects on

various cancer cells, including MDR cell lines, it was tested for itstoxicity on normal primary cells, including HUVEC and PBMC. Asshown in Fig. 1B , HUVECs were found to be more sensitive to thecytotoxic effects of CC-5079 than cancer cells. The IC50 for CC-5079on HUVEC was 0.17 nmol/L, which was 24- to 294-fold lower thanthat of cancer cells. This selectivity on HUVEC indicates that CC-5079 may be a potential antiangiogenic agent. The cytotoxicity ofCC-5079 was also tested on PBMC at concentrations ranging from 1to 100 Amol/L. Results (Fig. 1C) showed that CC-5079 did not affectunstimulated PBMCDNA synthesis but showed inhibitory effects onphorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulatedPBMC. These results suggest that CC-5079 has significant cytotoxiceffects on fast-proliferating cells but not on quiescent cells.Effect of CC-5079 on molecular events of cell cycle

progression of cancer cells. The strong antiproliferative activityof CC-5079 on cancer cells prompted us to test its effects on thecell cycle progression. As shown in Table 2, there was anaccumulation of MCF-7 cells with 4N DNA content (G2-M phase)and a concomitant decrease in cells in G0-G1 or S phase aftertreatment with either 20 nmol/L Taxol, 20 nmol/L colchicine, or20 to 100 nmol/L CC-5079. In contrast, the cell cycle distribution ofTaxol- and colchicine-treated MCF-7/Adr cells looked similar to theDMSO controls, whereas CC-5079 was equally effective in both celllines. The accumulation of cells with 4N DNA content increasedwith time followed by a decrease in cells with 4N DNA content andan increase in subdiploid cells at later time points (48-72 hours),indicative of apoptotic cells (data not shown). As expected, theeffect of CC-5079 on the cell cycle distribution was found to beindependent of MDR status of cells.We also assessed the effect of CC-5079 on the phosphorylation of

mitotic regulatory proteins and other molecular events involved incell cycle progression. As shown in Fig. 2A and B , treatment of

Antimitotic and Anti-TNF-a Effects of CC-5079




MCF-7 cells with CC-5079, Taxol, or colchicine for 24 hours resultedin phosphorylation of proteins involved in mitosis as judgedfrom slower migrating forms of the Cdc25C phosphatase or theantiapoptotic protein bcl-2. The changes in Cdc25C and bcl-2coincided with the appearance of phosphoepitopes recognized byMPM-2, an antibody that recognizes phosphorylated polypeptidesfound only in mitotic cells (20). In contrast, the MDR derivativeMCF-7/Adr cells seemed to be resistant to Taxol treatment, as therewas a significant decrease in phosphorylation of Cdc25C andproteins detected by MPM-2. Similar to Taxol and colchicine, CC-5079 also markedly induced the accumulation of cyclin B. Theseresults indicate that CC-5079 arrests the cell cycle and is capable ofinducing mitotic arrest in cells, which express P-gp and areresistant to known antimitotic drugs.Effect of CC-5079 on p53 and p21 expression, caspase

activity, and apoptosis of cancer cells. p53 protects mammalsfrom neoplasia by inducing apoptosis, DNA repair, and cell cyclearrest in response to a variety of stresses (21). When we examined

the effect of CC-5079 on the expression of p53 in several cancer celllines, we found that CC-5079 treatment of MCF-7 cells led to amarked increase in the level of p53 protein (Fig. 2A). Similar effectswere observed in cells treated with drugs, such as colchicine orTaxol. Unlike the MCF-7 cells, the MDR derivative MCF-7/Adr cellswere found to constitutively express high levels of p53. CC-5079,Taxol, and colchicine were unable to increase the level of p53protein in this MDR cell line. When p21 was examined in thesetreated MCF-7 cells, it was also appreciably induced by CC-5079 aswell as by Taxol and colchicine. In contrast, p21 was undetectableby Western blot analysis in MCF-7/Adr cells treated with orwithout these test compounds (Fig. 2A). These results indicate thatconstitutively expressed p53 in MCF-7/Adr failed to transactivatep21 expression whether cells were incubated in the presence andabsence of test drugs and that the antiproliferative effects ofCC-5079 in MCF-7/Adr cells are p53 independent.To determine whether the antimitotic effects of CC-5079

eventually leads to apoptosis of cancer cells, we examined the

Table 2. Effects of CC-5079, Taxol, and colchicine on cell cycle distribution in MCF-7, MCF-7/Adr, and A549 cells

Treatment* MCF-7 cells MCF-7/Adr cells A549 cells

G0-G1 S G2-M G0-G1 S G2-M G0-G1 S G2-M

DMSO 71 20 8.9 19 36 45 44 42 14

Taxol, 20 nmol/L 42 23 35 17 37 46 0.11 36 64

Colchicine, 20 nmol/L 33 23 45 18 36 46 44 17 39

CC-5079, 4 nmol/L 72 20 8.2 21 34 45 43 43 14CC-5079, 20 nmol/L 46 23 32 4.0 23 73 21 38 41

CC-5079, 100 nmol/L 34 10 56 3.0 22 75 0.91 7.6 91

*MCF-7, MCF-7/ADR, and A549 cells were treated with the indicated compounds for 1 day. Cells were then fixed, stained with PI, and analyzed by flow

cytometry. Percentages of cells in each cell cycle phase are shown. Representative of two independent experiments.

Table 1. Inhibitory effects of CC-5079 on proliferation of various cancer cell lines

Cell line Tumor origin P-gp* Growth inhibition,c IC50 (mean F SD, nmol/L)

Taxol Colchicine CC-5079

HT29 Colon 0 <0.10 2.8 F 0.31 21 F 0.23

HT-144 Melanoma 0 <0.10 31 F 0.22 50 F 4.2

HCT-116 Colon 0 0.30 F 0.026 20 F 1.8 17 F 1.1

A549 Non–small cell lung cancer 0 1.2 F 0.22 2.0 F 0.16 25 F 2.3NIH:OVCAR-3 Ovary 0 <0.10 5.8 F 0.65 4.1 F 0.23

PC-3 Prostate 0 3.0 F 0.32 19 F 2.2 25 F 0.12

HCT-15 Colon + 150 F 45 91 F 4.5 21 F 0.21MCF-7 Breast 0 0.50 F 0.12 3.0 F 0.65 6.9 F 0.34

MCF-7/Adr Breast + 7,100 F 520 920 F 88 17 F 1.1

MES-SA Uterus 0 1.2 F 0.11 2.1 F 0.22 11 F 1.3

MES-SA/MX2 Uterus + 2,800 F 330 31 F 0.26 12 F 0.88MES-SA/Dx5 Uterus + 1900 F 310 1100 F 98 16 F 1.3

*P-gp levels were reported previously and confirmed by flow cytometry after staining cells with anti-P-gp-FITC antibody. Scoring code: 0, not detected;

+, high-level expression.cCells were grown in the presence of test compounds for 3 days, and the growth was assessed by [3H]thymidine incorporation. All experiments weredone at least in two replicates, and IC50s were calculated from dose-response curves by nonlinear regression analysis.

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Figure 2. Effect of CC-5079 on molecular eventsof cell cycle progression and apoptosis.A, phosphorylation of G2-M regulatory proteinsand Cdc25C and expression of p53 and p21.Asynchronous MCF-7 and MCF-7/Adr cells weretreated with test compounds at indicatedconcentrations for 24 hours and protein washarvested. MPM-2 phosphoepitopes, Cdc25C,and p53 and p21 proteins were analyzed byWestern blot. B, phosphorylation of bcl-2 andaccumulation of cyclin B1. Asynchronous A549cells were treated with test compounds atindicated concentrations for 24 hours and proteinwas harvested. Bcl-2 and cyclin B1 proteins wereanalyzed by Western blot. C, activation ofcaspases. A549 cells were treated with test drugsat indicated concentrations for 30 hours.Caspase-3, caspase-8, and caspase-9 activitywas measured. D, induction of apoptosis. A549cells were treated with test drugs at indicatedconcentrations for 48 hours. Cells were collectedfor double staining (FITC-Annexin V and PI) andanalyzed with flow cytometry. Representative oftwo independent experiments.





ability of CC-5079 to activate caspase-3, caspase-8, and caspase-9in A549 cells. Caspase-3, considered an ‘‘executioner’’ caspase, isimplicated in the last and irreversible phase of the apoptoticcaspase pathway and is activated by upstream ‘‘initiator’’ caspases,such as caspase-8 and caspase-9 (22). Indeed, CC-5079 (25-400nmol/L), similar to Taxol (100 nmol/L), was able to induce aconcentration-dependent increase in activity of these caspases inA549 cells (Fig. 2C). A549 cells incubated with 100 nmol/L CC-5079showed a time-dependent increase in caspase-3 activity comparedwith untreated cells, reaching a maximum at 30 hours (data notshown). Fluorescein-labeled Annexin V binding in conjunction witha PI dye exclusion test was also used to discriminate intact cells(FITC�/PI�), apoptotic cells (FITC+/PI�), and necrotic cells(FITC+/PI+) following the treatment of A549 cells with CC-5079or Taxol. Exposure of A549 cells to 100 nmol/L Taxol or 100 to 1,000nmol/L CC-5079 for 48 hours generally resulted in an increase inthe percentages of apoptotic and necrotic cells and a reduction inthe percentage of intact cells when compared with the DMSOcontrol (Fig. 2D).

Effect of CC-5079 on tubulin polymerization and tubulinbinding of colchicine, vinblastine, and Taxol. Microtubules area major component of the mitotic spindle assembly, which pullsthe chromosomes apart at mitosis and then splits the dividing cellinto two. The majority of antimitotic agents induce mitotic arrestby interacting with tubulin. We tested CC-5079 for its effect ontubulin. The polymerization of purified tubulin at 37jC in thepresence of test compounds or DMSO control was monitoredspectrophotometrically. Without treatment of drugs (DMSOcontrol), tubulin subunits heterodimerize and self-assemble toform cylindrical microtubules in a time-dependent manner. Taxoltreatment enhanced tubulin polymerization, whereas microtubule-depolymerizing agents colchicine and vinblastine, as well as CC-5079, prevented tubulin polymerization (Fig. 3A). Results in Fig. 3Aalso indicate that CC-5079 inhibited tubulin polymerization in aconcentration-dependent manner.To test whether CC-5079 also affects microtubule polymerization

in cells, we treated A549 cells with or without test compounds for24 hours and subsequently fixed and stained with FITC-conjugated

Figure 3. Effect of CC-5079 on microtubules. A, tubulin polymerization in vitro. The ability of CC-5079 at indicated concentrations to inhibit polymerization of purifiedtubulin was assessed by turbidity change using spectrophotometry. DMSO was used as vehicle control. Taxol, vinblastine, and colchicine were used as control forpositive tubulin-targeting agents. B, microtubule polymerization in cancer cells. A549 cells were grown in medium containing 0.1% DMSO (a) or 40 nmol/L colchicine(b), 40 nmol/L Taxol (c ), 8 nmol/L CC-5079 (d), 40 nmol/L CC-5079 (e), 200 nmol/L CC-5079 (f ) for 24 hours. After treatment, cells were fixed. Microtubules werestained green with FITC-conjugated anti-h-tubulin antibody, and chromosomal DNA was stained red with PI. C, effect on tubulin binding of colchicine, vincristine, andTaxol. Tubulin binding was tested with a SPA-based competition assay. Representative of three independent experiments.

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anti-h-tubulin antibody and PI for in situ observation of microtu-bule network and nuclei with fluorescence microscopy. A549cells without treatment (Fig. 3B, a , DMSO control) exhibited thecharacteristic staining of individual microtubules with fluorescentanti-tubulin as indicated by a fine network ‘‘mesh’’ of microtubularmaterials. Nuclei of these control cells were intact and appearednormal as visualized by staining with PI. Taxol (40 nmol/L)–treatedcells (Fig. 3B, c) exhibited a significant increase of microtubulebundles in cytoplasm due to enhanced tubulin polymerizationand highly condensed chromatin. CC-5079-treated cells exhibitedcondensed chromatin and a decreased amount of microtubularmaterials, which almost completely disappeared in the presence of40 to 200 nmol/L CC-5079 presumably due to depolymerization oftubulin (Fig. 3B, e and f ). However, colchicine (40 nmol/L) and lowerconcentrations of CC-5079 (8 nmol/L) had only a slight effect onmicrotubules and nuclei (Fig. 3B, b and d).The binding sites for three tubulin ligands, Taxol, vinblastine,

and colchicine, form the pharmacologic map of the surface oftubulin; they each bind at separate noncompeting sites (15). Usinga competitive binding SPA, we determined if CC-5079 interacteddirectly with tubulin by binding to these ligand-binding sites. Wefound that CC-5079 competitively inhibited [3H]colchicine bindingto biotinylated tubulin (IC50, 0.8 Amol/L), whereas it did notcompete with [3H]vinblastine and [3H]Taxol (Fig. 3C). Thus, weconclude that the antiproliferative activity of CC-5079 is based onbinding to tubulin at the colchicine-binding site.Effect of CC-5079 on TNF-A production from human PBMC

and PDE4 enzymatic activity. Besides the antimitotic effect ofCC-5079, this compound was also tested for an effect on cytokineproduction from PBMCs. CC-5079 at nontoxic concentrations wasfound to inhibit TNF-a production from LPS-stimulated PBMC ina dose-dependent manner. In contrast, Taxol and colchicine hadno marked effect on TNF-a production at low concentrations. TheIC50 values for CC-5079, Taxol, and colchicine are 270 nmol/L,>100 Amol/L, and 2.0 Amol/L, respectively. Because PDE4 isan essential cAMP-metabolizing enzyme involved in immune andinflammatory cells for LPS-activated TNF-a responses, thesecompounds were tested in a PDE4 enzymatic assay. CC-5079 wasfound to inhibit enzymatic activity of PDE4 purified from U937cells in a dose-dependent manner (IC50, 350 nmol/L). However,neither Taxol nor colchicine had any significant effect on PDE4activity at concentrations up to 100 Amol/L, although colchicineweakly inhibited TNF-a production from PBMC. These additionalbiological activities differentiate CC-5079 from other known tubu-lin interactive agents. Statistical analysis of results from structure-activity relationships (SAR) screening of 50 analogues of CC-5079indicated that the inhibition of TNF-a from LPS-stimulated PBMCby CC-5079 and its analogues is correlated with their inhibitoryeffect on PDE4 activity (Fig. 4A), indicating that CC-5079 impairsTNF-a production by inhibiting PDE4 activity in monocytes.However, the inhibition of the growth of cancer cells and HUVECby CC-5079 (and its analogues) was found to be unrelated withtheir inhibitory effect on PDE4 activity (Fig. 4B).In vivo antitumor activity of CC-5079 in mouse xenograft

model. We have shown that CC-5079 has dual biological activities(i.e., inhibition of the in vitro growth of different human cancer celllines by tubulin binding and impairment of the TNF-a productionfrom activated PBMC by blocking PDE4 activity). To test whetherCC-5079 also affects growth of solid tumors in vivo , we evaluatedthe activity in the HCT-116 colorectal cancer model in CB17 SCIDmice. Exponentially growing HCT-116 cells were injected s.c. into

SCID mice. When the tumors reached f100 mm3, the mice weregiven i.p. with vehicle, CC-5079, or the positive control Camptosar,an anticancer drug specifically targeting DNA topoisomerase I (23).CC-5079 at 5 mg/kg inhibited tumor growth by 22%, whereas CC-5079 at 25 mg/kg showed a significant inhibition of HCT-116 tumorgrowth by 46% (P < 0.001; Fig. 5). The body weights of these micewere not significantly affected, when CC-5079 at these doses wasadministrated.

Discussion

In our systematic effort to discover novel anticancer andimmunomodulatory agents from synthetic small molecules, anovel class of compound with a diarylalkene structure has beensynthesized. These compounds show properties similar to ourpreviously reported selective cytokine inhibitory drugs but havegreater potency in inhibiting tumor cell growth. Distinct from theprevious class of cytokine inhibitory compounds, these novel

Figure 4. Relationship between effect of CC-5079 and analogues on cellproliferation, TNF-a production, and PDE4 enzymatic activity. A, inhibitory effectof CC-5079 and analogues on TNF-a production from PBMC is correlatedwith inhibition of PDE4 enzymatic activity. B, inhibitory effect of CC-5079analogues on cell growth is unrelated with inhibition of PDE4 enzymatic activity.Data from SAR screening of 50 analogues were analyzed with Pearsoncorrelation calculation using the GraphPad Prism program.





compounds are found to interact directly with tubulin, arrest thecell cycle, and induce apoptosis of tumor cells (24). In the presentstudy, we have characterized the activities of CC-5079, one of themost potent among these compounds. We first tested CC-5079 in acancer cell proliferation assay and found that CC-5079 inhibitedthe growth of various cancer cells with IC50 values at thenanomolar level (Table 1). We also tested if CC-5079 has anyantiproliferative effect on normal cells and found that CC-5079was 10- to 100-fold more effective in inhibiting proliferation ofendothelial cells than that of cancer cells, suggesting that CC-5079may be useful in antiangiogenic therapy of cancer and otherdiseases (25). We found that CC-5079 at nontoxic concentrationswas able to inhibit the capillary tube formation in vitro andadhesion and migration of endothelial cells (data not shown).Using the chick chorioallantoic membrane–layered expressionscanning, CC-5079 was found to inhibit the expression of avh3

significantly (26). Our data further confirm the antiangiogenicpotential of CC-5079. On the other hand, CC-5079 was found not toimpair the viability of PBMC at concentrations as high as 100Amol/L, although it did inhibit the proliferation of PMA plusionomycin–stimulated human PBMC.Our data clearly show that CC-5079 is efficacious in inhibiting

the proliferation of a broad range of cancer cells as well asendothelial cells. CC-5079 was found to significantly induce cellcycle arrest in G2-M phase. The antimitotic effect of CC-5079 wasfurther confirmed by the fact that treatment of cells with thiscompound triggered several molecular events involved in themitotic signaling cascade (20, 27). These included phosphorylationof some important mitotic regulatory proteins, such as Cdc25C,bcl-2, and MPM-2 epitopes, and accumulation of cyclin B.As a cellular gatekeeper for growth and division, the tumor

suppressor gene p53 plays an essential role in sensing various stresssignals and serves as a focal point of signal integration to decidewhether cells will undergo growth arrest or apoptosis (28). Severalantimitotic agents have been reported to induce p53 and inhibitcyclin-dependent kinases, p21WAF1/CIP1 (p21), and activate/inactivate several protein kinases, including Ras/Raf, mitogen-activated protein kinases, and p34Cdc2 (29). These kinases are

associated directly or indirectly with phosphorylation of bcl-2 (2).Similar to the microtubule inhibitors Taxol and colchicine, CC-5079induces p53 and p21 expression in cancer cells. It has beenestablished that phosphorylation of bcl-2 and the elevations of p53and p21 may lead to apoptosis (30). Treatment of cancer cells withCC-5079 induces activation of caspase-3, caspase-8, and caspase-9and eventually results in cell undergoing apoptosis as shown bybivariate FITC-Annexin V/PI flow cytometric analysis (Fig. 2D).The fact that CC-5079 induced G2-M arrest suggests that the

observed antiproliferative and apoptosis-inducing effect may berelated to its effect on mitotic spindles in cells. Indeed, we foundthat CC-5079 disrupts microtubule assembly. The in vitro tubulinpolymerization study shows that CC-5079 completely blockedmicrotubule assembly. The microtubule network in cytoplasm wasdrastically disrupted, when tumor cells were treated with CC-5079.Results from competitive binding studies clearly show that CC-5079binds to tubulin at site distinct from vinblastine and Taxol andblocks colchicine binding to tubulin, indicating that CC-5079affects tubulin dynamics by binding the colchicine-binding site.The dramatic antimicrotubule effect of CC-5079 suggests that it

has potential for cancer chemotherapy as an antimitotic agent.This is further supported by the in vivo data showing that CC-5079has activity in the HCT-116 colorectal cancer xenograft model(P < 0.001). Although antimitotic agents have been widely usedin the clinic to treat patients with neoplastic disease, a major draw-back is the loss of efficacy over time because of the developmentof resistance. Drug resistance often develops through the expressionof efflux pumps, such as P-gp and other MDR proteins (31).However, unlike colchicine, vinblastine, and Taxol, CC-5079 wasequipotent in parental cancer cells versus MDR variants as weobserved in cytotoxicity tests, cell cycle analysis, phosphorylationof mitotic regulatory proteins, and induction of p53 and p21expression. Therefore, CC-5079 may circumvent P-gp-mediateddrug resistance seen with most chemotherapeutic agents. CC-5079may represent a new class of anti-tubulin agents with propertiesthat may provide advantages over the other tubulin inhibitors.CC-5079 also potently inhibits TNF-a production from mono-

cytes by inhibiting cAMP-specific phosphodiesterase PDE4 with

Figure 5. Effect of CC-5079 on human colorectal cancer HCT-116growth in mouse xenograft. HCT-116 cells (2 � 106) wereinjected s.c. into CB17 SCID mice. When the tumors reachedf100 mm3, the mice were given i.p. with vehicle control, CC-5079(5 mg/kg), CC-5079 (25 mg/kg), or Camptosar (10 mg/kg) asdescribed in Materials and Methods. Points, mean changes oftumor volumes plotted against time from 10 mice; bars, SE.*, P < 0.001.

Cancer Research




submicromolar activity. The second messenger cAMP is involved ina multitude of cellular processes, including growth and differen-tiation. PDE4s are critical components of cAMP signaling (32).Inhibition of PDE4 activity can induce growth suppression,apoptosis, and p53 and p21 proteins in human acute lymphoblasticleukemia cells (33, 34). After statistical analysis of results fromtesting of CC-5079 and 50 analogues, we can conclude that theTNF-a inhibitory effect of CC-5079 is due to its suppressive effecton PDE4. However, its antiproliferative activity was found notto correlate with PDE4 inhibitory activity. There is convincingevidence that an increase in the cellular levels of cAMP andinhibition of PDE4 lead to the induction of apoptosis in cancer cells(34, 35). TNF-a is a prominent member of the multifunctional TNFsuperfamily and has important roles in immunity as well asinfluencing apoptosis and cell survival. CC-5079 inhibits TNF-aproduction from monocytes but induces apoptosis of cancer cells,indicating that the apoptosis-inducing effect of the compound ismediated by pathways other than TNF/death receptor pathway.Accumulating evidence implicates TNF-a in inflammatory path-ways that increase tumorigenesis (36, 37). TNF-a under specificconditions is a tumor promoter and helps to produce the toxiceffects associated with conventional cancer therapy (5, 37). ThePDE4 and TNF-a inhibitory effects of CC-5079, along with its

antimitotic effect, may contribute to its anticancer effects and givethis compound potential for therapy of some immunoinflamma-tory disorders.In summary, our studies suggest that CC-5079 is a unique

antimitotic and anti-TNF-a agent because of its two distinctbiological functions. The first is the ability to bind directly totubulin and to thereby perturb microtubule polymerization and thefunction of the spindle apparatus, which causes cells to arrest inmitosis and undergo apoptosis in cancer cells, whether cellsoverexpress P-gp or not. The second is the ability to inhibit PDE4and thus TNF-a production from monocytes. In view of these dualeffects, CC-5079 is a novel drug lead that might be particularlyeffective for the treatment of neoplastic and inflammatory diseases.CC-5079 and its analogues are currently under development foroncology by EntreMed, Inc. (Rockville, MD).

Acknowledgments

Received 6/15/2005; revised 9/19/2005; accepted 11/9/2005.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. William E. Fogler (EntreMed) and Bernd Stein (Celgene) for criticalreview of the article.

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2006;66:951-959. Cancer Res Ling-Hua Zhang, Lei Wu, Heather K. Raymon, et al. Production with Antitumor Activity

αTubulin Polymerization and Tumor Necrosis Factor-The Synthetic Compound CC-5079 Is a Potent Inhibitor of

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