Molecular Medicine 4: 376-383, 1998

Molecular Medicine© 1998 The Picower Institute Press

Original Articles

Curcumin Is an In Vivo Inhibitor ofAngiogenesis

Jack L. Arbiser,"2 Nancy Klauber,7 Richard Rohan,/Robert van Leeuwen,1 Mou-Tuan Huang,' Carolyn Fisher,3Evelyn Flynn,2 and H. Randolph Byers4'Department of Dermatology, Harvard Medical School, Boston,Massachusetts, U.S.A.2Department of Surgical Research, Childrens Hospital, HarvardMedical School, Boston, Massachusetts, U.S.A.3Department of Chemical Biology, College of Pharmacy, Rutgers-TheState University of New Jersey, Piscataway, New Jersey, U.S.A.4Department of Dermatology, Boston University School of Medicine,Boston, Massachusetts, U.S.A.Communicated by P. Talalay. Accepted April 9, 1998.

Abstract

Background: Curcumin is a small-molecular-weightcompound that is isolated from the commonly used spiceturmeric. In animal models, curcumin and its derivativeshave been shown to inhibit the progression of chemi-cally induced colon and skin cancers. The geneticchanges in carcinogenesis in these organs involve differ-ent genes, but curcumin is effective in preventing carci-nogenesis in both organs. A possible explanation for thisfinding is that curcumin may inhibit angiogenesis.Materials and Methods: Curcumin was tested for itsability to inhibit the proliferation of primary endothelialcells in the presence and absence of basic fibroblastgrowth factor (bFGF), as well as its ability to inhibitproliferation of an immortalized endothelial cell line.Curcumin and its derivatives were subsequently testedfor their ability to inhibit bFGF-induced comeal neovas-

cularization in the mouse cornea. Finally, curcumin wastested for its ability to inhibit phorbol ester-stimulatedvascular endothelial growth factor (VEGF) mRNA pro-duction.Results: Curcumin effectively inhibited endothelial cellproliferation in a dose-dependent manner. Curcuminand its derivatives demonstrated significant inhibition ofbFGF-mediated comeal neovascularization in themouse. Curcumin had no effect on phorbol ester-stimu-lated VEGF production.Conclusions: These results indicate that curcumin hasdirect antiangiogenic activity in vitro and in vivo. Theactivity of curcumin in inhibiting carcinogenesis in di-verse organs such as the skin and colon may be mediatedin part through angiogenesis inhibition.

IntroductionCurcumin is a carotenoid pigment found in therhizome of Curcuma longa, source of turmeric

Address correspondence and reprint requests to: Dr. Jack L.Arbiser, Department of Dermatology, Emory UniversitySchool of Medicine, WMB 5001, Atlanta, GA 30022.Phone: (404) 727-5872; Fax: (404) 727-5878; E-mail:jlarbiser@bics.bwh.harvard.edu

(1,2). Turmeric is a major component of the dietof the Indian subcontinent. Administration ofcurcumin to mice treated with skin and coloncarcinogens has been shown to result in a de-creased incidence and size of tumors comparedwith control mice (3-7). The exact mechanism ofcurcumin's chemopreventive activities is notfully understood. Curcumin has been demon-

J. L. Arbiser et al.: Curcumin Inhibits Angiogenesis

strated to inhibit several signal transductionpathways, including those involving protein ki-nase C, the transcription factor NF-kB, phospho-lipase A2 bioactivity, arachidonic acid metabo-lism, antioxidant activity, and epidermal growthfactor (EGF) receptor autophosphorylation(8-12). Precisely how inhibition of these path-ways relates to curcumin's antitumor effect isuncertain.

Colon and skin cancers are major publichealth problems in Westernized nations. Hered-itary colon cancers account for up to 20% ofcolon carcinoma in the United States. Severalgenes have been shown to contribute to coloncarcinogenesis in human disease and transgenicmice (13-15). These syndromes are usually au-tosomal dominant, and they include familial ad-enomatous polyposis and nonpolyposis syn-dromes. In mice, phospholipase A2 expressionhas been demonstrated to be a co-factor in ge-netically susceptible mice (15). In addition, dele-tion of the cyclooxygenase 2 gene in colon car-cinoma-prone Apc mice results in suppression ofintestinal polyposis (16). Loss of p53 and activa-tion of ras oncogenes appear later in tumor pro-gression (17,18). Human skin cancers constitutethe most common form of human neoplasia andare increasing in incidence, primarily as a resultof sun exposure. In contrast to colon carcinoma,mutations of the p53 gene are felt to arise early inultraviolet-induced cutaneous carcinogenesis.Given that curcumin has protective effectsagainst both colon and skin cancer, but the genesinvolved in tumor progression of skin and coloncancer differ, we hypothesized that the antitu-mor effects of curcumin may be due in part toangiogenesis inhibition.

Here we demonstrate that curcumin inhibitsbasic fibroblast growth factor (bFGF)-inducedproliferation of endothelial cells in vitro and an-giogenesis in vivo. We also tested the effect ofcurcumin analogs with known differential che-mopreventive activities on in vivo angiogenesis.Our findings indicate that inhibition of angiogen-esis may underlie in part the antitumor activityof curcumin in vivo.

Materials and MethodsEndothelial Proliferation Assays

Bovine capillary endothelial cells were isolatedaccording to the method of Folkman et al. (19)and were plated at a concentration of 10,000cells/well in gelatinized 24-well dishes. The pri-

mary endothelial cells were cultured in Dulbec-co's modified Eagle's medium (DMEM) supple-mented with 10% bovine serum and grown at37°C in 10% CO2. Twenty-four hours after plat-ing, cells were treated with curcumin in the pres-ence or absence of bFGF. After 72 hr of treat-ment, cells were counted using a Coultercounter. Cell counts for each condition were re-peated in triplicate and in the presence or ab-sence on 1 ng/ml bFGF. Similarly, MS1 (ATCCCCRL 2279) endothelial cells, which are a SV40large T antigen immortalized murine endothelialcell line (20), were also plated at a concentrationof 10,000 cells/well in nongelatinized 24-welldishes. MS1 cells do not require endothelial mi-togens for growth and were cultured in DMEMsupplemented with 5% fetal calf serum (FCS).Cells were counted after a 72-hr exposure tocurcumin with the same method used for thebovine capillary endothelial cells.

Evaluation of bFGF-Induced CornealNeovascularization by Curcumin andCurcumin Analogs

Pellets were prepared according to a modificationof the method of Kenyon et al. (21). An aqueoussolution of 80 ng of basic fibroblast growth factor(Scios Nova, Mountain View, CA) was evapo-rated to dryness under reduced pressure in thepresence of 10 mg of sucralfate (Bukh Meditec,Vaerlose, Denmark). Ten microliters of 12% hy-dron and 10 mg of curcumin or curcumin ana-logs were then added, and the homogenous mix-ture was deposited onto a sterile 15 x 15 mm3-300/50 nylon mesh (Tetko, Lancaster, NY) andair dried. Once the mixture was dry, the meshwas manually dissociated to yield 225 pellets.Each pellet contained 80 ng of bFGF and 44 jig ofcurcumin or curcumin analog. In our prior ex-perience (21), pellets containing Hydron in theabsence of bFGF have not caused neovascular-ization, so pellets prepared in the absence ofbFGF were not used in this study. The approxi-mate pore size was 0.4 x 0.4 mm. Both sides ofthe mesh were covered with a thin layer ofHydron.

C57BL6 male mice (5-6 weeks old) wereanesthetized with methoxyflurane prior to im-plantation of pellets and with 0.5% propara-caine. A central, intrastromal linear keratotomywas performed with a surgical blade, and a la-mellar micropocket was prepared according tothe method of Kenyon et al. (21). The pellet wasadvanced to the end of the pocket. Erythromycin

377

378 Molecular Medicine, Volume 4, Number 6, June 1998

ointment was placed on the operated eye to pre-vent infection. Eyes were examined by slit lampon days 3-6 after implantation under generalanesthesia. Corneal angiogenesis is assayedthrough two measurements. The first measure-ment, vessel length, is the length of the vesselfrom the corneal limbus as it grows toward thebFGF pellet. Clock hours is a measurement ofneovascularized area of the cornea. The cornea isviewed as a circle that can be divided as a clock,with a maximum of 12 hr. Thus, a measurementof 3 clock hours implies that one-quarter of thecornea is vascularized. This system of measure-ment was established by Kenyon et al. (21).

RNAse Protection for VEGFHaCaT keratinocytes (22) were grown in(DMEM) (JRH) supplemented with 5% FCS(Hyclone, Logan, UT) in 25 cm2 flasks. One hourprior to stimulation with 12-0-tetradecanoyl-phorbol- 1 3-acetate (TPA), cells were switched toserumless media supplemented with 10 ,uM cur-cumin or an equal quantity of ethanol (finalconcentration 0.1 %). TPA was added to a finalconcentration of 5 ng/ml and incubated for 3 hrat 370C. Cells were harvested and RNA extractedwith guanidinium thiocyanate/phenol.

A plasmid containing the coding region ofhuman vascular endothelial growth factor(VEGF) 121 was obtained from H. Weich (Uni-versity of Freiburg, Germany), and used to gen-erate P32-labeled antisense riboprobe as per man-ufacturers protocols (Ambion, Austin, TX).RNAse protection assays were performed accord-ing to the method of Hod (23). Protected frag-ments were separated on gels of 5 % acrylamide,8 M urea, 1 X Tris-borate buffer, and quantifiedwith a phosphorimager (Molecular Dynamics,Sunnyvale, CA). An 18 S riboprobe was includedin each sample to normalize for variations inloading and recovery of RNA.

Phase II Enzyme Induction

The ability of curcumin derivatives to inducephase II activities was measured by assaying qui-none reductase [NAD (P)H: (quinone-acceptor)oxidoreductase, EC 1.6.99.2] in murine Hepacl c7cells. Serial dilutions of curcumin, curcumin de-rivatives, and tetraphenylcyclopentadienone(TPCPD) were added, and the concentration ofcompound required to double the specific activ-ity (CD) was calculated according to the methodof Prochaska et al. (24).

Statistics

Significant differences between two groups weredetermined using an unpaired, two-tailed Stu-dent's t test. Results are expressed as the mean +standard error of the mean.

ResultsInhibitory Effect of Curcumin on EndothelialProliferation in the Presence or Absence of bFGF

Endothelial cells are stimulated to proliferate inthe presence of 1 ng/ml bFGF. Curcumin wasadded in concentrations ranging from 0.5 to 10AM to primary endothelial cells. A steep decreasein cell number was seen at 10 ,M. No evidenceof cytotoxicity was observed, and the number ofcells at the end of treatment was not significantlyless than the number of cells originally plated.This decrease was observed in both the presenceor absence of bFGF (Fig. 1). In addition, cur-cumin was able to inhibit the growth of endo-thelial cells immortalized by SV40 large T anti-gen, with a similar dose response as seen withprimary endothelial cells.

Curcumin Inhibits bFGF-Induced Neovascularizationin the Mouse Cornea

The ability of curcumin to inhibit bFGF-inducedcorneal neovascularization in vivo was studied.Pellets were prepared containing 80 ng of bFGFand curcumin, or a control aromatic ketone, tet-raphenylcyclopentadienone (TPCPD). TPCPDwas added to rule out the possibility that theinhibition of neovascularization due to curcuminwas not secondary to dilution. There was nodifference in neovascularization in mice contain-ing bFGF pellets in the presence or absence ofTPCPD (data not shown). Neovascularizationwas assessed by slit lamp at 5 days after implan-tation, and the corneas were photographed. Boththe vessel length and clock hours were signifi-cantly reduced in the presence of curcumin(Fig. 2).

Curcumin Analogs Inhibit bFGF-InducedNeovascularization in the Mouse Cornea

Curcumin analogs were assayed for their abilityto inhibit bFGF-induced corneal neovasculariza-tion as described above. All analogs showed in-hibitory activity, with demethoxycurcuminshowing the greatest activity on both clock hoursand vessel length, tetrahydrocurcumin having

J. L. Arbiser et al.: Curcumin Inhibits Angiogenesis 379

Effect of curcumin on endothelbalproliferation In the presence of bFGF

I.a

.0

ia

aU

0 0.5 1 2.5 5 10 uM curcumin

Effect of curcumin on endothellalprolIferatIon In the absence of bFGF

0 0.5 1 2.5 5 10 uN curcumin

Effect of curcumin on the proliferation ofMS1 endothellal cells

0 1 2.5 5 10 20 uM curcumin

Fig. 1. Effect of curcumin on the proliferationof capillary endothelial cells. Cells were grownin the presence or absence of bFGF and doses of cur-

cumin ranging from 0 to 20 ,uM curcumin. The y-

axis denotes the cell number after 72 hr. (A) Effectof curcumin on primary capillary endothelial cellsstimulated with 1 ng/ml of bFGF. (B) Effect of cur-

cumin on the proliferation of unstimulated primarycapillary endothelial cells. (C) Effect of curcumin on

immortalized endothelial cells. The error bars repre-

sent standard error of the mean.

the least effect on clock hours, and bisdeme-thoxycurcumin having the least effect on vessellength (Fig. 3). All of the curcumin derivatesshowed significant inhibition of bFGF-mediatedneovascularization compared with control pel-lets.

Effect of Curcumin on VEGF Production byTransformed Keratinocytes

HaCaT cells are derived from spontaneouslytransformed human keratinocytes (22). In orderto determine whether curcumin could inhibitproduction of angiogenesis factors by relevanttumor cells as well as directly inhibit endothelialfunction; we treated HaCaT cells with tetradeca-noylphorbol ester (TPA) in the presence or ab-sence of curcumin and assayed expression ofVEGF mRNA. TPA caused a 2.5-fold increase inVEGF mRNA, which was not inhibited by cur-cumin (Fig. 4). Thus the primary antiangiogeniceffect of curcumin is directly on endothelium,rather than inhibition of production of VEGF, animportant angiogenic factor.

Effect of Curcumin Derivatives on Induction of PhaseII Enzyme InductionSeveral plant-derived compounds with antican-cer and chemopreventative activities also showthe ability to induce phase II detoxifying en-zymes, including quinone reductase. We wantedto determine whether the antiangiogenic activi-ties of curcumin derivatives correlated with theability to induce quinone reductase activity. TheCD (concentration to double the specific activity)value for curcumin was 7.3 ,uM, 9.0 ,uM fordemethoxycurcumin, and 11.0 ,tM for bisde-methoxycurcumin. These CD values are approx-imately equal, but they differ significantly fromthat of tetrahydrocurcumin. Tetrahydrocur-cumin, the curcumin derivative with the leastantitumor activity, caused a 1.6-fold induction ofquinone reductase activity at 25 ,uM. However,TPCPD, which is an unsaturated aromatic ketonewith no anti-angiogenic activity, had a CD valueof 4.8 AM. Thus, antiangiogenic activity does notcorrelate with phase II activity.

DiscussionCurcumin is a nontoxic, small-molecular-weightcompound with established chemopreventiveactivity in areas of direct contact, such as the skin

380 Molecular Medicine, Volume 4, Number 6, June 1998

C D

ss

x

uU)

0.

0.

0.

0.

0.

Effect ot curcumin on vessel length

*-0

0 CurcuminW0Q

Fig. 2. Effect of curcumin on corneal neovas-

cularization. (A, B) The photograpih oI the leftshows a cornea containiing bFGF and curcumill,while the corInea on the right contains TPCPD, a

control substance, and an equal quantity (80 ng) ofl)FGF. The arrows point to a corneal feeder vessel,which is enlarged in the abseince of curcumin and is

and gastroiintestinial tract (3-7). The reasons forits efficacy as a chemopreventive agent are nlotfully understood. Curcumin has been shown to

possess several activities that may affect tuniiori-genesis in vivo. First, curcumin has been showinto inhibit tumor initiation by the carcinogeinsbenzo(a)pyrene and 7,12,-dimethylbenzantlhra-cene (DMBA), as well as ttimor promotion in-dtuced by phlurbol esters (6,7,12). Curcumin has

also been shown to inhibit phorbol ester-in-dtuced ornithinie decarboxylase, a marker of cel-lular proliferation and ttumorigeniesis, ilt IOitiSeskiin. In addition, curcumini has an inhibitoryeffect on expression of c-fos and c-juu1, oncogeInesthat form the transcriptioin factor AP-1 (8,10).These pathways also influence tumor cell prolif-eration in vivo, and curcuminin Imay have a dualeffect on inhibiting both tumor growth in vivo

* lPCPD0 Curcumin

Effect of curcumin on clock hours

atteniuated in the presenice of curcuLinii. (C) The bargrapIh shows the eflect of cLIrcLlflhiII and TPCPD onvessel leIngth. P < 0.05. (D) The bar graph slhows thieeffect of ctircuminii and TPCPD on clock hours, ameasuLre of area. The error bars represenit standarderror of the mean.

throtugh inhibiting ttunmor promotioni, as well asilihibitinig angiogeInesis in tumors that have al-ready undergonie the angiogenic switclh.

Basic fibroblast growth factor (bFGF) is apotent angiogenic factor. This factor has beenshown to be a potent stimulus for both endothe-lial proliferation and migration. The activity ofbFGF on endothelial cells may be due in partthough stimilatioin of protein kinase C (25). Thecorneal neovascularization assay, which mea-stures vessel length and density in response to abFGF pellet placed in the normally avascularcornea, has proven tiseful in the characteriza-tioIn of multiple angiogenesis inhibitors (21).Admiinistration of cturcumin or its analogswithini the pellet restulted in potent inhibitionof bFGF-indtuced corneal neovascullarization.This iinhibitioni was not due to dilution of bFGF,

J. L. Arbiser et al.: Curcumin Inhibits Angiogenesis

Effect of curcumin analogs on vessel length

ic.s

0

0

0

* CondO Cucumin* TerahyrocumurinO ocrcumin* Demehoxycurcumln

Curcumin analogs

Effect of curcumin analogs on clock hours

* Contoo c* CucuniinToftahydrocurcurnunO Bldem.toxycurcumin* Dwmfoxycurcumin

Curcumin analogs

Fig. 3. Effect of curcumin analogs on cornealneovascularization. (A) Effect of curcumin ana-logs on vessel length. All curcumin derivatives showa significant difference in vessel length from controlsat p < 0.05. (B) Effect of curcumin analogs on clockhours. All curcumin derivatives show a significantdifference in area from controls at p < 0.05.

as administration of a structurally related inac-tive compound, tetraphenylcyclopentadienone(TPCPD), has no effect on bFGF-induced cornealneovascularization. Intraperitoneal administra-tion of curcumin at doses up to 300 mg/kg didnot inhibit corneal neovascularization (data notshown); this may be due to the well-knownrapid metabolism of curcumin (26,27). Higherdoses of curcumin caused weight loss in mice.

Curcumin had a strong antiproliferative ef-fect on endothelial cells, with a steep curve, oc-

curring between 5 and 10 ,uM. This was trueboth in the presence or absence of bFGF, and thisinhibition could not be overcome by the immor-talizing ability of SV40 large T antigen.

Food-grade curcumin consists of 77% cur-

cumin, 17% demethoxycurcumin, and 3% bis-demethoxycurcumin. In a previous study, Huanget al. found that these three compounds were

able to inhibit phorbol ester-stimulated induc-

0CO1a

Curcumin

TPA+ +_ + +

Fig. 4. Effect of curcumin on VEGF mRNAproduction in HaCat cells. The intensity of bandsfrom VEGF RNAse protection were quantified bydensitometry and normalized for loading by quanti-fication of 18S RNA. The error bars represent stan-dard error of the mean.

tion of ornithine decarboxylase and promotionof mouse skin initiated with 7,12-dimethylbenz-anthracene (DMBA) (6). These derivatives alsoinhibited phorbol ester-mediated transformationof JB6 cells (8). The saturated derivative tetrahy-drocurcumin was less active than the unsatur-ated derivatives in these assays. These curcuminanalogs were placed into corneal pellets and as-sayed for angiogenesis inhibition. Pure cur-cumin, demethoxycurcumin, tetrahydrocur-cumin, and bisdemethoxycurcumin were allactive in inhibiting bFGF-induced corneal neo-vascularization, but to varying degrees. Cur-cumin, demethoxycurcumin, and bisde-methoxycurcumin are much more effectivechemopreventive agents than tetrahydrocur-cumin in vivo (6), although all of the derivativeshave antiangiogenic activity. This may be due toother activities of curcumin, such as the ability toinduce phase II detoxifying enzymes, which mayinhibit further tumor promotion. All of the un-saturated curcumin derivatives have approxi-mately equal potencies in induction of phase IIenzymes, whereas the fully saturated tetrahy-drocurcumin has little ability to induce phase IIenzymes.

Angiogenesis inhibitors may be divided intotwo classes. The first class, or direct angiogenesisinhibitors, refer to those agents which are rela-tively specific for endothelial cells and have littleeffect on tumor cells (28). Examples of theseinclude soluble VEGF receptor antagonists andangiostatin (29,30). Indirect inhibitors may nothave direct effects on endothelial cells but may

381

382 Molecular Medicine, Volume 4, Number 6, June 1998

down-regulate the production of an angiogenesisstimulator, such as VEGF (20,31). VEGF hasbeen shown to be up-regulated during chemi-cally induced skin carcinogenesis; this is likelydue to activation of oncogenes, such as H-ras (20,32,33). Examples of indirect inhibitors of angio-genesis include inhibitors of ras-mediated signaltransduction, such as farnesyltransferase inhibi-tors (33). The antagonism of bFGF-mediated cor-neal neovascularization by curcumin and its de-rivatives suggests that curcumin is a directangiogenesis inhibitor. The lack of inhibition ofTPA-mediated VEGF production further supportsthe role of curcumin as a direct angiogenesisinhibitor.

Chemopreventive agents are a diverse groupof compounds with multiple activities. These in-clude inhibition of proliferation, enhancement ofDNA repair, and induction of detoxifying en-zymes, such as quinone reductase, for the re-moval of mutagenic xenobiotics (34). Fotsis et al.have found that the chemopreventive agentgenistein can inhibit the proliferation of endo-thelial cells in vitro (35). We show that cur-cumin, another chemopreventive agent, is capa-ble of inhibiting bFGF-mediated angiogenesis invivo. The precise mechanism of how chemopre-ventive agents actually prevent neoplasia is notfully understood. Inhibition of angiogenesis mayunderlie in part the beneficial activity of chemo-preventive agents.

AcknowledgmentsJ. L. A. was supported by NIH grantR03AR44947 and grants from the Society forPediatric Dermatology, the Dermatology Foun-dation, and the Thomas B. Fitzpatrick ResearchAward from the KAO Corporation. We acknowl-edge Albena Dinkova-Kostova for her assistancewith the quinone reductase assays.

References1. Ammon HP, Wahl MA. (1991) Pharmacology of

curcuma longa. Planta Med. 57: 1-7.2. Stoner GD, Mukhtar H. Polyphenols as cancer

chemopreventive agents. (1995) J. Cell. Biochem.Suppl. 22: 169-180.

3. Huang MT, Lou YR, Ma W, Newmark HL, ReuhlKR, Conney AR. (1994) Inhibitory effects of di-etary curcumin on forestomach, duodenal, andcolon carcinogenesis in mice. Cancer Res. 54: 584 1-5847.

4. Rao CV, Rivenson A, Simi B, Reddy BS. (1995)Chemprovention of colon carcinogenesis by di-etary curcumin, a naturally occurring plant phe-nolic compound. Cancer Res. 55: 259-266.

5. Conney AH, Lysz T, Ferraro T, Abidi TF, Man-chand PS, Laskin JD, Huang MT. (1991) Inhibitoryeffect of curcumin and some related dietary com-ponents on tumor promotion and arachidonic acidmetabolism in mouse skin. Adv. Enzyme Regul. 31:385-396.

6. Huang MT, Ma W, Lu YP, Chang RL, Fisher C,Manchand PS, Newmark HL, Conney AH. (1995)Effects of curcumin, demethoxycurcumin, bisde-methoxycurcumin, and tetrahydrocurcumin on12-O-tetradecanoylphorbol- 13-acetate-inducedtumor promotion. Carcinogenesis 16: 2493-2497.

7. Huang MT, Deschner EE, Newmark HL, Wang ZY,Ferraro TA, Conney AH. (1992) Effect of dietarycurcumin and ascorbyl palmitate on azoxymetha-nol-induced colonic epithelial cell proliferationand focal areas of dysplasia. Cancer Lett. 64: 117-121.

8. Lu YP, Chang RL, Lou YR, Huang MT, NewmarkHL, Reuhl K, Conney AH. (1994) Effect of cur-cumin on 12-O-tetradecanoylphorbol- 13-acetate-and ultraviolet B light-induced expression of c-junand c-fos in JB6 cells and in mouse epidermis.Carcinogenesis 15: 2363-2370.

9. Singh S, Aggarwal BB. (1995) Activation of tran-scription factor NF-kappa B is suppressed by cur-cumin. J. Biol. Chem. 270: 24995-25000.

10. Huang TS, Lee SC, Lin JK. (1991) Suppression ofc-junIAP- 1 activation by an inhibitor of tumorpromotion in mouse fibroblast cells. Proc. Natl.Acad. Sci. U.S.A. 88: 5292-5296.

11. Korutla L, Cheung JY, Mendelsohn J, Kumar R.(1995) Inhibition of ligand-induced activation ofepidermal growth factor receptor tyrosine phos-phorylation by curcumin. Carcinogenesis 16: 1741-1745.

12. Rao CV, Simi B, Reddy BS. (1993) Inhibition bydietary curcumin of azoxymethanol-induced or-nithine decarboxylase, tyrosine protein kinase, ar-achidonic acid metabolism and aberrant crypt fociformation in the rat colon. Carcinogenesis 14:2219-2225.

13. Dietrich WF, Lander ES, Smith JS, Moser AR,Gould KA, Luongo C, Borenstein M, Dove W.(1993) Genetic identification of Mom-i, a majormodifier locus affecting Min-induced intestinalneoplasia in the mouse. Cell 75: 631-639.

14. Su LK, Kinzler KW, Vogelstein B, Preisinger AC,Moser AR, Luongo C, Gould KA, Dove WF.(1992) Multiple intestinal neoplasia caused by amutation in the murine homolog of the APC gene.Science 256: 668-670.

15. MacPhee M, Chepenik KP, Liddell RA, Nelson KK,Siracusa LD, Buchberg AM. (1995) The secretoryphospholipase A2 gene is a candidate for the

J. L. Arbiser et al.: Curcumin Inhibits Angiogenesis 383

MomI locus, a major modifier of ApcMin inducedintestinal neoplasia. Cell 81: 957-966.

16. Oshima M, Dinchuk JE, Kargman SL, Oshima H,Hancock B, Kwong E, Trzaskos JM, Evans JF,Taketo MM. (1996) Suppression of intestinal pol-yposis in Apc delta716 knockout mice by inhibi-tion of cyclooxygenase 2. Cell 87: 803-809.

17. Hinds PW, Finlay CA, Quartin RS, Baker SJ, Fe-aron ER, Vogelstein B, Levine AJ. (1990) Mutantp53 clones from human colon carcinomas coop-erate with ras in transforming primary rat cells: acomparison of the "hot spot" mutant phenotypes.Cell Growth Differ. 1: 571-580.

18. Redston MS, Papadopoulos N, Caldas C, KinzlerKW, Kern SE. (1995) Common occurrence of APCand K-ras gene mutations in the spectrum of co-litis-induced neoplasias. Gastroenterology 108: 383-392.

19. Folkman J, Haudenschild C, Zetter BR. (1979)Long-term culture of capillary endothelial cells.Proc. Natl. Acad. Sci. U.S.A. 76: 5217-5221.

20. Arbiser JL, Moses MA, Fernandez CA, Ghiso N,Cao Y, Klauber N, Frank D, Brownlee M, Flynn E,Parangi S, Byers HR, Folkman J. (1997) Onco-genic H-ras stimulates tumor angiogenesis by twodistinct pathways. Proc. Natl. Acad. Sci. U.S.A. 94:861-866.

21. Kenyon BM, Voest EE, Flynn E, Folkman J,D'Amato RJ. (1996) A model of angiogenesis inthe mouse cornea. Invest. Opthalmol. Vis. Sci. 37:1625-1632.

22. Boukamp P, Petrussevska RT, Breitkreutz D, Hor-nung J, Markham A, Fusenig NE. (1988) Normalkeratinization in a spontaneously immortalizedaneuploid human keratinocyte cell line. J. CellBiol. 106: 761-767.

23. Hod Y. (1992) A simplified ribonuclease protec-tion assay. Biotechniques 13: 852-853.

24. Prochaska HJ, Santamaria AB, Talalay P. (1992)Rapid detection of inducers of enzymes that pro-tect against carcinogens. Proc. Natl. Acad. Sci. U.S.A.89: 2394-2398.

25. Kent KC, Mii S, Harrington EO, Chang JD, Mal-lette S, Ware JA. (1995) Requirement for proteinkinase C activation in basic fibroblast growth fac-

tor-induced human endothelial cell proliferation.Circ. Res. 77: 231-238.

26. Ravindranath V, Chandrasekhara N. (1981) Invitro studies on the intestinal absorption of cur-cumin in rats. Toxicology 20: 251-257.

27. Wahlstrom B, Blennow G. (1978) A study on thefate of curcumin in the rat. Acta Pharmacol. Toxicol.43: 86-92.

28. Arbiser JL. (1997) Antiangiogenic therapy anddermatology: a review. Drugs Today 33: 687-696.

29. Kim KJ, Li B, Winer J, Armanini M, Gillett N,Phillips HS, Ferrara N. (1993) Inhibition of vascu-lar endothelial growth factor-induced angiogene-sis suppresses tumour growth in vivo. Nature 362:841-844.

30. O'Reilly MS, Holmgren L, Shing Y, Rosenthal RA,Moses M, Cao Y, Sage EH, Folkman J. (1994)Angiostatin: a novel angiogenesis inhibitor thatmediates the suppression of metastases by a Lewislung carcinoma. Cell 79: 315-328.

31. Gess B, Sandner P, Kurtz A. (1996) Differentialeffects of kinase inhibitors on erythropoietin andvascular endothelial growth factor gene expres-sion in rat hepatocytes. Pflugers Arch. 432: 426-432.

32. Larcher F, Robles AI, Duran H, Murillas R, Quin-tanilla M, Cano A, Conti CJ, Jorcano JL. (1996)Upregulation of vascular endothelial growth fac-tor/vascular permeability factor in mouse skincarcinogenesis correlates with malignant progres-sion state and activated H-ras expression levels.Cancer Res. 56: 5391-5396.

33. Kohl NE, Omer CA, Conner MW, et al. (1995)Inhibition of farnesyltransferase induces regres-sion of mammary and salivary carcinomas in rastransgenic mice. Nature Med. 1: 792-797.

34. Fahey JW, Zhang Y, Talalay P. (1997) Broccolisprouts: an exceptionally rich source of inducersof enzymes that protect against chemical carcino-gens. Proc. Natl. Acad. Sci. U.S.A. 94: 10367-10372.

35. Fotsis T, Pepper M, Adlercreutz H, Fleischmann G,Hase T, Montesano R, Schweigerer L. (1993)Genistein, a dietary-derived inhibitor of in vitroangiogenesis. Proc. Natl. Acad. Sci. U.S.A. 90: 2690-2694.