[CANCER RESEARCH 48, 5941-5946, November 1, 1988] Inhibitory Effect of Curcumin, Chlorogenic Acid, Caffeic Acid, and Ferulic Acid on Tumor Promotion in Mouse Skin by 12-O-Tetradecanoylphorbol-13-acetate Mou-Tuan Huang, Robert C. Smart, Ching-Quo Wong, and Allan H. Conney Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08855-0789 [M-T. H., A. H. C.]; Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 [M-T. H., R. C. S., C-Q. W., A. H. C.]; and Toxicology Program, North Carolina State University, Raleigh, North Carolina 27695-7633 [R. C. SJ ABSTRACT also evaluated the effects of the related compounds chlorogenic acid, caffeic acid, and ferulic acid as potential inhibitors of The effects of topically applied curcumin, chlorogenic acid, caffeic acid, and ferulic acid on 12-O-tetradecanoylphorbol-13-acetate (TPA)- tumor promotion. induced epidermal ornithine decarboxylase activity, epidermal DNA syn thesis, and the promotion of skin tumors were evaluated in female CD-I MATERIALS AND METHODS mice. Topical application of 0.5, 1, 3, or 10 iano\ of curcumin inhibited by 31, 46, 84, or 98%, respectively, the induction of epidermal ornithine Materials. TPA was purchased from CRC Inc., Chanhassen, MN. decarboxylase activity by 5 nmol of TPA. In an additional study, the DL-['"C]Ornithine (58 Ci/mmol) and [3H]thymidine (5 Ci/mmol) were topical application of 10 ¿tinolofcurcumin, chlorogenic acid, caffeic acid, purchased from Amersham Corp., Arlington Heights, IL. fra/w-Reti- or ferulic acid inhibited by 91, 25,42, or 46%, respectively, the induction noic acid was obtained from Hoffmann-La Roche Inc., Basle, Switzer of ornithine decarboxylase activity by 5 nmol of TPA. The topical land. Fluocinolone acetonide, nonradioactive DL-ornithine, curcumin, application of 10 iimol of curcumin together with 2 or 5 nmol of TPA inhibited the TPA-dependent stimulation of the incorporation of |'I l|- chlorogenic acid, ferulic acid, and caffeic acid were purchased from the Sigma Chemical Co., St. Louis, MO, and DMBA was purchased from thymidine into epidermal DNA by 49 or 29%, respectively, whereas lower Calbiochem-Behringer, San Diego, CA. Aquasol was purchased from doses of curcumin had little or no effect. Chlorogenic acid, caffeic acid, New England Nuclear, Boston, MA. Acetone and dimethyl sulfoxide and ferulic acid were less effective than curcumin as inhibitors of the TPA-dependent stimulation of DNA synthesis. Topical application of 1, were purchased from Burdick & Jackson Laboratories, Muskegon, MI. Arachidonic acid was purchased from Nu-Chek Prep Inc., Elysian, MN. 3, or 10 fimol of curcumin together with 5 nmol of TPA twice weekly for Animals. Seven-week-old female CD-I mice were purchased from 20 weeks to mice previously initiated with 7,12-dimethylbenz[a]anthra- cene inhibited the number of TPA-induced tumors per mouse by 39, 77, Charles River Laboratories, Kingston, NY, and kept in our animal facility at least 1 week before use. Mice were fed a Purina Laboratory or 98%, respectively. Similar treatment of mice with 10 nmol of chloro Chow 5001 diet ad libitum (Ralston-Purina Co., St. Louis, MO) and genic acid, caffeic acid, or ferulic acid together with 5 nmol of TPA kept on a 12-h light, 12-h dark cycle. Mice were provided drinking inhibited the number of TPA-induced tumors per mouse by 60, 28, or water ad libitum. The dorsal region of each mouse was shaved with 35%, respectively, and higher doses of the phenolic acids caused a more electric clippers at least 2 days before treatment with TPA or DMBA. pronounced inhibition of tumor promotion. The possibility that curcumin Only mice that did not show signs of hair regrowth were used. All could inhibit the action of arachidonic acid was evaluated by studying the compounds were applied to the dorsal shaved area of 8- to 9-week-old effect of curcumin on arachidonic acid-induced edema of mouse ears. The mice in 200 ß\acetoneor 200 ^1 of acetone:DMSO (90:10). topical application of 3 or 10 ¿tmolof curcumin 30 min before the Ornithine Decarboxylase Assay and Preparation of Epidermal I lo- application of 1 /imol of arachidonic acid inhibited arachidonic acid- mogenates. The preparation of epidermal homogenate and the deter induced edema by 33 or 80%, respectively. mination of ornithine decarboxylase activity were done as described previously (18). Mice were treated topically with 200 /il of acetone, INTRODUCTION with TPA in acetone, or with curcumin and TPA together in acetone. In experiments with chlorogenic acid, caffeic acid, and ferulic acid, The ground dried rhizome of the plant Curcuma longa Linn mice were treated with 200 n\ of acetone:DMSO (90:10), with TPA in has been used for centuries as a naturally occurring medicine acetone:DMSO, or with the test compound and TPA together in for the treatment of inflammatory and other diseases (1,2), and acetone:DMSO. Five h after treatment, the mice were sacrificed by it has also been used as a coloring agent and spice in many cervical dislocation, and the dorsal area of the skin was removed. In order to remove the epidermis from the dermis, the skins were plunged foods. The powdered rhizome is commonly called turmeric. into a 58°Cwater bath for 30 s, and then the skins were immediately Curcumin (diferuloylmethane; Fig. 1) has been identified as the major pigment in turmeric, and this substance has also been submerged in an ice water bath as described by Slaga et al. (19). The epidermis was removed from the dermis by gentle scraping and placed used as a spice, food preservative, and yellow coloring agent in 1 ml of 50 mM potassium phosphate, pH 7.7, buffer containing 2 (3-5). Curcumin, which is widely used in curry, is responsible HIMdithiothreitol and 0.1 mM EDTA. The epidermis was homogenized for the yellow color of curry. Turmeric contains 1-5% curcumin on ice for 30 s with a Tekmar polytron tissumizer at full setting. The while the curcumin content of turmeric oleoresin is approxi epidermal homogenate was centrifuged at 11,000 x g for 30 min at 4°C mately 40% (6). and the supernatant fraction was removed and stored overnight at Recent studies have indicated that several compounds that —20°Cpriorto the determination of ornithine decarboxylase activity possess antioxidant or antiinflammatory activity inhibit TPA1- as described earlier (18). Protein was determined by the method of induced tumors on mouse skin (7-12). Since curcumin has been Lowry et a/., using bovine serum albumin as the standard (20). reported to possess both antioxidant and antiinflammatory Quantitation of Epidermal DNA Synthesis. Mice were treated topi activity (13-17), we have evaluated the effect of curcumin on cally with 200 ii\ acetone, TPA in acetone, or curcumin and TPA TPA-induced tumor promotion on mouse skin, and we have together in acetone. Acetone:DMSO (90:10) was the solvent for exper iments with chlorogenic acid, caffeic acid, and ferulic acid. At 18 h after treatment, 10 ^1 of [3H]thymidine solution (5 Ci/mmol, 1 mCi/ Received 5/6/88; revised 7/22/88; accepted 7/29/88. The costs of publication of this article were defrayed in part by the payment ml) were injected i.p. into each mouse, and 40 min later the mice were of page charges. This article must therefore be hereby marked advertisement in sacrificed by cervical dislocation. The isolation of epidermal DNA and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. determination of the incorporation of [3H]thymidine into DNA was 'The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; DMBA, 7,12-dimethylbenz[a]anthracene; DMSO, dimethyl sulfoxide; HETE, done as previously described (18). hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid. Tumor Studies on Mouse Skin. The dorsal region of 7-week-old 5941 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. INHIBITION OF TPA-INDUCED TUMOR PROMOTION CH cate that curcumin is about 10-fold more active than the phe OO II II HO CH= CH-C-CH2-C-CH = CH nolic acids. The results of an additional study indicated that an i.p. injection of curcumin could inhibit the induction of orni CURCUMIN thine decarboxylase activity in mouse epidermis that occurred after the topical application of TPA. The i.p. injection of 40 or 120 /¿molof curcumin (dissolved in dimethyl sulfoxide) into HOOC-CH= CH mice l h before the topical application of 5 nmol TPA inhibited the induction of epidermal ornithine decarboxylase activity by FERULIC ACID 46 or 86%, respectively (data not shown). During the course of this study, it was found that curcumin in the vehicle precipitated out of solution in the abdominal cavity and thus may not have HOOC-CH=CH been completely bioavailable. Curcumin could be seen as yellow flecks in the abdominal cavity throughout the experimental CAFFEIC ACID period of 6 h. Effect of Curcumin, Chlorogenic Acid, Caffeic Acid, and Fer ulic Acid on TPA-induced DNA Synthesis in Mouse Epidermis. H OOC-CH = C Topical application of 1, 3, or 10 nmo\ of curcumin together HOOCJ 1 H with 2 nmol of TPA inhibited the TPA-induced increase in DNA synthesis by 4, 14, or 49%, respectively (Table 2, experi ment 1). The topical application of fluocinolone acetonide (2 nmol), a known inhibitor of TPA-induced DNA synthesis, CHLOROGENICACID inhibited the TPA-induced increase by 86%. Topical applica Fig. 1. Structures of curcumin, ferulic acid, cafTeicacid, and chlorogenic acid. tion of 1, 3, or 10 ¿¿molofcurcumin simultaneously with 5 female CD-I mice was shaved with an electric clipper.