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ANTICANCER RESEARCH 27: 363-372 (2007)

Comparative Cytotoxicity and ROS Generation by Curcumin and Tetrahydrocurcumin Following Visible-light Irradiation or Treatment with Horseradish Peroxidase TOSHIKO ATSUMI1, KEIICHI TONOSAKI1 and SEIICHIRO FUJISAWA2

Departments of 1Human Development and Fostering and 2Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan

Abstract. In order to clarify the cytotoxic mechanism of curcumin, a well-known chemopreventive agent, the cytotoxicity (by MTT method), intracellular glutathione (using GSH detection kit) and intracellular reactive oxygen species (ROS) levels (with a flow cytometer), were measured in curcumin- and tetrahydrocurcumin (TH-curcumin)-treated cancer (HSG) and normal (HGF) cells under two different oxidation conditions: irradiation with visible light (VL) and enzymatic oxidation with horseradish peroxidase (HRP)/H2O2. The cytotoxicity of curcumin was highly enhanced by VLirradiation, whereas that of TH-curcumin was enhanced by HRP/H2O2 treatment. The cytotoxicity of curcumin against HGF cells was greater than that against HSG cells. Curcumin significantly reduced the intracellular GSH level significantly under VL-irradiation, and increased it under HRP/H2O2, whereas TH-curcumin had no effect with either oxidation treatment. HRP/H2O2 treatment of TH-curcumin enhanced generation of ROS; in contrast, VL-irradiation of curcumin was considered to produce ROS preferably. In conclusion, curcumin was highly photo-toxic, caused a decrease in GSH and mediated ROS generation. In contrast, the cytotoxicity of TH-curcumin was enhanced by enzymatic oxidation. A lowlevel pro-oxidant intracellular milieu induced by TH-curcumin could be effectively useful for cancer prevention. Curcumin-1, 7-bis (4-hydroxy-3-methoxyphenol)-1,6heptadiene-3,5-dione (Figure 1), obtained from the rhizome of the plant Curcuma longa, is a naturally occurring pigmented component of the spice turmeric and

Correspondence to: Dr. Toshiko Atsumi, Division of Physiology, Department of Human Development and Fostering, Meikai University School of Dentistry, 1-1, Keyakidai, Sakado-shi, Saitama 350-0283, Japan. Tel: +81 49 279 2771, Fax: +81 49 287 4712, email: [email protected] Key Words: Curcumin, GSH, HRP, ROS, tetrahydrocurcumin, VL-irradiation.

0250-7005/2007 $2.00+.40

is responsible for the yellow color of curried foods. Curcumin possesses well-known anti-inflammatory and anti-carcinogenic activities (1, 2) and also is a chemopreventive agent (3, 4). Numerous previous studies have shown that curcumin induces apoptosis in many cells (4-8). It was also demonstrated that curcumin produces reactive oxygen species (ROS) to a much greater degree than other ortho-methoxyphenols (9) and recently reported that the phosphatidylserine externalization and loss of mitochondrial membrane potential, as early signs of apoptosis in curcumin-treated normal human gingival fibroblast (HGF cells) and human submandibular gland carcinoma cells (HSG cells), were responsible for the generation of ROS (10). Curcumin demonstrated phototoxicity towards several species of bacteria (11) and manmalian cells (12-14) in the presence of oxygen. Curcumin photo-generated singlet oxygen and reduced forms of molecular oxygen, i.e., ROS; however, the generation of ROS alone does not explain these biological results (12). The causal relationship between ROS and the cytotoxicity of curcumin has not been sufficiently defined. Oxidative damage is not reversible in vivo when the level of ROS surpasses the protective antioxidant defense systems of cells. In such events, reduced cellular glutathione (GSH) plays a crucial role in the rescue of cells from the induction of apoptosis and necrosis by buffering the endogenously induced oxidative stress that accompanies a marked depletion of the level of the reduced form of GSH and an increase in ROS production due to cellular redox changes (15-18). These findings led us to propose that the intracellular-redox condition brought about by curcumin and affecting ROS generation may result in cytotoxicity. Recently, tetrahydrocurcumin (TH-curcumin, Figure 1), one of the major metabolites of curcumin, has been a matter of focus with respect to its cancer-preventing activity (19-22). TH-curcumin is easily converted from curcumin during digestion in vivo (23). TH-curcumin exhibits similar physiological and pharmacological protective properties against oxidative stress to curcumin (19-22).

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ANTICANCER RESEARCH 27: 363-372 (2007)

Figure 1. Chemical structures of curcumin and TH-curcumin.

Curcumin has previously been evaluated as a potential photosensitizer for oral application with a halogen lamp used in dental clinics (14). This compound present in foods may directly affect the oral mucosa and gingiva, as well as the gastrointestinal tract. Recently, it was demonstrated that eugenol, an ortho-methoxyphenol, acts in a bimodulatory fashion, i.e., as an antioxidant and/or prooxidant under oxidative stress elicited non-enzymatically by VL-irradiation, or enzymatically by horseradish peroxidase/hydrogen peroxide (HRP/H2O2) treatment (24). Curcumin and THcurcumin, both of which are ortho-methoxyphenols, were expected to show similar behavior to oxidized eugenol. In the present study, a condition of experimental oxidative stress caused by either treatment with HRP/H2O2 or VL-irradiation was set up, according to our earlier study (24), and whether the causal relationship between the cytotoxicity and ROS generation is a function of the intracellular redox condition was investigated. The cytotoxicity, intracellular GSH level, and intracellular ROS generation by curcumin and TH-curcumin were evaluated by using HSG cells and HGF cells in the presence or absence of VL-irradiation or HRP/H2O2 treatment.

Materials and Methods Reagents. The following chemicals and reagents were obtained from the companies indicated: curcumin (1,7-bis (4-hydroxy-3methoxyphenol)-1,6-heptadiene-3,5-dione), used without further purification, from Tokyo Kasei Chem. Co., Tokyo, Japan; 5-(and 6)-carboxy-2’,7’-dichlorofluorescin diacetate (CDFH-DA) from Molecular Probes Inc., Eugene, OR, USA; GSH Detection Kit (Chemicon International Inc. Temecula, CA, USA), MTT [3-(4, 5 -dimethelthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] and Aqueous One Solution from Promega Co., Madison, WI, USA; and HRP from Wako Pure Industrials, Ltd, Osaka, Japan. THcurcumin was a gift from Professor Yokoe at Josai University. The chemical structures of curcumin and TH-curcumin are shown in Figure 1.

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Cells and cell culture. Human primary gingival fibroblast cells (HGF) were cultured using the method of Kawase et al. (25) from a fragment of the attached gingiva of a supernumerary tooth from a 7-year-old female patient who had undergone extraction of the tooth. The informed consent of the patient and her parents was obtained, as was ethical clearance for the study from the Ethics Committee of Meikai University School of Dentistry. The tissue was cut into 1 to 2 mm3 pieces, which were then washed twice with phosphate-buffered saline (PBS) supplemented with 100 U/ml penicillin and 100 Ìg/ml streptomycin and placed into tissue culture dishes. The explants were incubated for about 3 weeks at 37ÆC in culture medium consisting of Minimum Essential Medium · modification (·-MEM) containing 30% FBS, 100 U/ml penicillin, and 100 Ìg/ml streptomycin, in a humidified atmosphere of 5% carbon dioxide in air. The outgrowths were then sub-cultured by trypsinization and passage in ·-MEM supplemented with 10% FBS. The HGF cells, uniformly having the structure of fibroblasts, were used at passage numbers 5-7. The HSG cell line, derived from a human submandibular gland carcinoma (26) and a gift from Professor Dr. M. Satoh, Tokushima University, were cultured in MEM supplemented with 10% FBS. Cytotoxicity. HSG and HGF cells were seeded into 96-well plates at a density of 5x103 cells/ well in 0.1 ml of MEM or ·-MEM with 10% FBS and were cultured at 37ÆC for 2 days. One hour before the addition of test materials, the cells were washed twice with serum-free medium. A stock solution of 10 mM curcumin or THcurcumin was prepared in dimethylsulfoxide (DMSO). It was diluted with DMSO and added to the wells at a 1/100 volume (final concentration: 1 ÌM to 100 ÌM) and the cells were then incubated for 10 min. There was no significant difference in cell survival between cultures containing 1% DMSO and those without DMSO, consequently 1% DMSO-treated cells were used as the control. Two kinds of treatment were performed after the above-mentioned 10-min incubation: (i) cells were irradiated from a distance of 10cm with visible light (VL: 400 nm≤Ïmax 540 nm) for 10 min using the above-mentioned dental lamp, or (ii) 200 ÌM hydrogen peroxide (H2O2) and 2 Ìg/ml HRP were added to the cell cultures. One day after treatment, the number of viable cells was determined using the MTT colorimetric assay (27). The relative viable cell number was expressed as a ratio (percentage) of the number of viable in the experimental wells to that in the control (without test compound) wells. The CC50 (cytotoxic concentration for 50% cell death) was determined from the dose-response curve. Intracellular GSH. A GSH Detection Kit (Chemicon International Inc., Temecula, CA, USA) was used according to the manufacturer’s instructions. HSG cells were seeded into 6-well plates at a density of 2x105 cells/well in 2 ml of MEM with 10% FBS and were cultured at 37ÆC for 2 days. Before the addition of test compounds, the cells were washed twice with serum-free MEM, after which curcumin or TH-curcumin (10 ÌM, 30 ÌM or 100 ÌM) was added to the wells. Then 2 kinds of oxidative stress condition were established: (i) VL-irradiation for 10 min as described above or (ii) the addition of H2O2 (100 ÌM)/ HRP (1 Ìg/ml), as used in the detection of ROS levels (see below). The cells were incubated at 37ÆC for a total of 1 hour and then washed twice with PBS. Lysis buffer (0.1% Triton X) was added to the cells on ice. Ten minutes later the cells were collected with a rubber policeman. The lysate was centrifuged at 12,000 xg for 10

Atsumi et al: Cytotoxicity and ROS of Curcumin and TH-Curcumin

Figure 2. Cell survival of HSG (a, b) and HGF (c, d) cells in the presence of curcumin (a, c) or TH-curcumin (b, d) with or without VL-irradiation (10 min). HSG and HGF cells (5x103 in each well of a 96-well plate) were cultured for 2 days. Curcumin or TH-curcumin was added to the cells (1 ÌM to 100 ÌM) and 10 min later they were irradiated with VL from a dental lamp for 10 min (Ïmax: 540 nm) and then cultured for 1 day. Cell survival was measured using the MTT method described in the text. Data are presented as the mean±SD (n=8). There was a significant difference between curcumin and TH-curcumin under the non-oxidation condition for both HSG cells (a and b, p