DMBA and TPA were purchased from Sigma (St.Louis, MO, USA). Commercial curcumin (77% curcumin, 17% demethoxycurcumin, and 3% bisdemethoxy curcumin, Fig. ) was purchased from ICN Biomedical (Costa Mesa, CA, USA). Feed ingredients (casein, methionine, mineral and vitamin mixtures, cellulose and choline bitartate) were purchased from ICN Biomedical. All other chemicals were of AR grade.
Curcuminoid compounds Curcumin: R1 = R2 = OCH3 (77%) Desmethoxycurcumin: R1 = H, R2 = OCH3 (17%) Bisdesmethoxycurcumin: R1 = R2 = H (3%)
Male Swiss albino mice, 5 weeks old, were purchased from the National Laboratory Animal Center, Thailand. All diet ingredients were stored at 4°C prior to the preparation of diets. The mice were quarantined for 7 days and had access to modified AIN-76A diet. Following quarantine, all the mice were randomly distributed by weight into various groups and were housed individually under controlled conditions of a 12-h light/12-h dark cycle and 22 ± 2°C.
Diets and treatments
In all treatment groups the mice were fed a modified AIN-76 diet (control diet). The control diet had the following composition (g/100 g): 20 casein, 0.3 DL-methionine, 3.5 mineral, 1 vitamin, 5 cellulose, 5 corn oil, 32 corn starch, 33 sucrose, and 0.2 choline bitartrate. In some treatment groups, the control diet was modified by the addition of 1% or 0.2% curcumin (Table ). The incorporation of curcumin into the diet was done with a V-blender after curcumin was premixed with a small quantity of diet in a food mixer to ensure its uniform distribution. All feeds were pelleted to avoid stratification and to ensure uniform feed and curcumin intake in the treated animals. All control and experimental diets were prepared every 2 weeks in our laboratory and stored at -20°C before use. The mice had access to food and water at all times, and food cups were replenished with fresh diet three times weekly.
As indicated in Fig. , beginning at 6 weeks of age, groups of animals were fed the control diet or the experimental diet containing 1% or 0.2% curcumin. At 8 weeks of age, groups of animals were started on carcinogen treatment. The mice were sorted into the following treatment groups (20 mice/group). One group of mice were fed the control diet and treated with DMBA and TPA on the skin. Some groups of animal were fed the control diet supplemented with 1% or 0.2% curcumin and similarly treated with DMBA and TPA. The vehicle (acetone solvent) chemical control groups were fed the control diet or the curcumin diet and treated with acetone on the skin. Animals were maintained on control or curcumin diets until the termination of the experiment. The backs of mice were shaved with electric clippers 3 days before the first skin treatment and thereafter were shaved when needed for the duration of the experiment. Carcinogen or vehicle solvent (acetone) was applied to the dorsal shaved area.
Figure 6 Experimental design for evaluation of chemopreventive efficacy by curcumin. Carcinogen: 100 μg of DMBA in 100 μl of acetone in a single application. 2.5 μg of TPA dissolved in 100 μl of acetone, twice a week for 26 weeks. (more ...)
All of the animals which were subjected to tumor initiation and promotion received 100 μg of 7, 12-dimethylbenz [a]anthracene (DMBA) dissolved in 100 μl of acetone in a single application to the skin of the back. From 1 week after DMBA application, tumor promoter, 2.5 μg of 12-0-tetradecanoyllphorbol-13-acetate (TPA) dissolved in 100 μl of acetone, was applied to the same areas on mouse skin twice a week for 26 weeks (Fig. ). The vehicle control groups received only 100 μl of acetone to the skin of the back. Mice were weighed at weekly intervals. Tumors appearing on mouse skin were counted and measured by a Pierre Vernier caliper weekly throughout week 26. The average tumor volume was calculated as (length) × (width) × (height) π/6. The data were analyzed by Student's t-test and chi-square test.
Determination of c-fos and c-Ha-ras gene expression
After 26 weeks of TPA application, pooled tumors and non-tumorous mouse epidermis from each experimental group (control diet, 1 and 0.2% curcumin diet) were collected. The skins were excised and placed in a water bath at 60°C for 30s and then submerged in liquid nitrogen [21
]. The tumors were rapidly removed and frozen in liquid nitrogen and the non-tumorous epidermis was removed from the dermis by gentle scraping and then rapidly frozen in liquid nitrogen. The samples were stored at - 70°C until used for western blot analysis. The c-fos and c-Ha-ras proteins were analyzed by enhanced chemiluminescence (ECL) Western blot analysis using specific anti-p62 fos and anti-p21 ras antibodies, respectively.
Preparations of Ha-ras-p21 and fos-p62 proteins
The pooled tumors and nontumorous mouse epidermis from each experimental group (control diet, 1 and 0.2% curcumin diets) were washed with cold phosphate buffer saline and suspended in 1 ml of buffer A (20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 μg/ml aprotinin, and 10 μg/ml leupeptin). The suspension was homogenized in a glass homogenizer with 30 strokes and centrifuged at 700 g for 5 min to remove unbroken cells and nuclei. The nuclear pellet was used for fos preparation. The supernatant was further centrifuged at 100,000 g for 30 min. The membrane pellet was resuspended in 1 ml of buffer B (1% NP-40, 0.5% sodium deoxycholate, 0.1%SDS, 0.6 mM PMSF and 1 mM sodium orthovanadate in PBS pH 7.4), rocked for 1 h, and then centrifuged at 100,000 g for 30 min. The supernatant was used as membrane fraction for ras-p21 analysis and stored as aliquots at -80°C.
The nuclear extract was prepared by the method of Dignam et al.
] as follows: the pellet obtained from the low speed centrifugation of the homogenate was resuspended in buffer C (20 mM HEPES pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2
, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) and was stirred gently with a magnetic stirring bar for 30 min at 4°C and then centrifuged for 30 min at 25,000 g (Sorval SS34 rotor), and the supernatant was dialysed against 50 volumes of buffer D (20 mM HEPES pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) for 5 h. The dialysate was centrifuged for 20 min at 25,000 g (Sorval SS34 rotor) and the resulting precipitate discarded. The supernatant, designated the nuclear extract, was frozen as aliquots in liquid nitrogen and stored at -80°C.
The protein contents in both nuclear and membrane fractions were determined with Bio-Rad reagent.
Immunodetection and quantification of ras-p21 and fos p-62 using ECL Western blot
SDS-PAGE and Western transfers were carried out essentially by the methods of Laemmli [23
] and Towbin et al
]. Nuclear and membrane extracts corresponding to 25 μg of protein were solubilized in Laemmli's sample buffer. Samples were boiled for 3 min and resolved by extended electrophoresis on a 12.5% polyacrylamide mini- slab gel containing SDS with an overlay of 4% polyacrylamide, along with prestained SDS-PAGE molecular weight markers and the ras-p21 Western blot standards (Oncogene Science, Manhasset, NY). Electrophoretically resolved proteins were electrotransferred onto Hybond ECL membrane in a Trans-blot electrophoretic transfer cell (Bio-Rad Laboratories).
After transblotting the electrophoretically resolved proteins, the blots were blocked with 5% nonfat dry milk dissolved in Tris-buffered saline containing 0.2% Tween-20 (TBST). Blots were then incubated with specific monoclonal antibody to c-Ha-ras protein (Santa Cruz Biotechnology) diluted in TBST and reincubated with peroxidase-linked secondary antibody (antimouse IgG; Amersham) diluted in TBST containing 0.5% nonfat dry milk. The blots were then throughly washed in excess TBST and probed with the ECL. Western blot detection system (Amersham) using reflection autoradiography films was performed according to the manufacturer's instructions. The autoradiograms were scanned with an Image Master sharp laser densitometric scanner, and the peak areas representing ras-p21 bands were determined.
Western blot analysis of the fos protein was performed by the same protocol as described above except in the detection system, the specific antibody to c-fos p62 protein (Santa Cruz Biotechnology) was used instead of specific antibody to ras-p21 protein.