Effect of dietary curcumin and ascorbyl palmitate on azoxymethanol-induced colonic epithelial cell proliferation and focal areas of dysplasia

Cancer Letters, 64 (1992) 117 - 121 Elsevier Scientific Publishers Ireland Ltd.

117

Effect of dietary curcumin and ascorbyl palmitate on azoxymethanol-induced colonic epithelial cell proliferation and focal areas of dysplasia M.-T. Huanga, E.E. Deschnerb, and A.H. Conneya ‘Laboratory

for Cancer

Rutgers Uniuersity,

Research,

Piscataway

Institute and the GI &ruice, York NY 10021

Department

NJ 088550789

Memorial

of Chemical

(Received 24 February

Memorial

College

Sloan-Kettering

Cancer Center,

1275

of Pharmacy, Sloan-Kettering

York Avenue,

New

1992)

1.992)

Summary a

major

yellow

pigment of rhizomes of the plant Curcuma longa Linn., is commonly used as a coloring agent in foods, drugs and cosmetics. Ascorbyl palmitate is a lipid soluble derioatiue of ascorbic acid. Both curcumin and ascorbyl palmitate haue antioxidant activity and are potent inhibitors of I,?-O-tetradecanoyl-phorbol13-acetate-induced tumor promotion in mouse skin. The effects of dietary curcumin and ascorbyl palmitate on azoxymethanol (AOM)-induced hyperproliferation of colonic epithelial cells and the incidence of focal areas of dysplasia (FADS) were evaluated in female CF-1 mice fed an AlN 76A diet. Subcublneous injections of AOM (10 mg/kg body wt. once weekly for 6 weeks) caused hyperplasia and the formation of FADS in the colon. Administration of 2% curcumin

turmeric obtained ,from powdered

Correspondence to: M.T. Huang, Laboratory Research, Department of Chemical Biology macognosy, Piscataway

and Pharmacognosy,

of Digestiue Tract Carcinogenesis,

1992)

13 March

13 March

Curcumin,

Biology

and ‘Laboratory

Hospital,

Z.-Y. Wanga, T.A. Ferraro”

(USA)

(Revision received (Accepted

H.L. Newmarka,

College of Pharmacy, NJ 08855 0789, USA.

0304-3835/92/$05.00 Printed and Published

Rutgers

for Cancer and PharUniversity,

0 1992 Elsevier Scientific Publishers in Ireland

in the diet inhibited AOM-induced formation of FADS while administration of 2% ascorbyl palmitate in the diet did not demonstrate inhibition. This result suggests that dietary curcumin may inhibit AOM-induced colonic

neoplasia

in mice.

Key words: curcumin; ascorbyl palmitate; azoxymethanol; colonic epithelial cells; hyperproliferation; focal areas of dysplasia Introduction Curcumin (diferuloylmethane) is the major yellow pigment in curry, mustard and turmeric. Both curcumin and turmeric (the ground rhizome of the plant Curcuma longa Linn.) are widely used as spices and coloring agents in food. Curcumin itself is commonly used as a coloring agent in foods, drugs ai7d cosmetics [ 191. Curcumin possesses antioxidant and anti-inflammatory activities [9,11,16 - 181. We have previously reported that topical application of curcumin to the backs of CD-1 mice markedly inhibited Ireland Ltd

118

12-0-tetradecanoylphorbol-13-acetate (TPA)induced inflammation, ornithine decarboxylase activity, DNA synthesis, hyperplasia, hydrogen peroxide formation and tumor promotion in mouse epidermis [6 - 81. Curcumin also strongly inhibits epidermal cyclooxygenase and lipoxygenase activities in vitro [6]. In this paper, we report an inhibitory effect of dietary curcumin on azoxymethanol (AOM) induced formation of focal areas of dysplasia (FADS) in the colon of mice. Similar studies with ascorbyl palmitate, another potent inhibitor of TPA-induced tumor promotion in mouse skin, showed little or no effect. Materials and Methods Chemicals and diets AOM was purchased from Sigma Chemical Company (St. Louis, MO). Tritiated [3H]thymidine was obtained from New England Nuclear (Boston, MA). Curcumin (turmeric type 97) was obtained from Kalsec, Inc. (Kalamazoo, MI). This is food grade curcumin 17% contained 80% curcumin, that demethoxycurcumin and 3% bisdemethoxycurcumin. Ascorbyl palmitate ( > 8% purity; Food Chemical Codex grade) was obtained from Hoffmann - La Roche, Inc. (Nutley, NJ). The control diet was a basic AIN 76A semipurified diet (Table I). This diet was

Table 1. Composition

of AIN 76A diet.

Control diet ingredients

g/kg

Casein DL-methionine Corn starch Sucrose Cellulose Corn oil Ethoxquin Salt mix AlN-76 Vitamin mix AINCholine bitartrate

200.0 3.0 150.0 500.0 50.0 50.0 0.01 35.0 10.0 2.0

chosen to avoid natural feed components known to contain appreciable plant phenolics. The diets were prepared and pelleted by Teklad Laboratory Animal Diets (Madison, WI). Animal experiments Female CF-1 mice, 5 weeks old, were obtained from Charles River Laboratories (Kingston, NY). The mice were fed Purina Laboratory Chow 5001 and provided with drinking water ad libitum for 1 week after arrival in our laboratory. The mice were then randomized into three groups (12 mice per group) and fed the control AIN 76A diet, the 2% curcumin diet, or the 2% ascorbyl palmitate diet. Water was provided ad libitum. After 2 weeks on the various diets, one half (6 mice) of each group were injected S.C. with normal saline (vehicle control), while the other half were injected S.C. with AOM (10 mg/kg body wt.) in saline, once weekly for 6 weeks; all mice were continued on their respective diets. One week after the last AOM injection, each mouse was administered 25 &i [ 3H] thymidine (25 Ci/mmol) in 20 ~1 water by i.p. injection and was sacrificed 1 h later. Tissue preparation Immediately after animal sacrifice, the colon was removed and placed into 10% neutral buffered formalin. The lower 2 cm of distal colon was processed by dehydration and the tissue embedded in paraffin. Sections (3 pm each) were cut and mounted on slides. Slides were then rehydrated and dipped into NTB2 emulsion (Eastman-Kodak, Rochester NY). After a suitable time, the slides were developed and stained with hematoxylin and eosin Y. Focal

areas of dysplasia

scoring

FADS were scored by methods previously described [2 - 51. Five hundred pm of serialsectioned distal colon from each mouse was examined for the number of FADS. These atypical areas are composed of cells exhibiting loss of mucin and loss of nuclear polarity and as having nuclei of variable size and shape.

119

Table II. Effect of dietary curcumin colonic epithelial cells of CF-1 mice. Diet administered

and ascorbyl

Body wt. (9)

palmitate

Cells scored per mouse

on control and AOM-induced

Labeled cells per crypt

indices in

proliferative

Distribution of labeled cells (46)” L/3

M/3

U/3

Saline Control 2% curcumin 2% ascorbyl palmitate

27.5 zt 1.9 26.0 + 0.9 29.3 +z 1.6

1135 f 50 1246 zt 69 1193 f 33

2.1 f 0.6 4.2 zt 0.6’ 2.4 zrz0.4

78.2 zt 2.5 70.0 f 4.1 83.0 +z 3.9

21.8 + 2.5 30.0*4.1 17.0 f 3.9

0 0 0

AOM Control 2% curcumin 2% ascorbyl palmitate

29.8 f 0.9 28.3 f 2.6 29.0 + 1.8

1919 f 69 1654 + 56 1811 zt 68

8.9 ztz 1.0” 7.0 + 1.0” 7.3 zt 0.7”

66.5 zt 0.7” 69.5 zt 3.1’ 72.8 f 4.4

32.0 f 0.7” 29.2 ztz 3.1’ 26.2 zrz4.2

1.5 f 0.6’ 1.3 f 0.3” 1.0 l 0.4’

The data are expressed as the mean * S.E. from 6 mice. *Statistically different ,irom saline control group (P < 0.05). ‘Statistically differeni from saline control group (P < 0.005). “L/3, M/3 and U/3 represent the lower, middle or upper third of the crypt. l

FADS occur in otherwise normal mucosa of AOM-treated mice.

appearing

Proliferation scoring A total of 25 longitudinally sectioned crypts were analyzed per colon. The number and position of [ 3H] thymidine-labeled cells as well as the total number of cells per crypt was noted. A cell was considered labeled when at least 4 grains appeared over the nucleus. The labeling index is the percent of labeled cells per crypt. Statistics

Data were analyzed by Student’s t-test and analysis of variance where appropriate. Values are presented as the mean f S.E. Values with P < 0.02 were considered to be significant. Results and Discussion Oral administration of 2% curcumin or 2% ascorbyl palmitate in AIN 76A diet did not affect body weight compared to the control group fed an AIN ‘76A diet (Table II). No visible signs of toxicity from curcumin or ascorbyl palmitate were observed.

Subcutaneously administered AOM (10 mg/kg, once weekly for 6 weeks) induced increased proliferation of colonic epithelial cells and the expansion of the proliferative compartment to the middle and upper third of the crypts (Table II) as well as formation of FADS (Table III). No FADS were found in control mice or control mice fed the curcumin or ascorbyl palmitate diets. The incidence of AOM-induced FADS in mice on the control diet was 6.0 * 1.0 per 500 pm of the distal

Table 111. FADS in 500 pm of distal colon 1 week after the sixth injection of azoxymethanol. Diet

FADS per 500 pm

Size (crypts/FAD)

Control

6.0 zt 1.0

2% Curcumin

2.5 ZIZ0.6’

2 % Ascorbyl Palmitate

7.5 * 0.8

2.3 zt (range 2.0 f (range 2.3 zt (range

‘Statistically

different from control

(P

c

0.6 1 - 6) 0.4 1 - 4) 0.5 1 - 6)

0.02).

120

colon. Two percent curcumin in the diet inhibited the average number of AOM-induced FADS per 500 pm of distal colon by about 58% (P < 0.02). The antioxidant ascorbyl palmitate (2% in the diet), however, did not affect proliferation or the incidence of AOMinduced formation of FADS. While administration of dietary curcumin alone significantly increased the number of labeled colonic epithelial cells per crypt in the distal colon, dietary curcumin did not influence the AOM-induced increase. Compared with AOM controls, neither curcumin nor ascorbyl palmitate caused significant alteration in the number of labeled cells per crypt in AOMtreated mice (Table II). In this preliminary study, curcumin appears to be a promising dietary agent for the reduction of colon neoplasms. Curcumin has been shown in earlier studies to inhibit TPA-induced tumor promotion in mouse skin [8] and to be a potent inhibitor of cyclooxygenase and lipoxygenase activities in mouse epidermis [6]. Other agents that are inhibitors of arachidonic acid metabolism to eicosanoids have shown activity as inhibitors of colonic neoplasia, including piroxicam [ 141, indomethacin [lo], quercetin [5], rutin [5] and $-3 fatty acids from fish oils [4,12,13,15]. Further studies on the effect of dietary curcumin as a possible inhibitor of AOM-induced colonic neoplasia are in progress.

Susceptibility to 1,2-dimethylhydrazine induced colonic tumors and epithelial cell proliferation characteristics of Fl, F2 and reciprocal backcrosses derived from SWR/J and 4

5

6

7

8

9

10

11

Acknowledgments 13

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This work was supported by grants CA49756, CA08748 and CA46845 from the National Cancer Institute, United States Public Health Services.

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