Protective effects of benzyl isothiocyanate and sulforaphane but not resveratrol against initiation of pancreatic carcinogenesis in hamsters

Cancer Letters 241 (2006) 275–280 www.elsevier.com/locate/canlet

Protective effects of benzyl isothiocyanate and sulforaphane but not resveratrol against initiation of pancreatic carcinogenesis in hamsters Yuichi Kuroiwa, Akiyoshi Nishikawa *, Yasuki Kitamura, Keita Kanki, Yuji Ishii, Takashi Umemura, Masao Hirose Division of Pathology, National Institute of Health Sciences, 1-8-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Received 31 August 2005; received in revised form 19 October 2005; accepted 21 October 2005

Abstract Potential chemopreventive effects of naturally occurring agents were investigated using a new 16-week medium-term pancreatic carcinogenesis models in hamsters. Male 6-week-old Syrian hamsters were subcutaneously injected with 10 mg/kg body weight Nnitrosobis(2-oxopropyl)amine (BOP) four times within a week, and fed a diet supplemented with 80 ppm benzyl isothiocyanate (BITC), 80 ppm sulforaphane (SFN) or 10 ppm resveratrol (RES) during the initiation or post-initiation stages. For the initiation stage, each chemical was given for 3 weeks including 1 week before and after the BOP injections. With post-initiation exposure, the groups were changed from basal diet 1 week after the last BOP injection, and then fed each chemical for 14 weeks. All the animals were sacrificed after 16 weeks. The multiplicities of combined pancreatic lesions including atypical hyperplasias and adenocarcinomas were significantly decreased by BITC and SFN given in the initiation but not the post-initiation stage. On the other hand, RES, a naturally occurring inhibitor of cyclooxygenase-2 (COX-2) reported chemopreventive effects, failed to show significant effects on pancreatic carcinogenesis in either the initiation or post-initiation stages. Our data suggest that the naturally occurring isothiocyanates BITC and SFN can block BOP-initiation of hamster pancreatic carcinogenesis. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Pancreatic carcinogenesis; Hamster; Benzyl isothiocyanate; Sulforaphane; Resveratrol

1. Introduction Pancreatic cancer is a serious problem in Japan as in western countries, with 5-year survival rates being less than 25% [1,2]. Although a number of therapeutic approaches have been introduced, median survival after diagnosis is generally less than 2 years so that only late stage disease can be diagnosed [1,2]. Therefore,

* Corresponding author. Tel.: C81 3 3700 9819; fax: C81 3 3700 1425. E-mail address: [email protected] (A. Nishikawa).

effective preventive approaches against this aggressive disease are urgently required. Epidemiological studies have suggested that increased risks of pancreatic cancer are associated with tobacco, obesity and high consumption of fat, fish, pork or beef, and that decreased risks are associated with consumption of cruciferous vegetables [3–7]. A number of experimental studies have also support that certain dietary chemicals isolated from foodstuffs can protect against cancer. An important group of agents that have this property are the organosulfur compounds such as isothiocyanates (ITCs), abundant in cruciferous

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.10.028

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vegetables for which consumption has epidemiologically shown an inverse link with pancreatic cancer [3,5]. It has been reported that several ITCs can prevent tumour development in various organs of rodents treated with diethylnitrosamine [8,9], benzo[a]pyrene [8], 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone [10], N-nitrosobenzylmethylamine [11], methylazoxymethanol acetate [12], 7,12-dimethylbenz[a]anthracene (DMBA) [13], N-butyl-N-(4-hydroxybutyl)nitrosamine [14] and 2-amino-3-methylimidazo[4,5-b]pyridine [15] when given in the initiation stage or throughout the experimental period. Additionally, we previously reported that phenethyl isothiocyanate (PEITC) effectively inhibits N-nitrosobis(2-oxopropyl)amine (BOP)induced lung and pancreatic carcinogenesis in hamsters when given simultaneously with BOP in the initiation phase [16]. Indeed, ITCs thus appear to be potent chemopreventive agents in the pancreas [17,18]. In human pancreatic cancer cells, it has been reported that benzyl isothiocyanate (BITC) and sulforaphane (SFN) which are abundantly included in garden cress and broccoli, respectively, have anti-proliferative activity [19,20]. Another group of chemicals expected to have anti-cancer potency are plant derived anti-inflammatory polyphenolic pigments such as resveratrol (RES), found in grapes and also reported to exert antitumor activity [21], inhibit proliferation and induce apoptosis in human pancreatic cancer cells [22]. The BOP-pancreatic carcinogenesis model using hamsters has been extensively studied because of the histopathological similarities of the induced lesions with human pancreatic tumours [16,23–28]. Recently, we established a new 16-week medium-term pancreatic carcinogenesis assay model in hamsters which uses precancerous and cancerous lesions, atypical hyperplasias and adenocarcinomas, as endpoint lesions, and reported chemopreventive effects of a cyclooxygenase-2 (COX-2) inhibitor, nimesulide, given in the post-initiation phase [29]. In the present study, we investigated the chemopreventive effects of two ITCs (BITC and SFN) and an anti-inflammatory substance RES on pancreatic carcinogenesis using this model. 2. Materials and methods 2.1. Chemicals BOP was obtained from Nacalai Tesque Inc. (Kyoto, Japan) and dissolved in physiological saline just before injection. BITC, SFN and RES were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan), LKT Laboratories, Inc.

(St. Paul, MN, USA) and Alexis Co. (Lausen, Switzerland), respectively, and mixed into powdered standard basal diet Oriental MF (Oriental Yeast Co., Ltd., Tokyo). The concentration for RES was set at 10 ppm, which was earlier found to suppress rat mammary carcinogenesis induced by DMBA when given throughout the experimental period [30]. The concentration of SFN was set at 80 ppm, calculated from food consumption and the concentration given by gavage which suppressed development of azoxymethane-induced aberrant crypt foci in rat colon when given in both the initiation and post-initiation stages [31]. The concentration of BITC was set at 80 ppm in line with SFN. The BITC and SFN/diet admixtures were prepared once a week, while for RES preparation was twice a week because its stability in the diet was unknown. Bromodeoxyuridine (BrdU) was obtained from Sigma-Aldrich Co. (St. Louis, MO). 2.2. Animals A total of 150 male Syrian hamsters (Japan SLC, Inc., Shizuoka, Japan), 5-weeks old and weighing about 70 g at the commencement, were housed, five per polycarbonate cage, in an air-conditioned room at 23G2 8C and 60C5% humidity under a daily cycle of alternating 12-h periods of light and darkness. Standard basal diet and tap water were available ad libitum. After 1 week of acclimation period, the animals were used in the experiments. 2.3. Experimental design As shown in Fig. 1, 150 hamsters were divided into nine groups, each consisting of 20 or 10 animals. The animals of Groups 2–9 were s.c. injected with 10 mg/kg BOP dissolved in saline four times within a week while Group 1 controls received saline alone. To explain effects on initiation, they were fed basal diet alone (Group 2), or diets mixed with 80 ppm BITC (Group 3), 80 ppm SFN (Group 5) or 10 ppm RES (Group 7), for 3 weeks including 1 week before and after the BOP injections, and returned to be fed basal diet. For post-initiation exposure, the other four groups were fed basal diet until 1 week after the last BOP injection, and then diets containing the test substances for 14 weeks (Groups 4, 6 and 8). Sixteen weeks after the first injection, all animals were sacrificed. They were given BrdU (100 mg/kg) by i.p. injection once a day for the final 2 days and once on the day of termination, 2 h before being euthanatized. Four parts (gastric, duodenal and splenic lobes, and head portion) of each pancreas were carefully identified and collected, fixed in 10% buffered formalin solution, routinely processed to paraffin blocks and histopathologically investigated for the existence of atypical hyperplasias and adenocarcinomas in haematoxylin and eosin (H-E)-stained preparations. At autopsy, the weights of body, lung, liver and kidney were measured.

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all cells constituting atypical hyperplasias were counted, and labelling indices (LIs) were calculated as the percentage of cells positive for BrdU incorporation divided by the total number of cells counted. 2.6. Statistical evaluation Data for body and organ weights, multiplicity of pancreatic lesions and BrdU-LI were analysed by the Student’s t-test, and incidences of pancreatic lesions were analysed by the Fisher’s exact probability test.

3. Results

Fig. 1. Experimental design.

2.4. Immunohistochemical procedure For immunohistochemical staining of BrdU, after first denaturing DNA with 4 N HCl, sections were treated sequentially with normal horse serum, monoclonal mouse anti-BrdU (Becton, Dickinson & Co., Franklin Lakes, NJ) (1: 100), biotin-labelled horse anti-mouse IgG (1:400), and avidin-biotin-peroxidase complex (ABC). The sites of peroxidase binding were demonstrated by incubation with 3, 3 0 -diaminobenzidine tetrahydrochloride (Sigma-Aldrich Co.). Immunostained sections were lightly counterstained with haematoxylin for microscopic examination. 2.5. Cell proliferation quantification For each animal, epithelial cells in 10 normal pancreatic ducts were counted for incorporation of BrdU. Additionally,

No animals died before the termination at week 16. As summarized in Table 1, all BOP-treated groups showed significant (P!0.05 or 0.01) decreases in body weights and increases in relative kidney weights as compared to the saline-treated Group 1. Relative lung weights in Group 7 were significantly (P!0.01) decreased, and relative liver weights in Groups 3 and 7 were significantly (P! 0.05) increased. As compared to Group 2 treated with BOP alone, relative kidney weights in Groups 3 and 7, and relative lung weights in Group 7 were significantly (P!0.01) increased. Additionally, relative liver weights were significantly (P!0.05) higher in Group 3 than in Group 2. As shown in Table 2, the incidence of adenocarcinomas was significantly (P! 0.05) decreased in Group 5 as compared to Group 2, along with the multiplicity of adenocarcinomas and atypical hyperplasias. Group 3 also showed a significant (P!0.01) decrease in the multiplicity of atypical hyperplasias. As summarized in Table 3, lowered proliferative activities in normal pancreatic ducts were shown in Groups 3–7, as compared to the control Group 1, this being statistically (P!0.05 or 0.01) significant in Groups 5, 6 and 7. However, in pancreatic atypical hyperplasias, there were no differences in proliferative activity. Liver adenomas

Table 1 Final body and relative organ weights Group

Effective no. of animals

Body weight (g)

Lungs (g%)

Liver (g%)

Kidneys (g%)

1. Saline 2. BOP 3. BOPCBITC / Basal diet 4. BOP / BITC 5. BOPCSFN / Basal diet 6. BOP / SFN 7. BOPCRES / Basal diet 8. BOP / RES

10 20 20 20 20 20 20 20

177.9G9.8 157.9G17.1 # 154.2G23.6 ## 151.4G15.7 ## 149.8G17.6 # 154.5G14.5 # 158.4G12.8 ## 164.5G18.6 #

0.59G0.08 0.61G0.13 0.58G0.11 0.64G0.13 0.59G0.13 0.68G0.18 0.50G0.07 **## 0.59G0.12

4.57G0.36 4.69G0.48 4.99G0.41 *# 4.85G0.41 4.73G0.44 4.65G0.35 4.91G0.33 # 4.73G0.43

0.55G0.04 0.61G0.04 ## 0.66G0.08 **## 0.62G0.05 ## 0.63G0.05 ## 0.62G0.04 ## 0.64G0.03 **## 0.63G0.07 ##

#, ##: P!0.05, P!0.01 vs. Group 1, respectively. *, **: P!0.05, P!0.01 vs. Group 2, respectively.

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Table 2 Incidence and multiplicity data for pancreatic atypical hyperplasias and adenocarcinomas Group

1. Saline 2. BOP 3. BOPCBITC/Basal diet 4. BOP/BITC 5. BOPCSFN/Basal diet 6. BOP/SFN 7. BOPCRES/Basal diet 8. BOP / RES

Effective no. of animals

No. of animals with (%) AH

ADC

Total

AH

ADC

Total

10 20 20 20 20 20 20 20

0 (0) 17 (85) 12 (60) 16 (80) 15 (75) 20 (100) 16 (80) 17 (85)

0 (0) 8 (40) 3 (15) 4 (20) 2 (10)* 11 (55) 8 (40) 4 (20)

0 (0) 18 (90) 14 (70) 16 (80) 15 (75) 20 (100) 17 (85) 18 (90)

0.00G0.00 3.10G2.02 0.90G1.02** 2.15G1.95 1.65G1.35* 2.60G1.35 2.25G1.92 2.25G2.05

0.00G0.00 0.55G0.76 0.20G0.52 0.20G0.41 0.10G0.31* 0.60G0.60 0.55G0.76 0.25G0.55

0.00G0.00 3.65G2.48 1.10G1.02** 2.35G2.11 1.75G1.48** 3.20G1.61 2.80G2.50 2.50G2.14

a

No. of tumours/animal (MeanGSD)

*, **: P!0.05, 0.01 vs. Group 2, respectively. a AH, atypical hyperplasia; ADC, adenocarcinoma. Table 3 Cell proliferative activity in pancreatic atypical hyperplasias and normal ducts Group

Atypical hyperplasia

Normal ducts

No. of the lesions examined

BrdU-LI (%)

No.of the ducts examined

BrdU-LI (%)

1. Saline 2. BOP 3. BOPCBITC/Basal diet 4. BOP/BITC 5. BOPCSFN/Basal diet 6. BOP/SFN 7. BOPCRES/Basal diet 8. BOP/RES

N.P. 62 17 43 33 51 44 42

N.P. 17.51G9.93 17.79G11.54 18.25G10.53 15.85G13.54 19.27G10.83 17.35G13.86 18.82G10.02

10 10 10 10 10 10 10 10

4.32G2.83 13.85G6.80## 8.85G5.69 9.20G6.04 6.88G3.44** 7.05G1.69** 8.52G2.79* 10.25G6.21

##: P!0.01 vs. Group 1. *, **: P!0.05, P!0.01 vs. Group 2, respectively. N.P.: Not present.

of hepatocellular or cholangiocellular origin, lung adenomas and adenocarcinomas, and renal mesenchymal tumours were sporadically detected, but no significant intergroup variation was noted (Table 4).

4. Discussion In the present study, suppression of body weight and increase of relative kidney weights was observed

Table 4 Incidences of proliferative lesions in the liver, lungs and kidneys Organ

Group

1

2

3

4

5

6

7

8

Lesion

Effective no. of animals

10

20

20

20

20

20

20

20

0 0

4 0

3 0

2 0

1 0

4 1

0 1

3 1

0

0

0

0

0

1

0

0

0 0 0

7 2 2

3 0 0

5 1 2

5 4 1

5 1 1

5 4 1

3 3 1

0 0

0 1

1 1

0 0

0 0

0 0

0 0

0 1

Liver Cholangiocellular Atypical hyperplasia Adenoma Hepatocellular Adenoma Lung Hyperplasia Adenoma Adenocarcinoma Kidney Atypical tubular hyperplasia Mesenchymal tumour

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in all BOP treated groups, but these changes were not affected by the additional treatments. Statistically significant differences in relative organ weights were also only sporadically found. The incidence of tumours in the group treated with BOP alone was basically same as that in our previous study [29] and multiplicity was only slightly. Therefore, reproducibility of the 16-week hamster model was confirmed. In the present study, animals administered BITC or SFN simultaneously with BOP, demonstrated decreased incidences of atypical hyperplasias in pancreatic ducts, as compared with animals receiving BOP alone, and SFN also decreased the incidence and multiplicity of adenocarcinomas. However, with administration in the post-initiation phase, only a tendency for decrease in the multiplicity of neoplastic lesions was noted, without statistical significance. The results are consistent with our previous report that another ITC, PEITC, suppresses pancreatic carcinogenesis in hamsters only when it was given in the initiation-phase [16,27]. It is thought that chemopreventive effects of ITCs could be mainly associated with inhibition of the metabolic activation of carcinogens by cytochrome P450s (Phase I enzymes), coupled with strong induction of Phase II detoxifying and cellular defensive enzymes regulated by the transcriptional factor, Nrf2 [32,33]. It is thus consistent that BITC and SFN had influence only in the initiation phase. Regarding cell proliferative activity, the rise in the BrdULI in the epithelial cells of normal pancreatic ducts due to BOP treatment was decreased with BITC or SFN given not only in the initiation phase but also the post-initiation phase, in line with their anti-proliferative activity in human pancreatic cancer cells [19,20]. However, antitumor effects of BITC and SFN may not depend on suppression of cell proliferative activity and no effects were noted on atypical hyperplasias as precancerous lesions in the present study, possibly because the dose were relatively low. While previously reported PEITC in the initiation phase to suppress lung tumorigenesis in hamsters induced by BOP [16], no such modifying effects were here found in the liver, lungs or kidneys. Our recent study demonstrated that the COX-2 inhibitor, nimesulide substantially protects against BOP-induced pancreatic tumours in hamsters in line with decreased cell proliferative activity of pancreatic ducts when given in the post-initiation (promotion) phase of carcinogenesis in both 52 and 16 week models [18,25,29]. However, in the present study, another COX2 inhibitor, RES failed to show any effects on the development of atypical hyperplasias or adenocarcinomas when given in the initiation or post-initiation phases. This is unlikely to be due to a lowered detection

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ability of the 16-week model, given the findings for nimesulide. Additionally, nimesulide reduced the elevation in proliferating cell nuclear antigen (PCNA)labelling caused BOP in pancreatic ducts [25], whereas RES had only marginal effects. Selective COX-2 inhibitors are known to prevent growth of cancer cells [34]. So that the dose of RES applied might have been insufficient for protection, although it did suppress mammary carcinogenesity in rats [30]. Recently, nitric oxide (NO)-donating nonsteroidal anti-inflammatory drugs (NSAIDs) have attracted attention as a new type of NSAIDs other than selective COX-2 inhibitors for cancer prevention [35]. NO-donating aspirin was found to inhibit the growth of various cultured human cancer cells including a pancreatic cancer line [36]. Thus, NOdonating NSAIDs may be of interest as candidate chemopreventive agents against pancreatic cancer. In conclusion, our data suggest that BITC and SFN, naturally occurring ITCs, can inhibit initiation of BOPinduced pancreatic carcinogenesis. Additionally, the results provide further evidence in support of the efficacy of our medium-term model of pancreatic carcinogenesis in hamsters for detecting modifying factors on pancreatic carcinogenesis. Acknowledgements We thank Ms Maeda, M and Kaneko, A for expert technical assistance. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare, Japan. References [1] M. Yamamoto, O. Ohashi, Y. Saitho, Japan pancreatic cancer registry: current status, Pancreas 16 (1998) 238–242. [2] J. Stephans, J. Kuhn, J. O’Brien, J. Preskitt, H. Derrick, T. Fisher, et al., Surgical morbidity, mortality, and long-term survival in patients with peripancreatic cancer following pancreaticoduodenectomy, Am. J. Surg. 174 (1997) 600–603. [3] G.W. Olsen, J.S. Mandel, R.W. Gibson, L.W. Wattenberg, L.M. Schuman, A case-control study of pancreatic cancer and cigarettes, alcohol, coffee and diet, Am. J. Public Health 79 (1989) 1016–1019. [4] P.A. Baghurst, A.J. McMichael, A.H. Slavotinek, K.I. Baghurst, P. Boyle, A.M. Walker, A case-control study of diet and cancer of the pancreas, Am. J. Epidemiol. 134 (1991) 167–179. [5] H.B. Bueno De Mesquita, P. Maisonneuve, S. Runia, C.J. Moerman, Intake of foods and nutrients and cancer of the exocrine pancreas: a population-based case-control study in the Netherlands, Int. J. Cancer 48 (1991) 540–549. [6] D.T. Silverman, C.A. Swanson, G. Gridley, S. Wacholder, R.S. Greenberg, L.M. Brown, et al., Dietary and nutritional factors and pancreatic cancer: a case-control study based on direct interviews, J. Natl Cancer Inst. 90 (1998) 1710–1719.

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