Chemopreventive effect of S-methylmethane thiosulfonate and sulindac administered together during the promotion/progression stages of colon carcinogenesis

Bandaru S. Reddy2, Toshihiko Kawamori, Ronald Lubet1, Vernon Steele1, Gary Kelloff1 and Chinthalapally V. Rao

Division of Nutritional Carcinogenesis, American Health Foundation, Valhalla, NY 10595 and
1 Chemoprevention Branch, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892, USA


    Abstract
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 Abstract
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S-methylmethane thiosulfonate (S-MMTS), isolated from cauliflower and having antiproliferative activity, and the non-steroidal anti-inflammatory drug sulindac have been shown to inhibit chemically induced colon carcinogenesis when they are administered during the initiation and/ or post-initiation stages. The present study was designed to investigate the chemopreventive efficacy of 80 p.p.m. S-MMTS administered during the initiation and post-initiation stages and of S-MMTS and sulindac administered together at low doses (40 and 160 p.p.m., respectively) during the promotion/progression phases (late in the premalignant stage) of colon carcinogenesis. At 5 weeks of age, groups of male F344 rats were fed diets containing 0 (control diet) or 80 p.p.m. S-MMTS. At 7 and 8 weeks of age all rats except those in the vehicle-treated groups were given s.c. injections of 15 mg/kg body wt azoxymethane (AOM). Rats receiving the control diet and intended for the study of inhibition of colon carcinogenesis during the promotion/progression phases were continued on the control diet for 14 weeks after the second AOM treatment; they were then switched to experimental diets containing 80 p.p.m. S-MMTS, 160 p.p.m. sulindac or 40 p.p.m. S-MMTS plus 160 p.p.m. sulindac. The rats were maintained on their respective dietary regimens until 52 weeks after carcinogen treatment and were then killed. Colon tumors were evaluated histopathologically. Administration of 80 p.p.m. S-MMTS alone during the initiation and post-initiation stages and promotion/progression stages had no significant effect on colon tumor inhibition. In contrast, the administration of 160 p.p.m. sulindac during the promotion/progression stages did significantly inhibit total colon tumor multiplicity (P < 0.05). Moreover, co-administration of 40 p.p.m. S-MMTS with 160 p.p.m. sulindac during the promotion/progression stages suppressed the incidence and multiplicity of non-invasive adenocarcinomas (P < 0.05–0.01) and multiplicity of invasive and total adenocarcinomas of the colon to a significant degree (P < 0.05–0.01). These findings have potential clinical implications.

Abbreviations: AOM, azoxymethane; MTD, maximum tolerated dose; NSAIDs, non-steroidal anti-inflammatory drugs; S-MMTS, S-methylmethane thiosulfonate.


    Introduction
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 Abstract
 Introduction
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Colorectal cancer is one of the leading causes of cancer death in both men and women in western countries, including North America; it accounts for ~56 000 deaths annually in the USA (1). There is increasing evidence that increased consumption of fruits and vegetables, including green and yellow vegetables, and intake of certain non-nutritive food components reduce the risk of colon cancer (2,3). Recently, S-methylmethane thiosulfonate (S-MMTS), isolated from cauliflower (4), has been shown to inhibit colon tumor incidence when administered to F344 rats during the post-initiation phase of carcinogenesis (5). Although the exact mechanism by which S-MMTS exerts its anticarcinogenic properties remains largely unknown, inhibition was associated with the suppression of several cell proliferation biomarkers, including ornithine decarboxylase (5). Epidemiological studies indicate that chronic ingestion of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, appears to reduce the risk of colorectal cancer and adenomatous polyps (6,7). Laboratory research shows that NSAIDs such as aspirin, ibuprofen, piroxicam and sulindac protect against chemically induced colon carcinogenesis (811) as well as against spontaneous intestinal tumors in animal models (12,13). Studies in our laboratory have demonstrated that sulindac and piroxicam, given late but still during the premalignant stage (promotion/progression), significantly inhibit azoxymethane (AOM)-induced colon carcinogenesis (9,10); this is in line with a study in familial adenomatous polyposis patients who showed regression of colonic polyps upon administration of sulindac (14). Inhibition of colon carcinogenesis by NSAIDs is associated with modulation of endogenous prostaglandin synthesis, which plays a role in the control of neoplastic and non-neoplastic cell proliferation and of immune function and/or induction of apoptosis in colon tumors (12,15).

The above studies provide evidence that sulindac and S-MMTS inhibit colon carcinogenesis by different mechanisms. There is increasing interest in the use of combinations of low doses of chemopreventive agents that differ in their mode of action, rather than administering single agents, as a means of obtaining increased efficacy and minimized toxicity. The present study was therefore designed to evaluate whether low dose levels of S-MMTS and sulindac administered together were more effective than either agent alone during the promotion/progression stage of colon carcinogenesis.

AOM (CAS no. 25843-45-2) was purchased from Ash Stevens (Detroit, MI). S-MMTS (>98% pure) and sulindac (>98% pure) (Figure 1Go) were provided by the National Cancer Institute through the DCP Repository, McKesson Bioservices (Rockville, MD). Weanling male F344 rats were obtained from the Charles River Breeding Laboratories (Kingston, NY). The ingredients of semi-purified diet were purchased from Dyets Inc. (Bethlehem, PA) and stored at 4°C prior to preparation of AIN-76 diet and the experimental diets, containing sulindac and S-MMTS individually and in combination. The modified AIN-76A diet consisted of 20% casein, 0.3% DL-methionine, 52% corn starch, 13% dextrose, 5% corn oil, 5% alphacel, 3.5% AIN mineral mixture, 1% AIN vitamin mixture and 0.2% choline bitartrate (9). All control and experimental diets containing sulindac and S-MMTS were prepared weekly in our laboratory and stored in a cold room. Before feeding the experimental diets, purity and stability of the test agents was established. The purity of S-MMTS was analyzed by gas chromatography using a 3% OV-17 chromsorb column and that of sulindac was determined with HPLC by previously published methods (9). The purity of these agents was found to be >98%. Both agents were quite stable at room temperature and at 4°C. When experimental diets stored for 14 days in a cold room were analyzed for S-MMTS and sulindac, the recovery of these agents from the diets reached >95%.



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Fig. 1. Structures of S-MMTS and sulindac.

 
Prior to initiation of the efficacy study, the maximum tolerated dose (MTD) of S-MMTS was determined in male F344 rats. The MTD is defined as the highest dose that causes <=10% body weight decrement compared with the appropriate control diet group and does not produce mortality or any clinical signs of toxicity. At 5 weeks of age, groups of male F344 rats (six in each group) were fed the AIN-76A diet containing 0 and 100, 200, 250, 300 or 500 p.p.m. S-MMTS. Body weights were recorded once weekly for 8 weeks. The results of this short-term study suggested that the MTD of S-MMTS is ~100 p.p.m. (data not shown). The MTD of sulindac evaluated previously in our laboratory was 400 p.p.m. (16).

The experimental methods for the efficacy study were as described previously (8,9). Weanling male F344 rats were quarantined for 1 week and had free access to AIN-76A diet. As shown in Figure 2Go, beginning at 5 weeks of age, groups of animals in the initiation and post-initiation study had free access to their respective control and experimental diets containing 0 and 80 p.p.m. S-MMTS. The rats in the assays testing efficacy of these agents during the promotion/ progression stages were initially fed the control diet. Beginning at 7 weeks of age, the rats intended for carcinogen treatment were s.c. injected with AOM at a dose rate of 15 mg/kg body wt once weekly for 2 weeks; rats intended for vehicle treatment received an equal volume of normal saline. Starting 14 weeks after the second AOM treatment, groups of animals designated for intervention during the promotion and progression stages were transferred from control diet to the experimental diets containing 80 p.p.m. S-MMTS, 160 p.p.m. sulindac or 40 p.p.m. S-MMTS together with 160 p.p.m. sulindac. These dietary regimens were continued until termination of the experiment 52 weeks after the second AOM treatment. Body weights were recorded every week for the first 10 weeks and then every 4–6 weeks. As scheduled, all rats were killed by CO2 euthanasia. Colons were fixed in 10% buffered formalin, embedded in paraffin blocks and processed by routine procedures with hematoxylin and eosin staining. The sections were examined for tumor type according to the classification which is routinely used in our laboratory (8). Upon termination of this study, >90% of the colon tumors in the AOM-treated animals fed the control diet had developed into adenocarcinomas. All adenocarcinomas were classified as invasive or non-invasive. The non-invasive adenocarcinomas of the colon were those growing out of the mucosal layers into the intestinal lumen, whereas invasive adenocarcinomas were those penetrating the muscularis mucosa deeply into the intestinal wall and beyond (8). Body weights, colon tumor incidence (percentage of animals with tumors) and multiplicity (mean no. tumors/animal) were compared between the animals fed the control diet and those given experimental diets containing sulindac or S-MMTS or a combination of sulindac and S-MMTS using the {chi}2 test or t-test.



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Fig. 2. Experimental design for evaluation of chemoprevention efficacy of S-MMTS and sulindac against colon carcinogenesis. In the continuous feeding study (initiation and promotion stages), groups of male F344 rats were fed experimental diets containing 0 or 80 p.p.m. S-MMTS for 2 weeks prior to exposure to AOM, during treatment and until termination. For the promotion/progression study additional groups of animals who were on the control diet for 2 weeks prior to exposure of AOM, during treatment and until 14 weeks after AOM treatment were transferred to experimental diets containing 80 p.p.m. S-MMTS, 160 p.p.m. sulindac or 40 p.p.m. S-MMTS plus 160 p.p.m. sulindac and were on this regimen until termination. AOM was given to the animals s.c. at 7 and 8 weeks of age at 15 mg/kg body wt.

 
The body weights of all animals receiving the experimental diet with S-MMTS beginning 2 weeks prior to or during and after carcinogen treatment until termination of the study (initiation and post-initiation stages) and those fed the diets containing S-MMTS and/or sulindac beginning 14 weeks after carcinogen treatment until the end of the study (promotion/progression stages) were comparable with weights of those fed the control diet (data not shown). In vehicle-treated rats fed the experimental diets containing S-MMTS or sulindac there were no gross changes in any organs that would be attributable to toxicity of these agents.

The summary of the results in Table IGo indicates that administration of AOM induced colon adenomas in ~9% of rats and adenocarcinomas in 82% of animals fed the control diet. Because of the long-term nature of this study, most of the colon tumors had become adenocarcinomas. No tumors were found in vehicle-treated animals. Administration of 80 p.p.m. S-MMTS (80% MTD) during the initiation and post-initiation periods and during the promotion/progression stages had no significant effect on colon tumor inhibition, although the percentage of inhibition of non-invasive and invasive adenocarcinomas reached ~26–46%. Administration of 160 p.p.m. (40% MTD) sulindac during the promotion/progression stages significantly inhibited total colon tumor multiplicity (34% inhibition, P < 0.05). Incidence and multiplicity of invasive adenocarcinomas were reduced by 25 and 33%, respectively, when sulindac was given during the promotion/progression stages. It is noteworthy that administration of 40 p.p.m. S-MMTS (40% MTD) together with 160 p.p.m. sulindac (40% MTD) during the promotion/progression stages significantly inhibited the incidence of non-invasive adenocarcinomas of the colon (59% inhibition, P < 0.05) and also the multiplicity of non-invasive (71% inhibition, P < 0.01), invasive (39% inhibition, P < 0.05) and total adenocarcinomas of the colon (48% inhibition, P < 0.01).


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Table I. Chemopreventive efficacy of S-MMTS and sulindac administered individually and in combination during the promotion/progression stages of AOM-induced colon carcinogenesis
 
Evaluation of agents with different modes of chemopreventive action is of considerable interest because their testing can pave the way for the use of a most effective combination of agents, whose aggregate chemopreventive action would be significant while toxicity would be minimal. This study demonstrates for the first time that the NSAID sulindac and naturally occurring S-MMTS, administered together at 40% MTD levels during the promotion/progression period, increased the chemopreventive efficacy against tumorigenesis in the colon, as compared with the results when these agents were given individually at 40% MTD levels. The present results support our earlier observations on the co-administration of piroxicam and difluoromethylornithine, which also differ in their modes of action and when administered together induce greater inhibition of colon tumorigenesis than is achieved with individual doses of these agents (16). Our findings in this and in the previous study (16) have important clinical implications since they suggest potential usefulness of co-administration of these agents at low doses for individuals who are at high risk of colon cancer development, such as patients with polyps. In addition, our observations suggest that various combinations of agents should also be investigated in other tumor types.

With regard to the mode of chemopreventive action of sulindac, this agent not only inhibits prostanoid synthesis by acting on cyclooxygenase, but also modulates the activities of lipoxygenase and arachidonic acid uptake, NADPH oxidase and oxidative phosphorylation, which are known to play a role in inflammation and cell proliferation (17,19). In addition, the mechanism of action of sulindac also involves the restoration of apoptosis (12,20,21). S-MMTS has been shown to decrease colonic mucosal ornithine decarboxylase activity, a rate-limiting enzyme involved in polyamine biosynthesis and cell proliferation (5). Thus, the chemopreventive effect of S-MMTS and sulindac may be mediated through the modulation of arachidonic acid metabolism, polyamine biosynthesis, cell proliferation and apoptosis.

In conclusion, the present study demonstrates for the first time that co-administration of S-MMTS and sulindac during the promotion/progression stages significantly inhibits AOM-induced colon tumor development. It is important to exploit the potential efficacy of combinations of chemopreventive agents in human clinical trials.


    Acknowledgments
 
We thank Laura Nast for the preparation of this manuscript and Ilse Hoffmann for editorial assistance and the staff of the Research Animal Facility and Histopathology Facility for expert technical assistance. This work was supported by USPHS grants CA-17613 and CN-55150 awarded by the National Cancer Institute.


    Notes
 
2 To whom correspondence should be addressed Email: b_reddy{at}ix.netcom.com Back


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Received January 12, 1999; revised April 2, 1999; accepted April 12, 1999.