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Clinical Cancer Research 13, 7146, December 1, 2007. doi: 10.1158/1078-0432.CCR-07-0823
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Nutritional Modulation of Antitumor Efficacy and Diarrhea Toxicity Related to Irinotecan Chemotherapy in Rats Bearing the Ward Colon Tumor

Hongyu Xue1, Michael B. Sawyer1, Catherine J. Field2, Levinus A. Dieleman3 and Vickie E. Baracos1

Authors' Affiliations: Departments of 1 Oncology and 2 Agriculture, Food, and Nutritional Science, and 3 The Center of Excellence for Gastrointestinal Inflammation and Immunity Research, University of Alberta, Edmonton, Alberta, Canada

Requests for reprints: Vickie E. Baracos, Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2. Phone: 780-432-8232; E-mail: vickieb{at}cancerboard.ab.ca.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: To evaluate and compare the influence of dietary elements on cancer progression, chemotherapy efficacy, and toxicity, particularly severe, late-onset diarrhea related to irinotecan (CPT-11) treatment.

Experimental Design: We used laboratory rats fed a standardized basal diet, Ward colon tumor, and CPT-11 therapy for the study of CPT-11–induced diarrhea. Dietary interventions were selected from nutrients already established to modify other forms of colitis and which have been hypothesized to mitigate chemotherapy-induced gastrointestinal injury (glutamine, n-3 fatty acids, prebiotic oligosaccharides). Animals adapted to test diets were treated with CPT-11 at the maximum tolerated dose (125 mg/kg x 3 days) and diarrhea was followed continuously for 1 week.

Results: The inclusion of n-3 fatty acids in the diet (5%, w/w of total fat) suppressed tumor growth and enhanced CPT-11's efficacy; this treatment did not affect the incidence or severity of diarrhea. By contrast, oral glutamine bolus (0.75 g/kg) administered prior to each CPT-11 treatment reduced the incidence of severe diarrhea (34.1 ± 4.7% versus 53.8 ± 4.2%, P < 0.005) and decreased the area under the curve of diarrhea score (16.5 ± 1.0 versus 18.8 ± 0.5, P < 0.05). Identical results were obtained with i.v. bolus glutamine administration. Glutamine treatment did not alter CPT-11's antitumor efficacy. The addition of prebiotic oligosaccharides to the diet (8%, w/w of diet) did not mitigate the severity of diarrhea, and it raised the activity of β-glucuronidase in cecal contents, a key bacterial enzyme mediating CPT-11–related intestinal toxicity.

Conclusion: Our experiments suggest that glutamine and n-3 fatty acids might be potentially useful adjuncts to CPT-11 treatment.

Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin, CPT-11, Camptosar) is a semisynthetic derivative of camptothecin, a plant alkaloid. CPT-11 has emerged as a first-line drug for colorectal cancer and has also been shown to be effective in other malignancies (1, 2). The use of CPT-11 is mainly limited by its gastrointestinal toxicity, with diarrhea as its dose-limiting toxicity as a single agent. CPT-11 is converted by carboxylesterase to SN-38, the active metabolite responsible for both toxicity and antitumor activity (3). SN-38 is metabolized to a nontoxic glucuronide (SN38G) prior to biliary excretion; however, β-glucuronidase produced by the intestinal microflora, which reconverts SN-38G to SN38, is believed to play a critical role in mediating SN-38–related gut toxicity (3, 4).

One approach to improving the therapeutic index of a chemotherapeutic agent is to combine adjuvant factors to enhance antitumor efficacy and/or to reduce toxicities. Specific dietary elements have been attracting attention in this context, including the amino acid glutamine, n-3 polyunsaturated fatty acids (PUFA) eicosapentaneoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3) and prebiotic oligosaccharides (i.e., ref. 5). Prebiotic is the term used to define nondigestible fermentable food ingredients which stimulate the growth of selected intestinal bacteria (i.e., lactobacilli, bifidobacteria), that are important to the host's health (6). These nutrients have been clearly established to mitigate inflammation and tissue injury in chronic inflammatory bowel disease (7–9) and have been proposed to be potential modulators of gut injury related to cancer chemotherapy (10–15). Various mechanisms of these effects have been proposed as dietary influences may be exerted on multiple levels, including the physiology of cells and organs, signal transduction within cells, and the endocrine, immune, and gastrointestinal systems. However, little is known about the potential capacity of these dietary factors to modulate CPT-11–induced diarrhea. Prebiotics have attracted attention for managing diarrheas in other intestinal injuries that are related to the disruption of intestinal flora balance. In the specific case of CPT-11, it is unknown how prebiotic oligosaccharides may alter intestinal flora composition and the level of β-glucuronidase produced by intestinal bacteria, thereby influencing SN-38G metabolism.

The effects of diet on tumors and the host response to therapies remain poorly defined. The existing literature is hard to interpret due to a lack of a systematic approach. For any specific nutrient, the available literature encompasses diverse animal models, therapies, nutrient levels and basal (background) diets, making it quite daunting to perform a systematic interpretation that could be applied to humans. Almost all studies examined a single nutrient or nutrient class, so that the relative efficacy of different nutrients remains completely unknown. The application of clinically relevant antineoplastic therapy within a nutritionally relevant and controlled dietary design is required to move understanding forward in this area. The objective of our study was to comparatively assess the ability of glutamine, n-3 PUFAs, and prebiotic oligosaccharides to alter CPT-11's efficacy and to mitigate its dose-limiting toxicity, diarrhea. Emerging debates have revolved around the scheduling and routes of glutamine administration (16, 17), and we further compared glutamine incorporation in the diet with oral and i.v. bolus administration.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Features of the model system. The experimental system included an implanted tumor, CPT-11 chemotherapy [maximum tolerated dose as described by Cao et al. (18)], and specifically formulated basal diet and diet treatments. We selected CPT-11 chemotherapy in rats bearing the Ward colon tumor (18), which is particularly applicable for the study of severe late-onset diarrhea, the clinically relevant dose-limiting toxicity. The kinetics of acute and delayed diarrhea are clearly observed in the rat and is consistent with those observed in patients treated with CPT-11 (19). Tumors were s.c. introduced to enable the assessment of rate of tumor growth.

Semipurified diets permit the definition of the lipid, protein, and carbohydrate constituents of the diet. Our conception of dietary design in animal models is spelled out in detail in our recent article (20). In brief, diets are formulated to meet or exceed the nutrient requirements of laboratory rats and are based on the American Institute of Nutrition-76 modified basal diet with 40% of calories from fat. The modified fat component is formulated to be similar to typical North American dietary patterns in humans (40% of calories, polyunsaturated/saturated fat ratio of 0.35; Table 1 ; ref. 21). CPT-11 toxicity in this dietary background is similar to what has been reported for chow-fed animals.


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  		Table 1. Composition of the experimental diets

 
Savarese et al. (10) reviewed the possible prevention of chemotherapy toxicity by glutamine. Because several recent studies have shown that glutamine bolus treatment rescued rats from lethal endotoxin shock or hyperthermic stress (22, 23), we opted for the bolus dose used in those investigations (0.75 g/kg/d). Other experimental treatments were incorporated in the diet (Table 1). A glutamine-containing diet (2%, w/w) was initiated 7 days prior to the first dose of CPT-11 and continued thereafter; this was calculated to provide an identical daily amount of glutamine as was provided in the bolus. Control diets contained 1.1% (w/w) of total fatty acids as n-3 and met the essential fatty acid requirements of laboratory rats. Marine fish oil was used to formulate a diet containing 5% (w/w) total fatty acids as n-3; including 3.2% eicosapentaneoic acid and 0.8% docosahexaenoic acid (Table 1). To allow for adequate incorporation of n-3 PUFAs into plasma membranes, diet treatment was started 14 days prior to tumor implantation and continued thereafter. We also studied a chicory-derived prebiotic mixture consisting of inulin and oligofructose (1:1 w/w) at doses that reduced chronic colitis in HLA-B27 transgenic rats (24). Prebiotic treatment was initiated 14 days prior to tumor implantation, which is sufficiently long to increase beneficial flora, such as bifidobacteria and lactobacilli (25, 26), and was continued thereafter.

Animals and tumors. Animal use was reviewed and approved by the Institutional Animal Care Committee and conducted in accordance with the Guidelines of the Canadian Council on Animal Care. Female Fisher 344 rats (body weight, 150-180 g), 11 to 12 weeks of age, were obtained from Charles River. Rats were housed two per cage under aseptic conditions (positive air–pressured room, cages, bedding, and filter tops; handling under a laminar flow hood) in a temperature-controlled (22°C), and light-controlled (12 h light) room; water and food were available for ad libitum consumption. One week before chemotherapy, rats were separated into individual housing in wire-bottomed cages. The Ward colorectal carcinoma was provided by Dr. Y. Rustum (Department of Cancer Biology, Chair, Director of Institute Core Resources), Roswell Park Cancer Institute, Buffalo, NY (18). Tumor pieces (0.05 g) were transplanted s.c. into the left flank of the rats via trocar under slight isoflurane anesthesia. A major consideration in selecting this tumor site was to facilitate the continuous evaluation of tumor growth and response to CPT-11 treatment.

Drugs. CPT-11 was provided by Pfizer as a ready-to-use clinical formulation (20 mg/mL). Atropine (0.6 mg/mL), obtained from the hospital pharmacy, was in a clinically injectable formulation.

Experimental design. Acclimation to the semipurified diets was initially with laboratory rodent chow blended with our control diet (50:50 w/w) for 1 week, followed by allocations to the test diets (control, fish oil, prebiotic, glutamine). When tumors reached ~2.0 cm3, CPT-11 injections were initiated. The day when the first dose of CPT-11 was administered was designated as day 0.

Determination of the maximum tolerated CPT-11 dose. Tumor-bearing rats fed the control diet were randomized into four groups, each of which received three consecutive daily i.v. CPT-11 injections at 75 (n = 5), 100 (n = 9), 125 (n = 21), and 150 (n = 7) mg/kg/d. Seven days after the last dose of CPT-11 (day 9), rats were killed.

Study on the dietary modulation of the antitumor efficacy and toxicity related to the 3-day CPT-11 treatment at maximal tolerated dose. Tumor-bearing rats were fed control (CON), fish oil–enriched (FO), glutamine-enriched (GLN), and prebiotic-enriched (PRE) diets described in Table 1. In addition to the diet, each animal was given a bolus treatment (glutamine or sham). Bolus treatments were given by oral gavage or i.v. infusion, and consisted of glutamine (Sigma-Aldrich) or the same solution (vehicle) only. Rats treated with oral or i.v. bolus glutamine were fed the control diet throughout the study. Glutamine oral bolus (0.75 g/kg) was administered by gavage 30 min before each daily CPT-11 injection, whereas rats from other groups received isovolemic water as sham. Oral glutamine 3% (w/v) solution was made immediately before use, and was filtered with a 0.45-µm filter. Glutamine i.v. bolus (0.75 g/kg) was prepared immediately before use as a 3% (w/v) solution in lactated Ringer solution and filtered with a 0.45-µm filter. Intravenous bolus glutamine was administered via the lateral tail vein of anesthetized rats at a rate of 0.5 mL/min, immediately before each daily chemotherapy. All three modes of glutamine delivery (diet, i.v., and gavage) provided a total daily dose of 0.75 g/kg/d.

Eleven rats were allocated to each experimental treatment. At day 9, rats were killed and tissues/organs were collected. A separate set of animals (five from the control, and six from the oral bolus glutamine-treatment) were killed 6 h after the last injection of CPT-11. Colonic mucosa, tumor, and whole blood were harvested from these animals for assay of glutamine levels.

Outcome measures. Diarrhea was scored as 0—normal, normal stool or absent; 1—slight, slightly wet and soft stool with mild perianal staining; 2—moderate, wet and unformed stool with moderate perianal staining of the coat; and 3—severe, watery stool with severe perianal staining of the coat (27). Diarrhea was scored twice daily until day 7 and daily thereafter. Data are presented as incidence of diarrhea grade 3, total incidence of diarrhea grades 2 and 3 (27), and area under the curve of diarrhea score. The incidence of delayed diarrhea was calculated for each animal by counting the observations of a particular score(s) out of the total eight observations between days 3 and 7 when diarrhea developed to its full severity (27). The area under the curve of diarrhea score was calculated from the diarrhea score-time graph of each individual animal between days 3 and 7. All diarrhea severity assessments were conducted by one person who was blinded to experimental treatments.

Body weight was monitored daily. Tumors were measured at time points indicated in the figures, in three dimensions with a caliper, the length (L), width (W), and height (H). Tumor volume was calculated according to the following equation: tumor volume (cm3) = 0.5 x L (cm) x W (cm) x H (cm; ref. 28). Tumor volume doubling times were calculated as described (20). The relative tumor volume (R) for each tumor was calculated relative to its volume at the start of chemotherapy. The interaction between the diet and CPT-11 was expressed as a ratio of the relative tumor values between a given tumor (R) and the mean of all the control tumors (RCm), the formula of which is (R/RCm x 100%). Tumor growth inhibition was expressed as 1 – (R/RCm x 100%). When assessing the effects of diet alone on tumor growth, absolute volume value (V) was used in place of R because equal amounts of tumor tissue were assumed to be implanted into each animal.

Animals were killed by carbon dioxide asphyxiation followed immediately by exsanguination by cardiac puncture. Aliquots of heparinized whole blood were centrifuged at 4°C at 3,000 x g for 15 min. The plasma was removed and frozen at –70°C for later assessment of amino acid and fatty acid profiles. Tumor and tibialis anterior muscles were collected, weighed, and then immediately frozen in liquid nitrogen. The full length of the colon (after the ceco-colonic junction) was longitudinally cut into two halves, one of which was mounted on a wax strip and fixed in 10% (v/v) neutral buffered formalin. Cecal content, which was collected in aseptic conditions for β-glucuronidase assay, as well as mucosal tissue scraped off from the first 6-cm section of the other half of the proximal colon, were collected and immediately frozen in liquid nitrogen.

Glutamine/glutamate concentrations were determined using high-performance liquid chromatography. Amino acids were converted to their o-phthaldialdehyde derivatives using established methods (29). Calibration was done for every 10 samples using a commercially prepared standard. All samples were run in duplicate and averaged.

Plasma triglycerides were separated and fatty acid methyl esters were prepared from the scraped silica band using 14% (w/v) BF3/methanol reagent and separated by automated gas liquid chromatography (Varian CP 3800; Varian Instruments) on a fused silica BP20 capillary column (25 m x 0.25 mm internal diameter; Varian Instruments), as previously described (30).

The β-glucuronidase activity of cecal contents was determined by a modified method of Freeman (31). Briefly, samples were mechanically homogenized in 0.1 mol/L of potassium phosphate buffer (pH 6.8) and centrifuged to obtain supernatant fractions. A reaction mixture containing 0.02 mol/L of KPBS, 0.1 mmol/L of EDTA, 1 mmol/L of phenolphthalein-β-glucuronide, and 100 µg of total protein per microliter of homogenate was incubated for 30 min at 37°C and then stopped with three volumes of 0.2 mol/L of glycine buffer (pH 10.4). The liberated phenolphthalein was measured at 540 nm. β-Glucuronidase activity was expressed per gram of protein and 1 unit was defined as 1.0 g of phenolphthalein liberated from phenolphthalein glucuronide (pH 6.8) per hour at 37°C.

Formalin-fixed colon tissue was embedded in paraffin wax, sectioned, and stained with H&E for histopathologic examination (5). The sections were viewed by the same individual who was blinded to the treatment. All images were acquired under 200x magnification with MetaMorph 6.0 (Universal Imaging).

Data are expressed as mean ± SE. Statistical analyses were done using GraphPad Prism (GraphPad Inc.). Differences among groups were tested using one-way or two-way ANOVA on the effect of drug and/or diet. P < 0.05 was accepted as being statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Defining efficacy/toxicity profile of the CPT-11 regimen. We used diarrhea, the dose-limiting toxicity for CPT-11–based regimens, as the major end point. CPT-11 induced both early-onset diarrhea (within 12 h of each CPT-11 injection) as well as delayed diarrhea, which was of much greater intensity than early-onset diarrhea and which began 12 h after the third dose (Fig. 1A ). Early-onset diarrhea was transitory and could be largely alleviated by atropine (1 mg/kg s.c. injection immediately before each CPT-11 injection). Atropine did not affect the time course or severity of late-onset diarrhea (data not shown) and was given as a standard prophylactic treatment in the main study.


Figure 1
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  		Fig. 1. Antitumor activity and diarrhea toxicity related to CPT-11 treatment. A, time course of CPT-11-induced diarrhea. Top, tumor-bearing rats on the control diet received three daily i.v. injections of CPT-11 at 125 mg/kg/d. Diarrhea was scored twice per day between days 0 and 7 and once per day afterwards. Black arrows, single CPT-11 injections at 125 mg/kg. Triangles, time points when histologic examinations (bottom) were done, corresponding with the diarrhea dynamics. Bottom, photomicrographs of H&E-stained colonic sections (left to right) from rats killed before CPT-11 administration, 6 h, and 7 d (left to right) after the last dose of CPT-11 (125 mg/kg/d x 3 d). Magnification, x200. B, dose-dependent antitumor activity of the 3-d CPT-11 regimen. Tumor-bearing rats on the control diet received three daily i.v. injections of CPT-11 at ({blacksquare}) 75, ({blacktriangleup}) 100, ({blacktriangledown}) 125, ({blacklozenge}) and 150 mg/kg/d. Black arrows, single injection of CPT-11 at the specified dose. Y-axis, the relative tumor volume as compared to the baseline volume when CPT-11 therapy was initiated. Mean tumor values were compared at the indicated time points between treatments at various doses, and means at a certain time point that don't share a common symbol are significantly different (P < 0.05).

 
The effects of CPT-11 were dose-dependent (Fig. 1B and Table 2 ). A dose of 125 mg/kg/d produced severe but self-limiting delayed diarrhea with a minimal mortality rate consistent with clinical data; the 100 mg/kg/d dose did not consistently produce severe diarrhea, whereas the 150 mg/kg/d dose was associated with 43% mortality. Therefore, the maximum tolerated CPT-11 dose for the 3-day regimen was defined as 125 mg/kg. This dose provided the baseline profiles of antitumor efficacy and toxicity, on which the effects of different dietary treatments were tested.


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  		Table 2. Dose dependent toxicity profiles associated with 3-d CPT-11 regimen

 
Effects of diet treatments. CPT-11 treatment led to a small number of animal deaths (one from control, two from prebiotic-enriched, and one from fish oil–enriched diets), comparable with the mortality in the preliminary work.

The levels of glutamine and its immediate metabolite, glutamate, were assessed and because these amino acids are relatively rapidly metabolized, these observations were made 6 h after glutamine administration. Oral bolus glutamine treatment significantly raised glutamine levels in plasma (Table 3 ). Levels of glutamate were elevated in colonic mucosa, as would be expected due to the high glutaminase activity in mucosal tissue (32, 33). Oral bolus glutamine treatment did not affect the levels of glutamine or glutamate in the tumor.


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  		Table 3. Effects of oral bolus glutamine treatment on levels of glutamine and glutamate in colonic mucosa and tumor tissue, and glutamine level in plasma 6 h after CPT-11 treatment (125 mg/kg/d x 3 d)

 
Plasma triglyceride levels generally reflect dietary fat intake. The plasma triglyceride compositions of n-3 and n-6 fatty acids were not affected by chemotherapy (data not shown). At 7 days after the end of chemotherapy, in animals fed the control diet, neither eicosapentaneoic acid nor docosahexaenoic acid were detectable in plasma triglyceride. Fish oil feeding raised the levels of eicosapentaneoic acid and docosahexaenoic acid to 1.76 ± 0.64% and 1.76 ± 0.63% of total fatty acids, respectively (P < 0.05). Fish oil feeding also reduced plasma triglyceride levels of arachidonic acid from 2.80 ± 0.60% to 0.39 ± 0.28% (P < 0.005) of total fatty acids in triglyceride.

We determined β-glucuronidase activity in cecal contents before CPT-11 treatment, to study whether different diets affected the activity of this enzyme produced by intestinal microflora (Fig. 2 ). Feeding the prebiotic-enriched diet resulted in increased β-glucuronidase activity (P < 0.0001) as compared with the control diet, whereas the fish oil and glutamine-enriched diet did not affect β-glucuronidase activity.


Figure 2
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  		Fig. 2. Prebiotic oligosaccharides increase β-glucuronidase activity in cecal contents. Rats fed on different diets (CON, control diet; FO, fish oil diet; PRE, prebiotic diet; GLN, glutamine-enriched diet) were killed before CPT-11 administration, and β-glucuronidase activity was determined in the collected cecal contents. Animals treated with bolus glutamine had been on the control diet and did not receive glutamine bolus until prior to CPT-11 administration. The pre–CPT-11 enzyme activity in cecal content should be identical to that of the control diet group. *, P < 0.0001 prebiotic diet versus control diet.

 
In the fish oil–fed rats, there was a 33.0 ± 6.5% (P < 0.05) inhibition of tumor growth 16 days after tumor implantation compared with control diet animals. Fish oil lengthened tumor doubling times by 15.4% (3.87 ± 0.15 versus 3.35 ± 0.14 days, P < 0.02; Fig. 3A ). The prebiotic- and glutamine-enriched diets did not affect tumor growth (data not shown). CPT-11 therapy was initiated for each treatment group when the average tumor burden was of equal size (~1.3% of body weight). Fish oil combined with CPT-11 therapy retarded tumor regrowth after chemotherapy (two-way ANOVA, P < 0.05) and caused a greater inhibition on tumor growth (26.7 ± 9.1%, P < 0.05) compared with the combination of control diet and CPT-11 treatment. Neither prebiotic- nor glutamine-enriched diets affected tumor response to CPT-11 compared with the control diet (Fig. 3B).


Figure 3
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  		Fig. 3. Effects of fish oil–enriched diet on Ward colon tumor growth and antitumor efficacy of CPT-11 in vivo. A, fish oil diet inhibited Ward colon tumor growth in vivo. Fisher rats were implanted with Ward colon tumor and changes in tumor volume were followed: ({blacksquare}) control diet, ({blacktriangleup}) fish oil diet. B, fish oil diet enhanced the antitumor efficacy of CPT-11 treatment. Three daily i.v. injections of CPT-11 at 125 mg/kg/d (black arrows) were initiated when rats of all the dietary treatment groups had tumors of ~2.0 cm3 in volume. Y-axis, relative tumor volume as compared with the baseline volume when the chemotherapy was initiated: ({blacksquare}) control diet + CPT-11, ({blacktriangleup}) oral bolus glutamine treatment + CPT-11, ({blacktriangledown}) prebiotic diet + CPT-11, and ({blacklozenge}) fish oil diet + CPT-11. Points, means; bars, SE. Significant differences were found in the tumor volume change between control and fish oil dietary treatments both before chemotherapy as fish oil diet treated alone (A) and following CPT-11 treatment (B) via two-way ANOVA comparison (P < 0.05). *, P < 0.05 comparison for the tumor values at the indicated time points between control and fish oil diet treatments via Bonferroni posttests.

 
Animals fed the control diet developed late onset diarrhea of high incidence and cumulative severity (Fig. 4 and Table 4 ). Oral glutamine bolus significantly improved the diarrhea profile by decreasing the incidence of score 3 diarrhea and area under the curve of diarrhea score, and this was particularly prominent between days 3 and 7. Animals receiving oral bolus glutamine treatment also showed less body weight loss after CPT-11 treatment (Fig. 4 and Table 4). The i.v. bolus glutamine improved the diarrhea profile to an identical extent as the same dose by oral gavage (Table 4). When the planned glutamine dose was incorporated in the diet, the diarrhea scores for these animals were intermediate and were not significantly different from either the control animals or those administered the glutamine bolus. Tumor response to CPT-11 was identical with all three ways of glutamine administration (data not shown). Neither fish oil nor prebiotic treatment affected any dimension of the diarrhea severity (Table 4).


Figure 4
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  		Fig. 4. Effect of oral bolus glutamine on the time course of CPT-11-induced diarrhea. Tumor-bearing rats on different dietary treatments received 3 daily i.v. injections of CPT-11 at 125 mg/kg/d. Diarrhea was scored twice per day between days 0 and 7 and once per day afterwards. Black arrows, single CPT-11 injection at 125 mg/kg. ({blacksquare}) control diet + CPT-11, ({blacktriangleup}) oral bolus glutamine treatment + CPT-11.

 

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  		Table 4. Effect of dietary treatments on the toxicity profiles associated with CPT-11 treatment

 
At 6 h following the completion of CPT-11 treatment, there was not yet any obvious disruption of the architecture of colonic mucosa except for mild shortening of crypt length. No change in crypt number and goblet cell population was observed. At study completion, all rats had recovered from diarrheal symptoms, and rats treated with irinotecan showed rebound crypt hyperplasia and goblet-cell hyperplasia in the colonic mucosa (Fig. 1A). No difference was found with different diet treatments at these two time points (data not shown).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Modulation of disease and treatment response by dietary factors. Our experiments revealed that host x tumor x chemotherapy interactions could be modified by dietary modifications. Feeding n-3 PUFAs slowed tumor growth (per se), and also improved Ward colon tumor response to CPT-11 therapy. We also showed the ability of glutamine therapy to mitigate CPT-11–induced diarrhea, when this amino acid was applied as a dose bolus immediately prior to chemotherapy. By contrast, n-3 fatty acids and the prebiotic oligosaccharide mixture had no apparent efficacy in preventing CPT-11–induced diarrhea. Taken together, these results suggest that specific dietary modifications might increase the efficacy of therapy and mitigate its side effects. Future work employing the model system to assess for potential additivity or synergy of the effects of nutrient combinations will allow for the development of optimized dietary conditions, which may eventually be conducive to the escalation of chemotherapy doses within a specialized dietary background.

These systematic studies make it possible to study any beneficial (or deleterious) effects which may occur in response to specific dietary treatments over the progression of tumor and treatment. Our model allows a factorial approach incorporating host, tumor, and single or multiple dietary factors. Ours is the first study encompassing a controlled comparison of three classes of nutrients putatively able to alter tumor growth and host or tumor response to cytotoxic therapy. Prior work in this domain has generally been limited to subsets of the overall conception presented here. Usually, proponents of dietary modulation focus on a single dietary element. A variety of investigations of diet therapy and chemotherapy have been conducted in healthy, rather than tumor-bearing animals (14, 34, 35), and might not have been conducted in the range of the maximum tolerated dose of a specific drug (4, 5), and thus, will not entirely represent clinical settings. Where interaction between diet and chemotherapy toxicity has been studied, the clinically relevant outcome (diarrhea) has often been replaced by surrogate measures such as histology, body weight loss, and food intake (4, 18, 36), which may or may not be related to diarrhea severity.

In our model, only glutamine bolus treatment significantly reduced severe diarrhea. There are no prior reports regarding the therapeutic potential of glutamine for CPT-11–related diarrhea in an animal model. This otherwise nonessential amino acid is hypothesized to become conditionally essential during stress states in which the demand for glutamine outstrips its synthesis from endogenous precursors (37). Savarese et al. (10) and Ziegler (11) reviewed clinical studies supporting the prevention of chemotherapy toxicity by glutamine. Most glutamine supplementation was done during conditioning regimens of bone marrow transplant and focused on transplantation-related outcomes. There has been limited work exploring glutamine supplementation in colorectal cancer patients treated with 5-fluorouracil (38). Glutamine supplementation was reported in a small study series with five patients experiencing grade 4 diarrhea in response to CPT-11, which significantly improved upon glutamine administration (10).

We initially selected bolus glutamine treatment based on literature showing that this could substantially rescue rats from lethal endotoxemia, septic shock, or hyperthermia. These conditions are associated with intestinal inflammation and compromised gut barrier function (22, 23), similar to CPT-11 treatment. Although our initial approach of bolus administration was influenced by this thinking, there are several ways of glutamine administration that could be considered nutritionally relevant. The oral versus i.v. comparison is important because glutamine is subject to considerable first-pass metabolism in the small intestine, and i.v. administration is the more direct means of raising systemic concentrations. Our results suggest that oral and i.v. administration are of equal efficacy, provided that the glutamine was administered as a bolus. Incorporation of amino acids in diets is a conventional approach in experimental nutrition studies; however, in this study, this feeding paradigm did not show a clear ability to alter the severity of diarrhea. Mixing glutamine with the diet makes for a more continuous intake throughout the day and it may be that acutely and substantially raised tissue levels of glutamine achieved with the bolus treatment are required to evoke the mechanisms essential for this protection. Wischmeyer et al. (22, 23) related the bolus glutamine administration to the potentiation of innate cytoprotective heat shock response during septic and hyperthermic stresses. It would be of interest to examine this link in the context of CPT-11–related stress.

Data suggesting that high dietary intakes of n-6 PUFAs generally support tumor proliferation and that the corresponding n-3 fatty acids suppress tumor growth are becoming more abundant (12). This has been shown for diverse tumor types, and is also evident for the Ward colon tumor. Dietary supplementation with fish oils containing n-3 fatty acids has been reported to both enhance the cytotoxic effect of chemotherapy (i.e., doxorubicin, cisplatinum, and bleomycin) and to offer protection to host tissues (12, 13) in animal models. Hardman et al. (5) reported enhanced regression of MCF7 breast carcinoma xenografts by coadministration of fish oil with CPT-11, and our results confirm their conclusions with respect to the ability of fish oil to enhance the antitumor activity of CPT-11. Here, fish oil treatment did not alter the development of severe diarrhea in response to CPT-11. Hardman et al. (5) did not measure diarrhea, but showed some evidence for better preservation of intestinal epithelia in animals fed fish oil and treated with CPT-11. The planned time points in our study were respectively too early and too late to coincide with the time point when glutamine's effect on diarrhea was observed, and new work will be needed for histologic results. It should be noted, however, that well-preserved gut histology might not necessarily correlate with the amelioration of diarrhea, which is, in nature, a functional disorder as the net result of intestinal secretion and fluid absorption. For example, oversecretion of Cl– might be an important contributor to the pathogenesis of CPT-11–induced diarrhea, independent of intestinal epithelial destruction (27, 39, 40). There are other differences between our respective studies. Hardman et al. (5) used a comparatively long-term low-dose CPT-11 regimen; they used pure corn oil in their control diet; the very high n-6 fatty acid content of corn oil would be expected to increase inflammatory responses (41) and that may have made the response of the control animals to CPT-11 treatment worse than it would otherwise have been.

Prebiotic oligosaccharides are of interest for their anti-inflammatory effects in chronic bowel disease and in colon cancer prevention (9, 42). Feeding oligofructose and/or inulin reduced genetically or chemically induced colitis in animal models (24, 25), as well as in some emerging studies in human inflammatory bowel disease (43). There are only a few reports on the effects of dietary fibers on chemotherapy-induced intestinal toxicity, specifically with methotrexate and 5-fluoruracil (14, 15). The case of CPT-11 is unique. Biliary excretion of CPT-11 as its inactive glucuronide, and the reversal of this glucuronidation by enzymes produced by the intestinal flora, means that β-glucuronidase activity is influential in the development of CPT-11 toxicity. Although it is an emerging nutritional concept to treat certain types of refractory diarrhea using prebiotics (44, 45), and this would also seem relevant in methotrexate and 5-fluoruracil chemotherapy (15, 35), our results suggested that caution may need to be exercised when employing prebiotic treatment in conjunction with CPT-11. Prebiotic oligosaccharides doubled β-glucuronidase activity in cecal contents, which would be expected to increase the formation of SN38, the toxic metabolite of CPT-11, and thus, to aggravate rather than ameliorate intestinal injury. Microbiological studies are warranted to determine which glucuronidase-producing intestinal bacterial strains are affected by diet components and which bacterial strains worsen CPT-11–induced intestinal injury.

Our observations raise some issues of potential clinical importance. We are currently conducting a study of oral high-dose glutamine therapy in patients with colorectal cancer treated with CPT-11 in the follow-up of these animal studies and the case series of Savarese et al. (10). Our experimental results show that specific prebiotics fed at nutritionally relevant levels doubled β-glucuronidase activity in cecal contents. It is also known from experimental studies that when the intestinal bacterial load is significantly reduced by antibiotic treatment, CPT-11 toxicity is greatly diminished, suggesting that intestinal bacteria mediate CPT-11 toxicity (46, 47). It will thus be of interest to assess endogenous β-glucuronidase activity as a potential determinant of severe diarrhea in patients treated with CPT-11. There are no published data relating to this point. Considering the great heterogeneity of dietary (prebiotic) fiber intake in humans, it seems plausible that fiber intake could be an innate variant determining CPT-11–induced gut toxicity by affecting the formation of SN38 in the distal gastrointestinal tract. The dietary intake and physiologic levels of n-3 fatty acids in cancer patients are also incompletely characterized and could vary considerably (48). The possibility that fish oil could enhance the tumor response to CPT-11 treatment remains to be tested in patients, and this would require the supplementation of a sufficient dose and duration to achieve effective levels.

A rationale for the adoption of defined dietary conditions in animal models of cancer. A clearly defined and nutritionally relevant dietary platform is needed for preclinical research on host x tumor x chemotherapy interactions. Within the body of literature using various animal models to investigate dietary effects on toxicity/efficacy of chemotherapy, diversely varied background diets have been used. These range from elemental diets and semipurified diets to chow diets composed of natural ingredients (5, 14, 34). Elemental diets are made of purified triglycerides, free fatty acids, free amino acids, sugars, vitamins, and minerals. Semisynthetic diets are composed of purified proteins, starch, sugars, and defined lipid components from specified fats and oils. Laboratory animal chows are not of standard macronutrient composition and are formulated from plant materials (i.e., corn, soybean meal), and from meat meal, fish meal, and fats which are frequently undefined. The application of elemental diets in human nutrition is limited to i.v. feeding and to a few isolated cases of specialized nutritional requirements, and are not typically used in the nutritional support of patients receiving CPT-11 chemotherapy. Although chow diets are composed of ingredients used in foods, their exact composition is incompletely characterized, variable between batches and brands, and more importantly, chows are delivered premixed and thus cannot be modified. For this reason, semipurified diets are the most widely used in experimental research in clinical nutrition, as they are well-defined, permit systematic modulation of key nutrient classes, and are composed of whole proteins, complex carbohydrates, and lipid sources commonly consumed by humans.

A careful consideration of basal diet is indicated. It should meet known nutrient requirements for the animal species under study, and its nutritional relevance must be considered. This is particularly important with respect to the lipid composition of the diet because both the levels and type of dietary fat (including relative and absolute amounts of saturated, polyunsaturated, n-6 and n-3 fatty acids have all been shown to affect tumor growth, response to therapy, or toxicity). The amounts of fish oil in our experimental diets can be defined as nutritionally relevant as it showed efficacy in modulating intestinal and immune functions in studies using dosages that could realistically be achieved in clinical diets (49, 50). The basal diet we used was selected to be similar in composition to typical diets of North American populations in terms of overall fat level, polyunsaturated/saturated fat ratio, and n-6/n-3 ratio. Caution must be taken in interpreting studies which used diets with insufficient amounts of n-3 PUFA or a highly unphysiologic ratio of n-6 to n-3 PUFAs (5), as these are likely to induce n-3 essential fatty acid deficiency or are proinflammatory and/or promote tumor growth. There is a tendency to use a single oil or fat source, rather than to formulate a fat blend, as done here. For example, corn oil has been used as the sole fat source (5), and although a corn oil–based diet is not likely to induce an n-3 essential fatty acid deficiency, in a short feeding study, the skewed ratio of n-6 to n-3 PUFA in corn oil (i.e., polyunsaturated/saturated ratio of 40) might be supportive of enhanced tumor growth or increased inflammatory mediators (41).


   Acknowledgments
 
The authors thank Gail Hipperson and Dan McGinn for help in animal care; Abha Hoedl for excellent technical support; and Sue Goruk, Megan Pawlowicz, and Michelle Mackenzie for their help at various stages of this study.


   Footnotes
 
Grant support: Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, and the Crohn's and Colitis Foundation of Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/ 9/07; revised 7/11/07; accepted 7/23/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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