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3 Department of Oncology, 4 Department of Agriculture, Food and Nutritional Science, and 5 Center of Excellence for Gastrointestinal Inflammation and Immunity Research, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2
* To whom correspondence should be addressed. E-mail: vickieb{at}cancerboard.ab.ca.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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(P < 0.05); 2) increased reduced GSH (rGSH):oxidized GSH ratio (P < 0.05); 3) prevention of upregulated activity of a key bacterial enzyme (β-glucuronidase) in the cecal content that mediates CPT-11 intestinal toxicity (P < 0.05); and 4) increased proportions of CD3+CD8+ lymphocytes and memory CD8+ subset in mesenteric lymph nodes following CPT-11 therapy. By contrast, glutamine treatment did not alter CPT-11's antitumor activity, the amino acid concentrations, Hsp expression, or the ratio of rGSH:oxidized GSH in the tumor. Our data demonstrate a striking dichotomy in the response of tumor and host to oral glutamine administration, concurring with the concept that this nutrient may favorably alter the balance between the host and tumor.
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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8% of colorectal cancer patients treated with regimens containing CPT-11 and 5-fluorouracil (4). Glutamine protects the gut during a variety of stress conditions, including trauma, sepsis, burns, and several cancer chemotherapies (5-fluorouracil, methotrexate, doxorubicin) (5–9). Several potential mechanisms for this protection are proposed. Glutamine preserves glutathione (GSH) in the gut during oxidative stress during methotrexate chemotherapy and endotoxin challenge (10,11). Glutamine may also preserve gut barrier integrity and gut-associated and systemic immune competence (12). Glutamine (0.3–2 g·kg–1·d–1) is usually provided continuously in the diet in parenteral nutrition or mixed with enteral feed (7–9,11). Wischmeyer et al. (13,14) recently explored administration of glutamine as a single i.v. or oral bolus and a dose of 0.75 g/kg was optimally protective from lethal endotoxin shock or hyperthermia in rats (13,14). Wischmeyer et al. (13,14) suggest that additional protective mechanisms not seen with continuous feeding occur after bolus doses of glutamine, particularly the heat shock response, an inherent cellular cytoprotective response. Our recent work (15) provides the first direct evidence, to our knowledge, that high-dose oral bolus glutamine limits the development of severe diarrhea in rats treated with CPT-11.
There has been concern expressed that provision of glutamine may promote tumor growth, based on the prevalent notion that tumors have a propensity to use glutamine as a source of energy and of nitrogen for biosynthesis (16,17). The utility of nutritional treatments to reduce chemotherapy toxicity would depend upon a differential effect on the host and tumor, such that host tissues would be protected from injury and there would be no protection, or even sensitization, of tumor tissues to antineoplastic treatments. Although such differential effects on host and tumor would be ideal, it could equally be predicted that nutritional support might invoke cellular protective mechanisms in the tumor and may cause resistance to therapy. Based on these considerations, we designed a study to examine the effects of an oral bolus glutamine in tumor-bearing rats treated with CPT-11. The objective of our work was to determine the effect of this treatment on specific cytoprotective effectors, including heat shock protein (Hsp) and GSH, in the host and tumor.
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Materials and Methods |
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Animal use was 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), 11–12 wk old, were obtained from Charles River. Rats were housed 2 per cage in a temperature- (22°C) and light-controlled (12 h light) room and consume water and food ad libitum. One week before chemotherapy, rats were separated into individual housing in wire-bottom cages. The Ward colorectal carcinoma, which was originally chemically induced by azoxymethane in its host strain (Fisher rat) and is transplantable within that rat strain, was provided by Dr. Y. Rustum, Roswell Park Institute (18). Tumor pieces (0.05 g) were transplanted subcutaneously on the flank via trocar under slight isoflurane anesthesia. Tumor volume was estimated (cm3) = 0.5 x length (cm) x width (cm) x height (cm), as described (15). CPT-11 was provided by Pfizer as a ready-to-use clinical formulation. Atropine (0.6 g/L) was a clinical injectable formulation.
Diet
The diets used in this study are described in detail elsewhere (15). Briefly, semipurified diet was based on AIN-76 basal diet, with a modified fat component similar to a North American dietary pattern (40% of energy, polyunsaturated:saturated fat ratio of 0.35). Rats were initially fed Rodent Laboratory Chow (Harlan Teklad). During the adaptation period, this nonpurified diet was mixed with study diet (50:50 wt:wt) for 1 wk, followed by transition to a 100% semipurified diet for 2 wk before tumor implantation.
Experimental design
When tumor volume reached 2 cm3, 3 consecutive daily CPT-11 i.v. injections at 125 mg·kg–1·d–1), the maximal tolerated dose (15), were initiated. Atropine (1 mg/kg subcutaneously) was administered immediately before each CPT-11 injection to alleviate early-onset cholinergic symptoms (15).
Glutamine was administered by oral gavage (0.75 g·kg–1·d–1) 30 min before each daily CPT-11 injection. The sham treatment group was gavaged with isovolemic sterile water. Glutamine (Sigma) was made as a 3% (wt:v) solution immediately before use and filtered with a 0.45-µm filter. The first day of CPT-11 administration was designated d 0. Sham- and glutamine-treated rats (n = 6 per treatment) were killed on d 3, 6 h after the last injection of CPT-11 (6.5 h after last glutamine gavage). This time point was selected to capture early responses to glutamine treatment, especially Hsp expression. Additional sham- and glutamine-treated rats (n = 12 per treatment) were followed until d 9 for tumor response to CPT-11 treatment and to evaluate diarrhea. β-Glucuronidase activity was assayed in cecal content collected from glutamine- and sham-treated rats killed at these 2 time points (d 3 and d 9) and additionally before chemotherapy (d 0) (n = 6 per treatment).
Outcome measures
Diarrhea. A clinically comparable 3-point scale was adopted in grading the diarrhea (19); these assessments were conducted by a researcher unaware of the study treatments. Incidence of grade 3 diarrhea was calculated for each animal by counting observations of a particular score(s) out of the total 8 observations between d 3 and d 7 when diarrhea developed to its full severity (19). Area under curve of diarrhea score was calculated between d 3 and d 7 (15).
Sample collection and assays. Rats were killed by CO2 asphyxiation. Immediately after rats became unconscious, whole blood was collected via cardiac puncture into heparinized tubes. Cecal digesta content was collected under aseptic conditions. The full length of the colon was washed and longitudinally cut into halves, 1 of which was mounted on a wax strip and fixed in 10% (v:v) neutral buffered formalin and the mucosa was gently scraped off the other half and immediately frozen in liquid nitrogen. Tumor was excised intact, blotted, and weighed before the nonnecrotic portions were collected and frozen in liquid nitrogen.
Apoptosis in situ via terminal transferase-mediated dUTP nick end labeling. Apoptotic cells in colonic mucosa were identified with the DeadEnd Colorimetric terminal transferase-mediated dUTP nick end labeling (TUNEL) System (Promega) according to the manufacturer's instruction. In each section, 20–25 areas of colonic mucosa, each of which contained at least 10 crypts, were randomly selected to count TUNEL-positive cells under 400x magnification. The analysis was performed by a researcher who was unaware of the dietary treatment.
Hsp were detected by Western blot (13) using antibodies to Hsp70 (1:4000), Hsp90 (1:4000), Hsp25 (1:12,000), and corresponding horseradish peroxidase-conjugated secondary antibodies (Stressgen). Hsp were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech) and analyzed by autoradiography using a Typhoon 8600 Variable Mode Imager (Amersham Biosciences). The densitometry determination was performed using ImageQuant 3.2 software (Molecular Dynamics). Data shown are relative densitometric ratios against the value for heat shock cognate70 (1:5000, Stressgen), the cognate constitutively expressed form of Hsp70 used as the protein loading control.
Reduced GSH (rGSH) and GSH disulfide (GSSG) were determined with Oxford rGSH/GSSG kit (product no. GT-30), which features 1-methyl-2-vinyl-pyridinium trifluoromethane sulfonate as a scavenger of rGSH. Free amino acid concentrations were determined by HPLC after conversion to their o-phthaldialdehyde derivatives (20). Cysteine and proline are not detected by this method. We determined β-glucuronidase activity by a modified method of Freeman as described (15). The β-glucuronidase activity is expressed as U/g protein. One unit is defined as 1.0 µg of phenolphthalein liberated from phenolphthalein glucuronide per hour.
Mesenteric lymph node cell phenotype. Immune cells were isolated from the mesenteric lymph nodes (MLN) (21) and cells (200,000 cells per well) were phenotyped using 2 color direct immunofluorescence (22), using antibodies to CD3, CD4, CD8, CD25, CD45RA (BD Bioscience), and OX12 (Cedarlane). Streptavidin QR (Sigma) was added to all biotin-labeled antibodies. The percentage of cells expressing each of the antibody markers was determined by flow cytometry (FacScan, Becton Dickinson) (22). We attempted to perform every phenotype assay on each rat, but this was not always possible due to limited lymph node size; the actual number of rats utilized for each assay is indicated in tables.
Statistical analysis
Data are expressed as means ± SEM. The effect of glutamine on tumor growth following CPT-11 treatment was tested using 2-way ANOVA followed by Bonferroni post hoc tests (GraphPad Prism, GraphPad). Treatment differences in apoptotic index and immune phenotype were analyzed using 1-way ANOVA followed by post hoc Tukey's test (SPSS 12.0). For the other measures, the effects of glutamine were analyzed using the unpaired 2-tailed Student's t test for independent samples. P < 0.05 was considered significant.
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Results |
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Bolus glutamine administration differentially induces cytoprotective mechanisms in host tissues following CPT-11 treatment.
Amino acids were determined in samples taken 6.5 h after the last glutamine bolus. This time point was selected for detection of the Hsp response and would likely be past the peak glutamine concentrations; however, at this time, concentrations of glutamine, glutamate, and several other amino acids were raised in either plasma or colonic mucosa tissue (P < 0.05) (Table 1). Ornithine and citrulline were not detectable in colon or tumor, and in plasma their concentrations (ornithine, 35.8 ± 7.4 µmol/L; citrulline, 16.5 ± 0.8 µmol/L) were not affected by glutamine. Despite increased plasma concentrations, none of the amino acid concentrations in tumor tissue were altered by glutamine treatment.
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, in the colonic mucosa by 3.1-, 7.2-, and 3.8-fold, respectively, compared with sham treatment (Fig. 1). However, the abundance of these inducible Hsp in tumor tissue was not affected by glutamine treatment (Hsp25 relative density: 0.67 ± 0.20 for sham vs. 0.72 ± 0.16 for glutamine; Hsp90 relative density: 1.10 ± 0.08 for sham vs. 1.02 ± 0.12 for glutamine). Tumors did not contain any detectable Hsp70.
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6 h after CPT-11 treatments, so we also selected this time point. CPT-11 increased apoptotic index in both sham- (20.9 ± 2.1/mm2; P < 0.0005) and glutamine-treated (16.7 ± 2.8/mm2; P < 0.005) rats compared with healthy reference rats (2.3 ± 0.4/mm2) (Fig. 2). The sham- and glutamine-treated rats did not differ (P = 0.2).
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Phenotype of mesenteric lymph node cells was analyzed 7 d after the completion of CPT-11 chemotherapy (Table 3). There were numerous effects of CPT-11 treatment compared with rats that had not received chemotherapy. CPT-11 decreased the proportion of CD3+ T cells (primarily CD3+CD4+ cells, as suggested by the reduced CD3+CD4+:CD3+CD8+ ratio) and, accordingly, an increased proportion of B cells (OX12+) in MLN, irrespective of glutamine treatment. Although only a small proportion of cells expressed the IL-2 receptor, the proportion in CPT-11-treated rats was 30% lower due to proportionately less CD8+CD25+ cells (P < 0.05). CPT-11 also decreased the proportions of cytotoxic T cells (CD3+CD8+ cells) and antigen mature CD8+ cells (CD45RA–) as after CPT-11 compared with healthy controls (P < 0.05). Glutamine normalized the relative abundance of CD3+CD8+ cells and memory/antigen mature CD8+ cells (CD45RA–) to levels in healthy controls and did not affect the CD3+CD4+:CD3+CD8+ ratio or the proportion of CD8+CD25+ cells.
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Discussion |
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The potential for purified nutrients at high doses to "feed the tumor" or to confer tumor protection against cytotoxic therapy remains a prevalent notion. Tumors are said to be glutamine traps, and glutaminolysis has been proposed as a major energy-producing process in tumor cells (16,17). In tissue culture, tumor cells rely on glutamine for cell growth as an essential energy source and biosynthetic precursor (24,25). In animal models, however, the size, protein synthetic rate, and DNA content of tumors are not generally affected by supplemental glutamine (26,27). A majority of tested tumors indeed do not show net glutamine uptake from the arterial supply in vivo (28–30). Our findings principally agree with these studies and indicate that tumor amino acid pools remained strikingly unaffected by glutamine supplementation.
Overexpression of Hsp is thought to be involved in tumor chemoresistance by directly or indirectly permitting cancer cells to overcome chemotherapy-induced apoptosis (31,32). Here, the failure of glutamine to modulate tumor Hsp expression and GSH may simply result from the lack of adequate and sustained alteration of the tumor glutamine pool. Whereas only a single time point was studied, the concurrent alterations in amino acid concentrations in plasma and colon tissue would suggest that the selected time should have revealed alterations in tumor amino acid concentrations. Amino acid transport, metabolism, or both could potentially contribute to the insensitivity of tumor tissue to amino acid supplementation.
A multiplicity of glutamine actions in the tumor-bearing host. Glutamine bolus significantly improved CPT-11-induced diarrhea and we are currently pursuing a clinical investigation of this potential benefit of glutamine therapy. Because transient anorexia after CPT-11 chemotherapy was identical in sham- and glutamine-treated rats, feed intake-related effects on the examined endpoints could be excluded.
We know of no other published data on the effects of tumor and concurrent CPT-11 chemotherapy on plasma and tissue amino acids and our results suggest notably low levels of arginine and citrulline compared with published results for normal rats. There are suggestions in the literature that hypocitrullinemia could be a potential quantitative marker for intestinal damage caused by intense chemo- and radiation therapy (33,34). Diminished plasma citrulline could limit arginine synthesis through the intestinal-renal axis, resulting in decreased arginine concentration (35,36).
Glutamine administered orally is subject to extensive first pass metabolism in the small intestine and liver (37) and its actions may be due as much to secondary metabolites as to glutamine per se. The wide range of changes in plasma and colon amino acid concentrations (Table 1) reflects the sophisticated metabolic pathways of glutamine in vivo. Some products derived from glutamine metabolism, such as arginine, are important modulators of gut physiology and immunity (38). Consistent with previous findings, our glutamine bolus increased arginine concentration systemically and in the colon (39). T-cell depletion has been considered as the primary contributor to immunodeficiency related to high-dose chemotherapy (40) and both glutamine and arginine promote lymphocyte proliferation and activation (11). Our oral glutamine treatment attenuated the relative depletion of cytotoxic T cell, especially memory CD8+ cells in MLN associated with CPT-11 treatment. This may add to the competence of gut-associated immunity against invasion of pathogens present in the gut lumen.
CPT-11 is associated with oxidative stress in normal tissues with diminished GSH store (41). GSH synthesis may play a pivotal role in protecting from CPT-11-related toxicity (42). Glutamine (via glutamate) is a precursor for GSH synthesis and has been suggested to be limiting for GSH synthesis during stress (43). Glutamine treatment raised the ratio of rGSH:oxidized GSH in the colon tissue but at the same time lowered the reduced and total GSH stores in tumor. Previous work examining tumor-bearing rats treated with methotrexate suggests that glutamine decreased GSH content in tumor but increased it generally in host organs including heart, kidney, and intestine (10,44). These authors conjectured that the acidic tumor environment blocks GSH regeneration and ultimately depletes the GSH store in the tumor (10).
Glutamine increased Hsp25, 70, and 90 in colon mucosa, the 3 key inducible Hsp playing a central role in protecting cells by preserving the structure of normal proteins and removing damaged ones. This response varied somewhat between individual animals; however, this variability remains largely unexplained. Wischmeyer et al. (13) had also observed greater variability in the Hsp response of colon and ileum compared with lung and heart. The action of glutamine to induce Hsp may be direct, because there is evidence that Gln induces HSP in cells in culture (45,46). Induction of Hsp preserves the functional integrity of epithelia, i.e. electrolyte absorption/secretion, intestinal myoelectric activity, and mucosal barrier (47–49) and reduces production of pro-inflammatory mediators (48,50), which may in turn be involved in triggering diarrhea (19).
The protective ability of glutamine treatment may or may not have derived from alteration of apoptosis in colonic epithelium and our results on this point were equivocal. The improvement of diarrhea outcomes independent of gut apoptosis has been observed by other authors using keratinocyte growth factors (51,52). Glutamine could exert its benefit on CPT-11-induced diarrhea largely through other mechanisms that may more predominantly correct intestinal absorptive/secretory function.
Local deconjugation of SN-38 glucuronide catalyzed by β-glucuronidase produced by colonic microflora intensifies the epithelial exposure to SN-38 (53). We provide direct evidence that administration of CPT-11 affects colonic β-glucuronidase; a marked increase of the enzyme was detected 6 h after CPT-11 treatment. CPT-11 can increase levels of several β-glucuronidase-producing flora (Escherichia, Clostridium, and Staphylococcus species) within 1–6 h following treatment (54). The post-CPT-11 increase of the enzyme activity was abolished with glutamine treatment in a manner that might alleviate the exposure of intestinal epithelium to SN-38.
Glutamine induced Hsp and favorably altered GSH stores and β-glucuronidase activity in colon. The early induction of these effects suggests that they have the potential to contribute to the subsequent mitigation of severe diarrhea, which was most evident on d 4–7. It may be that 1 of these effects is primary; however, it is also possible that their simultaneous occurrence may be important. As suggested by Wischmeyer et al. (13,14), bolus glutamine may represent a novel therapeutic paradigm for preventing chemotherapy-related injury via boosting the inherent stress response of tissues vulnerable to the drug. A future objective of research in this context will be to further clarify the dose, schedule of administration, and additional dietary elements to optimize the protective response.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: H. Xue, M. B. Sawyer, C. J. Field, L. A. Dieleman, D. Murray, and V. E. Baracos, no conflicts of interest.
6 Abbreviations used: CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin; GSH, glutathione; GSSG, glutathione disulfide; Hsp, heat shock protein; MLN, mesenteric lymph node; rGSH, reduced glutathione; TUNEL, terminal transferase-mediated dUTP nick end labeling.
Manuscript received 24 October 2007. Initial review completed 26 November 2007. Revision accepted 9 January 2008.
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LITERATURE CITED |
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