Chemotherapeutic agents produce cytotoxicity via induction of apoptosis and cell cycle arrest. Rapidly proliferating cells in the bone marrow and intestinal crypts are highly susceptible to chemotherapy, and damage to these cellular compartments may preclude maximally effective chemotherapy administration. Glucagon-like peptide(GLP)-2 is an enteroendocrine-derived regulatory peptide that inhibits crypt cell apoptosis after administration of agents that damage the intestinal epithelium. We report here that a human degradation-resistant GLP-2 analogue, h[Gly2]-GLP-2 significantly improves survival, reduces bacteremia, attenuates epithelial injury,and inhibits crypt apoptosis in the murine gastrointestinal tract after administration of topoisomerase I inhibitor irinotecan hydrochloride or the antimetabolite 5-fluorouracil. h[Gly2]-GLP-2 significantly improved survival and reduced weight loss but did not impair chemotherapy effectiveness in tumor-bearing mice treated with cyclical irinotecan. Furthermore, h[Gly2]-GLP-2 reduced chemotherapy-induced apoptosis, decreased activation of caspase-8 and -3, and inhibited poly(ADP-ribose) polymerase cleavage in heterologous cells transfected with the GLP-2 receptor. These observations demonstrate that the antiapoptotic effects of GLP-2 on intestinal crypt cells may be useful for the attenuation of chemotherapy-induced intestinal mucositis.
Chemotherapeutic agents exert their cytoablative actions on rapidly proliferating cells via several different mechanisms,ultimately leading to cell cycle arrest and/or cellular apoptosis. The cytotoxic actions of chemotherapeutic agents are not tumor specific,and injury to normal cells in the bone marrow and intestinal crypt often complicates the treatment of patients with neoplastic disease(1, 2). Although molecules such as granulocyte-colony stimulating factor may be used to attenuate bone marrow toxicity after chemotherapy (3), no agents are currently available that selectively prevent chemotherapy-induced cell death in the intestinal crypt compartment. As a result, gastrointestinal toxicity characterized by severe mucositis and diarrhea often limits both the dose and duration of chemotherapy treatment, leading to reduced treatment effectiveness in susceptible patients.
Several intestine-derived molecules have been identified that maintain the integrity of the mucosal epithelium in part via prevention of apoptosis after intestinal injury. For example, intestinal trefoil factor promotes resistance to apoptosis after cellular injury in vitro (4), and intestinal trefoil factor-deficient mice exhibit enhanced susceptibility to intestinal injury and increased colonic epithelial cell apoptosis in vivo (5). Similarly, keratinocyte growth factor protects mice from chemotherapy and radiation-induced intestinal injury (6), and fibroblast growth factor 2, transforming growth factor β, cytokines,interleukin 11, and interleukin 15 reduce intestinal apoptosis in vivo (7, 8, 9, 10).
GLP-23is an intestinotrophic peptide secreted by enteroendocrine cells in response to intestinal injury (11, 12, 13). Exogenous administration of GLP-2 is trophic to the small and large intestinal epithelium in part via stimulation of crypt cell proliferation(14). Administration of GLP-2 to rodents with indomethacin-induced intestinal injury improves survival and reduces epithelial damage in part via inhibition of apoptosis in the crypt compartment (15). The antiapoptotic actions of GLP-2 prompted us to examine whether GLP-2 might ameliorate the extent of intestinal injury arising from chemotherapy administration in vivo.
MATERIALS AND METHODS
5-FU was obtained from Roche Laboratories. IRT(7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin)used in mice was a gift from Pharmacia Upjohn (Mississauga, Canada),and recombinant h[Gly2]-GLP-2 was kindly provided by NPS Allelix Corp. (Mississauga, Canada). Cell culture experiments were performed using IRT and forskolin obtained from Sigma (St. Louis, MO).
All experimental protocols were approved by the Animal Care Committee of the University Health Network–Toronto General Hospital. Experiments with IRT alone were performed in 8–9-week-old CD1 female mice (Charles River, Toronto, Canada). Experiments with 5-FU were carried out in 11–13-week-old BDF-1 female mice (Harlan, Toronto,Canada). Experiments using IRT treatment in BALB/c mice inoculated with CT-26 murine colon carcinoma cells were performed in 10-week-old female mice (Charles River). All mice were housed in plastic-bottomed wire-lid cages and maintained on a 12-h light/12-h dark cycle in a temperature-controlled room and given water and chow ad libitum.
For all animal experiments, mice received s.c. injection of either 0.5 ml of saline (PBS) or 10 μg of h[Gly2]-GLP-2, a human GLP-2 analogue (12) dissolved in 0.5 ml of saline, twice daily at 8 a.m. and 6 p.m. beginning 3 days before administration of either 5-FU (400 mg/kg) or IRT (280 mg/kg). For non-tumor-bearing mice, studies were carried out in adult CD1 female mice. For tumor-bearing BALB/c mice, CT-26 murine colon carcinoma cells(American Tissue Culture Collection) syngeneic to BALB/c mice were grown in monolayer cultures in DMEM (4.5 grams/liter glucose)supplemented with 5% FCS, 1 mm pyruvate (Life Technologies, Inc., Burlington, Canada), and penicillin G sodium (100 units/ml)/streptomycin sulfate (0.1 mg/ml; Sigma) in a humidified 5%CO2 atmosphere at 37°C as described previously (16). A single cell suspension with>90% viability was injected s.c. (5 × 105 cells) in the left flank region. Six days later, a 7-day treatment regimen was initiated [3 days of treatment with either 0.5 ml of saline (PBS) or 10 μg of h[Gly2]-GLP-2 administered s.c. twice daily at 8 a.m. and 6 p.m., followed by a 3-day regimen of IRT (100 mg/kg dose) or vehicle administered via the i.p. route once daily and a 24-h recovery period]. This 7-day regimen was repeated three times (n = 25 mice/group), at which point some animals in the control groups became moribund; hence, all mice were euthanized after receiving CO2 anesthesia 30 days after tumor implantation. The tumor IR was determined using the following equation: IR(%) = (1 − T/C × 100 where T and C represent tumor weights in IRT-treated (T) and untreated control (C) mice respectively. An IR of 58% was considered to represent an efficacious tumor response to IRT(17).
Intestinal cross-sections (4–6 μm) from each mouse were cut and stained with H&E, and intestinal micrometry was performed as described previously (15). The number of cells/hemi-crypt column and the number of surviving crypts/circumference were measured in both the small and large intestine at 12-h intervals(n = 5 mice/time point) after IRT administration as described previously (18, 19). Apoptotic cells within the small and large intestinal crypts were scored using the TUNEL assay and by their morphological appearance after staining with H&E. An apoptosis cell index was obtained on a positional basis for all intact half-crypts present in an entire jejunal and colonic cross-section per mouse, as described previously (20, 21, 22),24 h after the first dose of IRT. Extensive crypt damage precluded an accurate positional analysis of apoptotic events beyond this time point. All slides were scored in a blinded fashion.
Aliquots of whole blood and tissue homogenates obtained using sterile technique were plated on blood agar plates and incubated at 37°C for 48 h.
Whole blood samples were collected in venipuncture tubes containing EDTA and analyzed using an automated whole blood sorter calibrated for mouse samples. Blood smears were performed on all samples to confirm the automated analysis.
Intestinal lysates were centrifuged at 12,000 rpm for 30 min at 4°C and boiled for 5 min in sample buffer. Forty μgs of total protein were fractionated by discontinuous SDS-PAGE under reducing conditions and electrophoretically transferred onto Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech, Montreal, Canada) using standard techniques. Immunoreactive proteins were detected with a secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence commercial kit (Amersham Pharmacia Biotech) as described previously (23). Primary antibodies used included caspase-3 (1:5000 dilution; gift of R. Sekaly;Université de Montreal, Montreal, Canada), caspase-8 and caspase-9 (both at a 1:500 dilution; gift of T. Mak, University of Toronto, Toronto, Canada), p53 (1:500 dilution; Pab 246;Santa Cruz Biotechnology), PARP (1:4000 dilution; PharMingen,Mississauga, Ontario, Canada), and anti-actin (1:5000 dilution,Sigma). Densitometry was performed on blots exposed onto X-ray film(X-OMAT AR; Kodak Diagnostic Film) using a Hewlett Packard ScanJet 3p scanner and the NIH Image software.
Induction of Apoptosis in Transfected BHK Cells
BHK fibroblast cells containing the stably integrated pcDNA3.1 plasmid (BHK-pcDNA3; Invitrogen, Carlsbad, CA) or the identical plasmid containing the rat GLP-2 receptor (BHK-GLP-2R) were propagated as described previously (23). Cells were pretreated with either h[Gly2]-GLP-2 (20 nm) or forskolin (40μ m) before the addition of IRT (final concentration, 10μ m). Control cultures were treated identically in the absence of IRT, and the number of viable cells in each condition was measured using the Cell-Titer 96 aqueous assay kit (Promega, Madison,WI). Cells treated with IRT alone were fixed in 4%paraformaldehyde, and DNA was stained using 4′,6-diamidino-2-phenylindole (1 μg/ml; Sigma).
Measurement of Caspase-3-, Caspase-8-, and Caspase-9-like Enzymatic Activity
Enzymatic reactions were performed at 37°C using 150 μg of protein lysate, reaction buffer [50 mm HEPES (pH 7.4), 75 mm NaCl, 0.1%3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and 2 mm DTT), and Ac-DEVD-pNA (Calbiochem, San Diego, CA) to measure caspase-3-like protease activity, Ac-IETD-pNA (Biosource International, Camarillo, CA) to measure caspase-8-like protease activity, and Ac-LEHD-pNA (Biosource International) to measure caspase-9-like protease activity. Spectrophotometric detection of the chromophore pNA at 405 nm was used to quantify enzymatic activity.
Survival analysis was performed using the Fisher’s exact t test. Statistical differences between treatment groups were determined by unpaired Student’s t test or by ANOVA using n-1 post hoc custom hypotheses tests, as appropriate.
Administration of h[Gly2]-GLP-2 for 3 days before treatment with IRT significantly enhanced survival in CD1 mice (Fig. 1,A). The protective effect of h[Gly2]-GLP-2 was not restricted to a single chemotherapeutic agent or murine genotype because h[Gly2]-GLP-2 significantly enhanced survival in BDF-1 mice after administration of the antimetabolite 5-FU (Fig. 1 B).
To assess the efficacy of h[Gly2]-GLP-2 in tumor-bearing mice, CT-26 murine colon carcinoma tumor cells were injected into BALB/c mice and propagated in vivo. h[Gly2]-GLP-2 significantly enhanced survival after cyclical IRT administration to tumor-bearing mice (Fig. 1,C, P < 0.01,h[Gly2]-GLP-2/IRT-treated mice versus all other groups of mice). Mice receiving both h[Gly2]-GLP-2 and IRT (100 mg/kg) tolerated three times the amount of IRT before mortality was observed (Fig. 1,C). Furthermore, tumor-bearing mice given saline and IRT demonstrate progressive weight loss and lost 16% of their body weight over the entire duration of the experiment. Interestingly, h[Gly2]-GLP-2-treated mice demonstrated less weight loss between days 2–6 and 14–20 (data not shown; P < 0.05, mice treated with saline versus h[Gly2]-GLP-2 after IRT). Although h[Gly2]-GLP-2 enhanced survival and reduced weight loss, it did not impair IRT-induced tumor regression (Fig. 1 D).
Because chemotherapy administration may be associated with increased intestinal permeability and bacterial septicemia, we assessed bacterial infection in chemotherapy-treated mice. h[Gly2]-GLP-2-treated mice exhibited a significant reduction in bacterial culture positivity in all organs examined 96 h after IRT administration (Fig. 2,A, P < 0.05 for mice treated with saline versus h[Gly2]-GLP-2 after IRT). Furthermore, the bacterial burden (expressed as bacterial colonies/gram tissue) was significantly reduced in the liver and spleen (Fig. 2,B, P < 0.05, mice treated with saline versus h[Gly2]-GLP-2 after IRT) and in the blood (Fig. 2,C, P < 0.05, mice treated with saline versus h[Gly2]-GLP-2 after IRT)in h[Gly2]-GLP-2-treated mice. A significant leukopenia was observed in mice after IRT treatment, and the mean WBC count was modestly but significantly higher in h[Gly2]-GLP-2-treated mice (Fig. 2 D).
To assess the histological consequences of h[Gly2]-GLP-2 action in the setting of chemotherapy, we analyzed the crypt compartment of IRT-treated mice. Morphometric analysis revealed a significant reduction in both the number of crypts and the number of cells within each crypt in the small and large intestine after IRT treatment (Fig. 3, E, a–e). h[Gly2]-GLP-2 significantly reduced the rate of crypt loss in the jejunum (Fig. 3,A) and restored crypt cell number 96 h after IRT treatment (Fig. 3,B). Similarly, h[Gly2]-GLP-2 pretreatment prevented crypt loss and enhanced the crypt cell number in the colon (Fig. 3, C and D).
To understand the mechanisms by which h[Gly2]-GLP-2 protected the crypt compartment of the small and large intestine from IRT-induced injury, a temporal and spatial analysis of apoptosis in the crypt compartment was performed. The number of apoptotic crypt cells was markedly increased after IRT treatment and significantly reduced in mice pretreated with h[Gly2]-GLP-2 (data not shown). Because pluripotent stem cells within the crypt compartment are thought to reside at cell positions 3–5 in the small intestine and 1–3 in the colon, whereas the clonogenic potential stem cells reside at positions 6–8 in the small intestine and 5–7 in the colon (21, 22), a positional topographical assessment of apoptosis within the crypt compartment was performed. h[Gly2]-GLP-2 pretreatment significantly reduced apoptosis in the jejunum at crypt cell positions 4–5 (Fig. 4,A, P < 0.05, saline-versus h[Gly2]-GLP-2-treated mice). Similarly,h[Gly2]-GLP-2 reduced apoptosis in the colon at crypt cell positions 3–5, 7, and 8 (Fig. 4,B, P < 0.05, saline- versus h[Gly2]-GLP-2-treated mice). Furthermore, a significant reduction in procaspase-8 cleavage was observed in the colon of h[Gly2]-GLP-2-treated mice at both 72 and 96 h after IRT treatment (Fig. 4 C, P < 0.05 and P < 0.01 for saline- versus h[Gly2]-GLP-2-treated mice receiving IRT).
The small and large intestine is comprised of a mixed heterogeneous population of cell types that may be differentially affected by IRT. Because intestinal cell lines expressing the endogenous GLP-2 receptor have not yet been identified, we used BHK cells expressing the rat GLP-2 receptor (23) to examine the direct effects of GLP-2 on apoptosis in vitro. IRT induced apoptosis in BHK-GLP-2R cells as evidenced by detection of chromatin condensation and nuclear fragmentation (Fig. 5,A). A significant improvement in cell viability was observed in BHK-GLP-2R cells but not control BHK cells after pretreatment with h[Gly2]-GLP-2 for 36 h before IRT administration (Fig. 5,B). Analysis of caspase-8- and capase-9-like protease activity after IRT treatment was quantified by assessing cleavage of the substrates Ac-IETD-pNA and Ac-LEHD-pNA, respectively. h[Gly2]-GLP-2 treatment significantly reduced caspase-8-like enzymatic activity (Fig. 5,C; P < 0.05). In contrast, h[Gly2]-GLP-2 had no effect on the levels of caspase-9-like enzymatic activity in IRT-treated cells (Fig. 5,C). h[Gly2]-GLP-2 also reduced the IRT-induced cleavage of caspase-3 substrate Ac-DEVD-pNA and decreased procaspase-3 cleavage into the active p17 subunit (Fig. 5,D). Furthermore,h[Gly2]-GLP-2 also decreased the IRT-induced cleavage of PARP, a downstream substrate of activated caspase-3 (Fig. 5 E).
IRT hydrochloride, a potent DNA topoisomerase I inhibitor, is active against a broad variety of hematological and gastrointestinal neoplasms and induces apoptosis in both normal and neoplastic cell types (24, 25). Furthermore, IRT produces extensive intestinal toxicity that is manifested histologically as mucositis and clinically as both early- and late-onset diarrhea in both human(2, 26) and rodent studies (27, 28). Our results demonstrate that administration of h[Gly2]-GLP-2 significantly improves survival, reduces bacterial infection, and decreases intestinal damage in IRT-treated mice. The protective effects of h[Gly2]-GLP-2 are not restricted to a single class of chemotherapeutic agent because h[Gly2]-GLP-2 significantly increased survival in 5-FU-treated animals. Furthermore, h[Gly2]-GLP-2 also improves survival and reduces weight loss in IRT-treated tumor-bearing mice, demonstrating that the protective effects of GLP-2 are not diminished in the setting of active neoplasia.
The significant reduction in chemotherapy-associated mortality in h[Gly2]-GLP-2-treated mice may be explained in part by the reduction in circulating bacteremia. Recent experiments have demonstrated that GLP-2 reduces mucosal permeability in rats after major small bowel resection (29). Furthermore, GLP-2 markedly reduced circulating bacteremia and decreased bacterial infection in the liver and spleen in mice after indomethacin-induced intestinal injury(15). Although the precise mechanism(s) activated by h[Gly2]-GLP-2 leading to reduction in bacterial infection remains unknown, the demonstration that h[Gly2]-GLP-2 reduced macromolecule flux, decreased intestinal permeability, and markedly enhanced intestinal barrier function in GLP-2-treated mice (30)provides a clear link between GLP-2 action and reduced bacterial translocation in the setting of intestinal injury. Hence, it seems likely that GLP-2-mediated enhancement of intestinal barrier function contributes to the reduction in bacterial sepsis observed after IRT administration in h[Gly2]-GLP-2-treated mice.
The initial observation that GLP-2 exerts trophic actions in the intestinal mucosa was largely attributed to stimulation of crypt cell proliferation (11, 14). Although the number of identifiable cells undergoing spontaneous apoptosis in the normal intestinal crypt compartment is low, intestinal injury after exposure to ionizing radiation or chemical agents results in marked induction of apoptosis in the crypt compartment (6, 7, 8, 20, 21, 22, 31, 32). Our finding that GLP-2 reduces the percentage of apoptotic cells in the crypt compartment after chemotherapy is consistent with recent evidence demonstrating a marked reduction in crypt apoptosis after GLP-2 treatment of mice with indomethacin-induced enteritis(15). Hence, the available evidence suggests that GLP-2 maintains the integrity of the intestinal epithelium by both stimulating cell proliferation and inhibiting apoptotic cell death in the crypt compartment.
What are the mechanisms activated by GLP-2 signaling that confer resistance to apoptosis-mediated injury in the intestinal epithelium after IRT treatment? Consistent with studies demonstrating the importance of the CPP32 subfamily of caspases in the pathogenesis of IRT-induced apoptosis (24), we observed reduced IRT-mediated caspase-3 and PARP activation in BHK-GLP-2R cells after GLP-2 treatment in vitro. Furthermore, a significant reduction of procaspase-8 cleavage, as evidenced by inhibition of p18 subunit generation, was observed in GLP-2-treated intestine after IRT administration in vivo. In contrast, we did not observe any changes in the levels of caspase-9 after GLP-2 treatment of mice in vivo or of cells in vitro. These findings,summarized in Fig. 6, demonstrate for the first time that GLP-2 receptor signaling may be linked directly to specific cell survival pathways in heterologous cell types.
Although signaling through G protein-coupled receptors of the glucagon/GLP-1/GLP-2 receptor superfamily has not previously been reported to modify apoptotic pathways, recent experiments suggest an emerging link between G protein-coupled receptor signaling and cell death. Activation of the somatostatin receptor modulates pH-dependent cell death in heterologous cell types (33, 34), and signaling through the parathyroid hormone/parathyroid hormone-related protein receptor diminishes activation of apoptotic pathways in cells of the chondrocyte and osteoblast lineages (35). Although the cellular localization of intestinal GLP-2 receptor expression has not yet been identified, our data clearly suggest that intestinal cells expressing the GLP-2R are likely to be protected from cell death associated with exposure to genotoxic stress in vivo. Given the emerging importance of GLP-2 receptor signaling for preservation of intestinal mucosa in the face of external injury(15, 29, 36, 37), our findings provide a scientific rationale for exploring the therapeutic use of GLP-2 in settings characterized by induction of intestinal injury via activation of apoptosis in the mucosal epithelium in vivo.
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.
This work was supported in part by grants from the Medical Research Council of Canada and the Ontario Research and Development Challenge Fund. R. P. B. is a research fellow and D. J. D. is a Senior Scientist of the Medical Research Council of Canada. GLP-2 is the subject of a licensing agreement between the University of Toronto, Toronto General Hospital, D. J. D., and NPS Pharmaceuticals Corp., and D. J. D. is a consultant to NPS Pharmaceuticals Corp.
The abbreviations used are: GLP, glucagon-like peptide; PARP, poly(ADP-ribose) polymerase; IR, inhibition ratio;TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; BHK, baby hamster kidney; pNA, paranitroanilide; 5-FU,5-fluorouracil; IRT, irinotecan.