Intestinal glucuronidation protects against chemotherapy-induced toxicity by irinotecan (CPT-11) - PubMed (original) (raw)

Intestinal glucuronidation protects against chemotherapy-induced toxicity by irinotecan (CPT-11)

Shujuan Chen et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Camptothecin (CPT)-11 (irinotecan) has been used widely for cancer treatment, particularly metastatic colorectal cancer. However, up to 40% of treated patients suffer from severe late diarrhea, which prevents CPT-11 dose intensification and efficacy. CPT-11 is a prodrug that is hydrolyzed by hepatic and intestinal carboxylesterase to form SN-38, which in turn is detoxified primarily through UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed glucuronidation. To better understand the mechanism associated with toxicity, we generated tissue-specific Ugt1 locus conditional knockout mouse models and examined the role of glucuronidation in protecting against irinotecan-induced toxicity. We targeted the deletion of the Ugt1 locus and the Ugt1a1 gene specifically in the liver (Ugt1(ΔHep)) and the intestine (Ugt1(ΔGI)). Control (Ugt1(F/F)), Ugt1(ΔHep), and Ugt1(ΔGI) adult male mice were treated with different concentrations of CPT-11 daily for four consecutive days. Toxicities were evaluated with regard to tissue glucuronidation potential. CPT-11-treated Ugt1(ΔHep) mice showed a similar lethality rate to the CPT-11-treated Ugt1(F/F) mice. However, Ugt1(ΔGI) mice were highly susceptible to CPT-11-induced diarrhea, developing severe and lethal mucositis at much lower CPT-11 doses, a result of the proliferative cell loss and inflammation in the intestinal tract. Comparative expression levels of UGT1A1 in intestinal tumors and normal surrounding tissue are dramatically different, providing for the opportunity to improve therapy by differential gene regulation. Intestinal expression of the UGT1A proteins is critical toward the detoxification of SN-38, whereas induction of the UGT1A1 gene may serve to limit toxicity and improve the efficacy associated with CPT-11 treatment.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Liver hepatocyte specific deletion of the Ugt1a1 gene and its impact on bilirubin and SN-38 glucuronidation. (A) The location of the LoxP sequence (Ugt1 F/F) and the result of albumin-Cre–mediated deletion (Ugt1 ΔHep mice). (B) Ugt1 ΔHep mice accumulate 2–3 mg/dL TSB. (C) In the Ugt1 ΔHep liver, exons 3 and 4 are deleted in liver tissue as shown by RT-PCR analysis. Primers used are specific for exons 1 and 5. (D) Western blot analysis shows no expression of UGT1A1 in Ugt1 ΔHep MLMs. (E) Bilirubin glucuronidation analysis was performed in neonatal Ugt1 ΔHep MLMs (mean ± SEM). (F) SN-38 glucuronidation analysis was performed by using adult Ugt1 ΔHep MLMs (mean ± SEM). ***P < 0.001, Student t test.

Fig. 2.

Fig. 2.

Intestinal enterocyte-specific deletion of the Ugt1a1 gene and its impact on bilirubin and SN-38 glucuronidation. (A) The generation of villin-Cre–mediated deletion (Ugt1 ΔGI mice). (B) Ugt1 ΔGI mice have normal or undetectable levels of TSB. (C) In the intestines of Ugt1 ΔGI mice, exons 3 and 4 are deleted, as shown by RT-PCR analysis. (D) Western blot analysis shows no expression of UGT1A1 protein in Ugt1 ΔGI MIMs. (E) Bilirubin glucuronidation assay was conducted in neonatal Ugt1 ΔGI MIMs (mean ± SEM). (F) SN-38 glucuronidation activity in adult Ugt1 ΔGI MIMs (mean ± SEM). ***P < 0.001, Student t test.

Fig. 3.

Fig. 3.

CPT-11–induced lethality in Ugt1 F/F vs. Ugt1 ΔHep mice (A and B) and Ugt1 F/F vs. Ugt1 ΔGI mice (C–E). Age-matched adult male mice or littermates were used for these experiments. CPT-11 was administered to Ugt1 F/F, Ugt1 ΔHep, and Ugt1 ΔGI mice (10 mice per group) once per day for 4 constitutive days by i.p. injection. The respective doses are indicated above each graph. CPT-11–induced lethality was determined over the course of 3 wk. GraphPad Prism software was used to graph the survival vs. time curves and to calculate the P value.

Fig. 4.

Fig. 4.

CPT-11–induced weight loss and hematotoxicity in Ugt1 F/F, Ugt1 ΔHep, and Ugt1 ΔGI mice. Age-matched adult male mice or littermates were used for these experiments. (A) CPT-11 was administered to Ugt1 F/F and Ugt1 ΔHep mice (10 mice per group) at the 75-mg/kg dose once per day for 4 constitutive days by i.p. injection. Weight was monitored daily. After data collection, percentage of weight loss in comparison with the pretreatment was described as the mean ± SD. Two-way ANOVA was used for statistical analysis. (B) Percentage of weight loss in Ugt1 F/F and Ugt1 ΔGI mice after CPT-11 treatment at 10 mg/kg after the same dosing regimen as described. (C) Correlation of survival and weight loss. Data are described in a 2 × 2 contingency table. Two-tailed P value was analyzed by Fisher’s exact test.

Fig. 5.

Fig. 5.

CPT-11–induced intestinal damage in Ugt1 ΔGI mice. Intestinal tissue from untreated (Control) or treated Ugt1 ΔGI mice was analyzed on the fifth day after 4 consecutive days of treatment with 10 or 25 mg/kg CPT-11. Intestinal tissue was sectioned into duodenum, jejunum, ileum, and colon and the morphology of the intestines photographed after H&E staining.

Fig. 6.

Fig. 6.

(A) Intestinal epithelium cell proliferation resulting from CPT-11 treatment. Ugt1 F/F and Ugt1 ΔGI mice were treated with 10 or 25 mg/kg CPT-11 as outlined and cellular proliferation monitored by Ki67 (in green) and β-catenin (in red) immunohistochemistry for cell membrane staining, along with DAPI nuclear counterstain (blue). (B) Ki67, β-catenin, and DAPI immunofluorescences staining in colon tissues. (C) After treatment with irinotecan at 10 mg/kg, on day 5, jejunum total RNA was prepared from the Ugt1 F/F and Ugt1 ΔGI mice and used to quantitate the expression of cytokines IL-6 by RT and qPCR. (D) IL-6 expression was also determined in colon tissues from the CPT-11 treated Ugt1 F/F and Ugt1 ΔGI mice. Results were described as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, Student t test.

Fig. 7.

Fig. 7.

Gene expression patterns in normal tissue (N) and colon tumors (T). Both tumor tissues and the adjacent normal colon tissues were collected from CPC-APC mice, total RNA prepared, and gene expression monitored by RT followed by qPCR analysis. Each gene analysis was performed on five individual samples (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, Student t test. Gene expression is either repressed (A) or induced (B) in tumor tissue. (C) Xenobiotic nuclear receptor expression patterns.

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