The Cytotoxicity Of Irinotecan To Mouse Peritoneal Resident Macrophages And The Underlying Mechanism

Aim:CPT-11(Irinotecan) is a semisynthetic derivative of camptothecin which is used as a first-line chemotherapeutic agent in clinic. It has been reported that the metabolic product of CPT-11, SN-38, causes fragmentation of the chromosomal DNA and leads to apoptotic cell death by stabilizing topoisomerase I-DNA covalent complex in tumor cells. Unfortunately, induction of undesirable poisonous effects, such as vomiting, nausea or delayed diarrhea and even enteritis in the patients, has become a major obstacle for the clinical application of CPT-11 even though it can improve the effective chemotherapy. The underlying mechanism of CPT-11’s intestinal toxicity is not fully understood. It is unclear whether CPT-11 is cytotoxic to peritoneal resident macrophages. In this study, CPT-11 was administered intraperitoneally to mice, which induced enteritis as expected, and their peritoneal exudate cells were isolated for evaluation of their composition and phenotypes. We aimed to explore whether CPT-11 is toxic to peritoneal macrophages and what the underlying mechanism is. Considering the relationship of peritoneal macrophages, B-1cells and intestinal homeostasis, we also explored the effects of CPT-11 on B-1 cells. Our finding was likely to explain, at least partly, why CPT-11 treatment in cancer patients induces diarrhea and enteritis. Methods:1. In vitro: the effect of CPT-11 on RAW 264.7 cell proliferation was evaluated by WST-1 assay. Nuclei were revealed by Hoechst 33342 staining. Cell cycle was analyzed by propidium iodide(PI) staining together with flow cytometry. The regulation of CPT-11 on cleaved caspase-3, PARP and cell cycle-related proteins was determined by Western blotting.2. In vivo: CPT-11 was administered intraperitoneally to mice. The change of mouse body weight was monitored every day and the jejunum and colon were analyzed by histochemistry with hematoxylin and eosin(HE) staining. The expression of peritoneal macrophage markers(CD11b, F4/80, and MHC II) and B cell markers(CD19 and CD23) was analyzed by flow cytometry. Cell cycle distribution and cell apoptosis of peritoneal macrophages were analyzed by flow cytometry after PI staining. The expression of GATA6 and cleaved caspase-3 in the peritoneal macrophages was observed by immunofluorescence microscopy. The expression of cleaved caspase-3 and PARP in CPT-11-treated peritoneal macrophages was determined by Western blotting. Results:1. CPT-11 inhibited the proliferation of RAW 264.7 cells in a time- and dose-dependent manner, and the 50% inhibiting concentration(IC50) values of 24 hand 48 h-treatments were 81.36 μmol/L and 18.84 μmol/L, respectively, suggesting that CPT-11 was cytotoxic to RAW 264.7 cells. Under the action of CPT-11, RAW 264.7 cells and their nuclei became bigger. CPT-11 induced nuclear fragmentation(indicating cell apoptosis) in a dose-dependent manner. The pseudopodium protrusions as observed in control cells were disappeared after CPT-11 treatment and the cells became round compared with the control group. The ratios of cells at hypodiploid peak(apoptotic peak, indicating DNA degradation and cell apoptosis) and G2/M phase were increased with the increase of CPT-11 doses. Moreover, CPT-11 resulted in activation of caspase-3, decreased PARP levels, as well as increased levels of cyclin B1 but decreased levels of cyclin D1.2. CPT-11 treatment induced body weight loss in the mice and induced inflammation in the colon and jejunum. HE staining showed that the jejunum and colon generated inflammation lesions and lymphocytes invaded the site of inflammation.3. CPT-11 treatment eliminated the so-called large peritoneal macrophages and suppressed their expression of a vital transcription factor GATA6. The ratios of peritoneal macrophages at hypodiploid peak(apoptotic peak) and G2/M phase were also increased with the increase of CPT-11 doses. Mechanically, CPT-11 treatment resulted in activation of caspase-3 and the decrease of PARP expression in the peritoneal macrophages. Caspase-3 inhibitor z-DEVD-fmk attenuated CPT-11-induced loss of GATA6+ peritoneal macrophages and decrease of PARP in the mice.4. CPT-11 administration reduced the numbers of peritoneal B and B-1 cells. Conclusion:1. CPT-11 inhibited the proliferation of RAW 264.7 cells by arresting their cell cycle and induced apoptosis through the caspase-3 pathway.2. CPT-11 depleted GATA6+ peritoneal resident macrophages via inducing cell cycle arrest as well as caspase-3-dependent apoptosis.3. Depleting of GATA6+ peritoneal resident macrophages had consequently impaired the numbers and functions of peritoneal resident macrophages and peritoneal B-1 cells. Because of the suppression of their innate functions, the intestinal inherent immune function also was consequently impaired, this may be the main reason why CPT-11 induces diarrhea and enteritis during its chemotherapeutic application on tumor patients.