Effects of azathioprine and its metabolites on repair mechanisms of the intestinal epithelium in vitro.
Erosions and ulcerations of the intestinal epithelium are hallmarks of inflammatory bowel diseases (IBD). Intestinal epithelial cell migration (restitution) and proliferation are pivotal mechanisms for healing of epithelial defects after mucosal injury. In addition, the rate of apoptosis of epithelial cells may modulate intestinal wound healing. The purine antagonists azathioprine (AZA) and 6-mercaptopurine (6-MP) are widely used drugs in the treatment of IBD. In the present study, the hitherto unknown effects of AZA as well as its metabolites 6-MP and 6-thioguanine (6-TG) on repair mechanisms and apoptosis of intestinal epithelia were analysed. Intestinal epithelial cell lines (human Caco-2, T-84 and HT-29 cells, rat IEC-6 cells) were incubated with AZA, 6-MP or 6-TG for 24 h (final concentrations 0.1-10 microM). Migration of Caco-2 and IEC-6 cells was analysed by in vitro restitution assays. Caco-2 and IEC-6 cell proliferation was evaluated by measurement of [3H]thymidine incorporation into DNA. Apoptosis of Caco-2, T-84, HT-29 and IEC-6 cells was assessed by histone ELISA, 4'6'diamidino-2'phenylindole-dihydrochloride staining as well as flow cytometric analysis of Annexin V/propidium iodide (PI)-stained cells. Cell cycle progression was evaluated by PI staining and flow cytometry. Epithelial restitution was not significantly affected by any of the substances tested. However, proliferation of intestinal epithelial cells was inhibited in a dose-dependent manner (maximal effect 92%) by AZA, 6-MP as well as 6-TG. In HT-29 cells, purine antagonist-effected inhibition of cell proliferation was explained by a cell cycle arrest in the G2 phase. In contrast, AZA, 6-MP and 6-TG induced no cell cycle arrest in Caco-2, T-84 and IEC-6 cells. AZA, 6-MP as well as 6-TG induced apoptosis in the non-transformed IEC-6 cell line but not in human Caco-2, T-84 and HT-29 cells. In summary, AZA and its metabolites exert no significant effect on intestinal epithelial restitution. However, they profoundly inhibit intestinal epithelial cell growth via various mechanisms: they cause a G2 cell cycle arrest in HT-29 cells, induce apoptosis in IEC-6 cells and dose-dependently inhibit intestinal epithelial proliferation.
Schroll
Sebastian
S
Department of Gastroenterology, Hepatology, and Endocrinology, Medizinische Hochschule Hannover, Hannover, Germany.
Sarlette
Alexander
A
Ahrens
Katja
K
Manns
Michael P
MP
Göke
Michael
M
D000818:Animals; D017209:Apoptosis; D001379:Azathioprine / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D002453:Cell Cycle; D002460:Cell Line; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D015212:Inflammatory Bowel Diseases / Q000188:drug therapy / Q000473:pathology*; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D051381:Rats
D007166:Immunosuppressive Agents; D001379:Azathioprine