Bcr-Abl inhibitors, such as bosutinib and imatinib, are predominantly used for treatment of chronic myeloid leukaemia. However, lower gastrointestinal toxicity, such as diarrhoea, is a prevalent adverse drug reaction (ADR). For example, bosutinib and imatinib cause diarrhoea in up to 90% and 50% of patients, respectively.1 This can decrease patient quality of life, treatment efficacy and in severe cases cause patient hospitalisation. We aim to elucidate the mechanism of Bcr-Abl inhibitor-induced diarrhoea to help abrogate the aforementioned issues.Methods
Caco-2 cells (human colorectal cancer cells resembling small intestinal cells) were differentiated into monolayers of polarised enterocytes and utilised as an in vitro model. Cells were seeded into transwells and electrical resistance or flux of FITC-dextran (a fluorescently labelled polysaccharide) across the monolayer was measured to assess changes in paracellular permeability. Enteroids (small intestinal organoids) produced from male BALB/c mice were used as an ex vivo model. Changes in permeability of enteroids were determined by leakage of injected FITC-dextran out of the enteroid. Changes in mRNA levels, protein levels and protein localization of tight junction components were studied using RTqPCR, immunoblotting and immunofluorescence, respectively. Drug-induced cell death was assessed by CellTitreGlo and Toxilight assays for Caco-2 cells and enteroids, respectively. Results were analysed by ANOVA and are representative of ≥3 independent experiments.Results
25 µM bosutinib increased paracellular permeability of Caco-2 monolayers to ions and FITC-dextran (ANOVA, p<0.05), whilst imatinib was less effective at inducing this change. 10 µM bosutinib increased enteroid leakage (ANOVA, p<0.01) but 10 µM imatinib had no effect. All concentrations tested were sub-apoptotic.Results
In Caco-2 cells, bosutinib caused relocalization and decreased protein levels of intercellular junction proteins E-cadherin, Occludin and ZO-1. Bosutinib also transiently decreased mRNA levels of ZO-1 but not that of E-cadherin or Occludin. Imatinib did not alter mRNA levels, protein levels or localization of any of these proteins.Results
Endoplasmic reticulum (ER) stress is involved in intercellular junction degradation; 2 therefore, we assessed whether Bcr-Abl inhibitors could induce ER stress. However, no increase in ER stress markers BiP or CHOP were detected after bosutinib or imatinib treatment in Caco-2 cells.Conclusions
Decreased intestinal barrier integrity is likely an important factor in the aetiology of bosutinib-induced diarrhoea. This is potentially mediated by intercellular junction degradation. Understanding the mechanism by which Bcr-Abl inhibitors induce diarrhoea will aid in abrogation of diarrhoea ADRs.