Chitosan Oligosaccharide Prevents Afatinib-Induced Barrier Disruption and Chloride Secretion through Modulation of AMPK, PI3K/AKT, and ERK Signaling in T84 Cells

Abstract

Diarrhea is an important adverse effect of epidermal growth factor receptor-tyrosine kinase inhibitors, especially afatinib. Novel antidiarrheal agents are needed to reduce epidermal growth factor receptor-tyrosine kinase inhibitor-associated diarrhea to improve the quality of life and treatment outcome in cancer patients. This study aimed to investigate the anti-diarrheal activity of chitosan oligosaccharide against afatinib-induced barrier disruption and chloride secretion in human intestinal epithelial cells (T84 cells). Chitosan oligosaccharide (100 μg/mL) prevented afatinib-induced barrier disruption determined by changes in transepithelial electrical resistance and FITC-dextran flux in the T84 cell monolayers. In addition, chitosan oligosaccharide prevented afatinib-induced potentiation of cAMP-induced chloride secretion measured by short-circuit current analyses in the T84 cell monolayers. Chitosan oligosaccharide induced the activation of AMPK, a positive regulator of epithelial tight junction and a negative regulator of cAMP-induced chloride secretion. Moreover, chitosan oligosaccharide partially reversed afatinib-induced AKT inhibition without affecting afatinib-induced ERK inhibition via AMPK-independent mechanisms. Collectively, this study reveals that chitosan oligosaccharide prevents the afatinib-induced diarrheal activities in T84 cells via both AMPK-dependent and AMPK-independent mechanisms. Chitosan oligosaccharide represents a promising natural polymer-derived compound for further development of treatment for afatinib-associated diarrheas.

Chitosan Oligosaccharide Promotes Junction Barrier through Modulation of PI3K/AKT and ERK Signaling Intricate Interplay in T84 Cells

“…The cytotoxic activity on T84 cells was determined by MTT assays as previously described [ 17 ]. Briefly, 5000 cells were seeded in 96-well cell culture plates and cultured for 24 h at 37 °C.…”

“…The measurement of integrity of epithelial tight junction was determined as previously described [ 17 ]. Briefly, T84 cells (5 × 10 5 cells/support) were seeded on a Snapwell permeable support and cultured for 3–7 days to develop monolayers.…”

“…It has been reported that COS with an MW of ~5000 Da is the most potent polymer promoting junction barriers through adenosine 3′, 5′-cyclic monophosphate (AMP)-activated protein kinase (AMPK)-activation and extracellular Ca 2+ -dependent manners in T84 cell monolayers [ 16 ]. AMPK acts as a positive regulator of epithelial tight junction and a negative regulator of cAMP-induced chloride secretion [ 17 ]. COS-induced activation of AMPK is arbitrated by calcium-sensing receptor (CaSR)-mediated calcium release from the endoplasmic reticulum [ER] in the intestinal epithelial cells [ 16 ].…”

“…Previously, we have reported the anti-diarrheal activity of COS against afatinib-associated diarrhea in colorectal T84 cancer cells. COS promotes tight junction integrity and prevents afatinib-induced potentiation of cAMP-induced chloride secretion by AMPK activation [ 17 ].…”

“…Mutations or pathophysiological conditions disrupting the crosstalk between AKT and MAPK/ERK signalings lead to disease development through overactivation of the individual pathways [ 27 ]. We previously reported that COS promotes tight junction integrity through activation of AKT and inhibition of ERK pathways [ 17 ]. However, the exact intricate interplay mechanism between AKT and ERK signaling cascades mediated by COS in inducing barrier junction integrity remains unclear.…”