The impact of camptothecin treatment on the developing intestinal epithelial cell: Cellular and molecular mechanisms of cytotoxicity.

摘要

The major obstacle in clinical use of camptothecins family anticancer drugs is the unexpected and significant incidence of gastrointestinal toxicities, which limit the further evaluation of these drugs in clinical trials.; The aim of this dissertation was to determine the cellular and molecular mechanisms of intestinal epithelial cell toxicity induced by camptothecin family drugs. To accomplish this aim, Caco-2 cell line was used as an in vitro model of human intestinal epithelial cells. Cytotoxicity studies were performed in parallel with the in vivo complex of topoisomerase (ICT) bioassay (specifically measures topo-I DNA cleavable complexes induced by camptothecins) in Caco-2 cells at different stages of their development. Furthermore, toposomerase-I related parameters (e.g. topoisomerase-I activity and protein level) were examined in relation to cleavable complex formation during differentiation of epithelial cells. Moreover, the downstream cellular responses to camptothecins that occur after cleavable complex formation including (1) cell cycle perturbations, (2) repair of DNA damage, (3) ability of cells to undergo apoptosis, (4) activation of signaling pathways initiated by DNA damage, and (5) engagement of the enzymatic machinery that results in apoptosis were examined. The potential role of these events in increased sensitivity of the epithelial cells to camptothecins was evaluated. Our studies clearly demonstrated that cleavable complex formation reflects the topo-I mediated DNA damage and plays a central role in differential sensitivity of epithelial cells to camptothecins during their proliferation and differentiation. Comparison of cleavable complexes and topo-I protein levels also showed that these two parameters were highly correlated in our experimental system. Specifically, differentiated cells were less sensitive to topo-I mediated DNA damage induced by camptothecins compared to proliferating cells and this effect was associated with depressed topo-I protein levels and activity in differentiated cells and up-regulation of topo-I in proliferating cells. Based on these findings, we suggest that the mechanism of sensitivity to camptothecins results from the differential regulation of topo-I protein during the development of intestinal epithelial cells in vivo. Alternatively, changes in global chromatin accessibility as cells differentiate could restrict access to DNA, thereby reducing cleavages and DNA damage in differentiated cells.