The pyrrolizidine alkaloid lasiocarpine impairs cell cycle progression in vitro.

Abstract

1,2-Unsaturated pyrrolizidine alkaloids (PA) induce severe acute and chronic hepatotoxicity. Effects include an impairment of the cell cycle. To elucidate this effect, cell cycle progression was analyzed by flow cytometry, changes in cell and nucleus size, differential gene expression and protein phosphorylation patterns of regulatory key proteins in the Chinese hamster lung fibroblast cell line V79 and/or the human hepatoblastoma cell line HepG2, both overexpressing human CYP3A4 (V79_3A4/HepG2_3A4). Lasiocarpine, a potent PA representative, reduced the cell viability of human CYP3A4-overexpressing cell lines concentration-dependently. Microscopic observation showed a marked increase in cell and nucleus size of V79_3A4 cells after exposure to 10 µM lasiocarpine. In the human CYP3A4-overexpressing cell lines, cells accumulate in G₂/M phase after lasiocarpine treatment. Based on these findings, the gene expression pattern of cell cycle-related genes was investigated in HepG2_3A4 cells showing a decrease of e. g. WEE1, and CHEK1 and an increase of PAK1 and ATM. While results on cell cycle regulation at the level of gene expression are of limited relevance, protein phosphorylation plays an important role. Therefore, we also elucidated the protein phosphorylation status of regulatory key proteins. The results clearly indicate an induction of the DNA damage response and a late G2 arrest. In conclusion, an impairment of the cell cycle was observed. It correlates with the metabolic activation of lasiocarpine and is most likely mediated by adduct formation of the reactive pyrrole esters with DNA, leading to a disruption of cellular homeostasis and genomic instability.