Tangutorine induces p21 expression and abnormal mitosis in human colon cancer HT-29 cells

Abstract: A novel β-carboline alkaloid, tangutorine (benz[f]indolo[2,3-a]quinolizidine) was isolated from the leaves of Nitraria tangutorum L. [Duan JA, Williams ID, Che CT, Zhou RH, Zhao RH, Tangutorine: a novel β-carboline alkaloid from Nitraria tangutorum. Tetrahedron Lett 1999;40:2593–6], and its unique structural characters led us to initiate a study of its potential anti-proliferation activity. The in vitro treatment with low doses of tangutorine slightly stimulated the proliferation of human colon cancer HT29 cells until at concentrations higher than 6.25μg/ml when the cell numbers, cellular MTT reduction, and cell proliferation by 3H-thymidine incorporation decreased in a dose-dependent manner (IC50 =15μg/ml=48μM). Morphological studies of cells by fluorescence and electron microscopy did not show features for apoptosis but only large vacuoles, swollen mitochondria and dense cytoskeletal filaments bunching in the cytoplasm. Immunoblotting analysis revealed a dramatic induction of cyclin kinase inhibitor p21 as well as an inhibition of topoisomerase II expression at 25μg/ml tangutorine, thereby impeding cell progression from S to G2/M phase. Cells accumulated at G1 phase of the cell cycle at concentrations ≥50μg/ml tangutorine. Interestingly, some cells escaped from prolonged growth arrest without cell division and resulted in binucleated and polyploid G1 cells. Taken all results together, tangutorine induced a p21 suppression of all cyclins and their associated kinases, such as the topoisomerase II, and thus inhibited normal DNA replication and mitosis. [Copyright &y& Elsevier]