311 PUBLICATION Different cell cycle kinetic effects of N1N11-diethylnorspermineinduced polyamine depletion in four human breast cancer cell lines

Abstract

3-Hydrogenkwadaphnin (3-HK) (Fig. 1) is a daphnane-type diterpene ester isolated from the leaves of Dendrostellera lessertii (Thymelaeaceae) with differentiation and apoptotic potency among several leukemic cells without any measurable adverse effects on normal cells [Moosavi, M.A., Yazdanparast, R., Sanati, M.H., Nejad, A.S., 2005a. 3-Hydrogenkwadaphnin targets inosine 5′-monophosphate dehydrogenase and triggers post-G1 arrest apoptosis in human leukemia cell lines. Int. J. Biochem. Cell Biol. 37, 2366–2379]. In this study, we evaluated differentiating and apoptotic efficiency of a second new anti-proliferating agent from the same plant relative to 3-HK using acute myeloid leukemia (AML) KG1 cell line. 3-HK at 5–30 nM inhibited proliferation of KG1 cells after 24–96 h of treatment. NBT reducing assay and expression of cell surface markers (CD11b and CD14) confirmed that the inhibition of proliferation is associated with differentiation toward macrophage-like morphology. Regarding the relatively weaker potency of 3-HK in the induction of differentiation compared to that of the crude extract, we looked for additional compound(s) with similar properties in the crude extract. This effort led to isolation of the second compound from the leaves’ extract with higher differentiating potency. The new compound inhibited proliferation of KG1 cells by almost 48±3.1% after 72 h of treatment with a single dose of 1.5 μg/ml. The treated cells differentiated along the monocyte/macrophage lineage based on the morphological features apparent after Wright–Giemsa staining, phagocytic activity and expression of cell surface markers as analyzed by flow cytometry. On the other hand, the results indicated that exposure of KG1 cells to either 3-HK or the new compound for 3–4 days induced apoptosis as assayed qualitatively by acridine orange/ethidium bromide (Ao/EtBr) double staining, agarose gel electrophoresis and quantitatively by Annexin-V technique and sub-G1 DNA staining using flow cytometry. Based on the present data, these two active constituents of D. lessertii have the novelty of being further evaluated for pharmaceutical applications.