Potent induction of apoptosis by germacranolide sesquiterpene lactones on human myeloid leukemia cells

doi:10.1016/j.ejphar.2003.09.058

European Journal of Pharmacology

Volume 482, Issues 1–3, 15 December 2003, Pages 77-84

https://doi.org/10.1016/j.ejphar.2003.09.058 Get rights and content

Abstract

This paper studies the cytotoxic effect induced by four known natural sesquiterpene lactones (tatridin A, tamirin, reynosin, ineupatorolide A) and one synthetic derivative (tatridin A diacetate) on the myeloid leukemia cell lines HL-60 and U937. Tatridin A diacetate and ineupatorolide A were found to be the most cytotoxic compounds with growth inhibition caused by induction of apoptosis as determined by flow cytometry and microscopy of nuclear changes. The results reported here support the conclusion that apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase-1 and was also associated with an early release of cytochrome c from the mitochondria.

Introduction

Sesquiterpene lactones are natural products isolated from many plant families and most widely distributed within the Compositae. These compounds are known for their various biological activities, including cytotoxicity to tumor cells (Beekman et al., 1997). Covalent binding of sesquiterpene lactones to free sulfhydryl groups in proteins and interference with the functions of these macromolecules have been described Lee et al., 1997, Schmidt, 1997. For example, sesquiterpene lactones inhibit many enzymes involved in biological processes, such as DNA–RNA–protein synthesis (Schmidt, 1999). Although the precise mechanism of action of sesquiterpene lactones on growth inhibition is not quite clear, it is believed that α,β-unsaturated carbonyl compounds, in particular α-methylene lactones, exert their biological effect by acting as alkylating agents. These compounds react with nucleophiles, especially cysteine sulphydryl groups, by a Michael-type addition (Lyss et al., 1998). Thus, compounds with two α,β-unsaturated carbonyl groups may show considerable cytotoxic properties. The introduction of a more lipophilic residue (e.g. esterification of hydroxyl groups) should yield a compound with higher cytotoxic activity, although bulky groups near the reaction center could reduce the activity, i.e. there must be a balance between lipophilicity and steric hindrance. Moreover, conformational flexibility seems to be important in binding to the molecule target: a rather rigid molecule can lead cause important steric hindrance. In this study, all compounds present at least one α,β-unsaturated carbonyl group, the alkylating agent.

Physiological cell death occurs through an evolutionary conserved suicide process, termed apoptosis, which plays a considerable role in early development and homeostasis of adult tissues (Ameisen, 2002). Previous studies have demonstrated that apoptosis of human leukaemic cells can be induced with anticancer agents Makin and Dive, 2001, Makin and Dive, 2003. The main aim of this work was to investigate the cytotoxic effects of five sesquiterpene lactones (1 to 5) with wide structural variation in order to determine the structure–activity relationship (Fig. 1): the germacranolides tatridin A (1), diacetyl tatridin A (2), tamirin (desacetylchrysanolide, 3), and ineupatorolide A (4), and the eudesmanolide reynosin (5). We were also interested in investigating the possible mechanisms of action of these compounds on the growth inhibition/cell viability of human myeloid leukemia cells.

The present report demonstrates that exposure to sesquiterpene lactones of the germacranolide type elicits apoptosis on HL-60 and U937 cells as assessed by flow cytometry, induction of double-stranded DNA damage and appearance of apoptotic morphology. Also cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) and procaspase-3 processing occurred after treatment with sesquiterpene lactones. We further investigate the mechanism underlying tumor cell growth inhibition by these compounds and find mitochondria to be one of the primary targets of the sesquiterpenes' proapoptotic function.

Section snippets

Drugs and reagents

All sesquiterpene lactones were obtained from Canary Island endemic plants and were isolated according to published methods with minor modifications. Structural identities of sesquiterpene lactones were determined spectroscopically (proton nuclear magnetic resonance, infrared spectroscopy, mass spectrometry) as described previously: 1 (tatridin A), 2 (diacetyl tatridin A), 3 (tamirin), 4 (ineupatorolide A), 5 (reynosin). Tatridin A (1) was isolated from the aerial parts of Tanacetum

Sesquiterpene lactones induce apoptosis in human myeloid leukemia cells

All sesquiterpene lactones tested (Fig. 1) were found to inhibit the growth and cell viability of HL-60 and U937 cells in culture as determined by the 3-[4,5-dimethylthiazol-2-yl-]2,5-diphenyl tetrazolium bromide (MTT) dye-reduction assay (Table 1). Compounds 2 and 4 were found to be the most potent, while 1 and 5 were least potent. The potency of these sesquiterpene lactones on inhibition of proliferation in HL-60 cells were as follows: 2>4∼3>1∼5; meanwhile in U937 cells, the potency of these

Discussion

In this study, we have tested HL-60 and U937 cells for their sensitivity to different sesquiterpene lactones. In HL-60 cells, compound 2 was the most cytotoxic compound tested, probably due to its high lipophilicity. However, other factors, such as molecular geometry, the chemical environment of the target sulfhydryl and the presence of two α,β-unsaturated esters (although these groups have a low alkylating potency on their own) may also influence the activity of sesquiterpene lactones. The

Acknowledgments

This work was supported in part by a grant from University of Las Palmas de Gran Canaria (UNI 2000/13) (AR) and from the Canary Islands Government 156/99 (FE). We thank José Estévez (University Hospital of Gran Canaria) for his collaboration in the Western blot assays and Dr. Richard Brown for critical reading of the manuscript.

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¹: Both authors contributed equally to this work.

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Potent induction of apoptosis by germacranolide sesquiterpene lactones on human myeloid leukemia cells

Abstract

Introduction

Section snippets

Drugs and reagents

Sesquiterpene lactones induce apoptosis in human myeloid leukemia cells

Discussion

Acknowledgments

Brain Res.

Biochimie

J. Biol. Chem.

Phytochemistry

Phytochemistry

Trends Cell Biol.

Trends Mol. Med.

J. Immunol. Methods

Blood

Bioorg. Med. Chem.

Mutat. Res.

Trends Biochem. Sci.

Cell