Acetyl-11-Keto-b-Boswellic Acid Induces Apoptosis in HL-60 and CCRF-CEM Cells and Inhibits Topoisomerase I

Antiproliferative action of different pentacyclic triterpenes has repeatedly been reported, and some lipoxygenase inhibitors have been shown to induce cell death in various cell systems. Acetyl-11-keto-b-boswellic acid (AKBA) is a pentacyclic triterpene that inhibits 5-lipoxygenase in a selective, enzymedirected, nonredox, and noncompetitive manner. To investigate a possible effect of AKBA on leukemic cell growth, proliferation of HL-60 and CCRF-CEM cells was assayed in the presence of AKBA and a structural analog without effect on 5-lipoxygenase, amyrin. Cell counts and [H]thymidine incorporation were significantly reduced in a dose-dependent manner in the presence of AKBA (IC50 5 30 mM) but not amyrin. An additive effect of AKBA with the crosslinking of the CD95 receptor was also observed. Flow cytometric analysis of propidium iodide-stained cells indicated that the cells underwent apoptosis. This was confirmed by flow cytometric detection of sub-G1 peaks in AKBA-treated cells and by DNA laddering. However, because HL-60 and CCRF-CEM do not express 5-lipoxygenase mRNA constitutively, a mechanism distinct from inhibition of 5-lipoxygenase must account for the effect of AKBA. In a DNA relaxation assay with fX174RF DNA, AKBA inhibited topoisomerase I from calf thymus at concentrations of $10 mM. A semiquantitative cDNA polymerase chain reaction approach was used to estimate the relative level of expression of topoisomerases in both cell lines. The data suggest that induction of apoptosis in HL-60 and CCRF-CEM by AKBA may be due to inhibition of topoisomerase I in these cells. Programmed cell death (PCD) is a feature of major importance not only in normal animal development but also in the turnover and renewal of many different cell populations in the adult body (Ellis et al., 1991; Raff et al., 1993). Cells dying by PCD undergo typical morphological changes, which are easily distinguishable from necrosis caused by accidental cell death (Wyllie et al., 1984; Walker et al., 1988). PCD of lymphocytes is crucial in regulating immune responses and maintaining self-tolerance (Cohen et al., 1992). PCD is also induced in virus-infected or malignant cells by effector cells of the immune system, and inhibition of apoptosis by the bcl-2 gene product, for example, has been implicated in the development of cancer (Podack and Kupfer, 1991; Korsmeyer, 1992). Consequently, cytostatic agents influencing mechanisms of PCD have been described (Solary et al., 1994). Their clinical use has been limited by severe side effects (Rougier and Bugat, 1996); therefore, cytostatic drugs inducing PCD with low toxicity should be promising research tools. Frankincense extracts have been used in the traditional medicine of India and other countries since ancient times for the treatment of several diseases, including inflammatory disorders and cancer (Martinez et al., 1989). Today, the frankincense extract salai guggal is used in India for the treatment of rheumatic diseases with a minimum of side effects. A clinical trial on ulcerative colitis with an extract of Fig. 1. Chemical structures of AKBA and a-amyrin. Received for publication December 11, 1997. ABBREVIATIONS: AKBA, acetyl-11-keto-b-boswellic acid; CPT, camptothecin; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; HETE, hydroxy-6,8,11,14-eicosatetraenoic acid; HPETE, hydroperoxy-6,8,11,14-eicosatetraenoic acid; PCD, programmed cell death; PCR, polymerase chain reaction; PI, propidium iodide; PMNL, polymorphonuclear leukocyte; PGB2, prostaglandin B2. 0022-3565/99/2882-0613$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 288, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 288:613–619, 1999 613 at A PE T Jornals on N ovem er 2, 2017 jpet.asjournals.org D ow nladed from Boswellia serrata gum resin demonstrated effects comparable to those of standard medication (Gupta et al., 1997). The main compounds of frankincense, the gum resin of B. serrata and B. carterii, boswellic acids were identified as inhibitors of 5-lipoxygenase (Ammon et al., 1991) and human leukocyte elastase (Safayhi et al., 1997) and were shown to be direct, nonredox inhibitors of mammalian 5-lipoxygenases (Safayhi et al., 1992). Structure-activity relationships for acetyl-11keto-b-boswellic acid (AKBA) have been elucidated (Sailer et al., 1996). AKBA is a pentacyclic triterpene of the ursane type and inhibits 5-lipoxygenase in rat polymorphonuclear leukocytes (PMNLs) with IC50 5 1.5 mM. Recently, induction of apoptosis by 5-lipoxygenase inhibitors in chronic myelogenous leukemia cells (Anderson et al., 1995) by pentacyclic triterpenes in melanoma (Pisha et al., 1995) and different other cell lines, including leukemia, has been reported (for a review, see Mahato et al., 1992). In vitro, not clearly defined frankincense extracts showed inhibitory action on topoisomFig. 2. Effect of AKBA on cell survival. Cells were grown for 48 h in the presence of AKBA, amyrin, or 0.5% DMSO alone. Untreated cells served as control. Cells were counted in a Neubauer hemocytometer, and cell survival was determined by trypan exclusion test (n 5 3). Fig. 3. Effect of AKBA on [H]thymidine incorporation. Cells (1 3 10) were plated onto 96-well flat-bottom culture plates and then incubated with AKBA or amyrin for 4 h and for an additional 18 h after the addition of 37 kBq [H]thymidine per well. Untreated cells served as control. Cells were harvested on glass fiber filters, and [H]thymidine incorporation was assayed by liquid scintillation spectrometry (n 5 3). 614 Hoernlein et al. Vol. 288 at A PE T Jornals on N ovem er 2, 2017 jpet.asjournals.org D ow nladed from erase II (Wang et al., 1991). Our study aim was to investigate the effects of AKBA and its structural analog amyrin (Fig. 1) on two human leukemic cell lines, HL-60 and CCRF-CEM. AKBA was found to inhibit cell growth and to induce apoptosis in both cell lines. Because CCRF-CEM cells do not express 5-lipoxygenase mRNA constitutively (Jakobsson et al., 1992), another mechanism must account for the observed effects. This may also be true for HL-60 cells, which are not capable of producing 5-lipoxygenase metabolites unless differentiated with agents like dimethyl sulfoxide (DMSO), retinoic acid, or vitamin D3 (Kargman and Rouzer, 1989; Brungs et al., 1994). Because we could demonstrate that AKBA inhibits a mammalian topoisomerase I, we suggest that this mechanism could be responsible for the apoptosis-inducing effect of AKBA. Materials and Methods Chemicals. AKBA was obtained from the gum resin of B. carterii (olibanum) by extraction into ether, precipitation with barium hydroxide, acetylation to mixed anhydrides with acetic anhydride, cleavage of the mixed anhydrides, and crystallization of the mixture of acetyl-boswellic acids from methanol (Winterstein and Stein, 1932). AKBA was isolated from this mixture by C18 reversed-phase high performance liquid chromatography and characterized by infrared , H NMR, and mass spectroscopy and by its melting points as described elsewhere (Safayhi et al., 1992). Amyrin was purchased from Roth (Karlsruhe, Germany), and camptothecin (CPT) was from Sigma (Munich, Germany). Solutions were prepared in DMSO and diluted with RPMI 1640; the final concentration of DMSO in all assays was 0.5%. Anti-CD95 monoclonal antibody (clone CH-11) was obtained from Coulter Immunotech (Hamburg, Germany), and [H]thymidine was purchased from Amersham Buchler (Braun-