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Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets.

Abstract

Several lichen compounds, i.e. lobaric acid (1), a [beta]-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic [alpha]-methylene-[gamma]-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a [beta]-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 [micro]g/ml: 1 93.4[+ or -]6.62%, 2 98,5[+ or -]1.19%, 5 14.7[+ or -]2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose-response relationship in the range of 3.33-100 [micro]g/ml. According to the calculated I[C.sub.50] values the highest inhibitory activity was observed for the depsidone 1 (I[C.sub.50] = 28.5 [micro]M) followed by 2 (I[C.sub.50] = 77.0 [micro]M). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (I[C.sub.50] = 24.6 [micro]M).

[c] 2004 Elsevier GmbH. All rights reserved.

Keywords: 12-lipoxygenase; 12(S)-HETE; Lichen; Protolichesterinic acid; Lobaric acid; Baeomycesic acid; Usnic acid; Parietin

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Introduction

12-Lipoxygenases (12-LOXs) catalyse the initial reaction that leads to the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from arachidonic acid (Yamamoto et al. 1997). Recent reports show that 12-LOXs and their products have important functions in biological systems and seem to be involved in regulation of cell growth and proliferation, atherosclerosis and neurotransmission (Yoshimoto and Takahashi, 2002). The involvement of 12-LOX in malignant growth is suggested by overexpression in cancer cells and tissues and growth stimulation of malignant cell lines by 12-HETE (Natarajan and Nadler, 1998; Pidgeon et al. 2003). Indeed, a role has been suggested for platelet type 12(S)-LOX in carcinogenesis as well as angiogenesis and metastasation (Nie and Honn, 2002; Virmani et al. 2001; Timar et al. 2000; Nie et al. 2001).

Lichens represent a source of compounds unique in nature formed by symbiosis between fungi and algae. In traditional medicine some lichen species such as Iceland moss, Cetraria islandica, have been widely used for treating inflammatory conditions such as asthma and gastritis, as well as tuberculosis, without being associated with any adverse effects. For the present study five lichen compounds were selected from different chemical classes, i.e. a depsidone, aliphatic lactone, depside, dibenzofuran and anthraquinone. Some of these compounds had previously been shown to exhibit 5-lipoxygenase (5-LOX) inhibitory activity (Ingolfsdottir et al., 1994, 1996, 1997) and had furthermore been found to have anti-proliferative effects against malignant cell lines (Ogmundsdottir et al. 1998; Kristmundsdottir et al. 2002).

The aim of the present study was to evaluate the inhibitory activity of the selected lichen compounds on platelet-type 12(S)-LOX with respect to their known anti-proliferative and 5-LOX inhibitory effects. To the best of our knowledge, this is the first report of lichen compounds being investigated for 12-LOX inhibitory properties.

Experimental

Materials

Lichen compounds

Lobaric acid (1), (+)-protolichesterinic acid (2), (+)-usnic acid (3), parietin (4) and baeomycesic acid (5) were isolated from different lichen species as reported respectively elsewhere (Ingolfsdottir et al. 1996, 1994, 1997, 1998, Paulson et al. 2004). Structures of investigated compounds are presented in Fig. 1. The degree of purity for all lichen compounds was >95% as determined by NMR and HPLC analyses.

Methods

12(S)-LOX assay

The assay was carried out as described previously (Schneider et al., 2004). Peripheral venous blood from healthy volunteers was drawn into trisodium citratecontaining (9:1, v/v) blood collection tubes (BD Vacutainer Systems). The blood was centrifuged at 200g for 10 min at 20[degrees]C and platelet rich plasma (PRP) was collected. PRP was further centrifuged at 1200g for 15 min at 20[degrees]C and the platelet pellet was washed twice with PBS-buffer (Fluka) containing 1 mM EDTA (Fluka). Thereafter, the platelets were resuspended in PBS to a final concentration of 0.9 X [10.sup.8] platelets/ml. The platelet suspension was preincubated at 37[degrees]C for 7 min in the presence of 2 mM reduced glutathione (Sigma) with test solutions or controls. Test samples were dissolved in absolute ethanol (final ethanol concentration of maximum 2.5% in the assay mixture). Thirty-three micromolars arachidonic acid (Sigma) were added and the suspensions were incubated for another 7 min at 37[degrees]C. Reactions were stopped by addition of 2 M HCI and by cooling with ice. The 12(S)-lipoxygenase product, 12(S)-HETE, was quantified by 12(S)-HETE-EIA (Correlate-EIA[TM]-12(S)-HETE-kit, 96well, Assay Designs, Ann Arbor). Optical density was read on a microplate reader at 405nm and was used to calculate the concentration of 12(S)-HETE in comparison to a 12(S)-HETE calibration line. For control purposes selected samples were also quantified by RP-HPLC. The supernatants were purified by solid phase extraction (SPE) on Sep-Pak[R]-[C.sub.18] cartridges (Waters). The samples (methanol eluate) were evaporated under a stream of nitrogen and dissolved in absolute ethano! for HPLC-analysis (column: LiChroCART[R] 55-4 with Purospher[R] STAR RP-18e, 3[micro]m, mobile phase: A = water with 1% (v/v) 0.1 N [H.sub.3]P[O.sub.4]; B = C[H.sub.3]CN with 1% (v/v) 0.1 N [H.sub.3]P[O.sub.4]. Solvent gradient: 50% B to 90% B linear in 20 min, 90% B isocratic for 5 min flow: 1.0 ml/min, temperature: 22[degrees]C, detection: UV 240 nm). The 12(S)-HETE-concentrations were calculated in relation to a 12(S)-HETE-standard (Sigma). The results are means of at least three and in most cases four single experiments. Positive control measurements were performed with baicalein (Aldrich), I[C.sub.50] values (concentration causing 50% inhibition of 12(S)-HETE production compared to untreated control) were calculated by regression analysis of the mean results of four different concentrations (100, 33.3, 10, 3.33 [micro]g/ml). For baicalein I[C.sub.50] = 24.6 (EIA) and 22.2 [micro]M (HPLC), respectively, were calculated.

Results and discussion

Using a cell based in vitro assay in human platelets lichen metabolites 1-5 which were selected from different chemical classes were tested for platelet-type 12(S)-LOX inhibitory activity. At a concentration of 100 [micro]g/ml lobaric acid (1), (+)-protolichesterinic acid (2) and baeomycesic acid (5) showed inhibition of 12(S)-HETE production of 93.4 [+ or -] 6.62%, 98.5 [+ or -] 1.19% and 14.7 [+ or -] 2.76%, respectively. Hence, lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-LOX, whereas baeomycesic acid (5) showed only weak activity. (+)-Usnic acid (3) and parietin (4) were not active at this concentration. Lobaric acid (1) and (+)-protolichesterinic acid (2) were subsequently tested at different doses in the range of 3.33-100 [micro]g/ml. Both compounds showed a clear dose-response relationship. However, (+)-protolichesterinic acid (2) revealed a sharp decline in activity between 33.3 and 10 [micro]g/ml whereas lobaric acid (1) indicated a more attenuated decrease in inhibitory activity at reduced doses (see Fig. 2). According to the calculated I[C.sub.50] values the highest inhibitory activity was observed for the depsidone lobaric acid (1) (I[C.sub.50] = 28.5 [micro]M) followed by protolichesterinic acid (2), (I[C.sub.50] = 77.0 [micro]M). The activity of lobaric acid (1) was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (I[C.sub.50] = 24.6 [micro]M) (Sekiya and Okuda, 1982).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Lobaric (1) and protolichesterinic acids (2) are thus dual inhibitors of 5- and 12-LOXs, lobaric acid in both cases being more potent. However, baeomycesic acid (5) which proved to be a 5-LOX inhibitor with similar potency as lobaric acid (1), exerted only weak 12(S)-LOX inhibitory activity. In terms of anti-proliferative activity the order of potency is protolichesterinic acid followed by lobaric acid both with marked activity against several cell lines (Ogmundsdottir et al. 1998), but baeomycesic acid is only weakly active.

In a recent speculative review Shureiqi and Lippman (2001) proposed that during carcinogenesis a shift occurs in a dynamic balance between different LOX pathways, towards the pro-carcinogenic 5-, 8- and 12-LOX pathways and away from the anti-carcinogenic 15-LOX pathway. LOX pathways have received particular attention in pancreatic cancer, a form of cancer that still has a very poor prognosis, indicating involvement of 5- and 12-LOX, but not 15-LOX (Ding et al. 2003). Three LOX inhibitors, the non-specific inhibitor nordihydroguaiaretic acid, the 5-LOX inhibitor Rev-5901 and the 12-LOX inhibitor baicalain were all found to inhibit in vitro and in vivo growth of pancreatic cancer cell lines as well as inducing apoptosis. The 12-LOX inhibitor was somewhat less potent than the non-specific inhibitor (Tong et al. 2002).

Conclusion

In conclusion, marked 12-LOX inhibition was demonstrated for two lichen metabolites that were previously known to have 5-LOX inhibitory activity. Such substances are of particular interest in light of recent data indicating pro-carcinogenic effects of 5- and 12-LOX and anti-carcinogenic effects of 15-LOX.

Acknowledgements

IS gratefully acknowledges financial support by the School of Natural Sciences, University of Graz.

Received 30 October 2003; accepted 10 March 2004

References

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Ogmundsdottir, H.M., Zoega, G.M., Gissurarson, S.R., Ingolfsdottir, K., 1998. Anti-proliferative effects of lichen-derived inhibitors of 5-lipoxygenase on malignant cell lines and mitogen-stimulated lymphocytes. J Pharm Pharmacol 50, 107-115.

Paulson, A.S., Kristinsson, H., Ingolfsdottir, K., 2004. Unpublished results

Pidgeon, G.P., Tang, K., Cai, Y.L., Piasentin, E., Honn, K.V., 2003. Overexpression of platelet-type 12-lipoxygenase promotes tumor cell survival by enhancing [.sub.[alpha]][v.sub.[beta]]3 and [.sub.[alpha]][v.sub.[beta]]5 integrin expression. Cancer Res 63, 4258-4267.

Schneider, I., Gibbons, S., Bucar, F., 2004. Inhibitory activity of Juniperus communis on 12(S)-HETE production in human platelets. Planta Med 70, 471-474.

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Timar, J., Raso, E., Dome, B., Li, L., Grignon, D., Nie, D., Honn, K.V., Hagmann, W., 2000. Expression, subcellular localization and putative function of platelet-type 12-lipoxygenase in human prostate cancer cell lines of different metastatic potential. Int J Cancer 87, 37-43.

Tong, W-G., Ding, X-Z., Witt, R.C., Adrian, T.E., 2002. Lipoxygenase inhibitors attenuate growth of human pancreatic cancer xenografts and induce apoptosis through the mitochondrial pathway. Mol Cancer Ther 1, 929-935.

Virmani, J., Johnson, E.N., Klein-Szanto, A.J.P., Funk, C.D., 2001. Role of platelet-type 12-lipoxygenase in skin carcinogenesis. Cancer Lett 162, 161-165.

Yamamoto, S., Suzuki, H., Ueda, N., 1997. Arachidonate 12-lipoxygenases. Prog Lipid Res 36, 23-41.

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F. Bucar (a), I. Schneider (a), H. Ogmundsdottir (b), K. Ingolfsdottir (c,*)

(a) Institute of Pharmacognosy, University of Graz, Universitaetsplatz 411, A-8010 Graz, Austria

(b) Molecular and Cell Biology Research Laboratory, Icelandic Cancer Society, Reykjavik

(c) Faculty of Pharmacy, University of Iceland, Hagi, Hofsvallagata, 107 Reykjavik, Iceland

*Corresponding author. Tel.: +354-1-525-4582; fax: +354-1-525-4071.

E-mail address: kring@hi.is (K. Ingolfsdottir).
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Author:Bucar, F.; Schneider, I.; Ogmundsdottir, H.; Ingolfsdottir, K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Nov 1, 2004
Words:2167
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