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Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 255

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Studies on Cytotoxic and NeutrophilChallenging Polypeptides and Cardiac

Glycosides of Plant Origin

BY

SENIA JOHANSSON

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

Dissertation for the degree of Doctor of Philosophy (Faculty of Pharmacy) inPharmacognosy presented at Uppsala University in 2001

ABSTRACTJohansson, S, 2001, Studies on cytotoxic and neutrophil challenging polypeptides andcardiac glycosides of plant origin. Acta Universitatis Upsaliensis. ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Pharmacy 255. 74 pp. Uppsala.ISBN 91-554-5092-X

This thesis examines the isolation and characterisation (biological and chemical) ofpolypeptides from plants. A fractionation protocol was developed and applied on 100 plantmaterials with the aim of isolating highly purified polypeptide fractions from smallamounts of plant materials. The polypeptide fractions were analysed and evaluated forpeptide content and biological activities. A multitarget functional bioassay was optimisedas a method for detecting substances interacting with the inflammatory process of activatedneutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatorymediator, N-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activatingfactor (PAF), to induce exocytotic release of the enzyme elastase, which then wasquantified by photometric determination of the product p-nitroanilide (pNA) formed from achromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formationmore than 60%, and 3% stimulated formation.

Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from Phoradendrontomentosum. In addition, the cardiac glycoside digitoxin was isolated from Digitalispurpurea. All these substances expressed cytotoxicity and a neutrophil challenging activity.

Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong tothe group of thionins. All the peptides were evaluated for cytotoxicity in a human cell linepanel. Phoratoxin C was the most potent towards the cell lines (mean IC50: 160 nM), andwas therefore investigated further on tumour cells from patients. Correlation analysis of thelog IC50 values indicated a mechanism of action different from clinically used archetypalcytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours whencompared to the haematological cancer types. The phoratoxin C was 18 times more potenttowards the solid tumour samples from breast cancer cells (87 nM) compared to the testedhaematological malignancies.

The structure-activity relationship concerning cytotoxicity was evaluated for digitoxinand related cardiac glycosides. Digitoxin was shown to be potent, with the average IC50 37nM being within the therapeutic concentration used for cardiac congestion (13-45 nM).Digitoxin expressed selective toxicity towards solid tumours from patients compared tohaematological malignancies.

Senia Johansson, Division of Pharmacognosy, Department of Medicinal Chemistry,Faculty of Pharmacy, Biomedical Centre, Uppsala University, Box 574, SE-751 23Uppsala, Sweden

© Senia Johansson 2001

ISSN 0282-7484ISBN 91-554-5092-X

Printed in Sweden by Tryck & Medier, Uppsala 2001

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Ibland jag finner en klövermed fyra magiska blad.

Jag griper den ivrigt och spar denså barnsligt förtjust och glad.

Ni alla förnumstigt klokafår gärna åt dårskapen le.

Jag tror ändå att den mytenkan något av sanning ge:

att lycklig blir den som finnerdär andra inget kan se.

Docent Maj Levander-Lindgren

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List of Original Publications

This thesis is based on the following articles. They are referred to in the text by theirRoman numerals (I-IV).

I Claeson, P., Göransson, U., Johansson, S., Luijendijk, T. and Bohlin, L.Fractionation protocol for the isolation of polypeptides from plant biomass.J Nat Prod 1998, 61, 77-81.

II Johansson, S., Göransson, U., Luijendijk, T., Backlund, A., Claeson, P., andBohlin, L. A neutrophil multitarget functional bioassay in search of anti-inflammatory natural products. (2001)Submitted for publication

III Johansson, S., Gullbo, J., Lindholm, P., Ek, B., Thunberg, E., Samuelsson, G.,Larsson, R., Bohlin, L. and Claeson, P.Purification and Characterization of Novel Cytotoxic Polypeptides fromPhoradendron tomentosum. (2001)Submitted for publication

IV Johansson, S., Lindholm, P., Gullbo, J., Larsson, R., Bohlin, L. and Claeson, P.Cytotoxicity of digitoxin and related cardiac glycosides in human tumour cells.Anti-Cancer Drugs 2001, 12, 475-483.

Reprints made with permission from the publishers

Studies on Cytotoxic and Neutrophil ChallengingPolypeptides and Cardiac Glycosides of Plant Origin.

by Senia Johansson

Contents

1. Introduction 7

2. Background 8

Peptides in plants 8 Cyclic peptides 8 Macrocyclic peptides 8 Thionins 9 Protease inhibitors 12

Polymorphonuclear neutrophils 12 Physiological responses 12 Phagocytosis 13 Exocytosis 14 Elastase 14 Cytotoxic action of neutrophils 15

Some host defence mechanisms in plants and animals 15 Superoxide generation 15 Nitric oxide 16 Antimicrobial peptides 16 Programmed cell death or apoptosis 17

Cytotoxicity 18 Screening for cytotoxic natural products 18 Mechanism-based screening for cytotoxicity 19 Common mechanisms and resistance for cancer drugs 19

3. Aims of the Present Investigation 21

4. Experimental Bioassay Procedures 22 Neutrophil isolation 22 Elastase release assay 22 Superoxide production 22 Inhibition assay of the isolated elastase 23 Haemolytic test 23 Cell line panel 24 Measurement and data analysis of cytotoxic activity 24

5. Development of Methods for Isolation of Polypeptides from Plant Material 26 Plant material 26 Fractionation of polypeptides 26 Isolation of varv peptide A 28

6. A Multitarget Bioassay on the Neutrophil 30 Optimisation of a multitarget bioassay on the neutrophil 31 Validation 31 Identification of the extracellular release of superoxide, elastase and defensins 32 The evaluation of the activity 34

7. Isolation and Characterisation of Substances with Cytotoxic and 38 Neutrophil Elastase Releasing Activity Phoratoxins from Phoradendron tomentosum 38 Isolation and characterisation of peptides from Phoradendron tomentosum 39

Contents

Analysis of the reference materials phoratoxins A and B 41 The neutrophil challenging activity of phoratoxins 43 The cytotoxic evaluation of the fractions and the isolated peptides 44

Digitoxin and related cardiac glycosides 48 A bioassay guided isolation of digitoxin from Digitalis purpurea 48 The neutrophil challenging activity of digitoxin 49 The cytotoxic evaluation of digitoxin and related compounds 49

8. Summary and Concluding Discussion 53 The fractionation protocol 53 A multitarget bioassay on the neutrophil 53 Further characterisation of phoratoxins and digitoxin, biological and chemical 54

9. Concluding Remarks 57 Future perspectives 57

10. Acknowledgement 59

11. References 60

Appendix Abbreviations used in this thesis 74

Introduction

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1. Introduction

Plants and animals have diversified differently through evolution, but because ofthe same origin, is it thought that they might have the same ancestral proteins. Thedefence mechanism is one example where it might be evident. Studies of innateimmunity led to the discovery of common molecular mechanism used for hostdefence in plants, insects, and mammals (Bergey et al., 1996; Borregaard et al.,2000; Klessig et al., 2000; Marx, 1996). This includes the various receptors thatrecognise classes of microbial cell-surface molecules, the similar signaltransduction pathways that activate transcription of genes related to host defence,and the ubiquitous cationic peptides and proteins that act as antimicrobialeffectors. Among the diverse defensive chemicals that are synthesised in plants inresponse to herbivore or pathogen attacks are protease inhibitor proteins (Ryan,2000; Ryan et al., 1993). These proteins can inhibit proteases of pests andpathogens, limiting protein digestion and retarding both growth and developmentof the attacker in response to wounding. Protease inhibitor genes have recentlybeen proposed as modulators of programmed cell death (PCD) in plants (Solomonet al., 1999) and of defence by preventing unwanted cell death (Prasad et al.,1994).

Antimicrobial peptides have been identified as key elements in the innate hostdefence against infection. Recent studies indicate that the activity of antimicrobialpeptides may be decreased in cystic fibrosis, suggesting a major role of thesepeptides in host defence against infection. They have also been shown to killmammalian target cells and microorganisms by a common mechanism thatinvolves an electrostatic interaction with membranes (Florack and Stiekema, 1994;Hughes et al., 2000; Lehrer et al., 1993). One type of these antimicrobial peptidesis small (3-5 kDa), basic, and rich in cysteine. These are the mammalian, insect,and plant defensins. Another group of small cysteine-rich, highly basic peptidesthat are thought to play a role in the protection of plants against microbialinfection are the thionins (Florack and Stiekema, 1994; García-Olmedo et al.,1998).

Human neutrophils are responsible for our first line of cellular defence againstinvading pathogens. In this work, highly purified plant fractions were obtained bya fractionation protocol for the isolation of polypeptides from plant biomass. Theisolated purified plant fractions were further evaluated in the optimised neutrophilmultitarget functional bioassay. The substances responsible for the observed effectin the neutrophil bioassay were isolated from Phoradendron tomentosum andDigitalis purpurea. The active substances were found to be polypeptides andcardiac glycosides, respectively. To gain further insight, and in particular, in casesubstances from plants with the neutrophil challenging activity also have apotential role in inducing death of cancer cells, the isolated substances werefurther investigated for cytotoxicity in a mechanism-based cell line panel, and intumour cells from patients.

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2. Background

Peptides in Plants

Numerous peptides, such as hormones, neurotransmitters, and snake toxins, havebeen isolated from human and animal sources. In contrast, only a limited numberof peptides have been found in plants. Nevertheless, the isolation of peptides fromplant biomass has recently received attention for three main reasons. First, plantscontaining unique pharmacologically active peptides have been found within natural-products-based drug discovery programs (Gustafson et al., 1994; Witherup et al.,1994). Second, genetically transformed plants (”transgenic plants”) are now consideredan attractive and cost-efficient alternative to fermentation-based systems for productionof, for example, high value recombinant peptides (Goddijn and Pen, 1995; Whitelam,1995). Third, like animals, plants are known to make use of peptides as signalsubstances (Bergey et al., 1996; Marx, 1996). Examples of plant peptides are systemin,cyclic peptides, defensins, thionins, and several protease inhibitors.

Cyclic peptidesCyclic peptides are an important group of compounds with potent, specific biologicalactivities. A large number of small cyclic peptides have been isolated from plants.Small cyclic peptides occur to a great extent in the Caryophyllaceae and Rubiaceaefamilies. Cyclic peptides are also found in Jatropha spp. within the familyEuphorbiaceae (Horsten et al., 1996; van den Berg et al., 1995; van den Berg et al.,1996). Among the cyclic peptides that are found today, the most common biologicalactivities appear to be antitumor and cell-growth-inhibitory effects (Hamanaka et al.,1987; Itokawa et al., 1984). This might be misleading since the most prevalentbiological assays seem to be antitumour assays, and therefore, there might bemany other possibilities that have not yet been examined. However, theoctapeptides dichotomins from Stellaria dichotoma var. lanceolata exert bothcytotoxic and cyclooxygenase inhibitory activities (Morita et al., 1996).

Macrocyclic peptidesKalata B1 was isolated from the African plant Oldenlandia affinis based onethnopharmacological reports of African women making use of that plant foraccelerating contractions and childbirth (Gran, 1970). To obtain the drug kalata-kalata, natives boil the dried plant leaves to produce an extract, which is then sippedduring labour. Kalata B1 was isolated in the beginning of the 1970s from extractsof the plant, and was considered an uterotonic agent; however, not until 1995 wasthe primary structure of the peptide confirmed (Saether et al., 1995). The peptidecontains 29 amino acids and three disulphide bonds; and for a peptide this large, ithas the unusual feature of a cyclized backbone. Although cyclic peptides are producednaturally in microbes and plants, they are usually restricted to peptides with fewerthan 15 amino acids (Itokawa et al., 1997). Over the past few years, some othercyclic peptides with similar sizes and with cysteine residues have been reported.Together, they form a new family of macrocyclic peptides, recently named cyclotides(Craik et al., 1999).

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Most of these macrocyclic peptides have been found in plants from the Rubiceaefamily, of which Olenlandia affinis is a member. Circulin A and B were isolatedfrom Chassalia parvifolia (Gustafson et al., 1994); and cyclopsychotride A, fromPsychotria longipes (Witherup et al., 1994). All these peptides have a cyclic backbone,and sequences somewhat homologous with that of kalata B1. Violapeptide 1 fromViola arvensis Murray (Schöpke et al., 1993) was the first reported cyclotideisolated from the Violaceae family.

In addition to their cyclic nature, these macrocyclic peptides also have an inhibitortype of cysteine-knot motif in their disulphide connectivity (Daly et al., 1999a;Derua et al., 1996; Saether et al., 1995; Tam and Lu, 1997; Tam and Lu, 1998). Thismotif contains a bonding pattern of Cys I-IV, II-V, and III-VI, characterised by theCys III-VI disulphide bond threading through the other two to give a knottedmotif. The knotted motif together with small triple-stranded β-sheets has beenfound in peptides and proteins with diverse functions. These include ion channeltoxins, protease inhibitors, and growth factors (McDonald and Hendrickson, 1993;Pallaghy et al., 1995).

This cyclotide family is of particular interest because the individual members exhibit adiverse range of biological activities. The circulins exhibit anti-HIV activity(Gustafson et al., 1994); cyclopsychotride A inhibits neurotensin binding to cellmembranes (Witherup et al., 1994), and kalata B1 exhibits uterotonic activity.Originally, violapeptide I was discovered for its haemolytic activity (Schöpke etal., 1993); but recently, Daly et al. (1999b) and Tam et al. (1999) have shown thatin addition kalata B1, the circulins, and cyclopsychotride A have relatively weakhaemolytic activity. Circulin B and cyclopsychotride are antimicrobial againstboth Gram-positive and Gram-negative bacteria, whereas kalata B1 and circulin Aare specific for the Gram-positive Staphylococcus aureus. After a modification ofthe sole arginine residues in kalata B1, the antimicrobial activity was markedlydecreased, whereas the modification of arginine residue of circulin A resulted in nosignificant loss of activity. The function of these cyclic peptides within plants,although yet to be established, most likely involves a defence mechanism (Tam etal., 1999).

ThioninsThionins are low-molecular-weight proteins (5 kDa) occurring in seeds, stems, roots,and leaves of a number of plant species. Thionins can be divided into at least fourtypes depending on the plant species and tissue in which they occur, and on theoverall net charge and the number of amino acids and disulphide bonds present inthe mature protein (Garcia-Olmedo et al., 1989). Type 1 thionins, which are abundantlypresent in the endosperm in the family Poaceae, are highly basic, and consist of 45amino acids, 8 of which are in the central disulphide loop. Type 2 thionins havebeen extracted from leaves and nuts of the parasitic plant Pyrularia oubera(Vernon et al., 1985), and from the leaves of barley Hordeum vulgare (Bohlmannand Apel, 1987; Bohlmann et al., 1988). They consist of 47 and 46 amino acids,respectively, and are slightly less basic than the type 1 thionins. Both type 1 andtype 2 have eight cysteines that are involved in four disulphide bonds. Type 3thionins, which consist of 45 or 46 amino acids, 9 of which are in the centraldisulphide loop, have been extracted from leaves and stems of mistletoe species:

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Viscum album, Phoradendron tomentosum, Phoradendron liga, and Dendrophthoraclavata (Mellstrand and Samuelsson, 1974a; Samuelsson and Pettersson, 1970;Samuelsson and Pettersson, 1977; Thunberg and Samuelsson, 1982). Thesethionins, which have three disulphide bridges that are conserved with respect tothe previous types, are as basic as type 2 thionins. Type 4 thionins, which consistof 46 amino acids with three disulphide bonds, but neutral in charge, have beenextracted from seeds of Abyssinian cabbage (Crambe abyssinica) (van Etten et al.,1965). All four types of thionins appear to be highly homologous at the amino acidlevel. The cysteine residues and the tyrosine residue at position 13 are highlyconserved, except for the type 4 thionins.

Thionins are toxic to either Gram-positive or Gram-negative bacteria, fungi, yeast,and various mammalian cell types. Toxicity requires an electrostatic interaction ofthionins with the negatively charged membrane phospholipids, followed by eitherpore formation or a specific interaction with a certain domain in the membrane(Florack and Stiekema, 1994). The purely electrostatic interaction of thionins withmembranes, the first step in the exposure of toxicity, can be inhibited by divalentcations, such as calcium (Florack and Stiekema, 1994; Sauviat, 1990). Upon thionintreatment of mammalian cells, the specific interaction of thionins with certainphospholipids, mediating transduction of cellular signals, can explain the releaseof specific compounds along with the activation of calcium channels and specificenzymes (Florack and Stiekema, 1994). Cytotoxicity correlates well with ability toform ion channels. In fact, Hughes et al. (2000) propose that the primary mode ofaction for the toxicity of thionins is due to their ability to form ion channels in cellmembranes; and they go on to propose that the passage of ions through thechannel probably involves Tyr-13.

Antimicrobial activity has been demonstrated for several thionins (Bohlmann et al.,1988; Fernandez de Caleya et al., 1972; Florack and Stiekema, 1994; Molina et al.,1993; Terras et al., 1993; Terras et al., 1996), supporting the suggestion that thioninsare defence proteins (Fernandez de Caleya et al., 1972). Other evidence for such apossible function comes from observations that the expression of several thioninsis inducible by phytopathogenic fungi (Bohlmann et al., 1988; Boyd et al., 1994;Epple et al., 1995; Titarenko et al., 1993; Vale et al., 1994). Furthermore, high-levelexpression of viscotoxin A3 cDNA in transgenic Arabidopsis thaliana has beenshown to enhance the resistance against the pathogen Plasmidiophora brassicae(Holtorf et al., 1998).

ViscotoxinsViscum album L contains two groups of toxic components: lectins and viscotoxins(Samuelsson and Jayawardene, 1974). The peptides from the European mistletoe,Viscum album L, called viscotoxins, were first isolated and sequenced bySamuelsson and co-workers (Samuelsson, 1958; Samuelsson, 1961), and werefound to belong to the group of thionins. Five viscotoxins, A1, A2, A3, B and 1PS,have been isolated; and their primary structures are determined (Olson andSamuelsson, 1972; Orrù et al., 1997; Samuelsson, 1973; Samuelsson and Jayawardene,1974; Samuelsson and Pettersson, 1971; Samuelsson et al., 1968; Schrader and Apel,1991). In addition, cDNAs encoding viscotoxins from Viscum album L. have beenisolated and characterised for the full-length preprotein of viscotoxin A3 and for

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the precursor of viscotoxin B. Besides the viscotoxin domain, the precursor contains asignal sequence and a acidic polypeptide domain. Since both the negative charge andthe position of the cysteine have been conserved within the acidic domain,(Schrader-Fischer and Apel, 1993) propose that this may play an important role inkeeping the thionin inactive within the plant cell. Another cDNA of thioninprecursors in Viscum album L. has a highly divergent thionin domain with eightinstead of six cysteine residues, while the acidic polypeptide domain and the signalsequence is conserved among all of the variants (Schrader-Fischer and Apel, 1993).Viscotoxins contain three S-S bridges, Cys3-Cys40, Cys4-Cys32, and Cys16-Cys26(Orrù et al., 1997).

PhoratoxinsIn 1967 Samuelsson and Ekblad isolated for the first time phoratoxin (later calledphoratoxin A) from a Californian mistletoe, Phoradendron tomentosum (DC) Engelm.subsp. macrophyllum (Cockerell) Wiens. In 1974, Mellstrand and Samuelsson reportedthe amino acid sequence for this peptide, which consists of 46 amino acids tightlybound together by three disulphide bridges in the same way as for the viscotoxins(Mellstrand and Samuelsson, 1974a). Thunberg (1983) isolated phoratoxin B, whichwas shown to be very similar to phoratoxin A. Some known activities ofviscotoxins and phoratoxins are summarised in Table 1.

Table 1. Listing of various activities displayed by viscotoxins and phoratoxins.

Name Activity ReferenceViscotoxins Bradycardia, negative inotropic effect on heart,

vasoconstriction of vessels of cats upon injection(Rosell and Samuelsson, 1966)

Non-random binding to DNA (Woynarowski and Konapa, 1980)

Cytotoxic to human KB and HeLa cells in vitro (Konopa et al., 1980)

Inhibition of translation in hamster BHK-21 cellsin vitro

(Carrasco et al., 1981)

Altered ionic permeabilities and depolarisation ofthe membrane

(Andersson and Johannsson, 1973)

Enhanced E. coli-stimulated phagocytosis andrespiratory burst

(Stein et al., 1999)

Generation of reactive oxygen intermediates andinduced expression of mitochondrial Apo2.7molecules

(Büssing et al., 1999b)

Accidental cell death and generation of reactiveoxygen intermediates in human lymphocytes

(Büssing et al., 1999a)

Phoratoxins Bradycardia, negative inotropic effect on heart,vasoconstriction of vessels of cats upon injection

(Rosell and Samuelsson, 1966)

Toxic to mice upon intraperitoneal injection (Mellstrand and Samuelsson, 1973)

Hemolytic to human erythrocyte (Thunberg, 1983)

Depolarisation of the membrane on frog skeletalmuscle fibres

(Sauviat, 1990)

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Protease inhibitorsProtease inhibitor proteins are among the diverse defensive chemicals that aresynthesised by plants in response to pathogen attacks. These proteins can inhibitproteases of pests and pathogens, limiting protein digestion, and retarding bothgrowth and development of the attacker (Ryan, 2000). Plant protease inhibitors canhave a regulatory role in that, after wounding caused by insect chewing or duringchilling-induced oxidative stress, they can block PCD and thereby prevent suchdeath from going beyond what is wanted (Solomon et al., 1999).

Systemin, an 18-amino-acid polypeptide released from wound sites on tomatoleaves caused by insects or other mechanical damage, is the most powerful inducerof protease-inhibitor synthesis known. The wound-inducible protease inhibitors have awide range of specificities that include all four known mechanistic classes ofproteases: serine, cysteine, aspartyl, and metallo-carboxyl protease inhibitors (Ryan,2000). Numerous protein protease inhibitors have been isolated and identified.Many of these are directed towards serine proteases, and are strictly competitiveinhibitors, forming 1:1 complexes with the enzyme they inhibit. In these complexes,all activities of the enzyme are completely abolished. Each inhibitor is a substratefor the enzyme it inhibits, at a unique peptide bond called the reactive site peptidebond (Laskowski, 1986).

Squash inhibitors of serine proteases, built of 27-33 amino-acid residues and cross-linked with three disulphide bridges, form a uniform family of small proteins thatare highly stable and rigid. The reactive site peptide bond (P1-P1') is betweenresidue 5 (Lys, Arg or Leu) and 6 (always Ile). The major structural motif is adistorted, triple-stranded, antiparallel beta-sheet. A similar folding motif has beenrecently found in a number of proteins, including conotoxins, carboxypeptidaseinhibitor from potato, kalata B1 polypeptide, and proteins in some growth factors(Otlewski and Krowarsch, 1996). Protease inhibitors are of biochemical interests,not only because of their involvement in defence mechanisms, but also because oftheir involvement in carcinogenesis (Clawson, 1996), virus replication (Weller andWilliams, 2001), and inflammation (Konstan, 1998).

Polymorphonuclear Neutrophils

Physiological responsesThe polymorphonuclear neutrophil (PMN) is a central component of theinflammatory process, having the ability to migrate to the inflammation site and torelease toxic products such as proteolytic enzymes (Travis, 1988), reactive oxygenspecies (ROS) (Babior, 1984; Nathan et al., 1981; Weiss, 1980), and cationicproteins (e.g., defensins and cathelicidins) capable of killing invading pathogens(Chertov et al., 2000; Clark et al., 1976; Ganz and Lehrer, 1994; Zanetti et al.,1995). The targets of the PMN include bacteria, fungi, viruses, virally infectedcells, and tumour cells.

Before entering the tissue, neutrophils must adhere to the endothelium andsubsequently migrate through the vessel wall. This process is tightly regulated byadhesion molecules on neutrophils and endothelial cells at sites of inflammation

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(Cohen, 1994; Hiemstra et al., 1998). Within the tissue, neutrophils continue tomigrate along a gradient formed by locally produced chemotactic factors, whichinclude the bacterial product N-formyl methionyl-leucyl-phenylalanine (fMLP);secreted products of stimulated phospholipid metabolism, such as the plateletactivating factor (PAF) and leukotriene B4 (LTB4); and immunomodulatorymolecules, such as the cytokine interleukin 8 (Baggiolini and Dewald, 1986; Curfset al., 1997; Dewald and Baggiolini, 1986; Goldman and Goetzl, 1982; Marascot et al.,1984; Nardin et al., 1991; Proost et al., 1996; Schiffmann et al., 1975a; Schiffmann etal., 1975b; Williams et al., 1977).

Chemotaxis, the ability to migrate towards an attractant, is essential for themigration of neutrophils and macrophages to sites of tissue damage and infection(Bokoch, 1995). During migration to the site of inflammation, neutrophils maycontribute to tissue injury. Tissue injury occurs when neutrophils accumulate inunusually high numbers, when they receive inappropriate stimuli and/or when theactivity of their products is not adequately controlled (Dallegri and Ottonello,1997). Neutrophils are, for example, involved in the pathogenesis of variousinflammatory lung disorders, including chronic bronchitis and chronic obstructivepulmonary disease (Hiemstra et al., 1998).

PMNs are equipped with an array of preformed compounds that are stored in thevarious types of neutrophil granule, and that may be released upon stimulation.These compounds include serine and metalloproteinases, and non-enzymaticpolypeptides such as neutrophil defensins (Borregaard and Cowland, 1997). Inaddition, neutrophil stimulation also results in the synthesis and release of anumber of mediators, including reactive oxygen intermediates, lipid mediators,and cytokines that may contribute to injury (Hiemstra et al., 1998).

PhagocytosisThe neutrophil is one of the professional phagocytes in humans. The neutrophilmakes tight contact with its target, and its plasma membrane then flows around thesurface until the bacterium is completely enclosed. These intracellularcompartments are called phagosomes, where digestive and antibacterialcompounds are released. The phagosome minimizes the amount of extracellularfluid entering the phagosome with the bacterium, which means that the phagosomeis initially a very small space (Hampton et al., 1998) where the bacteria can besubjected to high concentrations of antimicrobial substances. Neutrophils alsoundergo a burst of oxygen consumption that is caused by an NADPH oxidasecomplex that assembles at the phagosomal membrane. This oxidative burst hasalso been shown to be essential for killing a number of microorganisms (Cohen,1994; Hampton et al., 1998). These agents are strongly anti-microbial but may alsocause damage by destructing surrounding tissue and inducing apoptosis in otherimmune reactive cells.

ExocytosisThe neutrophil has intracellular stores of both membrane proteins and solubleproteins that can be incorporated into the plasma membrane as a means ofadhesion to endothelium (secretory vesicles), for migration through basement

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membranes (gelatinase granules), and for phagocytosis, killing, and digestion ofmicroorganisms (specific granules and azurophil granules) (Figure 1). One reasonfor segregating the proteins into different subpopulations of granules is that someproteins cannot exist in the same compartment (Borregaard, 1997). For instance,azurophil granules carry terminally processed serine proteases, antibiotic proteins,and myeloperoxidase (MPO), all in a potentially active state (Gullberg et al.,1997). Specific granules, on the other hand, store metalloproteases and antibioticpeptides as inactive latent proforms, which are allowed after degranulation tobecome active first (Gullberg et al., 1997). Another reason is that the content ofdifferent granules is needed at different times and places (Borregaard, 1997). Theexocytosis of granules and vesicles cause not only a release of lytic enzymes, butconcurrently, increased cell response by production of reactive oxygenintermediates and chemotactic stimulation. These activities lead to the destructionof invading pathogens (Dahlgren and Karlsson, 1999; Gullberg et al., 1999).

Type ofgranule

Azurophilgranules

Specificgranules

Gelatinasegranules

Secretoryvesicles

Type ofcontents

MyeloperoxidaseAntibiotic proteins(e.g., defensins)Serine proteases(active)

LactoferrinAntibiotic proteins(e.g., cathelicidins)Metallo-proteinases(inactive)

Gelatinase AlkalinePhosphatasePlasma proteins

Type of Killing Killing Migration Adherencefunctions Digestion Migration

Chemotaxis Activation

Figure 1. Differences in tendency for extracellular release for some typical cell contents, such asazurophil, specific granules, gelatinase granules, and secretory vesicles. Although the granule subtypeshave overlapping functions to a great extent, distinct functions associated with the contents of variousgranules can be recognised.

ElastaseElastases are a group of proteases that possess the ability to cleave the importantconnective tissue protein elastin. Elastin, which has the unique property of elasticrecoil, is widely distributed in vertebrate tissue, and is particularly abundant in thelungs, arteries, skin, and ligaments (Werb et al., 1982).

Neutrophil elastase (NE; EC 3.4.21.37), a 29-kDa glycoprotein and a potent serineprotease (Travis, 1988), is stored in primary (azurophilic) granules of neutrophils(Borregaard and Cowland, 1997; Gullberg et al., 1999). Human neutrophilelastase can cleave a variety of substrates, including elastin and all other majorconnective-tissue proteins (Werb et al., 1982). Neutrophil elastase is essential forphagocytosis and defence against infection by invading microorganisms. Thepowerful proteolytic activity of neutrophil elastase is essential for migration ofneutrophils through connective tissue and for destruction of foreign bacterial

Extracellular release

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invaders (Bieth, 1988). Elastase can be extremely destructive if not controlled,because they can destroy many connective tissue proteins. Under normalphysiological conditions theses proteases are carefully regulated bycompartmentalization or by natural circulating plasma inhibitors. Any elastasethat reaches the circulation is quickly complexed by the natural inhibitors α1-protease inhibitor (α1-antitrypsin) and α2-macroglobulin (du Bois et al., 1991;Virca and Schnebli, 1984; Virca and Travis, 1984). The complexes are clearedfrom the plasma and degraded by the liver or by macrophages. When animbalance occurs because of deficiency, effective α1-protease inhibitors, orabnormally high levels of elastases, can then result in severe permanent tissuedamage. Neutrophil elastase has been linked to adult respiratory distresssyndrome, cystic fibrosis, rheumatoid arthritis, and other inflammatory disorders(Döring, 1994).

The cytotoxic action of neutrophilsHuman neutrophils become capable of lysing tumour cells when activated by avariety of stimuli (Dallegri et al., 1991). PMNs possess at least two mechanismsby which they can lyse mammalian target cells. One of these depends upon theproduction of toxic oxidative metabolites that are generated from the PMNsrespiratory burst (English and Lukens, 1983; Nathan et al., 1979). However asecond mechanism, often detected during studies of PMN-induced antibody-dependent cellular cytotoxicity, is independent of the respiratory burst (Dallegri etal., 1984; Katz et al., 1980) and is mediated by one or more toxic proteins releasedfrom PMN granules.

At leukaemia (Lichtenstein et al., 1988), pulmonary (Okrent et al., 1990), andendothelial (Okrent et al., 1990) targets for example, the most potent cytolyticproteins of PMN are four small cationic peptides termed defensins, or humanneutrophil peptides 1, 2, 3 and 4 (HNP 1-4). The human defensins are secreted byactivated PMNs (Ganz, 1987) and could therefore function as cytotoxins duringantibody-dependent cellular cytotoxicity. In addition, a synergistic cytotoxiceffect occurs when target cells are exposed to a combination of defensins andtoxic oxidants or cathelicidins (Lichtenstein et al., 1988; Nagaoka et al., 2000).

Some Host Defence Mechanism in Plants and Animals

Superoxide generationDuring phagocytosis of microbial intruders, neutrophils increase their oxygenconsumption through the activity of an NADPH-oxidase. This generatessuperoxide anions (O2

-) and hydrogen peroxide (H2O2). These oxygen metabolitesgive rise to other ROS that are strongly anti-microbial (Babior, 1978; Badwey andKarnovsky, 1980), but which also may cause damage by destructing surroundingtissue (Winrow et al., 1993) and induce apoptosis in other immune reactive cells(Dallegri and Ottonello, 1997; Gustafsson and Bengtsson, 1999). ROS have alsobeen shown to induce cell death in plants (Lamb and Dixon, 1997); and (Lamband Dixon, 1997; Van Camp et al., 1998) speculate that ROS, when involved inactivating cell death in plants, may be generated by an NADPH oxidase similar tothe ROS signalling system in neutrophils. Recently, plant homologs of NADPH

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oxidase components (gp91 phox and Rac) have been cloned (Keller et al., 1998;Torres et al., 1998). Further support for NADPH oxidase involvement in plant celldeath is the observation that inhibitors of the NADPH oxidase complex caninterfere with the induction of cell death in plants (Lamb and Dixon, 1997).Hydrogen peroxide has also recently been shown to act as a second messenger forthe induction of defence genes in response to wounding, systemin, and methyljasmonate (Orozoxo-Cárdenasa et al., 2001), where the expressions of severaldefence genes were inhibited by the neutrophil NADPH oxidase inhibitordiphenylene iodonium.

Nitric oxideNitric oxide (NO) has previously been shown to serve as key redox-active signalfor the activation of various mammalian defence responses, including theinflammatory and innate immune responses (Schmidt and Walter, 1994; Stamler,1994). In the immune system, ROS often function together with NO, as forexample, in macrophage killing of bacteria and tumour cells (Nathan, 1995;Schmidt and Walter, 1994). NO has also been implicated in the activation of plantdefences (Delledonne et al., 1998), where it interacts with ROS and salicylic acidto induce PCD and defence gene expression (Delledonne et al., 1998).

Antimicrobial peptidesAmong plant antimicrobial peptides, thionins (described above) were the first tobe found, whose activity against plant pathogens was demonstrated in vitro.Antimicrobial peptides are cationic and generally not cytotoxic at concentrationswhere they kill microorganisms (Hoffmann et al., 1999). They appear to killmammalian target cells and microorganisms using a common mechanism, whichinvolves initial electrostatic interactions with negatively charged target-cellsurface molecules (likely the head groups of polar membrane lipids), followed byinsertion into the cell membranes that they permeabilize, forming voltage-regulated channels (Hughes et al., 2000; Lehrer et al., 1993).

Among the group of antimicrobial effector molecules, the cysteine-rich defensinsare widespread in eukaryotic cells in plants, insects, and mammals. Defensinshave wide spectra of activity directed against various bacteria, fungi, andenveloped viruses (Hoffmann et al., 1999). Four defensin families have beenreported in eukaryotes: insect defensins, plant defensins, and α- and β-defensinsin mammals. Whereas mammalian defensin consists solely of β-sheets linked bydisulphide bridges (in a slightly different pattern for α- and β-defensins), insectand plant defensins have an α-helix, stabilized through disulphide bridging tostrongly twisted antiparallel β-sheets (Hoffmann et al., 1999).

Plant defensins, which have been isolated from several taxa, are 45-54 aminoacids residues in length and probably ubiquitous in the plant kingdom. Based ontheir amino acid sequences, four defensin groups or subfamilies have beenestablished, with structural differences seeming to correlate with differences inantimicrobial specificity. All known members of the plant defensin family haveeight disulphide-linked cysteines. The distribution of defensins in plant tissue isconsistent with their putative defence role. Thus, they have been identified inleaves, tubers, flowers, pods and seeds. Although preferentially located in

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peripheral cell layers, plant defensins have also been reported in the xylem, and instomatal cells. All these locations are where first contact and entry of pathogenstake place. No toxicity, so far, of plant defensins to animal or plant cells has beenfound (García-Olmedo et al., 1998).

Human neutrophil defensins are small (29-33 amino acids) cationic peptides thatwere first identified on the basis of their antimicrobial activity. Four defensinsfrom the human defensin family are known to be expressed in neutrophils,namely, the human neutrophil peptides HNP-1, HNP-2, HNP-3, and HNP–4(Ganz and Lehrer, 1994; Ganz et al., 1990; Lehrer et al., 1993). Human neutrophildefensins are stored in the azurophil granules of neutrophils (Borregaard andCowland, 1997; Gullberg et al., 1997), and transferred to the phagolysosome uponphagocytosis (Ganz, 1987). In vitro, these peptides kill a variety of bacteria,including Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli;many fungi; and some enveloped viruses (Ganz et al., 1985; Lehrer et al., 1989;Lehrer et al., 1993). In addition to their antimicrobial activity, defensins alsodisplay cytotoxic activity toward various eukaryotic cells and cell lines (Lehrer etal., 1993). Thus, when released from stimulated neutrophils, defensins maycontribute to tissue injury. Various other extracellular activities of defensins havebeen described (Lehrer et al., 1993). These activities include chemotactic activityfor monocytes and lymphocytes (Chertov et al., 1996; Territo et al., 1989) and theability to stimulate IL-8 synthesis in airway epithelial cells (van Wetering et al.,1997).

Tang et al. (1999) reported the isolation and characterization of a novel cyclicdefensin detected in neutrophils and monocytes from macaque rhesus monkeys.This defensin, named rhesus theta defensin 1 (RTD-1), is the product of twohomologous genes that generate two highly similar propeptides that areproteolytically cleaved, joined head to tail to form a unique cyclical structure, andtargeted to the granules of leukocytes. In contrast to classical defensins, theantimicrobial of RTD-1 is not inhibited by increased salt concentrations.

Recently, Zanetti et al. (1995) identified a novel family of antibacterial proteins,known as cathelicidins, found in mammalian (human, rabbit, bovine, porcine andguinea pig) neutrophils and stored in the specific and gelatinase granules.Cathelicidins-derived antimicrobial peptides range in length from 12 to about 100residues. These peptides are not inhibited by increased salt concentrations. Bothdefensins and cathelicidins are released extracellularly (Ganz, 1987; Nagaoka etal., 1997; Yomogida et al., 1997) by activated neutrophils. Although the activitiesof defensins are completely lost by increasing salt concentrations, it has beenshown that they work synergistically with cathelicidins to exert an enhancedantibacterial activity in the extracellular milieu by augmenting the membranepermeabilization of target cells (Nagaoka et al., 2000).

Programmed cell death or apoptosisApoptosis (or programmed cell death—PCD) describes the physiological processfor the killing and removal of unwanted or dangerous cells (Rich et al., 1999;Vaux and Korsmeyer, 1999), which constitutes the main form of cell death inanimals, plants, and, other organisms. Numerous stimulants can induce PCD. In

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many systems, oxidative stress was found to be involved either directly orindirectly (Levine et al., 1996; Payne et al., 1995). In plants, it has been shownthat H2O2 treatment induces PCD in soybean and Arabidopsis cell cultures(Desikan et al., 1998; Levine et al., 1996; Mazel and Levine, 2001). Theconserved similarity of both morphological and biochemical hallmarks suggest acommon cell death process in plants and animals (Greenberg, 1996).

The neutrophils are recruited to inflammatory sites in response to infection ortissue injury. However, many defence mechanisms employed by these cells arepotentially deleterious to host tissues. For example, excessive release of granuleproteases or production of ROS can damage tissue and can amplify theinflammatory response by a variety of mechanisms. Thus, it is important that theneutrophils are effectively and rapidly destroyed concomitant with the removal ofan inflammatory stimulus to avoid excessive tissue damage or chronicinflammation (Akgul et al., 2001; Savill and Haslett, 1995). The neutrophils areconstitutively programmed to die by apoptosis (Raff, 1992), which is an efficient,non-inflammatory method of removing aging leukocytes from inflammatory sites.

Although apoptotic neutrophils retain membrane integrity, they are non-functional, and lose the ability to degranulate, move by chemotaxis, or generate arespiratory burst. This shutdown in activity and loss of functional capacity,resulting from the disablement of their activation pathways, is aided by decreasedexpression of surface receptors, which prevents them efficiently bindingextracellular ligands. To complete the elimination process, the intact but inactiveapoptotic neutrophils are then phagocytosed by macrophages utilizing novelsurface recognition mechanisms, which fail to trigger a pro-inflammatorymacrophage response (Savill et al., 1993; Savill and Haslett, 1995). By contrast,”accidental” cell death or necrosis, triggered by noxious stimuli such as hypoxiaor cell poison, involves a loss of membrane integrity, which leads to the release ofpotentially toxic intracellular contents (Duvall and Wyllie, 1986), inducing aninflammatory response. Apoptotic cells, if not recognized and removed,eventually undergo secondary necrosis releasing damaging intracellular contentsand amplifying the inflammatory response. For example, non-ingested apoptoticneutrophils undergoing secondary necrosis release large quantities of the potentdegradative enzyme neutrophil elastase, which eludes endocytic clearance bymacrophages This process might underlie the development of chronicinflammatory conditions in which apoptotic cell load and phagocytic clearancemechanism are mismatched (Ward et al., 1999).

Cytotoxicity

Screening for cytotoxic natural productsThe use of crude natural products to treat cancer can be traced back to antiquity.Over 3000 species of plants have been reported to be used in some form of cancertreatment (Cassady et al., 1981). Modern use of pure natural drugs in cancerchemotherapy has a shorter history, tracing back about fifty years. Today there areabout 50 commercially available anticancer drugs (excluding endocrines) thathave been approved by US Food and Drug Administration (USFDA); and

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significantly, the drugs based on natural products represent almost 1/3 of the totalapproved agents. One example is taxol, a natural product derived from the treeTaxus brevifolia, which is used for the treatment of ovarian and breast cancer(Michaud et al., 2000).

Mechanism-based screening for cytotoxicityOne of the largest drug discovery efforts in the field of cancer therapy has beenperformed at US National Cancer Institute (NCI) starting in 1955. After a growingconcern with the narrow spectrum of antitumour activity of available drugs, andan increasing dissatisfaction with the clinical results for many of the mostpromising new investigational drugs (Marsoni et al., 1987), NCI began, in 1985,to phase out its in vivo P388 mouse leukaemia screen, and to replace it with apanel of cell lines (currently >60) representing the major forms of human cancer(Alley et al., 1988; Monks et al., 1991). A semiautomated non-clonogenic in vitroassay was selected for the analysis of growth inhibition and cytotoxicity. Thispanel has been shown to generate remarkably reproducible and characteristicprofiles of differential in vitro sensitivity for cytotoxic agents with differentmechanisms of actions (Boyd and Paull, 1995). This fingerprint profile canclassify, by the use of correlation analysis or advanced neural network, the agentsthat are related to specific groups, such as anti-metabolites, alkylators, topo IIinhibitors, the P-glycoprotein (P-gp), and the multidrug resistance-associatedprotein (MRP). By using the same general principles for data treatment, Dhar etal. (1996) (Dhar et al., 1996) showed that prediction of mechanisms of action ofanticancer drugs is also possible with a panel of only ten human cell lines,representing defined types of cytotoxic drug resistance.

Common mechanisms and resistance for cancer drugsMulti-drug resistance (MDR) is a phenomenon originally seen in cultured tumourcells that, following selection for resistance to a single anticancer agent, becomeresistant to a range of chemically diverse anticancer agents. These MDR cellsshow a decrease in intracellular drug accumulation due to active efflux bytransporter proteins. Two proteins have been shown to cause this type ofmultidrug resistance in human tumour cells, the 170 kDa P-glycoprotein and the190 kDa multidrug resistance protein. Both proteins belong to the ABCsuperfamily of efflux pumps. The transporter best characterised is the protein P-gp,which has been identified in many cancers and has been the target for agents ableto inhibit its action, thereby reversing resistance (Barrand et al., 1997) Morerecently, Cole et al. (1992) identified another transporter, MRP, whose gene isoverexpressed in a multidrug-resistant variant of the small-cell lung carcinomacell line NCI-H69. This cell line, unlike most tumour cell lines that are resistant tomultiple chemotherapeutic agents, did not overexpress the transmembranetransport protein P-glycoprotein (MDR1). MRP confers resistance to a broadrange of natural compounds and mediates the ATP-dependent membrane transportof gluthione S-conjugates of chemotherapeutic drugs (Zaman et al., 1994).

Glutathione conjugation and transport of glutathione conjugates of anticancerdrugs out of cells have been shown to work as a system in the detoxification ofmany anticancer drugs, by changing cellular metabolism. The major componentsof this system include glutathione (γ-glutamylcysteinyl glycine; GSH), GSH-

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related enzymes, and the glutathione conjugate export pump. GSH can combinewith anticancer drugs to form less toxic and more water-soluble GSH conjugates,the conjugation reaction is then catalysed by glutathione S-transferases (GSTs).The GSH conjugates of anticancer drugs can be exported from cells by theglutathione conjugate export pump or by MRP. GSH, glutathione-relatedenzymes, and the glutathione conjugate export pump (or MRP) have been foundto be increased or overexpressed in many drug resistant cells, and in tumour celllines isolated from patients whose tumours are clinically resistant to drug therapy(Lewis et al., 1988; Russo et al., 1986; Wolf et al., 1987; Zhang et al., 1998).Increased detoxification of anticancer drugs by this system may confer drugresistance, and has particularly been associated with alkylating agents. Theseagents, which are electrophilic in nature, spontaneously interact with the thiol ofreduced GSH (Calvert et al., 1998). Inhibition of this detoxification system is astrategy for modulation of drug resistance.

The nuclear enzymes, topoisomerase I and II (topo I and II), are critical for DNAfunction and cell survival. Topoisomerase II (topo II), which binds to double-stranded DNA, cleaves both strands, passes a second strand of DNA through thecleaved site, and rejoins the strands at the original site of cleavage. This processproduces a species of DNA altered only in its topological configuration. Playingan important role in replicational, recombinational, and transcriptional events, andbeing the key to cell growth processes, topo II enzymes participate in most aspectsof DNA metabolism (Bakshi et al., 2001). Topo II inhibitors (e.g., anthracyclinesand epipodophyllotoxins) are active against several types of tumours. However,treatment with these drugs often results in the development of MDR. Because(topo II)-active drugs have several different modes of action, several mechanismsof resistance have been implicated, including decreased activation, and increaseddetoxification by glutathione-dependent enzymes (Sinha, 1995).

Vinca alkaloids and taxanes, which are known antitubulin agents, are classicallyperceived as follows: vinca alkaloids depolymerise microtubules, therebyincreasing the soluble tubulin pool, whereas taxanes stabilize microtubules andincrease the microtubular mass. However, more recent data suggest that bothclasses of agents have a similar mechanism of action, involving the inhibition ofmicrotubule dynamics (Dumontet and Sikic, 1999). Resistance to vinca alkaloidshas been associated with the expression of P-gp, whereas the resistance to thetaxanes is supposed to be linked to alterations or mutations in tubulin (Bhalla etal., 1994; Haber et al., 1995).

Aims of the present investigation

21

3. Aims of the Present Investigation

This thesis is a part of a programme at the Division of Pharmacognosy aimed atdeveloping methods for isolation and characterisation of biologically activepolypeptides from plant materials. The specific aims of this investigation were

• to develop and validate a fractionation protocol for isolation of polypeptidesfrom plant materials;

• to optimise a human neutrophil model to serve as a multitarget bioassay toinvestigate the biological activity of the highly purified polypeptide fractions;

• to evaluate polypeptide fractions in the optimised neutrophil bioassay andfor the cytotoxicity in a human cell line panel;

• to further characterise, chemically and biologically, the constituents inDigitalis purpurea and Phoradendron tomentosum responsible for theobserved effects in the bioassays.

Experimental bioassay procedures

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4. Experimental Bioassay Procedures

Neutrophil isolationPMNs were isolated from heparinized blood samples of healthy volunteers bysequential dextran sedimentation and Ficoll-Paque (Amersham Pharmacia BiotechAB, Uppsala, Sweden) gradient centrifugation at 500 × g for 30 min at 20°C.Contaminating erythrocytes were removed by hypotonic lysis in ice-cold distilledwater for 21 s, followed by addition of 9 volumes of Ca2+ and Mg2+ free phosphate-buffered saline (PBS; SVA, Uppsala, Sweden). After centrifugation (500 × g for10 min, 4°C) the PMNs were resuspended at a concentration of (10 to 30) × 106

cells/ml in PBS containing 0.9 mM CaCl2 and 0.49 mM MgCl

2.

Elastase release assayAfter the optimisation of the assay as described in Paper II, the followingprocedure was used. In all experiments, the PMN suspension in each test tube(concentration of 106 neutrophils per 970 µl) was pre-incubated for 5 min at 37°Cwith cytochalasin B (5 µg/ml) (Sigma), 0.8 mM N-succinyl-L-alanyl-L-alanyl-L-valine-L-p-nitroanilide (SAAVNA) (Sigma), and the test solutions (100 µg/ml) orEtOH (0.1%). The challenge was started with 0.1 µM PAF or with 0.1 µM fMLP(all from Sigma). To allow a direct comparison, samples were tested in parallel forboth PAF- and fMLP-induced exocytosis, using PMNs from the same preparation.Also, test tubes without the addition of PAF/fMLP were run in parallel. Afterincubation at 37°C for 10 min, the reaction was stopped by addition of citric acid(2%), and the tubes were then centrifuged at 500 × g for 10 min. The absorbanceof each sample was recorded (405 nm) using a Shimadzu UV-VIS double-beamrecording spectro-photometer. All samples were run in duplicate. The absorbanceof the corresponding background tube (without inducer) was subtracted from thatof the sample. The amount of inhibition of fMLP or PAF formation of pNA was,as a percentage, the relative decrease in absorbance compared to that of fMLP orPAF alone (100%), using the formula

% Inhibition= {1-[(ABS(S+I)-ABS(S+B))/(ABS(I)-ABS(B))]} × 100

where, for the subscripts, S is the sample; I, the inducer; and B, the buffer.

Superoxide productionSuperoxide generation was measured by means of superoxide dismutase-inhibitable reduction of cytochrome C as described by Jones and Hancock (1994).Test tubes containing 100 µM cytochrome C (Sigma) in PBS (Ca2+ and Mg2+)solution were prewarmed at 37°C in a waterbath. The cell suspension was added toall the test tubes; and, to one of each pair was added the superoxide dismutase(SOD, Sigma) in PBS solution to give a final concentration of 100 µg/ml. Afterincubation for 5 min at 37°C, 0.1 µM of PAF, fMLP, or the test substance wasadded to each test tube. After 20 min incubation (37°C) in a shaking water bath,the reaction was stopped by 5 min of centrifugation (500 × g, 4°C). The amount ofthe cytochrome C reduction was measured by recording the absorbance at 550 nm.


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Inhibition assay of the isolated elastaseThe PMNs were isolated as described above. The cells were diluted with PBS(having Ca2+ and Mg2+) and 2.5% BSA to a concentration of (1.5 to 4) × 106

cells/ml. The PMNs were activated by the addition of cytochalasin B to a finalconcentration of 5 µg/ml and then incubated for 10 min at 37°C. Addition of PAFto a final concentration of 0.1 µM initiated the release of elastase from the PMNs.After incubation (10 min), the exocytosis was stopped by centrifugation of thesolution at 500 × g for 10 min at 20°C. After decanting the supernatant, the cellpellet consisting of the PMNs was discarded. The elastase-containing supernatantwas added to the test tubes containing either the test solution or 10% EtOH. Thebackground reference samples were inactivated by adding citric acid (2%). Thereaction was initiated with the addition of SAAVNA solution to a finalconcentration of 0.8 mM. After incubation at 37°C for 30 min, the reaction wasstopped by adding of citric acid (2%). The test tubes were centrifuged at 500 × gfor 10 min. The absorbance of all samples, which were run in duplicate, wasdetermined at 405 nm. The inhibition of elastase was then calculated as describedabove.

Inhibition assay of trypsinIn the method used, described by Shibata et al. (1986), a trypsin containingsolution is incubated with the test solution and N-benzoyl-DL-arginine p-nitroanilide (BAPNA). The reaction of trypsin with its substrate leads to formationof a coloured product, pNA, which can be quantified by UV-measurement.

Trypsin was dissolved in 0.001 M HCl and 0.02 M CaCl2 to a concentration of 200µg/ml. The trypsin containing solution was added to test tubes containing the testsolution, or EtOH (0.1%). After incubation at 37°C for 10 min, 0.01 M BAPNAsolution (0.01 M BAPNA in 0.05 M TRIS-HCl adjusted to pH 7.5) was added.The test tubes were incubated at 37°C for 10 min. After incubation, adding 30%HOAc stopped the reaction. The test tubes were centrifuged at 500 × g for 2 min.All samples were run in duplicate, and trypsin inhibition was measured andcalculated as above.

Haemolytic testHessinger and Lenhoff (1973) described this test. In brief, human erythrocytes,obtained from the blood of healthy volunteers, were separated from PMNs bysedimentation in a 10% solution of Dextran T-500 in 0.9% NaCl. The mixture oferythrocytes and dextran was centrifuged for 30 min (500 × g, 20°C); and thesupernatant, discarded. The remaining erythrocytes were washed with 50 ml ofKrebs-Henseleit solution and centrifuged for 10 min (500 × g, 4°C). Theerythrocyte concentration was adjusted such that the absorbance for totalhaemolysis, by the addition of a saponin from Quillaja bark (Sigma), was between1.2 and 1.4 at 570 nm. To each test tube, the test substance in Krebs-Henseleitsolution and the erythrocyte test solution were added. After incubation for 15 minat 37°C, the reaction was terminated by centrifugation for 10 min at 1000 × g and20°C. The absorbance of each sample was compared with a sample that waswithout blood (as a blank), and with saponin for total haemolysis, and with waterfor no haemolysis. The polypeptide fractions, which were run in duplicate, weretested at a concentration of 100 µg/ml.


24

Cell line panelThe cell line panel consisted of four sensitive parental cell lines, five drug-resistantsublines, and one cell line with primary resistance (see Table 2).

Table 2. The cell lines, their origins, and their mechanisms of resistance.Cell line Origin Mechanism of resistanceRPMI 8226-S Myeloma ParentalRPMI 8226-LR5 Myeloma *GSH-associatedRPMI 8226-Dox40 Myeloma *Pgp-associatedU-937 GTB Histiocytic lymphoma ParentalU-937 Vcr Histiocytic lymphoma *Tubulin-associatedNCI-H69 Small Cell Lung Cancer ParentalNCI-H69AR Small Cell Lung Cancer *MRP-associatedCCRF-CEM Leukaemia ParentalCEM-VM-1 Leukaemia *Topo II-associatedACHN Renal Primary resistant

* = subline. Abbreviations: topo II (topoisomerase II), MRP (multidrug-associatedprotein), P-gp (P-glycoprotein), GSH (glutathione).

Choice of cell lines in Table 2 proceeded as follows: for melphalan resistance, theRPMI-8226-LR5 cell line, proposed to be associated with increased levels ofglutathione (Bellamy et al., 1991; Mulcahy et al., 1994), was selected; fordoxorubicin (Dox) resistance, the RPMI 8226-Dox 40 cell line, which shows theclassical MDR phenotype with overexpression of P-gp 170 (Dalton et al., 1986;Dalton et al., 1989), was selected; for vincristine (Vcr) resistance, the U-937 Vcrcell line, suggested to be tubulin-associated (Botling et al., 1994), was selected; forDox resistance, the NCI-H69AR cell line, expressing a MDR phenotype proposedto be mediated by MRP (Mirski et al., 1987; Slovak et al., 1993), was selected;and finally, for teniposide resistance, the CEM-VM-1 cell line, suggested to betopo II associated, and expressing the atypical MDR phenotype (Danks et al.,1988; Danks et al., 1987), was selected. The drug resistance of the primaryresistant ACHN cell line is probably multifactorial (Nygren and Larsson, 1991).

Measurement and data analysis of cytotoxic activitySubstances were tested for cytotoxicity using the non-clonogenic 72-hourfluorometric microculture cytotoxicity assay (FMCA) (Larsson and Nygren, 1989;Larsson and Nygren, 1990). The FMCA is based on measurement of fluorescencegenerated by hydrolysis of fluorescein diacetate to fluorescein by cells with intactplasma membranes. Experiments with cell lines were repeated 2-3 times. Qualitycriteria for a successful analysis included a fluorescence signal in the control wellsof more than ten times mean blank value, and a mean coefficient of variation (CV)in the control and blank wells of less than 30%.

V-shaped 96-well microtitre plates were prepared with 20 µl per well of testsolution at ten times the desired concentration. Each plate contained threesubstances, using triplicate wells for each concentration and compound, six controlwells, six blank wells, and three wells for each positive (0,1% Triton X-100) andnegative (PBS) control.

To measure cell survival, a survival index (SI) was defined as the ratio offluorescence in experimental wells, and that in control wells (with blank valuessubtracted)—multiplied by 100 to reflect a percentage. The IC50 value, calculatedfor each compound, was defined as the concentration of the compound at SI equal


25

to 50%. A procedure similar to the COMPARE analysis described by Paull et al.,(1989) (using Pearson´s correlation coefficients) was used for comparingcompounds and rank-ordering them for their similarity to a “mean” profile asdescribed in Paper IV. For pairs of compounds (x and y), amount of linearcorrelation of log IC50 values was determined using Excel (Microsoft). In addition,the resistance factor (RF) was calculated for each compound, defined as the ratioof IC50 values, that for the resistant subline and that for its (sensitive) parental cellline. The pairs of resistant/parental cell lines used for RF calculations of P-gp,GSH, MRP, topo II, and tubulin associated resistance were RPMI 8226-Dox40/RPMI 8226-S, RPMI 8226LR5/RPMI 8226-S, NCI-H69AR/NCI-H69,CEM-VM-1/CCRF-CEM, and U-937 Vcr /U-937 GTB, respectively (Dhar et al.,1996).

Development of methods for isolation of polypeptides from plant material

26

5. Development of Methods for Isolationof Polypeptides from Plant Material

The use of pre-fractionated plant extracts instead of crude extracts conceivablyoffers advantages in finding bioactive compounds present only in small amounts inthe plant, and can be utilised to exclude or include activity from certain classes ofsubstances. Although peptides in plants are often present only in small amounts(e.g., 1 µg systemin was isolated from 28 kg tomato leaves) (Marx, 1996) , yetconvenient assays for purifying peptides in low concentrations have been lacking.In this study a fractionation protocol for isolating peptides from a small amount ofplant material was developed (Paper I).

Plant materialThe plants were selected to represent a major part of the angiosperms withemphasis on medicinal plants, reputed Swedish anti-inflammatory plant taxa andplants known to contain peptides. The plant materials were mainly collected inUppland, Sweden, including the Uppsala University Botanical Garden. Voucherspecimens have been deposited at the Uppsala University Herbarium (UPS). Freshplant material was dried at 55-60°C to preserve it, and to prevent enzymaticdegradation of peptides. Previously reported plant peptides, containing severaldisulphide bonds, appear to be largely heat stable (Gran, 1973; Samuelsson, 1973),and several are reportedly stable to aqueous boiling. The dried, powdered plantmaterial was stored at –20°C until used.

Fractionation of polypeptidesFractionation of plant materials was done in five steps to obtain a highly purifiedfraction (fraction P). Before applying the plant material to the protocol, each stepwas optimised and analysed. Figure 2 shows the flowchart of the fractionationprocedure, applied to 100 plant materials.

Step 1 and 2: ExtractionPlant material was first extracted in dichloromethane; and then, in 50% EtOH.Although polypeptides are insoluble in dichloromethane, yet ubiquitous lipophilicsubstances, such as chlorophyll, lipids, and other low-molecular-weight substances(e.g., terpenoids, phenylpropanoids, etc.) are extracted and thus removed. Theextraction with 50% EtOH was chosen because it does not extract mostpolysaccharides (Beutler et al., 1993; Thunberg and Samuelsson, 1982) orenzymes, and the solubility of polypeptides is generally better in the 50% mixturethan in pure water or alcohol. Furthermore, such extractions need no preservationfrom microbial growth.

Step 3: Removal of tannins by filtration through polyamideAfter being acidified by HOAc to a final concentration of 2%, the 50% EtOHextract was passed through a column of polyamide, to remove the containedpolyphenols (tannins). The binding of tannins to the polyamide is highly pH-dependent, so lowering the pH from 7.5 to 3 leads to strong hydrogen bonding,which occurs between polyphenolics and polyamide. Thus, the tannins are


27

practically irreversibly bound to the column. The polypeptides were eluted withEtOH 50%/HOAc 2%.

Figure 2. Fractionation protocol for isolation of polypeptides from plant biomass.

Step 4: Size-exclusion chromatography on Sephadex G-10For this gel filtration step, several experiments were performed to establish asuitable mobile phase. The solution EtOH 50%/HOAc 2%/NaCl 0.2 M was chosen.The high-molecular substances (>700 Da) appeared in the void volume, and thebasic amino acids eluted in the low-molecular fraction by including the 0.2MNaCl. The plant extracts were readily dissolved by the combination EtOH (50%)

Removal of lipophilicconstituents

Pre-extraction withdichloromethane

Removal of salts andpolysaccharides

Solid-phase extraction onRP18 material

Size-exclusion chromato-graphy on Sephadex G-10

Plant MaterialDried, Powdered

Dichloromethane ExtractPlantResidue

EthanolExtract

Tannin-FreeExtract

Extraction of peptides

Removal of tannins

Extraction withethanol (50%)

Filtration throughpolyamide

High-Molecular-Weight Fraction

PolypeptideFraction

Removal of low-molecularweight compounds

Low-Molecular-Weight Fraction

Salts and Polysaccharides


28

and HOAc (2%), and the non-specific interactions between plant pigments and thegel were largely suppressed.

Step 5: Desalting by solid-phase extraction on C18 materialThe high-molecular-weight fraction described above contained, besides polypeptides,a large amount of NaCl and polysaccharides. The salt and the polysaccharideswere removed by solid-phase extraction (SPE) using a C18 reversed-phase column.Experiments with commercially available polypeptides (oxytocin, gramicidin,bacitracin, and insulin) showed that they are not eluted in 50 mM NH4HCO3, butsalt and polysaccharides were easily removed. The polypeptides were then elutedfrom the column in three steps by adding 20%, 50%, and 80% EtOH in 50 mMNH4HCO3, respectively. None of the reference peptides required higher concentrationsof ethanol to be eluted. Evaporating in vacuo and then lyophilising the combinedeluates yielded a polypeptide fraction (fraction P).

Isolation of varv peptide ATo further validate the fractionation protocol, the procedure was applied to plantmaterial of Viola arvensis (Violaceae), which had been reported to contain a cyclic29-residue polypeptide, viola peptide-I (Schöpke et al., 1993). From the hydrolysisof fraction P of Viola arvensis, free amino acids were obtained; and from the RP-HPLC chromatogram of the fraction, several peaks with UV spectra consistentwith tryptophan-containing peptides was shown.

Figure 3. Semipreparative RP-HPLC of the polypeptide fraction obtained from Viola arvensis.The peak labeled A in the chromatogram corresponds to varv peptide A. The semipreparativeHPLC was performed with a Shimadzu system, equipped with an SPD-M10Avp photodiode arraydetector (Shimadzu, Kyoto, Japan), and a 250 × 10 (i.d.) mm Dynamax column (C18, 5µm, poresize 300Å). Mobile phase: 40% CH3CN-i-PrOH (6:4)/ 0.1% TFA (adjusted to pH=2.25 byaddition of NH4OH). UV detection at 215 nm. Flow rate 4 ml/min.

The same isolation strategy was applied to a larger amount of plant material (asdescribed in Paper I). The substance corresponding to the major peak in thechromatogram at 215 nm was collected and designated varv (Viola arvensis)peptide A (Figure 3). Quantitative amino acid analysis of the hydrolysate of varvpeptide A indicated the amino acid composition shown in Table 3. The calculatedaverage mass of a linear peptide with this composition would be between 2901.3Da (2Asn; 1 Gln) and 2904.3 Da (2 Asp; 1 Glu). Mass spectrometry of varv peptideA, however, provided a lower molecular weight of 2879.4 Da, suggesting the


29

peptide to be macrocyclic, like the previously reported viola peptide-I (Schöpke etal., 1993).

Varv peptide A was then reduced with mercaptoethanol and subsequently alkylatedwith 4-vinylpyridine. Cleavage of the alkylated peptide with endoproteinase Glu-Cresulted in a single linear product, consistent with the opening of a macrocyclicring. The linear product was subjected to automated Edman degradation, defining2 Asx and 1 Glx from the quantitative amino acid analysis as 2 Asn and 1 Glu (Table3, Paper I). Consistent with the amino acid sequence obtained for the linearproduct, varv peptide A had the following structure:

cyclo- (-Thr-Cys-Val-Gly-Gly5-Thr-Cys-Asn-Thr-Pro10-Gly-Cys-Ser-Cys-Ser15-Trp-Pro-Val-Cys-Thr20-Arg-Asn-Gly-Leu-Pro25-Val-Cys-Gly-Glu-)

With this amino acid composition, the calculated average mass of a linear peptideis 2902.3 Da. Assuming the peptide to be macrocyclic (-18 Da), and its six cysteineresidues to be engaged in intramolecular disulphide bonds (-6 Da), its molecularweight is 2878.3 Da. This value agrees with the molecular weight 2879.4 Da forthe varv peptide A, experimentally determined by means of MALDI-TOF MSanalysis.

Varv peptide A and the previously reported viola peptide-I share a very highdegree of sequence homology. They differ only at two amino acid positions. TheTrp and the Arg residues in varv peptide A are substituted for an Arg residue andan X (unidentified amino acid) residue, respectively, in the published sequence ofviola peptide-I (Schöpke et al., 1993).

Table 3. Amino acid analysis of varv peptide A isolatedfrom Viola arvensis.

Amino acidResidues from

amino acid analysisResidues fromsequencinga

Asx 2,0 2 (2 Asn)Thr 3,7 4Ser 2,0 2Glx 1,0 1 (Glu)Pro 2,9 3Gly 4,9 5Cys 5,2b 6c

Val 3,0 3Leu 1,0 1Arg 1,0 1Trp 1,0d 1

a A total of 29 residues were determined by sequencing.b Half-cystine was determined as cysteic acid , based ona separate sample, following oxidation with performic acid.c Cys was determined as (pyridylethyl)cysteine, followingalkylation with vinylpyridine. d Trp was determinedphotometrically.

A multitarget bioassay on the neutrophil

30

6. A Multitarget Bioassay on the Neutrophil

The multitarget functional bioassays, in which observed effects cannot be attributeddirectly to a specific mode of action, encompasses assays on whole animals,isolated organs, and intact cells. For example, an observed relaxation of the isolatedguinea pig ileum can be due to mechanisms involving cell membrane receptors,second messengers, or ion channels, or due to other mechanisms. Single targetbioassays, on the other hand, are typically tests run on an isolated enzyme or areceptor (receptor binding). For such assays, which are highly specific, the observedeffect can be directly attributed to a specific mechanism, such as enzymeinhibition or receptor affinity.

Dewald and Baggiolini (1987) developed an assay to detect PAF-antagonists. Inthat assay, neutrophils were incubated with the antagonist under evaluation, PAF,and with a chromogenic substrate SAAVNA for released elastase. Elastase reactswith SAAVNA, which leads to formation of a coloured product, p-nitroanilide(pNA), which can be quantified photometrically (Bieth and Wermuth, 1973;Wenzel et al., 1980). This detection of pNA formed by elastase released from achemoattractant-stimulated neutrophil can be described as a multitarget functionalbioassay (Claeson and Bohlin, 1997), where both known and previously unknownpotential drug targets are present. An observed inhibition of formation of pNA canbe due to elastase inhibition, for example, or due to cytotoxic effects on the cell, oraffinity for a receptor on the neutrophil (Figure 4). By the same method,chemoattractant activity of a test compound is also possible, seen as increasedamount of pNA formed. In Paper II, the evaluation of the originally describedassay conditions is described, with the aim of optimising the bioassay formultitarget functional screening of natural products. Polypeptide fractions(fraction P) of 100 plant materials were investigated in the, thus optimised,neutrophil multitarget bioassay (Paper II).

Figure 4. Schematic picture of the neutrophil, viewed as a multitarget functional bioassay withvarious possible modes of action for test substances. Both inhibiting and stimulating effects canbe detected.

pNAp-nitroanilide

Elastaseinhibition

PAF/fMLP

ELASTASE

Suc-Ala-Ala-Val-pNA(SAAVNA)

PAF and fMLPreceptors

Other receptors

Intracellular signalling

Cytotoxic effects

NEUTROPHIL


31

Optimisation of a multitarget bioassay on the neutrophilThe bioassay method was optimised with respect to the formation of pNA. Throughoutthe optimisation procedure, the aim was to select conditions to facilitate thecomparison of activities of polypeptide fractions and test compounds towards PAFor fMLP induced release of pNA. As described in Paper II, the followingparameters were optimised:

• cytochalasin B concentration• SAAVNA concentration• PAF and fMLP concentration

The formation of pNA in PAF and fMLP-induced PMNs required cytochalasin B.The established optimal concentration of cytochalasin B for priming was 5 µg/ml(Figure 3a, Paper II). Concentration-response curves for the inducers PAF andfMLP were recorded and tested in parallel using PMNs from the same preparation.Equal biological responses for PAF and fMLP, reaching approximately 80% ofmaximum, were obtained at a concentration of 0.1 µM (Figure 3c, Paper II), whichwas chosen as the assay condition. Amount of formation of pNA depended on theSAAVNA concentration (Figure 3b, Paper II). For 0.1 µM PAF and fMLP, pNAreached a maximum at a concentration of 0.8 mM of SAAVNA, which was chosenas the assay condition (Figure 3d, Paper II). To validate the assay, thus optimised,one additional agonist, interleukin-8 (IL-8), was evaluated under the test conditionsdescribed. The same concentration as that for PAF and fMLP (0.1 µM) can beused to induce elastase release from neutrophils by IL-8 (Figure 4, Paper II).

ValidationWhen reference compounds were evaluated in the optimised assay; different modes ofaction were clearly detected (Table 4). The PAF-antagonist ginkgolide BN 52021(Földes-Filep et al., 1987) exhibited a selective inhibitory effect on PAF, as expected,with the IC50 value (Table 4) being 72 µg/ml, which is consistent with previouslyreported values for inhibition of PAF by the ginkgolide BN 52021 (Dewald andBaggiolini, 1987). At the highest tested concentration (100 µg/ml), ginkgolide wasunable to inhibit the fMLP inducer. The elastase inhibitor α1-antitrypsin (Virca andSchnebli, 1984) [IC50: 10 µM (PAF), 11 µM (fMLP)] had a non-selective inhibitoryeffect.

Table 4. Reference substances tested in theoptimised assay.

SubstancesPAF IC50

µg/mln fMLP IC50

µg/mln

α1-antitrypsin 10 9 11 9Trypsin-inhibitor 10 9 8 10BN 52021 72 12 - 11


32

Identification of the extracellular release of superoxide, elastase and defensinsSuperoxide generation by the neutrophilsThe generation of the superoxide anion O2

- in PAF (or fMLP) activated PMNs wasmeasured. Comparing the two agonists, fMLP induced a higher increase in theproduction of O2

- than did PAF (Figure 5). This result agrees with reports in theliterature, indicating that PAF only weakly stimulates superoxide generation (EC50

below 10 µM) of neutrophils, whereas fMLP is more potent (EC 50: 48 nM) (Turneret al., 1994).

0

0,1

0,2

0,3

0,4

0,5

∆ ABS

0,00

01

0,00

1

0,01 0,1 1

10

100

log conc. µM

fMLP

PAF

Figure 5. Superoxide production by PMNs when stimulated by the addition of differentconcentrations of PAF (o) and fMLP (u). Each point is the mean absorbance value ± SEM ofthree independent experiments run in duplicate.

SDS-PAGE of exocytosed proteins from stimulated neutrophilsThe PMNs were isolated and stimulated as described above. The PMNs werepelleted by centrifugation, and the supernatant was saved. The supernatant wasdiluted 50% with 2X treatment buffer (0.125 M tris-Cl, 4% SDS, 20% v/v glycerol,0.2 M DTT, 0.02% bromophenol blue, pH 8.8), boiled for 2 min, and stored in afreezer. SDS-PAGE was run (180V, 40mA and 1.5h) on a Hoefer miniVE(Amersham Pharmacia Biotech, Uppsala, Sweden). The running buffer was SDS(sodium dodecyl sulphate) containing tris-glycine. Amersham Pharmacia BiotechLow Molecular Weight Calibration Kit, containing proteins of 94, 67, 43, 30, 20,and 14 kDa, was used as the molecular weight marker.

The sample was run on a 10%T gel with ReadySol 40% (Amersham PharmaciaBiotech, Uppsala, Sweden) and polymerised with ammonium-persulfate, catalysedby TEMED. The gels were stained by means of a silver staining kit protein(Amersham Pharmacia Biotech, Uppsala, Sweden). As the results show (Figure 6),the sample prepared from the neutrophils contains enough proteins to bevisualised.

The biggest spot is albumin (at 67 kDa), which is shown to be present in allsamples. In the sample preparations, albumin is added to the assay, but also


33

exocytosed by the neutrophils. The purecommercially available elastase showsmore than one band, which is explainedby the occurrence of more than oneglycosylation (Wilde et al., 1990).Bands cluster in the sample, because alot of proteins are released that havemolecular weights in the same range aselastase and have more than oneglycosylation form, (Wilde et al., 1990).The patterns for the stimulated andunstimulated neutrophils do not differsignificantly. The silver staining is not aquantitative staining method, but aqualitative one. Silver staining, althoughvery sensitive method, doesn’t changewhen the concentration of the protein

Figure 6. SDS-PAGE of the exocytosedproteins from the PMNs. The lanes are fromthe left: weight marker, proteins exocytosedfrom PAF-stimulated neutrophils,unstimulated neutrophils, human elastase(Sigma).

increases over a certain limit, which was probably the case, since some elastase isreleased spontaneously from neutrophils. Although coomassie blue staining is aquantitative method, it is less sensitive than silver staining.

The release of defensins from fMLP-stimulated neutrophilsThe human PMNs were isolated, as described above in experimental procedures,from 100 ml heparinized blood samples. The cells were activated by addingcytochalasin B to a final concentration of 5 µg/ml, and then were incubated for 10min. To initiate release, the PMNs were stimulated by the addition of fMLP to afinal concentration of 0.1 µM. After incubation (10 min), the exocytosis was stoppedby the addition of 30% acetic acid, and then the solution w as centrifuged at 500 × gfor 5 min. The supernatant, after centrifugation, was diluted and lyophilised. Afterbeing lyophilised, it was extracted with 80% MeOH on ice at a room temperatureof 8°C for 8h.

Figure 7. RP-HPLC of fMLP-stimulated extract of neutrophils. A linear gradient from 10%CH3CN/0.1% TFA to 60% CH3CN/0.1% TFA was used. UV detection, at 215nm; flow rate, 0.3ml/min.


34

Table 5. Comparison of measured masses (MH+) froman enriched defensin fraction from human neutrophilsand calculated masses of HNP-1, -2, -3, and 4.

Collect Measured massesMALDI-TOF MS (MH+)

Calculateda

B 3443.13372.23487.3

3442.1 (HNP 1)3371.0 (HNP 2)3486.1 (HNP 3)

D 3711.1 3709.5 (HNP 4)

The RP-HPLC chromatogram of the enriched defensin fraction (Figure 7) includedseveral peaks, with UV spectra consistent with tryptophan-containing peptides.Fractions were collected and analysed by MALDI-TOF MS. By MALDI-TOFMS, fraction B was shown to contain three peptides with masses 3443.1, 3372.2,and 3487.3 MH+ (Table 5), in agreement with masses calculated for the neutrophildefensins HNP-1, -2, and -3. In fraction D, the mass 3711.1 MH+ was found, inagreement with the calculated for HNP 4.

The evaluation of the activityThis study includes an introductory classification of polypeptide fractions, sortingthem into mechanistic classes that can facilitate prioritising the fractions P forfuture studies. Figure 8 provides an outline of the classification procedure. The100 polypeptide fractions (fraction P) according to the fractionation protocol, werefirst tested for inhibition of PAF- and fMLP-induced pNA formation, and forability to induce elastase release in the absence of challenge with PAF or fMLP.Inhibitory polypeptide fractions were classified as being either selective inhibitors(of PAF or fMLP) or non-selective inhibitors (of pNA formation).

Figure 8. Schematic representation of the evaluation of the activity of the polypeptide fraction.

Further activitycharacterisation

Polypeptide fractionInactivePolypeptide fractioninduced elastase release

PAF-inducedelastase release assay

fMLP-inducedelastase release assay

Non-selective PAF and fMLPinduced elastase inhibition

Selective fMLP-antagonist

Selective PAF-antagonist

Trypsin inhibition assayElastase inhibition assayHaemolysis assay


35

Polypeptide fractions that were potent non-selective inhibitors of pNA formation(>80%) were further evaluated for direct inhibitory effect on isolated elastaseenzyme. To roughly evaluate selectivity of elastase inhibition, polypeptidefractions were also tested for ability to inhibit the enzyme trypsin. In addition,each fractions P was tested for haemolytic activity to determine whether it coulddamage cells.

The activity of fraction PAfter optimisation, the assay was operated successfully for 100 polypeptide fractions(listed in Table 2, Paper II) from 96 species, representing 46 families of 26 orders,where both PAF and fMLP were used to induce exocytosis.

The results from the screening (Paper II) are presented in a manner that linksrecorded biological activity to phylogenetic information (see Figure 9). Of the 100tested polypeptide fractions, 41% inhibited pNA formation more than 60%; 18%non-selectively inhibited pNA formation less than 30%; and 3% stimulatedformation of pNA, without the challenge of PAF or fMLP. In the orderMalpighiales, 4 of the 8 tested species inhibited pNA formation less than 30%;whereas in the family Violaceae, 3 of 4 tested species inhibited formation less than30%, and one, Viola patrinii, stimulated formation (Table 2, Paper II).

In other orders, such as Lamiales and Brassicales, a comparably high proportion ofthe tested species showed prominent inhibitory activity. For Lamiales, 11 out of 17polypeptide fractions inhibited pNA formation by more than 60%, and 2 enhancedformation. The fraction P of Nymphoides peltata of Menyanthaceae inhibited pNAformation of PAF-induced elastase release 36 ± 14%, and fMLP-induced elastaserelease 90 ± 3%, which may indicate selective inhibition by fMLP receptors.However, since all the polypeptide fractions contain a mixture of substances, morethan one active compound is possible, and therefore the presence of selectiveinhibitors in fractions, which can influence and modify non-selective inhibition,cannot be ruled out.

Haemolytic testA lysis test on erythrocytes was used to determine whether the polypeptidefractions at a concentration of 100 µg/ml could damage the cells, which may affectthe release of elastase from the neutrophil. The cell-damaging effect on erythrocytes isnot connected to elastase release by PMNs, but might, as a first step, facilitatedetermining whether the fraction or substance is damaging cells at the concentrationused in the neutrophil assay. At extract concentration 100 µg/ml, haemolysisoccurred in 7 of the polypeptide fractions tested (Table 2, Paper II).

Inhibition of the isolated elastaseThe polypeptide fractions that non-selectively inhibited pNA formation more than80%, were tested for ability to inhibit, in a single target bioassay, the isolatedenzyme elastase. For comparison, other polypeptide fractions were included asnegative controls. The polypeptide fractions inhibited the isolated elastase in thesame concentration range as they inhibited pNA formation (Table 2, Paper II),which indicates that the observed effects in the whole-cell assay are due toinhibition of the isolated enzyme elastase.


36

PTERIDOPHYTA FILICOPSIDA Filicales Dryopteridaceae, 47/1700 1 Polypodiaceae, 33/700 1 Pteridaceae 1CONIFEROPHYTA CONIFEROPSIDA Pinales Pinaceae, 12/220 1GINKGOPHYTA GINKGOOPSIDA Ginkgoales Ginkgoaceae, 1/1 1MAGNOLIOPHYTA ANGIOSPERM ROOT undetermined to order Nymphaeaceae, 6/75 1 Ceratophyllales Laurales Magnoliales, 6 Magnoliaceae, 7/165 1 MONOCOTYLEDONS Acorales Alismatales Asparagales, 29 Alliaceae, 30/850 2 Dioscoreales Liliales, 9 Colchicaceae, 15/165 1 Pandanales Arecales Poales, 16 Poaceae, 668/9500 2 Comellinales Zingiberales EUDICOTYLEDONS Ranunculales, 7 Papaveraceae, 23/230 1 Ranunculaceae 4 Proteales Caryophyllales, 26 Caryophyllaceae, 87/2300 1 Didiereaceae, 4/11 1 Polygonaceae, 46/1100 1 Santalales, 5 Santalaceae, 41/925 2 Saxifragales, 13 Crassulaceae, 33/1100 1 ROSIDS Geraniales, 6 Geraniaceae, 11/700 1 Malpighiales, 31 Linaceae, 14/250 1 Salicaceae, 2/435 1 Violaceae, 20/800 4 Oxalidales Fabales, 4 Fabaceae, 642/18000 3 Rosales, 11 Rosaceae, 95/2825 2 Urticaceae, 48/1050 1 Cucurbitales, 7 Cucurbitaceae, 119/775 1 Fagales, 8 Betulaceae, 6/110 3 Myrtales Brassicales, 15 Brassicaceae, 365/3250 4 Caricaceae, 4/33 1 Resedaceae, 6/80 1 Malvales Sapindales, 7 Burseraceae, 17/540 3 ASTERIDS Cornales Ericales, 24 Ericaceae, 107/3400 2 EUASTERIDS I Garryales Gentianales, 5 Apocynaceae, 480/4800 3 Gentianaceae, 78/1225 1 Rubiaceae, 630/10200 12 Lamiales, 17 Acanthaceae, 229/3450 5 Gesneriaceae, 139/2900 2 Lamiaceae, 252/6700 5 Oleaceae, 24/615 1 Plantaginaceae, n/ad 3 Solanales, 5 Boraginaceae, 130/2300 1 Convolvulaceae, 56/1600 2 EUASTERIDS II Aquifoliales Apiales, 7 Apiaceae, 446/3540 1 Asterales, 13 Asteraceae, 1528/22750 5 Menyanthaceae, 5/40 2 Dipsacales, 6 Caprifoliaceae, 5/210 1 Valerianaceae, 10/300 1

Low Medium High

•••

••

•

•

• •

•

••

•••• •

•••• •

••

••

••• **

••••• ••

•••

••••••

•••

• •

•• ••

• ••••••• ••••••••••••

• •••••

**••

••

•• ••••

•••

•

••

<30% 30-60% >60%

Figure 9. The biological activity of fraction P (•) in the neutrophil elastase release assay. Theactivity is linked to the systematic position and distribution of taxa included in the study. Thephylogenetic relationships are according to APG (1998) (Bremer et al., 1998) and theNCBI/GenBank taxonomy section. A numbers following an ordinal name indicates theapproximate number of included families; a number following a familial name indicates theapproximate number of genera and species, respectively. An ordinal name in bold typefaceindicates an order represented in the study.*These fractions stimulated pNA formation.


37

Inhibition of trypsinSome of the plant materials were also tested against the enzyme trypsin, to give anindication of the enzyme selectivity of the observed inhibition of elastase.Inhibition of trypsin by polypeptide fractions of Reseda luteola was high (97%).The fraction P of Rubus idaeus and Tabernaemontana dichotoma inhibitedelastase (80% and 77%), significantly more than they inhibited trypsin (8% and11%). These results suggest that some of the polypeptide fractions studied mightcontain selective protease inhibitors.

Stimulating activityExamination of the fraction P of Digitalis lanata, Digitalis purpurea, and Violapatrinii revealed an unusual and unexpected effect on the formation of pNA. Thefraction P from these plants significantly increased formation of pNA, in aconcentration-dependent manner (Figure 8, Paper II), independent of challengewith PAF or fMLP. Furthermore, these polypeptide fractions enhanced the pNAformation as much or more than exocytosis induced by either agonist, PAF orfMLP. The formation of pNA was dependent in these cases on the presence ofcytochalasin B, and was not inhibited by the PAF-antagonist ginkgolide BN52021; but instead the presence of α1-antitrypsin decreased the amount of pNAformed. Bioassay-guided fractionation of the specific fraction P (substances withmolecular weights >700 Da) of the leaves of Digitalis purpurea Ehrl. led to theisolation of digitoxin, as described in chapter 7.

Isolation and characterisation of substances with cytotoxic and neutrophil elastase releasing activity

38

7. Isolation and Characterisation of Substances withCytotoxic and Neutrophil Elastase Releasing Activity

After a primary fractionation and activity evaluation, the constituents responsiblefor the observed effects in Phoradendron tomentosum (Paper III) and Digitalispurpurea (Paper IV) were further characterised (chemically and biologically).

Phoratoxins from Phoradendron Tomentosum

The first phoratoxin was isolated in 1967 by Samuelsson and Ekblad from Phoradendrontomentosum (DC) Engelm. subsp. macrophyllum. This peptide was later renamedphoratoxin A. In 1974 the amino acidsequence was determined. Phoratoxin Aconsists of 46 amino acids (Mellstrand andSamuelsson, 1974a) tightly bound togetherby three disulphide bridges in the same wayas for the viscotoxins (Mellstrand andSamuelsson, 1974b).

Thunberg (1983) isolated phoratoxin Bfrom P. tomentosum. Three lyophilisedextracts were investigated. The leaves fromP. tomentosum subsp. macrophyllum(Cockerell) Wiens grown on Juglanshindsii Jepson extracts (extract A), and theleaves (extract B) and twigs and stems(extract C) from P. tomentosum subsp.macrophyllum grown on Populus fremontiiS. Wats. These three plant materials fromP. tomentosum L. were homogenised andextracted in 2% acetic acid. Theconcentrated extract was evaporated invacuo, lyophilised, and stored at –20°C(Mellstrand and Samuelsson, 1973;Samuelsson and Ekblad, 1967). The driedextract was diluted with distilled water,with the pH set to 5.0. The solution wasdiluted with water until the conductivitywas slightly lower than the acetate bufferemployed. The solution was passed througha cellulose phosphate column and washedwith 20 column volumes of 0.1 M NaOAc(pH 5.0). The adsorbed substances wereeluted with 0.1 M NaOAc containing 0.8 MNaCl (pH 5.0).

Figure 10. Separation of basic proteinson a column of SP- Sephadex C-25eluted with a linear gradient of sodiumphosphate buffer. A, B, and C representseparation of basic proteins from extractsA, B, and C respectively. Reproducedfrom Thunberg (1983).


39

Fractions were collected and concentrated in vacuo. To remove the salt, the fractionwas applied to a column (Sephadex G-25). The high molecular fraction was collected,evaporated in vacuo, and passed through a cation-exchange column (SP-SephadexC25), and then eluted in a linear gradient from 0.05 M sodium phosphate (pH 5.05)to 0.3 M sodium phosphate (pH 6.0). Fractions I-V were collected from the extract A-C (Figure 9), desalted and lyophilised (Thunberg, 1983). In extract A, from whichphoratoxin A was previously isolated, was shown to be a mixture of at least twopeptides, since a half valine residue was found. Phoratoxin B was isolated frompeak III in extract B (Thunberg, 1983). The sequences of phoratoxins A and B areshown in Figure 12.

Recent progress in the chromatography and sequence analysis of peptides has markedlyimproved the separation of peptides. One standard method today for separatingpeptide mixtures is RP-HPLC with gradients of increasing concentrations of acetonitrilein the presence of trifluoroacetic acid. With modern instruments and columns,complex peptide mixtures can be separated. Difficult separations are addressed bymodifying the gradient slope or organic eluant composition. Further improvementsin resolution are often needed, requiring fundamental changes in mobile phasecomposition or selection of complementary chromatographic separation mechanisms.

Isolation and characterisation of peptides from Phoradendron tomentosumThe fractions IV and V from extract B (see Figure 10) (Thunberg 1983) were furtherfractionated. The peptides isolated from fraction IV were named phoratoxin C andphoratoxin D. The molecular weights of phoratoxins C and D, experimentally determinedby mass spectrometry, were 4879.3 MH+ and 4311.2 MH+, respectively.

The peptides from fraction V were the previously described phoratoxin B, and twonovel peptides named phoratoxins E and F. The molecular weights were 4893.0MH+ (phoratoxin B), 4879.2 MH+ (phoratoxin E), and 4873.2 MH+ (phoratoxin F).

The results from the amino acid analyses of the isolated peptides are shown inTable 6. The primary sequences of phoratoxins B-F were determined unambiguouslyby means of a combination of procedures: Edman degradation, trypsin enzymaticdigestion, and electrospray ionization MS/MS sequencing. To gain information onthe C-terminal part of the molecule, the tryptic fragments of the pyridylethylatedpeptide (Table 7), or a fragment of the native peptide, was analysed by MS/MS(illustrated in Figure 11). Cysteine was identified as carbamidomethyl cysteine afteralkylation with iodoacetamide. The MH+ of the carbamidomethylated peptides (Table6) exceeded that of the native peptides by 343.2 Da. This corresponds to the mass ofsix carbamidomethylated cysteines, 57.03 Da for each derivatized cysteine, whichindicates the presence of six cysteine residues in the native peptides. Thepreviously isolated phoratoxin B and the novel phoratoxins C, E, and F consist of46 amino acid residues; and phoratoxin D has 41 amino acid residues, all with sixcysteines, similar with the earlier described phoratoxins A and B, which belong tothe group of thionins. The sequences of the isolated peptides are shown in Figure12, where in addition, alignment with other known thionins is shown.


40

Table 6. The amino acid composition and molecular weights of the phoratoxin peptides. For eachpeptide, the residues from amino acid analysis are listed to the left and the residues fromsequencing to the right.

Amino acidPhoratoxin B Phoratoxin C Phoratoxin D Phoratoxin E Phoratoxin F

Asp/Asn (D/N) 4.0 4 (2 N) 3.2 3 (2 N) 3.1 3 (2 N) 4.18 4 (2 N) 4.4 4 (2 N)Thr (T) 4.8 5 6.0 6 5.0 5 5.09 5 5.1 5Ser (S) 4.9 5 5.0 5 4.2 4 5.2 5 5.2 5Pro (P) 1.9 2 1.8 2 2.2 2 2.1 2 2.0 2Gly (G) 5.9 6 6.3 6 5.3 5 6.4 6 5.0 5Ala (A) 2.0 2 2.1 2 2.0 2 2.1 2 2.9 3Cys (C) 4.8a 6b 5.8a 6b 5.2a 6b 5.2a 6b 5.2a 6b

Val (V) - - - 1.1 1 -Ile (I) 3.5 4 3.7 4 3.6 4 2.7 3 3.8 4Leu (L) 1.0 1 1.0 1 1.0 1 1.2 1 2.0 2Tyr (Y) 1.0 1 1.0 1 0.9 1 1.0 1 1.0 1Phe (F) 1.0 1 1.0 1 1.0 1 1.0 1 -His (H) 1.0 1 1.0 1 1.0 1 1.0 1Lys (K) 3.9 4 4.1 4 4.2 4 4.3 4 4.2 4Arg (R) 2.9 3 3.0 3 3.0 3 3.1 3 3.0 3Trp (W) 1.0c 1 1.0c 1 1.0c 1 1.0c 1No aa:s 46 46 41 46 46

Mass, native(MH+ measured)

4893.0 4879.3 4311.2 4879.2 4873.2

Mass, native(calculated)

4892.3 4878.3 4310.1 4878.3 4872.3

Mass, alkylated(MH+ measured)d

5235.4 5221.1 4653.3 5221.6 5215.8

Mass, alkylated(calculated)e

5234.5 5220.5 4652.2 5220.5 5214.5

a Half-cystine was determined as cysteic acid, using a separate sample following oxidation withperformic acid. b Cysteine was determined as (pyridylethyl) cysteine, following alkylation with 4-vinylpyridine. c Tryptophan was determined photometrically d Measured mass of peptide alkylated byiodoacetamide in excess. e Calculated mass of fully C-carbamidomethylated peptide.

Table 7. Masses of theoretical trypsin digests of phoratoxins B-F are compared with experimentallyobserved MS fragments. For each peptide, the calculated masses are listed to the left and the measuredmasses (MH+) to the right.

Phoratoxin B Phoratoxin C Phoratoxin D Phoratoxin E Phoratoxin FFragment1

18-28 1092.6 1093.6 1092.6 1093.6 1092.6 1093.6 1078.6 1079.6 1072.6 1073.62-10 939.4 940.4 939.4 940.3 939.4 940.3 939.4 940.4 939.4 940.311-17 883.4 884.5 883.4 884.4 883.4 884.4 883.4 884.4 883.4 884.440-46 819.3 820.3 805.3 806.3 805.3 - 819.3 820.3 819.3 820.334-39 618.4 619.4 618.4 619.4 618.4 619.4 618.4 619.4 618.4 619.429-33 507.3 508.3 507.3 508.3 507.3 508.3 507.3 508.3 507.3 508.3

1 The tryptic fragments, in order of decreasing molecular weights.


41

Figure 11. The C-terminal MS/MS spectrum of the [M+4H]4+ ion at m/z 1378.5 of the native peptide,phoratoxin C. The Y series ions Y1 through Y13 are consistent with the sequence IISGTKCDSGWTH.

Analysis of the reference materials of phoratoxins A and BAnalyses were made on the previously isolated phoratoxins A and B that had beendeponated in a deep freezer of the Division of Pharmacognosy by Eva Thunbergand Gunnar Samuelsson. According to the HPLC and MS analyses, both referencematerials contained several substances, and had very similar chromatogram/ spectra.The C-terminal of the reference substances was also examined using MS/MS. Bothphoratoxins A and B were shown to have the C-terminal sequence DSGWDH.Thunberg (1983) reported that phoratoxin A probably was a mixture of at least twopeptides based on the low value of valine obtained in the amino acid analyses.Here, the reference material of phoratoxin B was further fractionated on RP-HPLC, with a linear gradient from 20% CH3CN/ 0.2% TFA to 40% CH3CN/ 0.2%TFA. The two main peaks isolated. The native peptides (3 nmol) were subjected toreduction and alkylation with iodoacetamide. The reaction products weresequenced by automated Edman degradation, and the amino acid sequences,confirmed by sequencing using MS/MS. The results from the amino acid analysisand Edman degradation are shown in Table 8.

The molecular weights of the major peptides isolated from reference material ofphoratoxin B were determined, and found to be 4879.1 MH+ (peak 1) and 4892.7MH+ (peak 2). Sequencing by means of Edman degradation of the alkylated peak 1yielded 41 residues from the N-terminal end: KSC*C*P TTTAR NIYNT C*RFGGGSR PVC*AK LSGC*KIISGTKC*D. A tryptic fragment gave the C-terminalsequence DSGWDH at m/z 438.6 [M+2H]2+ after MS/MS analysis.

Peak 2 yielded an N-terminal sequence of 41 residues, KSC*C*PTTAR NIYNTC*RFGG GSRPI C*AKLS GC*KII SGTKC*D, by Edman degradation. The nativepeptide fragment gave the partial C-terminal sequence KIISGTKC*DSGWDH at m/z873.5 [M+6H]6+ by MS/MS analysis.


42

Table 8. The amino acid composition and molecular weights of thephoratoxin peptides isolated from the reference material of phoratoxin B.For each peptide, the residues from amino acid analysis are listed to the leftand the residues from sequencing to the right.

Amino acidPeak 1 Peak 2

Asp/Asn (D/N) 4.0 4 (2 N) 4.1 4 (2 N)Thr (T) 4.6 5 4.7 5Ser (S) 5.0 5 4.9 5Pro (P) 1.9 2 2.0 2Gly (G) 6.0 6 5.9 6Ala (A) 2.0 2 2.0 2Cys (C) 5.6a 6b 5.6a 6b

Val (V) 1.0 1 - -Ile (I) 2.6 3 3.4 4Leu (L) 1.0 1 1.0 1Tyr (Y) 0.9 1 0.9 1Phe (F) 0.9 1 1.0 1His (H) 1.1 1 1.0 1Lys (K) 4.0 4 3.9 4Arg (R) 3.0 3 2.9 3Trp (W) 1.0c 1 1.0c 1No aa:s 46 46

Mass, native(MH+ measured)

4879.1 4892.7

Mass, native(calculated)

4878.3 4892.3

Mass, alkylated(MH+ measured)d

5221.5 5236.9

Mass, alkylated(calculated)e

5221.4 5235.4

a Half-cystine was determined as cysteic acid, using a separate samplefollowing oxidation with performic acid. b Cysteine was determined ascarbamidomethyl(cysteine), following alkylation with iodoacetamide.c Tryptophan was determined photometrically d Measured mass of peptidealkylated by iodoacetamide in excess. e Calculated mass of fully C-carbamidomethylated peptide.

Peak 1 is very similar to the reported sequence of phoratoxin A, but peak 1 containsAsp instead of Asn at position 45. Peak 1 has the same sequence as phoratoxin E,which was isolated in the present study. Peak 2 has the same sequence as the previouslyreported phoratoxin B.

So in conclusion, the reference material of phoratoxin B, isolated by Thunberg (1983),was shown to be a mixture of peptides with the sequences given below in Figure12 as phoratoxin B (peak 2 of Table 8) and phoratoxin E (peak 1 of Table 8). Thereference material of phoratoxin A contained a mixture of peptides very similar tothose of the phoratoxin B reference material, according to the MS analysis. Therelative amount of peak 1 (phoratoxin E) was higher in the reference material ofphoratoxin A than in that of phoratoxin B. Phoratoxin E is probably identical tothe previously reported phoratoxin A, since the reference material of phoratoxin Acontained the same C-terminal sequence as phoratoxin E. However, this remainsunclear since the original reference material of phoratoxin A (Mellstrand andSamuelsson, 1973) is no longer available.


43

Alignment of Phoratoxins with Other ThioninsFor several members of the thionin group, amino acid sequences were aligned(Figure 12). The sequences are currently available in literature (EMBL and SWISS-PROT). The primary structures of the phoratoxins share a high degree ofhomology with the viscotoxins and the α-and β-thionins.

10 20 30 40 | | | |

Phoratoxin A KS CC PTTTA R NIYNT CR FG G GSRPV C AKLSG C KIISGTK C DSGWNH-

Phoratoxin B KS CC PTTTA R NIYNT CR FG G GSRPI C AKLSG C KIISGTK C DSGWDH-

Phoratoxin C KS CC PTTTA R NIYNT CR FG G GSRPI C AKLSG C KIISGTK C DSGWTH-

Phoratoxin D KS CC PTTTA R NIYNT CR FG G GSRPI C AKLSG C KIISGTK C D------

Phoratoxin E KS CC PTTTA R NIYNT CR FG G GSRPV C AKLSG C KIISGTK C DSGWDH-

Phoratoxin F KS CC PTTTA R NIYNT CR LA G GSRPI C AKLSG C KIISGTK C DSGWDH-

Viscotoxin A1 KS CC PSTTG R NIYNT CR LT G SSRET C AKLSG C KIISAST C PSNYPK-

Viscotoxin A3 KS CC PNTTG R NIYNT CR LT G APRPT C AKLSG C KIISGST C PS-YPDK

Viscotoxin A2 KS CC PNTTG R NIYNT CR FG G GSREV C ASLSG C KIISAST C PS-TPDK

Viscotoxin 1-Ps KS CC PNTTG R NIYNT CR FG G GSREV C ARISG C KIISAST C PS-YPDK

Viscotoxin B KS CC PNTTG R NIYNT CR LG G GSRER C ASLSG C KIISAST C PS-YPDK

Ligatoxin A KS CC PSTTA R NIYNT CR LT G TSRPT C ASLSG C KIISGST C DSGWDH-

Denclatoxin KS CC PTTAA R XXYXI CR LP G TPRPV C AALSG C KIISGTG C PPGYRH-

α1-purothionin KS CC RTTLG R NCYNL CR SR G AQK-L C STVCR C KLTSGLK C PKGFPK-

α2-purothionin KS CC RSTLG R NCYNL CR AR G AQK-L C AGVCR C KISSGLS C PKGFPK-

β-purothionin KS CC KSTLG R NCYNL CR AR G AQK-L C ANVCR C KLTSGLS C PKDFPK-

α-hordothionin KS CC RSTLG R NCYNL CR VR G AQK-L C AGVCR C KLTSSGK C PTGFPK-

β-hordothionin KS CC RSTLG R NCYNL CR VR G AQK-L C ANACR C KLTSGLK C PSSFPK-

secalethionin KS CC KSTLG R DCYDL CR GR G AEK-L C AELCR C KITSGLS C PKDFPK-

Crambin A TT CC PSIVA R SNFNV CR LP G TPEAI C ATYTG C IIIPGAT C PGDYAN-

Crambin B TT CC PSIVA R SNFNV CR LP G TSEAA C ATYTG C IIIPGAT C PGDYAN-

Figure 12. Alignment of amino acid sequences of the members of the four different types of thioninscurrently available from literature (i.e., the EMBL, and SWISS-PROT databases). The one-letter codefor amino acids is used. The sequences were derived from phoratoxin A and B (Mellstrand andSamuelsson, 1974a; Thunberg, 1983); viscotoxin A1, A2, A3, B and 1-Ps (Olson and Samuelsson,1972; Orrù et al., 1997; Samuelsson, 1973; Samuelsson and Pettersson, 1971); ligatoxin (Thunberg andSamuelsson, 1982); denclatoxin (Samuelsson and Pettersson, 1977); α1-, α2- and β-purothionin (Jonesand Mak, 1976; Mak and Jones, 1976; Ohtani et al., 1975; Ohtani et al., 1977); α- and β-hordothionin(Hernández-Lucas et al., 1986); secalethionin (Békés et al., 1982); and crambin A and B (Teeter et al.,1981; Vermeulen et al., 1987).

The neutrophil challenging activity of phoratoxinsThe phoratoxins fractions II, III, and IV from Thunberg, 1983, were tested in theneutrophil elastase release assay. The fraction V showed 48% and 38% inhibition(PAF and fMLP induced, respectively) of the pNA formation at the concentration100 µg/ml (Table 9). The isolated peptides from this fraction V (phoratoxins B, E,and F) were further evaluated in the neutrophil elastase release assay. PhoratoxinC was included for comparison.


44

Table 9. Inhibiting activity (%) of the different fractions and phoratoxin B(isolated by Thunberg, 1983) on the pNA formation in the neutrophil elastaserelease assay at a concentration of 100 µg/ml.

PAF±SEM fMLP±SEMPhoratoxin B -1,5± 9 8.2± 8Fraction II 7.2± 6 -1.7± 3Fraction IV 23± 3 22,5± 2Fraction V 48± 5 38± 7

The phoratoxins B, C, E, and F were tested in the neutrophil elastase release assayat three different concentrations. Phoratoxin D was not tested due to the limitedamount available. In this assay, the phoratoxins showed an enhanced formation ofpNA in a concentration-dependent manner as seen in Figure 13. At the highesttested concentration, 20 µM, they all increased the pNA formation in the assay.The initial observed inhibition of theformation of pNA in fraction V may becaused by the presence of severalphoratoxins. This excess of “inducers”might influence the formation of pNAby desensitising the receptors, which isoften seen for agonists (Venter, 1994),or might in some other way give falseindication of inhibition. In an earlierstudy (Mellstrand, 1974), phoratoxin Adid not inhibit the enzyme trypsin; andin this study, the phoratoxins did notinhibit the elastase. Because the closelyrelated viscotoxins affect the exocytosisof neutrophils by inducing the generationof ROS (Stein et al., 1999), the releaseof elastase from neutrophils was thereforeexpected.

0

0,1

0,2

0,3

∆ ABS

0,1 1

10

100

log conc. µM

PTX F

PTX E

PTX C

PTX B

Figure 13. Amount of pNA formation fromneutrophils in the response to phoratoxins B, C,E, and F in different concentrations. Each pointis the mean absorbance value ± SEM for fourexperiments. PTX (phoratoxin).

Parallel samples of PMNs were pretreated with phoratoxins in several concentrations,and then assayed for fMLP induced elastase release. PMNs pretreated with phoratoxinswere still able to increase pNA formation after challenge with the inducers PAF orfMLP.

The cytotoxic evaluation of the fractions and the isolated peptidesCytotoxic activity in the cell line panelThree fractions II, IV, and V, and the five isolated peptides, phoratoxins B-F, fromP. tomentosum, induced a concentration-dependent decrease in cell viability. TheIC50-values for the three fractions ranged from 0.43 µg/ml to 41.4 µg/ml (Table10), with fraction II being the least potent (IC50 ranging from 2.2 to 41.4 µg/ml)and fraction IV (IC50 ranging from 0.43 to 3.9 µg/ml) being the most potent. Fromfraction IV, phoratoxins C and D were isolated; and from fraction V, phoratoxinsB, E, and F were isolated. The IC50-values for these isolated mistletoe toxins(Table 10) ranged from 0.038-0.83 µM where phoratoxin C (mean IC50: 0.16 µM),


45

being the most potent, and phoratoxin F (mean IC50: 0.4 µM), the least. In thesmall-cell lung cancer cell lines NCI-H69 and NCI H69AR, the phoratoxins wereequipotent to each other. The phoratoxins showed high potency towards the cellline NCI-H69, whereas the potency for NCI-H69AR was much lower.

Table 10. IC50 values in the cell line panel for the fractions and the isolated peptides.

Cell line

Fraction IIµg/ml

Fraction IVµg/ml

Fraction Vµg/ml

PTX BµM

PTX CµM

PTX DµM

PTX EµM

PTX FµM

RPMI 8226-S 19.0 1.7 3.6 0.35 0.18 0.27 0.43 0.45RPMI 8226- LR5 8.4 1.0 2.2 0.30 0.18 0.27 0.38 0.36RPMI 8226-Dox 40 41.4 3.9 7.6 0.37 0.13 0.23 0.43 0.51U-937 GTB 9.8 1.4 2.2 0.21 0.092 0.15 0.35 0.31U-937 Vcr 8.2 1.0 2.1 0.18 0.10 0.14 0.29 0.23NCI-H69 2.2 0.43 1.8 0.038 0.040 0.045 0.038 0.038NCI- H69AR 10.9 2.3 2.9 0.23 0.23 0.24 0.26 0.30CCRF-CEM 14.6 2.1 4.6 0.33 0.20 0.38 0.45 0.50CEM-VM-1 12.0 2.0 3.1 0.32 0.15 0.22 0.34 0.42ACHN 41.1 3.1 7.5 0.59 0.34 0.64 0.58 0.83mean 16.8 1.9 3.8 0.29 0.16 0.26 0.36 0.40

Abbreviations: PTX (phoratoxin).

Correlation analysis of the phoratoxinsThe fractions and the isolated peptides were tested in the cell line panel andcompared with one another (Table 11, part a). Correlations between isolatedpeptide with isolated peptide were high (0.82-0.99), whereas correlations betweenfraction and peptide were moderately high (0.55-0.86). The highest correlationwas between phoratoxin B and phoratoxin F (0.99), and the lowest correlation wasbetween phoratoxin E and the fraction V, from which it was isolated (0.55). Thisshows that the correlation between fraction and pure substances is lower whenmany compounds are present in the fraction. Fraction V, which contained threepeptides, was shown to exhibit the lowest correlation to the isolated peptides(0.55-0.69), whereas fraction IV (containing two peptides) was shown to have aslightly higher correlation with the pure peptides (0.73-0.85). This may complicatethe predication of a known resistance mechanism during the isolation procedure.However, the pure substance will show if a known resistance mechanism isinvolved (Dhar et al., 1996). The correlation of the phoratoxins with standarddrugs (Table 11, part b) was low (R<0.35). Further, the 10 highest correlations (R)with cytotoxic standard drugs and experimental agents with known targets,previously tested in the cell line panel, were ranked (Table VI, Paper III).Surprisingly, digitoxin gave overall the highest correlation to the phoratoxins.

Resistance factors in the mechanism-based cell line evaluationThe resistance factors (RFs) were calculated for the Pgp, topo II, MRP, GSH, andtubulin resistance mechanisms (Table 12). The isolated phoratoxins were assignedthe highest RF values (ranging from 5.3 to 7.8) for the MRP-associatedmechanism, whereas the fraction V obtain the lowest value (1.6). The overall lowRFs for the other resistance mechanisms examined (<3) indicate minimaldependence on these mechanisms.


46

Table 11. Correlation coefficients (R), obtained in the cell line panel, for analysing relationshipsof the log IC50 values of the fractions and the isolated phoratoxins (a) among themselves and (b)with standard drugs.

PTX B PTX C PTX D PTX E PTX F Fraction IV Fraction VaPTXB 1.00 0.88 0.94 0.97 0.99 0.85 0.69PTXC 0.88 1.00 0.96 0.82 0.87 0.73 0.56PTXD 0.94 0.96 1.00 0.89 0.94 0.77 0.64PTXE 0.97 0.82 0.89 1.00 0.98 0.78 0.55PTXF 0.99 0.87 0.94 0.98 1.00 0.85 0.68Fraction IV 0.85 0.73 0.77 0.78 0.85 1.00 0.86Fraction V 0.69 0.56 0.64 0.55 0.68 0.86 1.00bDoxorubicin 0.11 0.34 0.28 -0.02 0.09 0.39 0.54Vincristine -0.26 0.03 -0.10 -0.41 -0.29 0.01 0.17Cytarabine -0.07 0.10 0.01 -0.18 -0.12 -0.01 0.20Melphalan -0.01 0.18 0.05 -0.07 -0.05 0.01 0.01Topetecan 0.00 0.32 0.15 -0.14 -0.04 0.20 0.26

Abbreviations: PTX (phoratoxin).

Table 12. Mechanistic resistance factors (RFs) for the fractions and the isolated phoratoxins.

Fraction II Fraction IV Fraction V PTX B PTX C PTX D PTX E PTX FP-pg 2.2 2.4 2.1 1.1 0.7 0.8 1.0 1.1GSH-associated 0.4 0.6 0.6 0.8 1.0 1.0 0.8 0.8Topo II- associated 1.2 1.1 1.5 1.0 1.3 1.8 1.3 1.2Tubulin 0.8 0.7 1.0 0.8 1.1 1.0 0.8 0.7MRP 4.9 5.4 1.6 6.1 5.7 5.3 7.0 7.8Resistance factor = (IC50 resistant cell line)/ (IC50 parental cell line). Abbreviations: PTX(phoratoxin), P-gp (P-glycoprotein), GSH (glutathione), topo II (topoisomerase II), MRP(multidrug-resistance associated protein).

The activity in primary tumour cells from patientsThe IC50 values obtained in primary tumour cells ranged from 0.58 to 38.2 µg/mlfor the fractions (Table 13). As in the cell line panel, fraction IV was the mostpotent fraction, and fraction II, the least potent.

Table 13. Comparison of the IC50-values (µg/ml) of the fractions for tumourcells from patients.

PBMC CLL BCRatio

CLL/BCRatio

PBMC/CLLFraction II µg/ml 25.5 38.2 4.4 8.7 0.66Fraction IV µg/ml 4.5 4.4 0.58 7.6 1.0Fraction V µg/ml 7.3 4.8 2.2 2.2 1.5

PBMC (peripheral blood mononuclear cells), CLL (chronic lymphocytic leukaemia),BC (breast carcinoma).

The activity of phoratoxin C was further characterised in 25 human tumoursamples, 16 solid and 9 haematological. The IC50-values of phoratoxin C (TableVIII, Paper III) ranged from 87 nM to 2.1 µM. Interestingly, phoratoxin C wasassigned the highest activity in the four breast-carcinoma samples with an IC50 of87 nM. Phoratoxin C was selectively toxic to the solid tumour samples relative tohaematological tumour samples, shown in Table VIII by the ratio H/S of the meanIC50 value for the haematological tumours and that mean for the solid tumours


47

(H/S=2.8). Phoratoxin C was pronouncedly selectively toxic to the solid tumoursamples from breast carcinoma (BC) relative to the haematological (CLL) tumoursamples (CLL/BC=18.4), and selectively toxic to the breast cancer cells relative tothe normal peripheral blood mononuclear cells (PBMCs) (PBMC/BC=8.0).

Table 14. Comparison of the IC50-values (µM) of phoratoxin C and D for tumour cells frompatients.

PBMC CLL BC OvcaRatioCLL/BC

RatioCLL/Ovca

RatioPBMC/CLL

PTX C µM 0.7 (n=4) 1.6 (n=5) 0.087 (n=4) 0.3 (n=5) 18.4 5.3 0.44PTX D µM 0.7 (n=2) 0.86 (n=2) 0.36 (n=2) 0.56 (n=3) 2.4 1.5 0.81Abbreviations: PTX (phoratoxin), PBMC (peripheral blood mononuclear cells), CLL (chroniclymphocytic leukaemia), BC (breast carcinoma), Ovca (ovarian carcinoma).

Phoratoxin D was preliminarily tested against some of the tumour samples (Table14). Comparison of the IC50s (IC50 values) for phoratoxin C and D shows that theyare equipotent against PBMC. For the tumour cells, phoratoxin D is twice aspotent as phoratoxins C against CLL, whereas in reverse, phoratoxin C is twice aspotent against ovarian carcinoma, and four times as potent against breastcarcinoma. Comparing the ratios CLL/BC, CLL/Ovca, and PBMC/CLL,phoratoxin C was more selectivity toxic to solid tumour patient samples than wasphoratoxin D. The ratio CLL/BC is 18.4 for phoratoxin C and 2.4 for phoratoxinD. Phoratoxin C and D consist of 46 and 41 amino acid residues. Except forhaving a longer sequence, the amino acid sequence for phoratoxin D is the same asthat for phoratoxin C, all the way through amino acid residue 41. This indicatesthat the C-terminal sequence is important to the higher selectivity observed forphoratoxin C. However, this should be further investigated.

Comparison with the other 46 amino-acid-residue phoratoxins tested in the cellline panel, phoratoxin C is the only one that differed in the C-terminal sequence byhaving a Thr at position 45 instead of an Asp. Whether or not the Thr45 wasimportant for the higher selectivity against the solid tumour cells remains unclear.For pharmacological activity, the loop in the amino acid sequence formed by theCys16–Cys26 bridge is important. By modifying the conserved arginine residuesat positions 17 and 23 within this loop, Thunberg and Samuelsson (1983)(Thunberg et al., 1983) showed that these arginines are essential to the toxicity ofthe phoratoxins. Within this loop, phoratoxin F has the amino acid residues Leu18and Ala19, whereas phoratoxins B-E have Phe18 and Gly19. In the cell line panel,potencies of the phoratoxins vary only little, but may still vary considerably inselectivity, as seen for phoratoxin D compared to phoratoxin C. However, this alsomust be further investigated.


48

Digitoxin and Related Cardiac Glycosides

Cardiac glycosidesLong ago William Withering (1741-1799) recognised the usefulness of foxglove(Digitalis) extracts, particularly for the treatment of congestive heart failure. But,perhaps less well-known and surprisingly, the drug prior to Withering wasgenerally applied by inunction as a plaster or ointment in treating headaches andswellings, as well as cancerous skin conditions (Groves and Bisset, 1991).

Cardenolides and bufadienolides are regarded cardiac glycosides because bothincrease the contractile force of the heart by inhibiting the enzyme Na+, K+-ATPase.The molecular basis for the increased force of contraction is largely due to anincrease in cytosolic Ca2+ during systole, which increases the velocity and extentof muscle shortening. The toxicity associated with cardiac glycosides may resultfrom excessive intracellular Ca2+ that causes a transient late depolarisation followedby an after contraction. Both cardenolides and bufadienolides have previouslybeen tested for cytotoxicity in a variety of tumour models (Cassady et al., 1981;Hembree et al., 1979; Inada et al., 1993; Kelly et al., 1965; Koike et al., 1980;Repke et al., 1995; Shiratori, 1967).

A bioassay guided isolation of digitoxin from Digitalis purpureaExamination of the fractionated plant extracts of Digitalis lanata, Digitalispurpurea, and Viola patrinii (Figure 7, Paper II) revealed an unusual and unexpectedeffect of increased formation of pNA, as mentioned in chapter 6 and described inPaper II. For further investigation of this effect, D. purpurea was chosen due to thelimited amount of V. patrinii and D. lanata available. The fraction P of D. purpureawas dissolved in a mobile phase of aqueous 25% CH3CN and 0.1% TFA, and thenwas fractionated on a semipreparative HPLC system as described in Paper IV. Thecolumn was eluted for 30 minutes using a linear gradient from 25% CH3CN/0.1%TFA to 75% CH3CN/0.1% TFA. Ten fractions were collected and tested in eightconcentrations in the neutrophil elastase release assay. Two of the fractions, F6 andF8, were capable of enhancing the formation of pNA, as seen in Figure 14, whichalso highlights the importance of testing in several concentrations in the bioassay-guided isolation procedure. At the highest tested concentration (100 µg/ml), F6 wasthe most active compound. Testing at several concentrations, F8 was the most potentfraction, and was then further purified by HPLC. The most potent fraction, whichwas homogeneous, was unambiguously identified as the cardiac glycoside digitoxin,using spectroscopic methods. The 1H NMR spectrum (recorded at 600 MHz) andmass spectrum (obtained by the Electro Spray Ionization technique) were inagreement with spectrums of an authentic sample of digitoxin (Sigma).

The neutrophil challenging activity of digitoxinDigitoxin’s concentration-response for enhancement of pNA formation was bellshaped (Figure 15). In the assay, the maximal pNA-formation enhancement byisolated digitoxin was lower than that of the inducers PAF, fMLP, and the originalextract of D. purpurea. The substantial enhancement by the fraction P was possibly asynergistic effect involving more than one cardiac glycoside.


49

-0,25

0

0,25

0,5

0,75

1

∆ ABS

0,00

1

0,01 0,

1 1

10

100

log conc. µg/ml

F8

F6

Figure 14. The fraction P of Digitalis purpureawas separated into ten fractions F1-F10 on a RP-column. Each fraction was tested for activity inseveral concentrations of neutrophil elastaserelease. Of these ten fractions, two showed a dose-dependent activity, F6 (n) and F8 (¡). FractionF8 was shown to be the most potent fraction in theelastase release. Digitoxin, the most potentcompound in the elastase release, was laterisolated from F8.

0

0,05

0,1

0,15

0,2

∆ ABS

0,1 1

10

100

log conc. µM

Digitoxin

Figure 15. Amount of pNA formation fromneutrophils treated with several concentrationsof the isolated digitoxin. Each point is the meanabsorbance value ± SEM of three independentexperiments.

The cytotoxic evaluation of digitoxin and related compoundsCytotoxic activity in the cell line panelThe fractions P of D. lanata and D. purpurea were tested in the cell line panel. TheIC50 values for these fractions ranged from 1.1 to 5.8µg/ml (D. lanata) and from 0.63to 1.5 µg/ml (D. purpurea). D. purpurea was further separated into two neutrophilchallenging fractions, F6 and F8, which were also tested in the cell line panel. Thepotencies for these fractions were higher than the potency of fraction P. The IC50

values for the fraction F6 ranged from 0.20 to 1.4 g/ml; and for fraction F8, from0.20 to 0.81 µg/ml. Digitoxin was isolated from the fraction F8.

The unexpected finding that digitoxin expressed potent cytotoxicity prompted usto investigate the structure-activity relationship of cardiac glycosides. For this, thecytotoxicities of digitoxin and six related compounds were compared. All seven ofthe related compounds (Figure 1, Paper IV)—five cardenolides (digoxin,digitoxin, lanatoside C, ouabain and digitoxigenin), one bufadienolide(proscillaridin A), and one saponin (digitonin)—induced a dose-dependentdecrease in cell viability. The IC50 values for these compounds (Table 15) rangedfrom 6.4 to 7000 nM, with digitonin, (IC50 ranging from 1070 to 7000 nM; Table15) being the least potent, and proscillaridin A (IC50 values ranging from 6.4 to 76nM), the most potent. These results agree with literature data showing that thecytotoxic activity of cardenolides is generally weaker than the correspondingbufadienolides having the same steroidal skeletons (Kamano et al., 1998).


50

Table 15. IC50 -values (nM) of cardiac glycosides and digitonin for the human tumour cell line panel.

Cell lineDigitoxin Digitoxigenin Digoxin Lanatoside C Ouabain Proscillaridin A Digitonin

mean IC50

cell line

RPMI 8226-S 34 242 84 220 73 13 1280 278RPMI 8226-LR5 25 201 64 150 57 15 1310 260RPMI8226-Dox 40 59 395 172 339 148 20 1070 315U-937 GTB 32 251 68 142 66 6.4 1880 349U-937 Vcr 36 344 74 133 63 6.4 1100 251NCI-H69 12 635 56 137 70 66 2600 511NCI-H69AR 31 200 41 154 45 10 4730 744CCRF-CEM 25 283 49 127 57 <6.4* 2650 457CCRF-VM-1 40 441 63 190 74 9 4430 750ACHN 76 658 125 602 126 76 7000 1237

mean IC50

compounds37 365 80 219 78 23 2805

*Since the IC50 value is below the tested concentrations, the lowest tested concentration is used(Boyd and Paull, 1995). Each IC50 value is calculated from the mean of 2-3 experiments, allperformed in triplicate.

In most cases, the order of potency (high to low) of these substances on the cellline panel was proscillaridin A, digitoxin, ouabain, digoxin, lanatoside C,digitoxigenin, and digitonin. An exception to this was observed in the cell lineNCI-H69, where digitoxin was more potent than proscillaridin A. In the renaladenocarcinoma cell line ACHN, digitoxin was equipotent with proscillaridin A.Also, in the cell lines NCI-H69, CCRF-CEM, and its sublines H69AR and CEM-VM-1, digoxin showed a lower IC50 value than that of ouabain. The order ofcytotoxic potency of the cardiac glycosides found in this study virtually parallelsthe inhibitory potency of the cardiac glycosides on the Na+/K+- transportingATPase from human cardiac muscle from data in the literature (Schönfeld et al.,1986). The cytotoxic concentrations are higher than the concentrations required toinhibit Na+/K+-ATPase activity in human cardiac muscle; but for digitoxin, theaverage IC50 (37 nM; Table 15) is still within the therapeutic concentration usedfor cardiac congestion (13-45 nM) (Gilman et al., 1985). For the bufadienolideproscillaridin A, which was found to be of even higher potency (average IC50 23nM), the cytotoxic concentration is not within the range of therapeutic plasmaconcentration (0.9-1.1 nM) used for this drug in the treatment of heart congestion.

Correlation analysis of the cardiac glycosidesThe correlation analysis of the seven natural compounds with one another (Table3, part a, Paper IV) showed that the correlation of digitoxin with digoxin, ouabain,and lanatoside C was moderately high, whereas correlation with digitoxigenin,proscillaridin A, and digitonin was much lower. The highest correlation wasbetween digoxin and ouabain (0.946). The correlation of those natural compoundswith standard drugs (Table 3, part b, Paper IV) was very low to moderately high(–0.3 to 0.7). Digitoxin and digoxin were less correlated with standard drugs thanproscillaridin A. The low correlations to standard drugs in our database (Dhar etal., 1996) support the idea that the cardiac glycosides may act by cytotoxicmechanisms other than those of standard drugs.

Resistance factors of the cardiac glycosides in the mechanism-based cell line evaluationThe RFs were calculated for the P-gp, topo II, MRP, GSH, and tubulin resistancemechanisms (Table 4, Paper IV). The overall low RFs for the substances (<3)


51

indicate minimal dependence on the resistance mechanisms examined here.Proscillaridin A was more active against the MRP-expressing resistant sublineNCI-H69AR than to its parental cell line NCI-H69 (RF=0.15).

The structure-activity relationship for the digitoxin and related cardiac glycosidesThe structure activity relationship was investigated for digitoxin and the relatedcardiac glycosides. Digitoxigenin is the aglycone of digitoxin. This structuraldifference caused marked differences in the cytotoxicity patterns of digitoxin anddigitoxigenin. Virtually no significant correlations were found between the activityprofiles of these two compounds. Moreover, for the cell line panel tests,digitoxin’s additional three digitoxose sugar units increased its mean potency 6.5to 50 times compared to that of digitoxigenin, suggesting that an intact glycosideis essential for potent cytotoxic activity. The correlation of digitoxin with otherintact cardenolides (i.e., digoxin, ouabain, and lanatoside C) was moderately high,which may indicate (as might be expected) a similar mode of action for thesecompounds. The highest correlation occurred for digoxin and ouabain. Only lowcorrelation of the activity patterns of digitoxin with that of proscillaridin A,digitoxigenin, and digitonin was found.

Lanatoside C and digoxin are structurally closely related in that digoxin can beobtained from lanatoside C by hydrolytic removal of the acetyl and glucosemoieties. This structural difference in the sugar part of the compounds wasmanifested in a three–times-higher mean potency of digoxin compared tolanatoside C, approximately.

The activity of the cardiac glycosides in primary tumour cells from patientsThe cardiac glycosides were tested against tumour cells from patients. The IC50

values obtained in primary tumour cells ranged from 17 to 7050 nM (Table 16).As in the cell line panel, proscillaridin A was the most potent compound, anddigitonin, the least potent. The ratios between the haematological and the solidtumour samples (CLL/Ovca; CLL/BC) were calculated (Table 16). In most cases,the solid tumour samples (Ovca, BC) were more sensitive than the haematological(CLL) sample, which in turn was, in several cases, more sensitive than PBMC, itsnormal counterpart. The solid tumour toxicity was most pronounced for digitoxinand digoxin, while proscillaridin A, lanatoside C, and digitoxigenin showed noselectivity for haematological or solid tumour samples.

Table 16. Comparison of the IC50 values (nM) of cardiac glycosides and digitonin for tumourcells from patients.

Digitoxin Digitoxigenin Digoxin Lanatoside C Ouabain Proscillaridin A DigitoninPatient samplesPBMC (n=3) 106 1000 327 740 277 26 4640Ovca (n=2) 55 339 232 335 229 17 2637BC (n=2) 65 489 127 316 147 21 4786CLL (n=3) 150 527 520 424 358 18 7050RatioPBMC/CLL 0.71 1.90 0.63 1.75 0.77 1.44 0.66CLL/Ovca 2.73 1.55 2.24 1.27 1.56 1.06 2.67CLL/BC 2.31 1.08 4.09 1.34 2.44 0.86 1.47PBMC (peripheral blood mononuclear cells), Ovca (ovarian carcinoma), BC (breast carcinoma), CLL(chronic lymphocytic leukaemia). Each IC50 value is calculated from the mean of 2-3 experiments, allperformed in triplicate.


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Early epidemiological studies show that congestive heart-failure patients who receivecardiac glycosides have a tendency to develop breast tumours of lower growthpotential than untreated patients (Stenkvist et al., 1980; Stenkvist et al., 1979). Theobserved activity is proposed to be associated with an oestrogen-like effect (Stenkvist,1999; Stenkvist et al., 1982; Stenkvist et al., 1980; Stenkvist et al., 1979) of cardiacglycosides. This hypothesis conflicts with our result in which cardiac glycosidesinhibited the growth not only of the cells of breast carcinoma, but of all tumourcells tested (Table 15 and 16). Other papers also report (Cove and Barker, 1979;Falconer et al., 1983) that it seems unlikely that oestrogen-like properties ofdigoxin are of major importance in the growth suppression of breast tumours.Digitoxin has also recently been shown to induce apoptosis (Haux et al., 1999a),and to inhibit prostate (Haux et al., 2000) and breast cancer cell lines in therapeuticconcentrations used in the treatment of heart congestion (Haux et al., 1999a).Recent data on the potential of cardiac glycosides as anticancer agents (Haux,1999; Haux et al., 1999a; Haux et al., 1999b; Haux et al., 2000; Stenkvist, 1999) showthat digitoxin inhibits proliferation and induces apoptosis in various cell lines. Inanother recent study, the cardiac glycosides digoxin, ouabain, and oleandrin wereshown to induce apoptosis in metastatic human prostate adenocarcinoma cells, theeffect of which was linked to their abilities to induce sustained Ca2+ increases inthe cells (McConkey et al., 2000).

Summary and concluding discussion

53

8. Summary and Concluding Discussion

The fractionation protocolWhen this study was initiated, the overall aim was to develop methods for isolationand characterisation of biologically active polypeptides from plant material. Thefractionation protocol was developed to detect biologically active polypeptidespresent in small amounts in plants, and was validated by the isolation of themacrocyclic varv peptide A. Today, we have isolated varv peptides B-H fromViola arvensis (Göransson et al., 1999), and recently Craik et al., (1999) reportedthe discovery of several peptides from Viola odorata, Viola hederaceae, andOldenlandia affinis. There is approximately 40% sequence conservationthroughout the known sequences.

The bioassay-guided isolation of the cardiac glycoside digitoxin (765 Da) showedthat some modifications of the fractionation protocol would be advantageous.Further complementary analysis (e.g., LC-MS) and chromatographic mechanisms(e.g., Sephadex LH-20, cation exchange chromatography) may be included in theisolation of peptides to avoid the isolation of other high molecular weightsubstances (>700 Da) with both polar and lipophilic characters that are able toelute in the void volume in the gel filtration step with Sephadex G-10 .

A multitarget bioassay on the neutrophilThe utility of a neutrophil multitarget functional bioassay may provide importanthints on how substances from plants interact with the neutrophil. From the assay, itis possible to make several predictions. First, an inhibition at the receptor levelwill be by different biological responses in the elastase release, if differentinducers are used. In that case, receptor inhibition may be worth investigating.Second, if non-selective inhibition is observed, enzyme inhibition can be investigated.Last, if a stimulating activity is observed, receptor binding studies or functionalpharmacological experiments with various known receptor binding antagonists canbe performed. Thus, a multitarget bioassay might provide opportunities to findnew agents, with known as well as new mechanisms of action, involved inneutrophil interaction.

Biological activity linked to phylogenetic informationAlthough screening of natural products remains a major method of discoveringnew drugs, newer techniques of rational drug design, computer-aided drug design,and combinatorial synthesis promise to broaden the scope of compounds availablefor screening. To address this, the results were presented in a manner that linksrecorded biological activity to phylogenetic information. With increasedacceptance that the distribution of chemical substances and substance classes inplants is not purely random, a logical consequence is that, in order to efficientlyexplore the chemical diversity of plants, one must also explore the biological andevolutionary diversity. Furthermore, the results will be useful if sampling isexpanded, in order, for example, to select groups of plants with comparably goodpotential for the discovery of additional active substances (e.g., macrocyclicpeptides), or to select groups of plants that have not been fully investigated.


54

The activity of fraction PThere has been growing interest in isolation and characterisation of properties ofleukocyte elastase inhibitors because the inhibitors may be used as therapeuticagents to prevent chronic inflammatory diseases (Barnes, 1999). Plants are knownto produce protease inhibitors in their defence against pathogens, and may thereforeserve as an important source of protease inhibitors. In this study, the fractionationprotocol was applied to 100 plant materials, which were tested in a neutrophilmultitarget functional bioassay. Of these 100 polypeptide fractions (fraction P),41% showed more than 60 % inhibition, and most of them were shown to be aform of inhibition on the enzyme elastase.

The fraction P of Crambe maritima (Brassicaceae), Momordica charantia(Curcurbitaceae), and Viscum heyneanum (Santalaceae) showed high activity inthe neutrophil elastase release assay. M. charantia and V. heyneanum also highlyinhibited isolated elastase, 85% and 99% respectively. These polypeptide fractionsmost likely contain peptides. For example, the thionin crambin has been isolatedfrom another species in Brassicaceae, Crambe abyssinica. The trypsin inhibitors(MCTI) and elastase inhibitors (MCEI) have been isolated from M. charantia(Hamato et al., 1995; Hara et al., 1989). Recently, two new macrocyclic peptidesderived from Momordica cochinchinensis have been reported (Felizmenio-Quimioet al., 2001; Hernandez et al., 2000), MCoTI-I and MCoTI-II, which are trypsininhibitors comprising 34 amino acids. This suggests that, besides linear peptides,macrocyclic peptides are also common in plants. The extensive data set for theneutrophil elastase release assay, and especially for inhibition of isolated elastase,shows that plants may be an important source of new protease inhibitors.

Further characterisation of phoratoxins and digitoxin, biological and chemicalThe mistletoes are known to produce peptides. In the neutrophil elastase releaseassay, Viscum heyneanum and Phoradendron tomentosum were tested, and shownto inhibit the formation of pNA. In previous research within this department,mistletoe peptides have been isolated, including isolation of phoratoxins A and Bfrom Phoradendron tomentosum. In this study we reinforced and extended thisprevious work (Lecomte et al., 1987; Mellstrand, 1974a; Mellstrand, 1974b;Mellstrand, 1974c; Mellstrand and Samuelsson, 1973; Mellstrand and Samuelsson,1974a; Mellstrand and Samuelsson, 1974b; Rosell and Samuelsson, 1966; Sauviat,1990; Thunberg et al., 1983) on peptides from Phoradendron tomentosum.Methods for isolation of peptides have much improved during the past years. Inthis study, the structurally closely related phoratoxins, in some cases only differingin one amino acid, were isolated. The isolated phoratoxins B-F were all basic,containing 6 cysteine residues; and except phoratoxin D, all were shown to have46 amino acids. Phoratoxin D, which had 41 amino acids, might be an artifact or atruncated peptide.

Cytotoxic and neutrophil challenging activitySerendipitous discoveries have always played an important role in science,especially in the search for new drugs or new targets (Kaul, 1998; Kubinyi, 1999;Mueller and Scheidt, 1994), and recently played a role in the followingdiscoveries. By bioassay-guided fractionation, the cardiac glycoside digitoxin wasisolated as the most potent compound, both in cytotoxicity and in neutrophil


55

challenging activity. Digitoxin was shown to be cytotoxic in therapeuticconcentrations used for cardiac congestion (13-45 nM) (Gilman et al., 1985).Phoratoxins are also known to be cardiac active (Rosell and Samuelsson, 1966),and excessive intracellular Ca2+ is known to have a potential role for both cardiacglycosides and thionins cytotoxicity (Beeler, 1977; Evans et al., 1989). Interestingly,when a database of drug response patterns of more than 100 additionalinvestigational agents was searched for similarities with the phoratoxins, digitoxin,had the highest correlation coefficients. Notably, in this respect, the unusualstimulatory effects of phoratoxins on granulocyte exocytosis are found as well forthe cardiac glycoside digitoxin. Both digitoxin and the phoratoxin C were hereshown to act selectively against solid breast carcinoma cells from patients, byratios 2.3 and 18, respectively, and thus to be less toxic to the normal peripheralblood mononuclear cells (PBMCs) than to the breast cancer cells.

The discussion above raises the question of whether the observed effect on theneutrophil could be a cytotoxic activity. Necrotic neutrophils release elastaseextracellulary while apoptotic cells retain the toxic products within the granules.Further, neutrophils undergoing cell death are incapable of responding to furtherstimuli (Savill, 1997; Savill et al., 1993; Savill and Haslett, 1995). However, in thisstudy, neutrophils pretreated with digitoxin or phoratoxins were shown to be stillcapable of responding when the inducer fMLP was added. In contrast, theneutrophils pretreated instead with digitonin, which is known to lyse membranes,were shown to be unable to respond to the added inducers fMLP, suggesting thatcardiac glycosides possess a mechanism of action other than just the cell-membrane lysing effect of digitonin. In addition, there is a large difference in theincubation time between the neutrophil elastase release assay (15 min) and thecytotoxic assay (72 hour).

As mentioned above, phoratoxin C (Paper III) and digitoxin (Paper IV) were shown toact selectively against solid breast carcinoma. This observation is encouraging,since drugs with high solid tumour activity are rare (Fridborg et al., 1996; Larsson etal., 1994; Nygren et al., 1994). The main reason for the failure of cytotoxic therapies istheir insufficient selectivity for tumours. For example, treatments with radiation oralkylating agents perturb many functions that are common to all cells. The moreselective cytotoxic drugs, for instance, methotrexate, taxol, and etoposide, perturb thefunctions of specific macromolecular targets (dihydrofolate reductase, microtubules,and topoisomerase II), but these targets are present in both normal and malignantcells (Gilman et al., 1985). In our cytotoxic assay, established chemotherapeuticagents are generally more active against haematological compared to solid tumours(Csoka et al., 1995a; Csoka et al., 1995b; Dhar et al., 1998; Nygren et al., 1994).Most standard and experimental agents show a ratio well below unity. For example,one FMCA-based study with eighteen clinically used cytotoxic drugs showed thatamong those drugs, only cisplatinum reached a ratio >1 (approx 1.3) of responders,solid relative to haematological tumors in vitro (Fridborg et al., 1999). Furthermore,this estimate was shown to positively correlate with solid tumour activity. Theobserved selectivity ratios, far above 1 for digitoxin and phoratoxin C, are notable.If this antitumour activity can be translated into the in vivo situation, thesecompounds may serve as lead prototypes for developing new classes of anticanceragents with improved activity against solid tumour malignancies.

Concluding remarks

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9. Concluding Remarks

The recent field of innate immunity shows that nature, despite its enormousdiversity of species, very often uses the same basic principles for solving relatedproblems in defence against micro-organisms. Certainly, plants and mammals areorganised very differently for carrying out their essential functions, but manysimilarities exist in the way they resist attacks by micro-organisms.

A variety of bioactive secondary compounds, as well as defensive proteins, areusually considered to be a plant’s front line defence against all but the mostspecialised herbivores and pathogens. These products, which are directly toxic tothe consumer, serve as antiherbivore or antimicrobial defence compounds. Thesesubstances include the cardiac glycosides, saponins, protease inhibitors, cyclicpeptides, and thionins (Howe and Westley, 1988; Ryan et al., 1993; Ryan, 2000;Tam et al., 1999; Wink et al., 1993). In human, the neutrophils are considered tobe the front line in defence against the micro-organisms by releasing differenttoxic products. In this thesis, a large number of plant extracts, including those ofDigitalis purpurea and Phoradendron tomentosum, are shown to interact with theneutrophil.

Digitoxin and the phoratoxins induced the exocytotic release of elastase from theneutrophils, and most likely the release of other antimicrobial substances as well.Beside the exocytosis inducing effect, these natural products have been shown tohave cytotoxic action on cancer cells. The cytotoxic mechanism of the isolatedneutrophil stimulating substances in this study is unclear.

Digitoxin is cytotoxic in vitro at a therapeutic plasma concentration used forcardiac congestion, a concentration that seems to produce no bad side effects inpersons without cardiac disease (Grossmann et al., 1998). For the final answer asto whether digitoxin has a place in medical oncology, clinical studies must bedone. Because the safety profile of the drug is well known, the threshold forimplementing new evaluations of the drug should be low.

Future perspectivesThis thesis has shown that substances involved in plant defence may be potentiallyuseful in the development of new drugs in the treatment of inflammatory diseasesand cancer. To conclude this thesis, I propose as future studies the following:

• to further fractionate plant extracts, which were shown to have high inhibition onthe neutrophil elastase release assay (This would probably results in isolation ofprotease inhibitors. However, some modifications in the isolation procedure maybe necessary.)

• to further investigate the defensins in the neutrophils (The preliminary resultindicates a large number of peptides in the neutrophils, and so far only four humanneutrophil defensins have been reported in the literature.)

Concluding remarks

57

• to further investigate the fraction P of Viola patrinii, which belongs to the familyViolaceae that has been shown to contain a large number of macrocyclic peptides(The fraction P of Viola patrinii was shown to induce the release of elastase.)

• to investigate the mechanism behind the neutrophil challenging activity ofphoratoxins and digitoxin

• to investigate if there is a relationship between the neutrophil elastase releasingproperties and the cytotoxic activity

• to further investigate the mechanism of action for phoratoxins cytotoxic activity,and to investigate if the phoratoxins are capable of inducing the release ofdefensins from neutrophils, and whether this release differs from fMLP or PAF-induced exocytosis

• to further investigate the clinical potential of phoratoxins

• to investigate digitoxin in clinical studies on patients with breast cancer

Acknowledgement

58

10. Acknowledgement

The present work was carried out mainly at the Division of Pharmacognosy,Department of Medicinal Chemistry, Faculty of Pharmacy, Uppsala University,Sweden.

This thesis has only one author listed, but behind it stand a number of wonderfulpeople, without whom this would not have been possible, therefore

I wish to express my deepest gratitude to:

Associate Professor Per Claeson, my supervisor, for valuable discussions, and hissupport, and for sharing his great knowledge with me;

Professor Lars Bohlin, for valuable discussions, encouragement, and unfailingsupport;

Professor Gunnar Samuelsson and Dr Eva Thunberg for making available thefractions and reference materials of phoratoxins;

Dr Teus Luijendijk for excellent collaboration, and for his knowledge andfriendship;

Professor Rolf Larsson and Mr Joachim Gullbo at the Clinical Pharmacology,Institution of Medical Science, for valuable and fruitful discussions in thecytotoxic experiments;

Dr Bo Ek and Dr Åke Engström for fruitful discussions about MS analyses andEdman degradation;

Dr Håkan Tunón , for being my first supervisor at the department during my yearas a student, and for introducing me to the neutrophil assay, and for many goodlaughs during coffee breaks;

Dr Paul Johnson for excellent linguistic revision and his positive and supportivemanner;

Professor Finn Sandberg for his steadfast interest in the ongoing research projects;

My co-authors, for a fruitful collaboration;

Special thanks to Mr Ulf Göransson for being a very good friend and office mate,and for sharing a lot of interesting scientific as well as non-scientific discussions,and many good laughs through the years. Keep up the good spirit!

Acknowledgement

59

The incredible PhD student group at the department, Mr Ulf Göransson, MsTherese Ringbom, Mrs Petra Lindholm, Ms Erika Svangård, Ms Ulrika Huss, MrSonny Larsson, Mr Wimal Pathmasiri, and Mr Martin Sjögren for friendshipduring good and bad times, laughs, jokes, parties, trips, coffee breaks, club nightsat club ASA and scientific discussions. Extra thanks to Camilla, Magnus, Johnny,Micke, and Anders who has reminded us about life outside the department;

All my friends during the years at the division of Pharmacognosy, Mr RilwanAdewunmi, Dr Anders Backlund, Mrs Maj Blad, Ms Adriana Broussalis, MsRegina Bruggisser, Dr Jan G Bruhn, Dr Ubonwan Pongprayoon Claeson, MsLotta Flodmark, Mr Thomas Idowu, Dr Tiina Kummala, Dr Shisheng Li, DrBoling Liu, Dr Hans Naumann, Dr Premila Perera-Ivarsson, Dr Ylva Roddis, DrLaura Segura, Dr Åke Stenholm, Mrs Kerstin Ståhlberg, Dr Mervi Vasänge, MrsSolveig Österman and of course my “brother” Dr Hesham El-Seedi.

My students Ms Marjam Karlsson, Ms Elinor Ingels, Ms Åsa Björklund, MrsKerstin Ekehov-Karlsson, Ms Eva-Stina Larsson, Mr Ola Skvagerson, Ms LisbetLauny, Ms Petra Johansson (for a whole year), Mr Joel Solberg, Ms KatherehArabshshi, Mr Sonny Larsson and Mr Martin Lidell for good collaboration andgreat company in the laboratory and during coffe breaks.

My appreciation goes to all those good friends that I have come to know duringthese years, and whose names are not mentioned here, but will always remain inmy good thoughts.

All my other lovely friends and relatives for your interest, and support, and goodtimes, and for giving me a most pleasant time outside BMC. Special thanks toMia, my biggest fan (according to herself), who have encouraged me by sendingfunny emails to make me not so lonely in front of the computer. I won´t eat thatrabbit…

My mother Solweig who has always encouraged me to follow my own way, and inbelieving that I´m able to succed at whatever I choose. My father Torsten, whoalso has encouraged me and helped me in all sorts of ways, as for example, in thegrowing of Digitalis purpurea (which I´m sorry to say, never needed to be used).My brother Thomas for providing me with music during the years, and for yoursupport, and helping me with the posters. My late grandmother Daisy, a wonderfulperson, who already in my childhood showed me the importance of neutrophils.

Peter, my best friend and my love, for your endless faith in me, your support, andfor just being you!

Stockholm, August, 2001

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Appendix

Abbreviations used in this thesis

BAPNA N-benzoyl-DL-arginine p-nitroanilideBC Breast carcinomaBSA Bovine serum albuminCLL Chronic lymphocytic leukaemiaDox DoxorubicinFMCA Fluorometric microculture cytotoxicity assayfMLP N-formyl methionyl-leucyl-phenylalanineGSH GlutathioneHPLC High performance liquid chromatographyIC50 Concentration that gives 50% inhibitionIL-8 Interleukin 8LTB4 Leukotriene B4

MDR Multidrug resistanceMel MelphalanMRP Multidrug resistance proteinMS Mass spectrometryNCI National Cancer InstituteNMR Nuclear magnetic resonanceOvca Ovarian carcinomaPAF Platelet activating factorPBMC Peripheral blood mononuclear cellsPBS Phosphate buffered salineP-gp P-glycoproteinPMN Polymorphonuclear neutrophilspNA p-nitroanilideR CorrelationRF Resistance factorROS Reactive oxygen speciesRP Reversed phaseSAAVNA N-succinyl-L-alanyl-L-alanyl-L-valine-L-p-nitroanilideSEM Standard error of the meanS I S urvival indexSOD Superoxide dismutaseTFA Trifluoroacetic acidTopo II Topoisomerase IIUV Ultraviolet spectrometryVcr Vincristine

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