Studies on the Effects of Plant and Food Constituents on

Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 294

Studies on the Effectsof Plant and Food Constituents

on Cyclooxygenase-2Aspects in Inflammation and Cancer

BY

ULRIKA HUSS

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2003

in vitro

Division of Pharmacognosy, Uppsala University, SE-751 23

Printed in Sweden by Universitetstryckeriet, Uppsala 2003

7

Effects of Plant and Food Constituents on Cyclooxygenase-2

Introduction

Plants are remarkable factories of chemical compounds, referred to as phytochemi-cals. Every plant synthesises a diverse array of phytochemicals. These compounds partake in a variety of roles in plant life including maintenance of physiological functions, and defence against enemies such as bacteria, fungi, attacking insects, and plant eating animals (Dixon, 2001; Schultz, 2002). Phytochemicals have also been utilised by humans since ancient times, when the plants based on experience, started to be used as medicines to cure different diseases. The plants were initially used in unmodifi ed form, later as extracts, and in the 19th century, advances in chemistry made it possible to isolate the active compounds of some medicinal plants. A large number of the pharmaceutical agents used today contain natural compounds, including those with various modifi cations of the original molecule (Kinghorn, 2001). In addition, bioactive plant compounds have served as templates for several synthetic drugs, and as precursors used in the production of semi-syn-thetic drugs (Newman et al., 2003; Verdine, 1996; Wessjohann, 2000).

The biological effects of phytochemicals in our daily diet have been poorly studied, with the exception of the contents of plant-derived nutrients such as fats, fi bres, and carbohydrates. Phytochemicals such as plant secondary metabolites, including fl avonoids, terpenes, and alkaloids, are regarded as non-nutritive com-pounds in food. Hence, the impact of these compounds on human health has been neglected, although they occur in large amounts and varieties in fruits and veg-etables. Recently, however, numerous compounds that occur in our diet have been found to be biologically active, and several of these substances are now considered to promote health (Andlauer and Fürst, 2002). This category of food that includes physiologically active food components is believed to provide health benefi ts beyond basic nutrition, and today there is a large interest to capitalise on benefi cial effects of phytochemicals in our diet. This is exemplifi ed by the development and market-ing of so-called functional foods, which include beverages and food enriched with phytochemicals, suggested to be benefi cial for human health.

This evolution in our way of looking at drugs and health promoting substances is well illustrated by the topic of the present work: anti-infl ammatory effects of plant and food constituents. Historically, willow bark (Salix) was used for its anti-

8

Ulrika Huss

COOH

OH

COOH

O C H3

O

Salicylic acid Acetylsalicylic acid

Figure 1. From plant to commercial drug – willow bark was traditionally used for its anti-infl ammatory properties, today several pharmaceutical preparations containing acetylsalicylic acid, are marketed as anti-infl ammatory drugs. (Salix from C.A.M. Lindeman Bilder ur Nordens Flora, 1905)

Today we know that this compound specifi cally inhibits the cyclooxygenase (COX) family of enzymes, which play a key role in regulation of infl ammation. The cyclooxygenase enzymes catalyse the fi rst two steps in the conversion of arachidonic acid (AA) to prostaglandins (PGs), which in turn mediate infl ammation, fever, and pain (Vane and Botting, 2001). In addition, one of the PGs produced, prostaglan-din E

2 (PGE

2), is thought to be involved in the aetiology of cancer (Vane et al.,

1998). Hence, substances inhibiting the cyclooxygenase enzymes are important in the search for new anti-infl ammatory and anti-cancer drugs, and nature is a valu-able source for fi nding such molecules.

infl ammatory properties, which later resulted in isolation and synthesis of the pure compound salicylic acid, and its acetylated derivative acetylsalicylic acid, commonly known as aspirin (fi gure 1) (Vane and Botting, 1996).

9


Cyclooxygenase enzymes

The cyclooxygenase family

The cyclooxygenase family comprises three known members that catalyse prosta-glandin biosynthesis, namely, cyclooxygenase-1, -2, and -3 (COX-1, COX-2 and COX-3).

The fi rst identifi ed enzyme in the COX family, COX-1, is normally constitutively expressed, and is present in almost all tissues at constant levels (Smith, 1992; Smith et al., 1996). The metabolites of arachidonic acid (AA), derived from COX-1, are responsible for maintaining basic physiological conditions in the body, as in the case of cytoprotection of the gastric mucosa (Vane et al., 1998).

Recently, several splice variants of COX-1 mRNA have been reported (Chan-drasekharan et al., 2002; Schwab et al., 2003). Besides COX-1, a second protein (found translated) possesses catalytic activity. This enzyme was named COX-3 (Chandrasekharan et al., 2002). Yet it remains to be investigated where COX-3 is produced in vivo, and how it contributes to physiological and pathophysiological conditions.

The second isoenzyme, COX-2, is in contrast to COX-1, an inducible isoform triggered by various stimuli including infl ammatory agents, growth factors, and tumour promoters (Herschman, 1996). The induced enzyme is involved in pro-duction of PGs that promote the infl ammatory process. COX-2 is also expressed constitutively, in a few tissues, such as in the brain and in the kidney (Vane and Botting, 2001).

COX-1 and COX-2 – from gene to protein

COX-1 and COX-2 are genetically independent proteins, with genes located on human chromosomes 9 and 1, respectively (Funk et al., 1991; Jones et al., 1993). COX-1 is a housekeeping gene, approximately 22 kb in length, and contains 11 exons and 10 introns, whereas the COX-2 gene is about 8 kb, and contains 10 exons and 9 introns (Bakhle, 1999). The COX-2 gene has a promoter that contains several binding sites for different transcription factors including two nuclear factor-κB sites (NF-κB) (Appleby et al., 1994; Hinz and Brune, 2002). Consequently, the expres-sion of COX-2 can be regulated by a wide variety of mediators, including those involved in infl ammation.

The gene for COX-1 is transcribed as a 2.8 kb mRNA, whereas the gene for COX-2 is transcribed as 4.6, 4.0, and 2.8 kb mRNA variants (Tanabe and Tohnai, 2002). The COX-1 and COX-2 proteins are nearly the same length, about 600 amino acids, and share 60% identity in their amino acid sequence (Garavito and Mulichak, 2003). The enzymes are membrane-bound, and COX-1 is found pri-marily in the endoplasmic reticulum, whereas COX-2 is located in both endoplas-mic reticulum and nuclear membrane (Morita et al., 1995). The molecular weight

10

Ulrika Huss

NSAIDs SelectiveCOX-2inhibitors

valineisoleucine

COX-1 COX-2

of COX-1 is ~67 kDa, and the COX-2 protein is observed as variants, from 68 to 72 kDa. Both enzymes occur as homodimers (Garavito and Mulichak, 2003). A comparison of COX-1 and COX-2 is shown in table 1.

Table 1. Comparison of COX-1 and COX-2.

The overall structures of the enzymes are very similar. The structure consists of three domains: an N-terminal epidermal growth factor domain, a membrane-bind-ing motif, and a C-terminal catalytic domain containing the peroxidase and cyclo-oxygenase active sites (Kurumbail et al., 1996).

The cyclooxygenase active site lies in the end of a narrow hydrophobic channel that runs from the membrane-binding surface of the enzyme into the interior of the molecule. One notable difference between the COX-1 and COX-2 active sites is a one-amino-acid exchange at position 523 within the hydrophobic channel. For COX-2, the amino acid isoleucine is replaced by the smaller valine, and thus a side-pocket opens up in the active site of COX-2 (fi gure 2) (Browner, 1996). This structural feature has been exploited for fi nding selective COX-2 inhibitors. Com-pared to non-selective inhibitors, the COX-2 inhibitory drugs that are now being marketed consist of larger molecules with substituents that can extend into the side pocket.

Parameter COX-1 COX-2

Chromosome 9 1

Gene size 22 kb 8 kb

mRNA 2.8 kb 2.8, 4.0, 4.6 kb

Protein ~67 kDa 68-72 kDa

Localisation Nuclear membrane, ER Nuclear membrane, ER

Figure 2. Schematic view of the active sites of COX-1 and COX-2.

Prostaglandin biosynthesis

The enzymes COX-1 and COX-2 possess two distinct enzymatic activities, that of cyclooxygenase and peroxidase. Because of these two distinct enzymatic activi-

11


ties, the cyclooxygenase enzymes should actually be referred to as prostaglandin synthases-1 and -2 (PGHS-1 and PGHS-2). In general, however, cyclooxygenase is used to refer to both enzymatic activities. COX-1 and COX-2 catalyse the con-version of AA to PGs via a two-step process involving the cyclooxygenase and per-oxidase activities (fi gure 3) (Kulmacz et al., 2003). In the fi rst step, two molecules of O

2 are added to AA via the cyclooxygenase activity, generating prostaglandin

G2 (PGG

2). In the second step, PGG

2 is then rapidly reduced to prostaglandin H

2

(PGH2) by the peroxidase activity of the enzyme. The produced PGH

2 is quickly

converted to PGs (e.g., prostaglandin D2, E

2, PGF

2α), tromboxane A2 (TXA

2), or

prostacyclin (PGI2), by cell and tissue specifi c synthases (Vane and Botting, 2001).

COOHC H3

COOH

C H3

O

O

OOH

COOH

C H3

O

O

OH

AA

PGG2

PGH2

cyclooxygenase

peroxidase

synthases

COOH

TXA2

C H3

PGE2

OH

PGD2

O

PGF2a

HO

PGI2

Figure 3. Major pathways of prostaglandin biosynthesis

Cyclooxygenase inhibitors, the so-called non-steroidal anti-infl ammatory drugs (NSAIDs), are typically prescribed to control pain and infl ammatory disorders. NSAIDs decrease PG biosynthesis by inhibiting both COX-1 and COX-2. Conse-quently, side effects, such as gastrointestinal ulcers and bleeding commonly occur after long-term use of NSAIDs (Brun and Jones, 2001). The fact that COX-2 was found to be strongly associated with infl ammation, and COX-1 was found to be responsible for maintaining physiological processes, was the starting point in the search for specifi c COX-2 inhibitors. Compounds were sought that possess anti-infl ammatory effects equal to that of NSAIDs, but with decreased gastrointestinal side effects.

12

Ulrika Huss

Cyclooxygenase-2

COX-2 and prostaglandins – involvement in infl ammation and cancer

Infl ammation is a part of the body´s defence system, which acts to remove and repair damaged tissue or to neutralise harmful agents (Maslinska and Gajewski, 1998). This system can be activated by several stimuli, including mechanical and chemical injuries, and entities or events that invoke defence against microorgan-isms. The stimuli activate various enzymatic pathways and the release of various mediators, including prostaglandins synthesised via COX-2 (Nathan, 2002).

Prostaglandin E2 (PGE

2), one of the major metabolites produced in prostaglandin

biosynthesis, is an important mediator of infl ammation, by contributing to induc-tion of fever, increasing vascular permeability and vasodilation, and enhancing pain and oedema caused by other agents such as bradykinin and histamine (Vane and Botting, 2001).

Besides being involved in infl ammation, COX-2 and PGE2 have recently been

found to be engaged in tumour development. Different types of cancer including human lung, colon, and breast cancer, have been found to express high levels of COX-2 (Ermert et al., 2003; Koki and Masferrer, 2002; Soslow et al., 2000). Also, enhanced levels of PGE

2 have been observed in a variety of tumours (Earnest et al.,

1992; Rigas et al., 1993; Zweifel et al., 2002). The mechanisms behind the con-tribution of COX-2 in the aetiology of cancer are complex. The mode of action of COX-2 may include multiple mechanisms that may act at different stages in tumour development. Over-expression of COX-2 and high levels of PGE

2 have been found

to inhibit apoptosis, to increase cell proliferation, to promote angiogenesis, and to increase the invasiveness of malignant cells (Dempke et al., 2001; Koki and Masfer-rer, 2002; Leahy et al., 2000; Leahy et al., 2002; Masferrer et al., 2000; Souza et al., 2000).

COX-2, colon cancer, and diet

The development of colorectal cancer is a multistage process (Ross, 2002). Colorec-tal tumours occur as a result of genomic alterations, which cause disturbance in cells, including activation of oncogenes and inactivation of suppressor genes (Fearon and Vogelstein, 1990). In the colon, under normal circumstances, there is a dynamic balance between cell renewal and shedding of cells at the surface of the colonic crypt. During colonic tumourigenesis, the balance between cell renewal and cell death are disturbed. When apoptosis fails, an accumulation of cells at the top of the crypts (hyperproliferation) occurs, which will further form an adenomatous polyp (fi gure 4). It has been observed that most of the carcinomas in colon and rectum arise from adenomatous polyps (Fournier and Gordon, 2000).

13


Studies imply that COX-2 is involved in this process, because both elevated levels of COX-2, and also, signifi cantly increased levels of PGE

2 have been detected in

colon cancer tissue, compared to normal mucosa (DuBois et al., 1996; Kargman et al., 1995; Rigas et al., 1993; Sano et al., 1995). As previously discussed, COX-2 and PGE

2 are believed to affect several cellular mechanisms in cancer development,

including angiogenesis, apoptosis, and cell invasiveness. Yet, detailed investigations have to be performed, to elucidate how this enzyme and PGE

2, contribute in colon

cancer development.

Figure 4. Genomic alterations in colon cells may initiate the development of colorectal cancer. The process begins with hyperproliferation of colonic mucosa, followed by development of adenomatous polyps, ending up with carcinoma.

Colon cancer is one of the most common malignancies in the Western world (Reddy, 2000). Diet has long been considered as one important factor that may affect the process of cancer development (Willet, 1989). Compounds in food may initiate or promote colon cancer, or on the other hand, may retard, block, or reverse the development of colon cancer. Our diet consists of a complex mixture of compounds that may have diverse effects on the many forms of cancer, hence the number of possible interactions between specifi c dietary compounds and cancer is large.

One approach to reduce the mortality of colorectal cancer involves long-term use of compounds (naturally occurring in diet or synthetic compounds) that after inges-tion can prevent cancer development in the large bowel (Reddy and Rao, 2002). This strategy, called chemoprevention, can be used to prevent the development of adenomatous polyps and their subsequent progression to colorectal cancer. The COX-2 enzyme, up-regulated in colon cancer, is one possible target for chemo-prevention. A specifi c COX-2 inhibitor, celecoxib, found to reduce the numbers of adenomatous colorectal polyps in patients with familial adenomatous polyposis (FAP), is now used for chemoprevention of colon cancer. A number of the natural compounds identifi ed in vitro to be inhibitors of COX-2, occur in our daily diet, and hence may possibly affect the regulation or enzymatic activity of COX-2 in colon. Hence, we have initiated studies with the intention of increasing the general knowledge of the effects of diet-related compounds on COX-2 and colon cancer.

Normal colonmucosa

Hyperplasia Smalladenoma

Largeadenoma

Cancer

14

Ulrika Huss

COX-2 inhibitors – clinical effects

The identifi cation in 1990 of the inducible isoform of cyclooxygenase, COX-2, resulted in an intensive search for selective COX-2 inhibitors during the following decade. At that time, COX-2 became a novel target for anti-infl ammatory drugs. The minor differences in the active sites of COX-1 and COX-2 have been exploited to design selective COX-2 inhibitors with anti-infl ammatory properties, but with decreased side effects.

Today, there are fi ve selective COX-2 inhibitors, recently launched commercially in Sweden (fi gure 5).

SO2C H3

O

O

Rofecoxib

SO2NH2

NN

F3C

Celecoxib

SO2NH2

ON

C H3

Valdecoxib

S

ON

C H3

Parecoxib sodium

C H3

N

N

SO2C H3

C H3

Cl

Etoricoxib

N

O

C H3

Na+-

Drug Indications

Arcoxia® arthros and rheumatoid arthritis

BextraTM arthros, rheumatoid arthritis, and menstrual pain

Celebra® arthros and rheumatoid arthritis

DynastatTM

injectionshort-term treatment of postoperative pain

VIOXX® arthros and rheumatoid arthritis

Figure 5. Chemical structures of the fi ve selective COX-2 inhibitors marketed in Sweden.

The two fi rst selective drugs marketed were celecoxib and rofecoxib, prescribed for arthrosis and rheumatoid arthritis. Subsequently, the COX-2 selective drugs etori-coxib, valdecoxib, and parecoxib have been introduced in Sweden (table 2).

Table 2. Indications for selective COX-2 inhibitors.

These selective COX-2 inhibitors are marketed as drugs that have the same anti-infl ammatory benefi ts as traditional NSAIDs, but with a reduced risk of gastroin-testinal complications. The gastrointestinal safety profi les of celecoxib and rofecoxib have been evaluated in large clinical trials (Bombardier et al., 2000; Silverstein et al., 2000). These compounds showed benefi ts compared with traditional NSAIDs in

15


the incidence of serious gastrointestinal complications (Micklewright et al., 2003). However, more studies are necessary to evaluate the impact of these compounds on renal and gastrointestinal functions.

Besides being a key target for anti-infl ammatory drugs, COX-2 has recently become a potential target for prevention and treatment of tumour progression. Studies have shown that long-term use of aspirin and other NSAIDs decreases the risk of developing colon cancer (Thun et al., 2002). As previosly discussed, a similar protective effect has been demonstrated for the COX-2 selective compound celecoxib, found to reduce the numbers of adenomatous colorectal polyps in patients with familial adenomatous polyposis (FAP) (Phillips et al., 2002, Steinbach et al., 2000). Recently, the US Food and Drug Administration has approved this drug for reduction and regression of polyps in this group of patients.

Search for natural COX-2 inhibitors

In 1996, when our research program for COX-2 inhibitors in plants started, only a few compounds of natural origin were reported to be COX-2 inhibitors. At pres-ent, a wide variety of natural compounds have been reported for COX-2 inhibitory effects (Perera et al., 2001). Today, COX-2 is known to contribute in several patho-physiological processes (O´Banion, 1999), of which the involvement in cancer has made COX-2 to a new target in the fi eld of cancer research. Consequently, beyond the search for new COX-2 inhibitors, the involvement of COX-2 in cancer is now one of the major driving forces, rather than fi nding compounds with increased selectivity.

Therefore, besides the search for new COX-2 inhibitory compounds in plants, we have initiated studies to increase the general knowledge of the molecular mecha-nisms and effects of COX-2 inhibitors of natural origin, including their impact on COX-2 in colon cancer cells, and their cytotoxic effects on human cancer cells in vitro.

16

Ulrika Huss

Aim of the present study

This study is a part of an ongoing project to identify COX-2 inhibitors of natural origin, with emphasis of Swedish plants traditionally used in various ways against infl ammatory related conditions, or used as food. This thesis focuses on the infl u-ence of plant and dietary-related compounds on COX-2. The recent identifi cation of COX-2 as a possible target for anticancer drugs has expanded the overall scope of the project to include anticancer effects of the studied plants and compounds.

The specifi c objectives of this study were the following:

• to develop a bioassay suitable for evaluation of the effects of plant compounds on COX-2 catalysed prostaglandin E

2 biosynthesis

• to establish a cell model suitable for investigation of the possible effects of identi-fi ed inhibitors of COX-2 catalysed prostaglandin biosynthesis on cell levels of COX-2 and iNOS mRNA, protein, and enzymatic activity

• to evaluate the effects on COX-2 of common plant constituents, with emphasis on dietary compounds, and to investigate the possible effects of human faecal water and of compounds therein, with the intention of increasing our general knowledge about the infl uence of diet and faecal water on COX-2, and also their roles in the aetiology of colon cancer

• to investigate the cytotoxic effects of natural inhibitors of COX-2 on a human cancer cell line panel

17


Bioassays for evaluation of COX-2 inhibition

A wide variety of bioassays have been developed to evaluate the inhibitory effects of compounds with synthetic or natural origin, on COX-1 and COX-2, in order to fi nd compounds that preferentially inhibit COX-2. Consequently, variation in experi-mental conditions is high between bioassays, which nevertheless can be divided into two main categories: fi rstly, bioassays for fi nding compounds that inhibit enzymatic activity of COX-2, and secondly, bioassays for fi nding compounds that can suppress de novo synthesis of COX-2.

The assays used in the search for inhibitors of COX-2 enzymatic activity differ substantially in several respects, including reaction time, concentration of substrate, source and amount of enzyme, use of co-factors, and incubation time with drugs (Pairet and van Ryn, 1998). Also, the detection systems used for measuring the inhibition of a drug on enzymatic activity vary greatly. For example, the inhibi-tion of enzymatic activity could be measured continuously in real-time using a polarographic oxygen electrode to register the amount of oxygen that is used during conversion of AA to PGG

2 (Wang et al., 1999). However, more often a stop-time

assay is used instead, with a separate step for detection of produced prostaglan-dins. Methods frequently used to measure the amount of produced prostaglandins include HPLC, radioimmunoassay (RIA), enzyme immunoassay (EIA), or scintil-lation counting (if a radiolabelled substrate is used) (Kase et al., 1998; Kim et al., 1999; Noreen et al., 1998b; Subbaramaiah et al., 1998). The inhibitory effect of a compound on enzymatic activity can also be measured in an intact cell system (Kim et al., 1999).

Also, in the assays used in the search for inhibitors of de novo synthesis of COX-2, there are large variations, including cell types, and the stimuli used to induce COX-2 (Pairet and van Ryn, 1999). A variety of human and animal cells are used along with a variety of stimuli, which commonly are lipopolysaccharide (LPS) or cytokines, such as interleukin-1 (Pairet and van Ryn, 1999). Inhibitors of protein synthesis may interact at several levels by inhibiting transcription, by affecting post-transcriptional processes (including mRNA transport, control of stability, and translation), and by regulating the rate of protein degradation (fi gure 6).

18

Ulrika Huss

Figure 6. Possible targets for inhibitors of de novo synthesis of COX-2.

Commonly used methods to study the effects of compounds in cells include the COX-2 luciferase assay to evaluate COX-2 promoter activity (Subbaramaiah et al., 2001), Northern blot to identify and quantify the amount of produced mRNA in cells, and Western blot to evaluate COX-2 protein levels (Guan and Morrison, 1999a and 1999b).

Development of a scintillation proximity assay-based method for detection of natural COX-2 inhibitors (paper II)

The radiochemical in vitro method for measuring inhibition of cyclooxygenase catalysed prostaglandin biosynthesis developed by White and Glassman (1974), and further optimised by Noreen and co-workers (1998a), was the starting-point for developing the scintillation proximity assay (SPA) based COX-2 system. Briefl y described, the original radiochemical assay relies on COX-1 or COX-2 catalysed conversion of 14C-AA to radiolabelled PGs. The PGs produced are separated from the unmetabolised AA by column chromatography, and then the total amounts of PGs are quantifi ed as a group using scintillation counting (fi gure 7).

Cytoplasm

Nucleus

Pre-mRNAmRNA

DNA

Transcription

capping

splicing Degradation

Translation

N

CProcessing

Enzyme

Cofactors

Compound/extract

[14C]-AA

HCl

[14C]-PGs

Activation5 min, 4°C

Incubation10 min, 4°C

Reaction15 min, 37°C

Termination

Separation

Scintillation counting

[14C]-AA

Figure 7. Schematic picture of the radiochemical in vitro assay.

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This time-consuming separation step is the rate-limiting step in the assay. To increase the method’s throughput capacity in evaluating natural COX-2 inhibitors, the radiochemical assay was further developed. The goals were to eliminate separa-tion steps, to increase the throughput of compounds and extracts, and to enable automation of the system. Moreover, since data indicates that in particular PGE

2 is

involved in infl ammatory conditions and in certain types of cancer (Earnest et al., 1992; Uefuji et al., 2000), being able to quantify PGE

2 individually has become

essential. The scintillation proximity assay technique was selected because it allows specifi c detection of PGE

2 without separation steps, and it is suitable for automa-

tion.

Principles of the scintillation proximity assay

The scintillation proximity assay is a radioisotopic assay technique that has become widely used in high-throughput screening (HTS) because it has several advantages, such as requiring no separation steps and being easily automated (Sittampalam et al., 1998). The SPA technique is based on capturing radiolabelled target molecules on the surfaces of scintillant-containing microspheres. When bound to the micro-spheres, the radioactive β-decay of those molecules is close enough to reach the scintillant core, resulting in light emission that is detectable with a β-scintillation counter (Cook, 1996). Any unbound radiolabelled molecules will be too distant to a microsphere to transfer energy, so no signal occurs. Thus, no separation of bound and unbound radioisotopes is necessary. A commonly used radioisotope in SPA is tritium (3H), because it releases its energy in the form of β-particles with an average pathlength of 1.5 µM, suitable for SPA (Cook, 1996).

Development of the scintillation proximity assay-based detection system

In order to avoid non-specifi c binding of the labelled AA to the SPA beads, unla-belled AA was used in the enzymatic reaction instead of radiolabelled AA. Conse-quently, we had to use an indirect measurement of the produced PGs, as in a RIA system (Paper II). A specifi c amount of radiolabelled PGE

2 was allowed to compete

with unlabelled PGE2 from the enzymatic reaction, for the binding sites of antibod-

ies against PGE2. Thereafter, SPA beads precoated with a secondary antibody were

added to capture the PGE2 antibodies (fi gure 8). As in RIA, a standard curve was

used to quantify the amounts of produced PGE2.

LightSPA beadwith anti-PGE2

bound

PGE2

[3H]-PGE2

Figure 8. The principle of the scintillation proximity assay.

20

Ulrika Huss

When a sample shows inhibition, a high signal is detected, because a low amount of PGE

2 produced in the bioassay competes with the radiolabelled PGE

2. Con-

sequently, samples without inhibitory effects cause a low signal. Tritium labelled PGE

2 was used as tracer because 3H isotopes are ideal in SPA systems, due to the

pathlength of emitted β-particles. The detection part of the assays was optimised with respect to amount of antibody, SPA beads, and radiolabelled PGE

2. The detec-

tion range obtained (5 to 500 pg PGE2/well) was well suited for the analysis because

the amounts of PGE2 in the aliquots from the bioassay did not exceed 200 pg/well.

Modifi cation of the enzymatic reaction conditions

Compared to the original radiochemical assay, a number of parameters were changed. Figure 9 shows a schematic view of the SPA-based assay. To enable automation of the SPA-based assay, the temperature for the enzymatic reaction was changed from 37°C to room temperature. The reaction time was decreased from 15 to 6 min. This time point is at the beginning of the steady-state level of the velocity curve of the enzymatic reaction. Preferentially, the stop-time are set before reaching the steady-state level. However, to obtain a robust and reproducible assay for analysis of large numbers of samples, the stop-time was set at 6 min. Thus, an underestimation of the potency of the inhibitors might occur. Further, the stop reagent was changed to sodium bisulfi te, because HCl affected the antibodies.

Enzyme

NaHSO3

[3H]-PGE2

Activation, 5 min

Incubation, 10 min

Reaction, 6 min

Termination

Equilibration,10 min

AA

Compound/extract

Cofactors

PGs

Anti-PGE2

SPA beads

Scintillation counting

Incubation, 90 min

Figure 9. A schematic view of the SPA-based assay.

Finally, the kinetic parameter Km was estimated (1.0 µM). The method was

validated with known inhibitors of COX-2 of natural and synthetic origin. The most potent inhibitor (based on IC

50 values) was NS-398 (0.16 µM), followed by

rofecoxib (0.76 µM), indomethacin (3.9 µM), ursolic acid (86 µM), oleanolic acid (87 µM), and acetylsalicylic acid (780 µM). The rank order of compounds was essentially the same as for the original assay (Noreen et al., 1998a; Ringbom et al.,

21


1998), but the IC50

values were lower. This might be a consequence of the shorter reaction time and the specifi c detection of PGE

2. The SPA-based assay was used to

evaluate the COX-2 inhibitory effects of common natural compounds from several substance classes (Paper II). Of the 49 compounds tested, three were found to dose-dependently inhibit the enzyme: cinnamaldehyde, eugenol, and pyrogallol (further discussed in chapter “COX-2 inhibitory effects of common plant compounds”).

Establishment of a cell model for studying effects on COX-2 and iNOS protein synthesis

Macrophages are involved in the host defence mechanism against infections caused by bacteria and viruses (Aderem, 2002; Djaldetti et al., 2002). To study the anti-infl ammatory effects of natural compounds in an intact cell system, LPS-activated murine RAW 264.7 macrophages were used as a cell model. This cell line is com-monly used in anti-infl ammatory studies, because treatment with different stimuli, including LPS, trigger the cells to express high levels of COX-2 and of inducible nitric oxide synthase enzyme (iNOS) (Dirsch et al., 1998; Weisz et al., 1994).

In parallel with the cyclooxygenase enzyme family, there also exist constitutive isoforms of nitric oxide synthase (NOS), namely, endothelial NOS and neuronal NOS. These constitutive enzymes produce nitric oxide (NO), to maintain physi-ological functions, including regulation of vasodilatation and neurotransmission (Weinberg, 2000). Like COX-2, iNOS plays an important role in mediation of infl ammation. The iNOS enzyme is involved in chronic infl ammatory processes, such as rheumatoid arthritis (Weinberg, 2000).

As earlier mentioned, several infl ammatory stimuli, such as cytokines and the bac-terial endotoxin LPS, induce iNOS in various cells (Robbins and Grisham, 1997). The iNOS enzyme catalyses the conversion of L-arginine to NO, a potent infl am-matory mediator, which is unstable, and is converted to various nitrogene oxides, including nitrite (Perez-Sala and Lamas, 2001; Robbins and Grisham, 1997). Studies have shown that co-regulation occurs between the nitric oxide synthase and COX-2 systems (Patel et al., 1999; Perez-Sala and Lamas, 2001). The NO produced by iNOS regulates COX-2 activity and expression, but the metabolites produced by COX-2, may also infl uence iNOS expression (Perez-Sala and Lamas, 2001). Many natural compounds including apigenin, kaempferol, and pristimerin have been reported to inhibit iNOS (Dirsch et al., 1997; Liang et al., 1999), which motivate the search for inhibitors of natural origin.

Time-course of mRNA and protein levels of COX-2 and iNOS, and levels of PGE

2 and nitrite

To establish the time for stimulation of cells with LPS, the time dependency of induction of COX-2 and iNOS mRNA levels was investigated using a concentra-tion of 5 µg/ml of LPS (fi gure 10). Following cell lysis and RNA purifi cation, levels of COX-2, iNOS, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)

22

Ulrika Huss

were assessed in 20 µg of total cellular RNA by Northern blot analysis (appendix). Time-course experiments by Northern blot analysis showed that COX-2 mRNA was visible after a 1 h treatment with LPS, and that it increased, reaching a maxi-mum at 20 h. An induction of iNOS was observed after a 5 h treatment with LPS, which also was found to be maximal induction.

Figure 10. Time-course of iNOS and COX-2 mRNA levels in RAW 264.7 cells stimulated with LPS (5 µg/ml). GAPDH was used as a contol for equal loading of the samples.

The protein levels of COX-2 and iNOS were assessed by Western blot analysis (appendix). Time-course experiments showed that, after a 5 h treatment of cells with LPS, the COX-2 and iNOS proteins were clearly visible, and that maximum levels were reached after 21 h and 10 h, respectively (data not shown). Further, COX-2 protein was detected in both cytosolic and nuclear protein fractions, whereas iNOS protein was mainly found in the cytosolic fraction. COX-2 is reported to be distrib-uted both in the nucleus and in the cytosol (Patel et al., 1999), consistent with our fi ndings.

To investigate the effects on COX-2 and iNOS enzymatic activity in cells, the amount of PGE

2 and nitrite in cell supernatant was measured using the RIA and

Griess assays, respectively (appendix). Time-studies showed that the amount of PGE

2 and nitrite produced in LPS-stimulated (1 µg/ml) RAW 264.7 cells was not

detectable after a 5 h treatment, whereas after a 20 h treatment, a 20-fold increase in levels of PGE

2 and a 40-fold increase in levels of nitrite were observed.

Dose-response analysis of LPS induction of mRNA levels of COX-2 and iNOS

In dose-response experiments studying LPS mediated induction of mRNA, RAW 264.7 cells were treated 5 h with different concentrations of LPS. Stimulation with LPS resulted in dose-dependent induction of the mRNA levels of COX-2 and iNOS, in the RAW 264.7 cells. COX-2 mRNA was induced after a 5 h treatment with 0.5 µg/ml LPS, while iNOS mRNA was detectable after stimulation with 0.1 µg/ml of LPS (fi gure 11). Both enzymes were clearly detectable after stimulation with 1 µg/ml LPS, and reached maximum levels at 5 µg/ml.

23


Figure 11. Northern blot showing the mRNA levels of iNOS, COX-2, and GAPDH in RAW 264.7 cells stimulated with different concentrations of LPS for 5 h.

Established experimental conditions

In experiments, in which the effects of compounds on mRNA levels were studied, RAW 264.7 cells were simultaneously exposed to LPS (1 µg/ml) and to the test compound for 5 h, which made it possible to investigate both inhibitory and stimu-latory effects of compounds on the expression of COX-2 and iNOS. Moreover, in experiments investigating the effects of compounds on protein levels, cells were treated with LPS and the test compound, for either 5 h or 20 h, enabling detection of differences due to a short or long time treatment. The effects of compounds on PGE

2 and nitrite levels, were studied after a 20 h co-treatment with LPS.

The cell model was used to assess the effects of two fatty acids, eicosapentaenoic acid and all-(Z )-5-thia-8,11,14,17-eicosatetraenoic acid (compound 2), on COX-2 and iNOS mRNA, protein levels, and on enzymatic activity (further discussed in chapter “Studies of the effects of fatty acids on COX-2 and iNOS mRNA, protein levels, and enzymatic activity”).

Conclusion

As tools for fi nding new COX-2 inhibitors in plants, two bioassay systems were established. One system, to detect inhibitors of COX-2 catalysed prostaglandin bio-synthesis, was developed for rapid screening of large number of samples. By using SPA technology, we obtained a rapid COX-2 assay with a high capacity, for evaluat-ing inhibitors of COX-2 catalysed PGE

2 biosynthesis. The SPA-based assay has a

short handling time (30 min), compared with the original radiochemical assay with a handling time of 3 hours. In addition, the method can easily be automated because only pipetting steps are required. The assay was found to be suitable for screening of COX-2 inhibitors with natural origin.

24

Ulrika Huss

The second system, consisting of a cell model, was aimed to detect natural com-pounds that interact with different steps in protein synthesis of COX-2 or iNOS. The cell system was optimised regarding treatment time and concentration of LPS, to obtain a system suitable for investigation of both inhibitory and stimulatory effects of natural compounds on the expression of COX-2 and iNOS. Using the cell model, the effects of compounds could be analysed at several different levels, including mRNA, protein, PGE

2, and nitrite. We found the cell model to be suit-

able as a tool in the search for COX-2 and iNOS inhibitors. One possible approach for increasing the capacity of the model for testing compounds is to determine the amount of produced PGE

2 and nitrite in the cell supernatant before performing

Northern or Western blot experiments, using the RIA method and the Griess assay. Decreased levels of PGE

2 and nitrite may refl ect a reduction of protein synthesis

as well as an inhibition of the enzymatic activity. The RIA method and the Griess assay are rapid screening methods, compared with Northern and Western blot experiments. If decreased levels of PGE

2 or nitrite are observed, then the compound

could be further subjected to an analysis of its effects on mRNA and protein levels.

25


Strategies in the search for bioactive compounds in plants

Two commonly used strategies to detect bioactive compounds in plants include screening of isolated plant compounds and bioassay-guided isolation of plant extracts (Pezzuto, 1997). Bioassay-guided isolation of an active plant extract is a classical method for proceeding from a crude extract to a pure compound with biological activity. The plant extract is separated successively, and the activity of each fraction is assessed by the bioassay. The most active fractions are further sepa-rated and biologically tested, until pure active compounds are isolated. Both strate-gies, bioassay-guided isolation and screening of pure plant compounds, have been applied in the present work, in the search for COX-2 inhibitors of natural origin.

Plants with COX-2 inhibitory effects

As a part of the search for new COX-2 inhibitory compounds, a variety of plants have been assessed for their inhibition of COX-2 catalysed prostaglandin biosyn-thesis. Of those plants, two Swedish plants, Plantago major L. and Urtica dioica L., were selected for bioassay-guided fractionation by means of COX-2 inhibition (fi gure 12).

Figure 12. The plants, Plantago (groblad) and Urtica (brännässla).

Besides being found to inhibit COX-2, these plants have been reported to have traditional uses or other pharmacological activities, which might be correlated with inhibition of COX-2 (ESCOP, 1997; Samuelsen, 2000; Wagner et al., 1989).

26

Ulrika Huss

COX-2 inhibitory principles in Plantago major L. (paper I)

Plantago major L., a plant widely used for its anti-infl ammatory quality, was sub-jected to bioassay-guided fractionation (Paper I). The triterpenoid ursolic acid was previously isolated as a COX-2 active principle in a hexane extract of the herbal parts (Ringbom et al., 1998). In this study, the dichloromethane extract of P. major was assessed for bioassay-guided separation, and again ursolic acid, but also the tri-terpenoid oleanolic acid, was isolated as an COX-2 active compound. In addition, two fatty acids, linoleic acid (LA) and α-linolenic acid (α-LNA), were identifi ed as COX-2 inhibitors. The fatty acids, palmitic acid (PA) and myristic acid (MA), were also identifi ed in the extract, but did not inhibit COX-2.

COX-2 inhibitory principles in Urtica dioica L.

At the same time as the work with Plantago major was proceeding, bioassay-guided fractionation of Urtica dioica L. was performed. Extracts of U. dioca have been tra-ditionally used for treatment of rheumatic conditions (ESCOP, 1997; Obertreis et al., 1996). These extracts have been reported to exhibit anti-infl ammatory effects by means of inhibiting COX-1 and 5-lipooxygenase (Obertreis et al., 1996), and to inhibit NF-κB activation, a transcription factor that is activated for instance in rheumatoid arthritis (Riehemann et al., 1999). The aerial parts of U. dioica were successively extracted with hexane, dichloromethane, acetonitrile, and water, in a soxhlet apparatus. Of the extracts (tested at a concentration of 100 µg/ml), the hexane extract had the most potent inhibitory effect on COX-2 (44±1%). Bioas-say-guided fractionation of this extract resulted in isolation of a mixture of unsatu-rated fatty acids as active compounds (appendix). The plant is known to contain several unsaturated fatty acids, including linoleic acid and palmitic acid (Kraus et al., 1991).

Conclusions

Of the plant extracts that we have assessed for COX-2 inhibitory effect, we found that preferentially the non-polar extracts (hexane and dichloromethane) possess COX-2 inhibitory effects. Out of 55 non-polar extract tested, about half inhibited COX-2 by more than 30% (tested at 100 µg/ml), whereas of 130 polar extracts tested, only 5% were found to inhibit COX-2 by more than 30%. One might spec-ulate that fatty acids are partly responsible for the COX-2 inhibition observed in the non-polar extracts, since they are common in plants. Fatty acids were COX-2 active in P. major, U. dioica, and in Vaccinium myrtillus L. (Stenholm, 2001), and they were also present in COX-2 active extracts of Syringa vulgaris L. and Vincetoxicum hirundinaria Med. (Ringbom, 2002). This prompted us to set up a dereplication protocol for these compounds. Early identifi cation of fatty acids in plant extracts will facilitate bioactivity-guided fractionation of the extracts and thus increase the

27


likelihood of fi nding novel compounds with COX-2 inhibitory effects, and thus a dereplication protocol has been developed (Pathmasiri, (In manuscript); Stenholm, 2001).

Natural compounds affecting COX-2

The search for natural compounds with COX-2 inhibitory effects is rapidly progress-ing. In 2001, we presented a review in which reports (from 1991 to 1999) of natural compounds and plant extracts with effect on COX-2 are summarised (Perera et al., 2001). In the review, 65 isolated compounds affecting COX-2 are discussed. The compounds are reported to affect COX-2 at several levels, including inhibition of enzymatic activity, or by altering the levels of COX-2 mRNA or protein.

Among the natural compounds that infl uence COX-2 summarised in the review, we note that several are commonly abundant in plants, including catechin, querce-tin, oleanolic acid, and ursolic acid. One of the most thoroughly studied substance classes, regarding effects on COX-2, is that of the fl avonoids, but a wide variety of terpenes have also been reported to affect COX-2. Nonetheless, some unusual compounds, such as ajoene, a thioester in garlic (Allium sativum L.) (Dirsch and Vollmar, 2001); C-phycocyanin, a biliprotein from the blue green algae Spirulina platensis (Reddy et al., 2002 and 2003); γ-mangostin, a xanthone-derivative in mangosteen (Garcinia mangostana L.) (Nakatani et al., 2002); and tryptanthrin, an alkaloid found in Isatis tinctoria L. (Danz et al., 2001), are also examples of COX-2 inhibitory principles.

COX-2 inhibitory effects of natural and modifi ed fatty acids (paper I)

Fatty acids are important nutrients that commonly occur in plants and fi sh-oils, and also in animal fat (Dommels et al., 2002; Teitelbaum and Walker, 2001). As a part of our daily diet, fatty acids may infl uence several biological targets in the body, including the cyclooxygenase enzymes. The fatty acids are classifi ed into series, ω -3, ω -6, or ω -9, depending on the localisation of the fi rst double bond, counting from the methyl end of the fatty acid (Kelley, 2001). Fatty acids in the ω -3 series are essential for normal growth and development, and are believed to be important in preventing and treating several disorders including infl ammation and cancer (Simopoulos, 2002). A typical Western style diet is rich in ω -6 fatty acids such as AA (20:4 ω -6) and linoleic acid (18:2 ω -6), and contains low levels of ω -3 fatty acids, including eicosapentaenoic acid (20:5 ω -3) and α-linolenic acid (18:3 ω -3) (Calder, 2002). The dietary intake of fatty acids infl uences the fatty acid composi-tion of phospholipids in the cell membranes (Calder, 2002). As a consequence, the amount of AA in cell membranes may be affected, and hence also the production of prostaglandins.

As earlier concluded, bioassay-guided fractionation of the plants Urtica dioica L. and Plantago major L. resulted in isolation of fatty acids as some of the cyclo-oxygenase inhibitory principles (Paper I). The dichloromethane extract of P. major

28

Ulrika Huss

contained linoleic acid (LA) and α-linolenic acid (α-LNA), which were found to inhibit COX-2 and COX-1, whereas palmitic acid (PA) and myristic acid (MA) were found inactive. Further, the effects of the isolated fatty acids were compared with the COX-1 and COX-2 inhibitory effects of a variety of natural occurring fatty acids, including oleic acid (OA), stearic acid (SA), pentadecanoic acid (PDA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), and were also com-pared with the effects of six semi-synthetic fatty acids (compound 1 – 6) (Flock et al., 1999) synthesised from EPA and DHA (fi gure 13) (paper I).

Figure 13. Chemical structures of fatty acids assessed for inhibition of COX-1- and COX-2-catalysed prostaglandin biosynthesis.

Of the 15 fatty acids tested, only compound 2, EPA, DHA, and α-LNA were found to be more potent inhibitors of COX-2 than the COX-2 selective reference compound NS-398 (IC

50 of 53 µM) (table 3). Noticeably, all compounds were less

potent inhibitors of COX-1 than the reference compound indomethacin (IC50

of 1.3 µM).

Table 3. Effects of natural and modifi ed fatty acids on COX-1 and COX-2 catalysed prostaglandin biosynthesis. a Maximum dissolved concentration.

COOH

COOH

COOH

COOH

S COOH

S COOH

O COOH

COOH

COOH

COOH

COOH

COOH

COOH

S COOH

O COOH

O COOH

PDA

4PA

6

5

3SA

MA

DHA

EPA

1

2

α-LNA

LA

OA

4710

12

13

14

18

17

20

AA19 11 9 8 6 5 3

216

15

Compound COX-2 COX-1 COX-2IC50 (µM) COX-1

5-Thiaeicosatetraenoic acid (2) 3.9 26 0.2Eicosapentaenoic acid (EPA) 7.1 13 0.5Docosahexaenoic acid (DHA) 9.8 15 0.7α−Linolenic acid (α−LΝΑ) 12 93 0.1

NS-398 53 >100a <0.5Linoleic acid (LA) 94 170 0.65-Thiaeicosatetraenoic acid (4) 120 81 1.43-Thiaoctadecatetraenoic acid (1) 160 84 1.9Indomethacin 164 1.4 1173-Oxaheneicosapentaenoic acid (6) 180 50 3.63-Oxaoctadecatetraenoic acid (5) >500 >500 -3-Oxaoctadecatetraenoic acid (3) >500 >500 -Myristic acid (MA) >500 >500 -Oleic acid (OA) >500 >500 -Palmitic acid (PA) >500 >500 -Pentadecanoic acid (PDA) >500 >500 -Stearic acid (SA) >500 >500 -

29


Of the natural fatty acids, EPA, DHA, and α-LNA (ω -3 fatty acids) were the most potent inhibitors of COX-2. These compounds were essentially equipotent, all with IC

50 values of about 10 µM. The ω -6 fatty acid LA was a less potent inhibitor

of COX-2 catalysed PG biosynthesis (IC50

of 94 µM), and the monounsaturated fatty acid OA (ω -9), and the saturated fatty acids, SA, PA, PDA, and MA, exhibited no COX-2 inhibitory effect up to 500 µM.

Of the semi-synthetic fatty acids, compound 2 was the most potent COX-2 and COX-1 inhibitor. This compound was also the only synthetic analogue that was more potent than its origin. In comparison, the two other fatty acids (containing a thioether moiety), compound 4 and compound 1, were approximately 30 to 40 times less potent inhibitors of COX-2. Noticeably, the only structural difference between compound 2 and compound 4 is the position and confi guration of the double bound at C7 and C8. Compound 1 and compound 4 were found to be equi-potent inhibitors of COX-1. Of the three semi-synthetic fatty acids that contain an ether function (i.e., compound 3, 5, and 6), only compound 6 was found to inhibit the enzymes.

Of all fatty acids tested, α-LNA and compound 2 showed most selectivity toward COX-2, whereas compound 6, the fatty acid with the longest chain (21 atoms), was the fatty acid showing most selectivity toward COX-1.

Both COX-1 and COX-2 are able to oxygenate fatty acids other than AA, whereas in general, COX-2 is more liberal in accepting alternative substrates. The fatty acids EPA, α-LNA, and LA are proposed to act as substrates of the cyclooxygenase enzymes (Laneuville et al., 1995; Smith and DeWitt, 1995). In contrast, DHA is suggested to be an inhibitor of COX-1 and COX-2, without being a substrate (Smith and DeWitt, 1995). The ω -3 fatty acid, EPA, is reported to be metabolised by the cyclooxygenase-enzymes to PGE

3, a metabolite that is biologically less active

than the PGE2 produced from AA (Dommels et al., 2002). By competing with AA

in the prostaglandin biosynthesis, ω -3 fatty acids may cause a decrease in produc-tion of pro-infl ammatory prostaglandins. Thus, a diet rich in ω -3 fatty acids and with a low concentration of ω -6 fatty acids, including AA and the precursors of AA, is hypothesised to by benefi cial for patients with rheumatoid arthritis (Calder and Zurier, 2001).

Studies of the effects of fatty acids on COX-2 and iNOS mRNA, protein levels, and enzymatic activity

Two inhibitors of COX-2 catalysed prostaglandin biosynthesis, eicosapentaenoic acid (EPA) and the chemically modifi ed analogue, compound 2 (Paper I), were assessed using the murine macrophage cell model (previously described). The pos-sible effects of these compounds on mRNA, protein levels, and enzymatic activity of COX-2 and iNOS in cells were investigated.

30

Ulrika Huss

COX-2 and iNOS – mRNA levels

RAW 264.7 cells were treated simultaneously with LPS (1 µg/ml) and with EPA or compound 2 (0.1–100 µM) for 5 h. Dexamethasone (1 µM) was used as a positive control. None of the fatty acids altered the COX-2 or iNOS mRNA levels (fi gure 14). The positive control, dexamethason, completely inhibited the expression of both COX-2 and iNOS.

Figure 14. In RAW 264.7 cells, none of the fatty acids altered mRNA levels of COX-2 and iNOS, relative to the LPS-treated control, shown by Northern blot. RNA was isolated from cells treated with LPS (1 µg/ml) and with fatty acid (0.1-100 µM) or dexamethasone (1 µM) for 5 h. The fatty acids were dissolved in 0.1% DMSO. Unstimulated cells were used as a control. GAPDH was used as a control for equal loading of the samples.

COX-2 and iNOS – protein levels

Preliminary experiments demonstrated that cells treated with LPS and compound 2 (0.1–100 µM) for 5 h show a decreased protein level of COX-2 after treatment with the highest concentration of compound 2 (100 µM) (fi gure 15). Complementary experiments, including treatment with EPA, will be performed. After a 20 h treat-ment with LPS and with EPA or compound 2 (0.1–100 µM), the levels of COX-2 and iNOS protein were unaltered (data not shown), whereas dexamethason inhib-ited the protein levels of both COX-2 and iNOS.

Figure 15. Western blot showing the protein levels of COX-2, iNOS, and β-actin in cells treated with LPS (1 µg/ml) and compound 2 (0.1-100 µM) for 5 h. Unstimulated cells were used as controls, as were LPS-stimulated cells with 0.1% DMSO. β-actin was used as a contol for equal loading of the samples.

31


COX-2 and iNOS – enzymatic activity

Experiments showed that dexamethasone (10 µM) inhibited PGE2 levels (97±0.4%)

and nitrite levels (84±7%) effectively, whereas the fatty acids revealed no inhibition of the production of nitrite at the tested concentrations. EPA (1 µM) inhibited PGE

2

synthesis moderately (41%±15). Complementary experiments with higher concen-trations will be performed.

COX-2 inhibitory effects of common plant compounds (paper II)

Plant constituents from common substance classes were assessed for their inhibitory effects on COX-2 catalysed PGE

2 biosynthesis, using the SPA-based assay (Paper

II). A variety of compounds from different substance classes, including alkaloids, anthraquinones, fl avonoids, phenylpropanes, steroids, and terpenes (table 4), were evaluated with the intention of increasing our knowledge about common plant constituents that affect COX-2, in order to facilitate the search for novel COX-2 inhibitors in plants.

Table 4. Compounds assessed for inhibition of COX-2 catalysed PGE2 biosynthesis.

Compound Inhibition(%)

Compound Inhibition (%)

Alkaloids SteroidAtropine - Ouabain -Caffeine - TerpenesEmetine HCl - Abietic acid -Nicotinic acid - Betulin -Pipecolic acid - α-Bisabolol -(-)-Scopolamine HCl - (+)-Camphor -Stachydrine HCl - Citronellal -Theophylline - DL-Citronellol -Trigonelline HCl - β-Escin -

Anthraquinone Eucalyptol -Sennoside B - (R )-(+)-Limonene -

Flavonoids α-Lupeol -Chrysin - (-)-Menthol -Luteolin - Thymol -Myricetin 62 ±7 OtherNaringenin - Arbutin -Rutin - Hydroquinone -

Phenylpropanes Gallic acid -Caffeic acid 32 ±16 Pyrogallol 57 ±7Chlorogenic acid - (-)-Quinic acid -Cinnamaldehyde 45 ±7 α-Terthienyl -Coumarin - Thioctic acid -p-Coumaric acid - Shikimic acid -3,4-Dimethoxycinnamic acid - Compounds marked – showed less than 30 %Esculin - inhibition.Eugenol 40 ±7Isovanillic acid -Podophyllotoxin -Scopoletin -Umbelliferone -

32

Ulrika Huss

Of the 49 compounds screened for COX-2 inhibition, fi ve compounds, pyrogal-lol, cinnamaldehyde, eugenol, myricetin, and caffeic acid, were detected as hits (i.e., more than 30% inhibition at 100 µM). Dose-response experiments showed that eugenol and pyrogallol were about equipotent inhibitors, with IC

50 values of 129

and 144 µM, respectively. Cinnamaldehyde was less potent (IC50

of 245 µM), and myricetin and caffeic acid showed no dose-dependent inhibition. The potency of the three natural compounds found to inhibit COX-2 was rather weak compared with the two selective COX-2 inhibitors, NS-398 and rofecoxib (IC

50 of 0.16 µM

and 0.76 µM). Both eugenol and pyrogallol have phenolic moieties, whereas cin-namaldehyde lacks the phenolic entity (fi gure 16).

Figure 16. Chemical structures of compounds inhibiting COX-2 catalysed PGE2

biosynthesis.

A large number of compounds reported to inhibit the cyclooxygenase enzymes have a phenolic moiety, including a variety of fl avonoids (Chi et al., 2001, Dewhirst, 1980; Lee et al., 2001; Wang et al., 1999). Several mechanisms are suggested to be involved, by which the phenolic compounds inhibit the enzymatic activity, includ-ing interference or competition for the AA binding site, and by reducing the enzyme (Alanko et al., 1999; Bakovic and Dunford, 1993; Thompson and Eling, 1989). Noticeably, phenolic compounds are also reported to stimulate the activity of the cyclooxygenase enzymes. It is proposed that these compounds could enhance the enzymatic activity by protecting the enzyme from self-catalysed inactivation or by acting as reducing substrates for the intermediate oxidized form of the enzymes (Alanko et al., 1999; Johnson and Maddipati, 1998). Yet, it remains to be eluci-dated in detail, which mechanisms are involved, beyond the observed inhibitory and stimulatory effects of phenolic compounds.

Conclusion

The natural occurring fatty acids, α-LNA, LA, DHA, and EPA, and the semi-synthetic analogues, compounds 1, 2, 4, and 6, inhibited COX-2 catalysed pros-taglandin biosynthesis. These compounds are suggested to act as substrates for the cyclooxygenase enzymes, or as competitive inhibitors. In cells, after a 5 h treatment with compound 2 (100 µM) and LPS, the COX-2 protein level in those cells was decreased, whereas after a 20 h treatment, the level of COX-2 protein was unaltered. The reappearance of COX-2 protein after 20 h may be a result of poor stability of the fatty acid, but may also refl ect a reversible effect of compound 2. Hence it would

H2C

O C H3

O H

CH

C H2

O H

O H

H C CH

C H

OO H

EugenolPyrogallol Cinnamaldehyde

33


be of further interest to study the effects of compound 2 on COX-2 protein levels at several time points between 5 to 20 h.

In the set of common natural compounds evaluated for COX-2 inhibition, the two phenolic compounds, eugenol and pyrogallol, were found to inhibit COX-2 catalysed prostaglandin biosynthesis. As previously discussed, phenolic compounds have been suggested to inhibit the COX-2 enzyme by several mechanisms, includ-ing that of competitive inhibition. Moreover, the phenylpropane cinnamaldehyde, lacking a phenolic entity, was found to inhibit COX-2.

34

Ulrika Huss

The infl uence of diet on COX-2 (paper III)

Human faecal water

Faeces normally consist of about three-fourths water and one-fourth solid material, including protein, inorganic material, undigested food residues, bile pigments, and sloughed epithelial cells (Guyton, 1992). The aqueous phase of faeces, called faecal water, can be obtained by high-speed centrifugation of total faeces (Rafter et al., 1987; Venturi et al., 1997). The compounds in faecal water are believed to be more likely to exert effects on the colonic cells than the compounds bound to the solid parts of faeces (Rafter, 2002; Venturi et al., 1997). Thus, current interest is devoted to the role of faecal water in studies examining the mechanisms underlying the infl uence of diet on colon cancer. One possible target for the compounds in faecal water is COX-2.

Previous studies indicate that compounds in human faecal water are able to induce or inhibit COX-2 promoter activity in human colonic cells (Glinghammar and Rafter, 2001). We have therefore investigated the effects of faecal water on COX-2, by means of inhibitory or stimulatory effects on COX-2 catalysed pros-taglandin biosynthesis, and on COX-2 protein levels in a human colon cancer cell line (Paper III). In this initial study, faecal samples were collected from vegetar-ians, because their diet high in phytochemicals, are believed to decrease the risk of developing colon cancer (Almendingen et al., 1995), a process that are thought to be infl uenced by COX-2. Also, a variety of compounds in edible plants have been reported to affect COX-2 mRNA and protein levels, and to affect enzymatic activ-ity as well (Perera et al., 2001).

Being a complex biomass, faecal water is likely to containing a broad range of chemical constituents. A number of compounds have earlier been identifi ed in faecal water, including the bile acids; deoxycholic acid (DCA), 5α-deoxycholic acid, chenodeoxycholic acid, cholic acid, and also fatty acids (Rafter et al., 1987; van Faassen et al., 1993). The chemical composition of faecal water is not yet fully inves-tigated. Therefore, further analyses were performed to increase the knowledge of faecal water’s chemical content (Paper III). In this study, we focused on the content of phenolic compounds, as these compounds are common in fruits and vegetables, and a number of those are also reported to exhibit anti-infl ammatory properties by inhibiting COX-2 (Perera et al., 2001).

Effects of faecal water on COX-2

Of twenty samples assessed for their effects on COX-2 catalysed prostaglandin bio-synthesis, two inhibited the enzyme weakly (20%) and two were found to stimulate enzymatic activity (fi gure 17).

35


Figure 17. Effects of fecal water samples on COX-2 catalysed prostaglandin biosynthesis. Two fecal water samples inhibit COX-2 weakly (20%), and two samples appear to stimulate the enzymatic activity. Diagram bars represent mean values ± SEM (n=3).

To investigate the effects of faecal water on COX-2 protein levels, colon cancer cells (HT-29) were incubated with faecal water for 2 h and further cultivated for 16 h. Some of the faecal waters caused an induction of COX-2 protein in HT-29 cells, and one sample decreased protein levels slightly (fi gure 18 a and b).

-40

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Sample

FW

a)

C DCA fw 8 fw 9 fw 12

b)

C DCA fw13 fw14 fw18 fw20

Figure 18 a and b. HT-29 cells treated for 2 hours with DCA, (250 µM), or faecal water (1:80 dilution). DCA was used as positive control for COX-2 induction. Untreated HT-29 cells (C). Faecal water (FW).

Noticeably, in vivo, there is large variation between individuals with respect to exposure time of faecal water in the colon. Thus, it might be valuable to study the effects of faecal water on COX-2 in colon cancer cells after incubation with faecal water, at various time points. A more potent effect on COX-2 may not appear until after 15 to 25 h treatment.

36

Ulrika Huss

Chemical content of faecal water

A broad range of phenolic compounds was detected in human faecal water, of which a number of compounds were directly related to a dietary intake of fruit and vegetables. The major part of compounds was identifi ed as metabolites of ingested fl avonoids and other phenolic substances. Flavonoids were only abundant in low concentrations (less than 3 µM). There was also a large variation regarding the concentrations of phenolic compounds between the samples analysed. This might refl ect individual variations in intake of fruit and vegetables, but also individual dif-ferences in bacterial enzymatic activity in the colon.

Effects of dietary-related and faecal-water-related compounds on COX-2

In attempt to fi nd the compounds responsible for the inhibitory and stimulatory effects on COX-2 enzymatic activity and on COX-2 protein levels in colon cancer cells, we investigated the effects on COX-2 of compounds that were detected in faecal water. The main groups of compounds tested were fl avonoids, phenolic acids, and bile acids. The colonic epithelial cells constitute the interface between the ingested food and the blood. Hence, compounds in the luminal content and in the blood stream can affect the epithelial cells. Therefore, in addition to compounds found in faecal water, compounds abundant in edible plants were included.

Hitherto, we have investigated 18 compounds for their infl uence on COX-2 catalysed prostaglandin biosynthesis (table 5). For fi ve of those compounds, which occurred in high concentrations in faecal water, also their abilities to induce or decrease COX-2 protein levels were further studied: ferulic acid; 3-hydroxyphen-ylacetic acid; 3-phenylpropionic acid; 3,4-dihydroxy-phenylacetic acid; and 3-(4-hydroxyphenyl)-propionic acid (fi gure 19 a and b).

a)

C DCA 1 2 3

b)

C DCA 4 5

Figure 19 a and b. HT-29 cells treated for 24 hours with: DCA, 250 µM; (1) 3-phenylpropionic acid, 100 µM; (2) 3-hydroxyphenylacetic acid, 100 µM; (3) 3,4-dihydroxyphenylacetic acid, 100 µM; (4) 3-(4-hydroxyphenyl)-propionoic acid, 100 µM; (5) ferulic acid, 100 µM. Untreated HT-29 cells (C).

37


The compounds tested alone affected neither COX-2 enzymatic activity nor COX-2 protein levels. Thus, the compounds responsible for the observed effects of faecal water on COX-2 have yet to be determined. Faecal water samples are complex mixtures of compounds, therefore it is likely that the observed effects are due to a synergistic action of several compounds. Alternatively, other non-phenolic com-pounds may be responsible for the observed effects.

Table 5. Effects of diet-related compounds on COX-2 catalysed prostaglandin biosynthesis (n=3).

Compound Conc.(µM)

Inhibition (%)

Flavonoids

Daidzein 500 -5.9±3.8

Genistein 500 1.3±3.1

Hesperetin 500 11.5±2.9

Kaempferol 100 14.4±3.5

Bile acids

Chenodeoxycholic acid 100 4.7±2.7

Cholic acid 100 -2.7±1.8

Deoxycholic acid 100 7.4±3.7

Lithocholic acid 100 9.4±1.4

Taurocholic acid 500 -1.9±3.9

Phenolic acids

Benzoic acid 100 -9.6±5.5

Ferulic acid 100 0.6±5.2

Gallic acid 100 -3.5±3.5

3-phenylpropionic acid 500 0.6±3.0

3-OH-phenylacetic acid 500 -0.5±3.5

3,4-diOH-phenylacetic acid 300 -3.9±5.8

3-(4-OH-phenyl)-propionic acid 300 1.6±8.3

Procathechuic acid 100 -2.0±4.9

Other

Butyric acid 100 -6.9±4.7

Conclusion

Our diet plays an important role in the development of colon cancer. The com-pounds in food and in faecal water, have the ability to effect targets in the colon in positive or negative ways. Human faecal water samples are complex biomasses, with a broad range of compounds, including various phenolic substances. To fully under-stand faecal water’s possible role in cancer development, it remains to subject faecal water to further analysis, regarding their chemical content and also their biological effects.

The compounds tested in this study, did not individually affect COX-2. It is likely that compounds in faecal water act in synergy. Thus, it may be valuable to study the effects on COX-2, assessing combinations of compounds. Besides evaluating the

38

Ulrika Huss

effects on COX-2 of pure compounds identifi ed in faecal water, bioactivity-guided isolation of faecal water provides an alternative way to fi nd the compounds respon-sible for the observed effects on COX-2.

Although the effects of faecal water on COX-2 catalysed prostaglandin biosynthe-sis and protein levels were weak, the cumulative exposure of colonic cells to faecal water, from a lifetime perspective, may make these seemingly small effects impor-tant in the development of colon cancer.

39


Cytotoxic effects of NSAIDs and COX-2 inhibitors of natural origin (paper IV)

COX-2 inhibitors and NSAIDs are suggested to become important tools in the pre-vention and treatment of cancer. In order to increase the general knowledge of the possible anti-cancer effects of natural COX-2 inhibitors, an investigation of their cytotoxicity on human cancer cell lines was carried out (Paper IV). The compounds included in the study exhibited different structural features, but all have the ability to inhibit COX-2 (fi gure 20).

Figure 20. Chemical structures of the eight compounds assessed for cytotoxicity.

The compounds were assessed for cytotoxicity using a human cancer cell-line panel comprising four sensitive parental cell lines, fi ve drug resistant sublines, and one cell line with primary resistance (table 6) (Dhar et al., 1996).

Table 6. Origin and mechanism of resistance of the cell lines (according to Dhar et al.1996).

SO2C H3

O

O

Rofecoxib

COOH

O C H3

O

Acetylsalicylic acid

H2C

OCH3

OH

CH

C H2

OH

OH

H C CH

C H

OOH

EugenolPyrogallol Cinnamaldehyde

N

ClO

C H3

COOHH3C O

Indomethacin

O O

OCH3

OH

H3C O

HO

Curcumin

OH

OH

HO

Resveratrol

Of the compounds evaluated, six out of eight cyclooxygenase inhibitors were found to decrease cell survival in one or several of the ten cell lines. Two of the syn-thetic compounds, indomethacin and rofecoxib, and four of natural origin, namely, three phenolic (curcumin, resveratrol, and pyrogallol), and one phenylpropane (cinnamaldehyde), induced a dose-dependent decrease in cell viability in one ore several of the cell lines (table 7), whereas the two compounds, acetylsalicylic acid and eugenol, did not effect cell survival at the tested concentrations. The diarylhep-

Parental line Resistant line Origin Selecting agent Mechanism ofresistance

ACHN -- renal adenocarcinoma -- Primary MDR

CCRF-CEM CCRF/VM-1 T-cell leukaemia Teniposide Topo II-associated

RPMI 8226S RPMI 8226/LR5 myeloma Mephalan GSH-associated

RPMI 8226/S RPMI 8226/DOX40 myeloma Doxorubicin Pgp-associated

NCI-H69 H69-AR small cell lung cancer Doxorubicin MRP-associated

U-937 GTB U-937-vcr histiocytic lymphoma Vincristine Tubulin-associated

40

Ulrika Huss

tanoid, curcumin, a naturally occurring compound in the spice turmeric (Curcuma longa L.), was the most potent compound, and in addition, was found to affect cell survival in all ten cell lines.

Table 7. The cytotoxic effects of the natural and synthetic COX-2 inhibitors. The cytotoxicity is represented as estimated IC

50 values (µM) of compounds for the human cell-line panel.

Compound ACHN CCRF-

CEM

CCRF-

VM-1

8226S 8226

LR5

8226

DOX

NCI-

H69

NCI-

H69AR

U-937

GTB

U-937

Vcr

ASA >500 >500 >500 >500 >500 >500 >500 >500 >500 >500

Cinnamaldehyde >200 92 50-100 120 >200 >200 100-200 50-100 >200 >200

Curcumin 47 10-50 27 10-50 5-10 10-50 31 10-50 26 24

Eugenol >200 >200 n.d. >200 >200 >200 >200 >200 >200 >200

Indomethacin >50 >50 >50 >50 >50 >50 >50 10-50 >50 >50

Pyrogallol >200 100 >200 87 100-200 118 44 >200 160 >200

Resveratrol >100 >100 n.d. 71 57 10-50 >100 >100 58 70

Rofecoxib >50 >50 >50 >50 >50 >50 >50 10-50 >50 >50

n.d., not determined

Conclusion

COX-2 inhibitors are implicated as important tools in the prevention and treatment of cancer; yet the mechanisms by which these compounds exert their anti-cancer effects are not fully understood. Thus, the present study contributes to increase the general knowledge of the effects on cancer cells of natural and synthetic COX-2 inhibitors. Four of the natural COX-2 inhibitors decrease cell survival in a variety of cell lines in the human cell-line panel. The natural occurring diarylheptanoid, curcumin, was the most potent compound, and in addition, decreased cell survival in all ten cell lines. Of the synthetic compounds evaluated, indomethacin and rofecoxib were found to affect cell survival, but only in the NCI-H69AR cell line. Future studies will reveal whether the observed cytotoxic effect is correlated to an interference with a cyclooxygenase-dependent pathway.

Noticeably, studies have shown that NSAIDs can enhance the effects of anti-cancer drugs such as doxorubicin in certain cancer cell lines (Duffy et al., 1998; Roller et al., 1999). Hence, at present we have initiated a study in which the effects of a combination treatment of natural COX-2 inhibitors and the anti-cancer drug doxorubicin will be investigated.

41


Concluding Remarks

The knowledge about dietary plant constituents and their infl uence on COX-2 and cancer is still in its initial stage. One way to expand this knowledge is to investigate the effects of plants constituents and faecal water on COX-2 at various cellular levels. Thus, there is a vast need for different methods to evaluate the effects on COX-2 of plants and isolated natural compounds.

The COX-2 catalysed prostaglandin E2 biosynthesis, based on scintillation prox-

imity-assay technology, is a rapid method with a high sample capacity for evaluating inhibition of COX-2 enzymatic activity (Paper II). To study in depth the effects of compounds, the cell model enables investigation of the effects on COX-2 and iNOS at several levels, including that of mRNA, protein, prostaglandin E

2, and nitrite.

Bioassay-guided isolation of the plants, Plantago major L. and Urtica dioica L., resulted in isolation of fatty acids as inhibitors of COX-2 enzymatic activity (Paper I). Structurally related fatty acids assessed for COX-2 inhibition revealed variation in potency. One fatty acid, compound 2, decreased COX-2 protein levels in the murine macrophage cell line after a 5 h treatment. However, further studies are nec-essary to investigate in depth the effects of fatty acids on COX-2 protein synthesis in cells. Two phenolic compounds were also found to inhibit COX-2 enzymatic activity (Paper II). Fatty acids and phenolic compounds are commonly occurring in plants. These compound are often regarded solely as compounds interfering with different targets in bioassays. They may not be suitable for development of conventional drugs, but as natural parts of our diet, may exhibit important effects on different biological targets, thus worthwhile to study in depth. Nevertheless, dereplication of these compounds from plant extracts, will increase the likelihood of identifying new COX-2 inhibitory compounds in plants.

Compounds in our food are believed to affect the development of cancer, a process involving COX-2. The infl uence of diet on COX-2 in relation to colon cancer was investigated by studying the effects of faecal water’s on COX-2 in a colon cancer cell line (Paper III). In this pilot study, faecal water from twenty vegetarians were assessed. A vegetarian diet, rich in fruit and vegetables, includes a wide variety of phytochemicals that are likely to exert various biological effects in the colon. The faecal water samples in our study, exhibited both inhibitory and stimulatory effects on COX-2. Although, the observed effects of faecal water’s on COX-2 were weak, the cumulative effect over time may be important for the development of colon cancer. A follow-up study to investigate the effects of a new collection of faecal water from the same vegetarian group may reveal important information about possible day-to-day variations. Future studies might also comprise a larger study population of vegetarians, but also include a group of omnivores to see whether the observed effects follow a specifi c dietary pattern. Study populations with a controlled diet may possibly increase our insight into the effects of specifi c food categories.

42

Ulrika Huss

The identifi cation of COX-2 as a possible target for anticancer drugs challenged us to study the cytotoxic effects of COX-2 inhibitory compounds (Paper IV). Of the compounds assessed for cytotoxicity, curcumin was the most potent compound, in addition decreasing cell survival in all ten human cancer cell lines. A prospect for future studies is to further investigate whether the cytotoxicity is mediated via an infl uence on COX-2.

In conclusion, this thesis has increased the general knowledge of the effects on COX-2 of commonly occurring plant constituents, including fatty acids and phe-nolic compounds. As natural parts of our diet, these compounds, abundant in fruits and vegetables, may affect several targets in our bodies, including COX-2.

43


Acknowledgement

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

I wish to express my sincere gratitude to:

My supervisor Premila Perera, I am deeply grateful that I had the opportunity to share in your knowledge of both science and spirituality. Empathy is the most precious gift in life.

My supervisor Lars Bohlin, thank you for valuable discussions and support, for providing excellent working facilities, and also for giving me the opportunity to fi nd my own way.

My supervisor Mervi Vasänge, thank you for your enthusiasm, for guiding me in “the knowledge of writing”, and for always encouraging me.

My collegues and “offi ce-mates”; Therese Ringbom, for being an outstanding team-member, for all the knowledge that you have shared during these years, and for a never failing support; and Erika Svangård for being such an amazing person “sparkling with energy”, always willing to help everyone (and also being an excellent “travel-mate”, is there any language you are not able to speak? -“Vamos a la playa”). Along the road of PhD life, we have shared many “ups and downs”. I am grateful for the time we have spent together, I have learnt so much from you both.

The “senior PhDs”; Ulf Göransson for good advice, never hesitating to solve problems or take part in building “strange investigations”, and for your ironic encouragement in a silent Norrlandic way; and Senia Johansson for all the words of wisdom you have shared. Thank you for your support and willingness to share your knowledge.

Per Claeson, Jan G. Bruhn, Finn Sandberg, Gunnar Samuelsson, Anders Backlund, Wenche Rolfsen, Kerstin Ståhlberg, Maj Blad, and Siv Berggren, for your inspiring discussions and support.

The “team of pharmacognosy”; Åke Stenholm (thank you for your thoughtfulness), Wimal Pathmasiri, Charlotte af Klercker, Petra Lindholm, Sonny Larsson (you are an outstanding well of wisdom and kindness), Erik Hedner, Anders Herrmann (keep up the good spirit), Sofi a Lindgren, Martin and Mia; for your support and fi nding time for both scientifi c and maybe not so scientifi c discussions.

My co-authors, for a fruitful collaboration.

The members of the Division of Pharmaceutical Biochemistry, for introducing me to molec-ular biology and for always patiently sharing your knowledge. Malin Hobro-Söderberg thank you for your friendship, you know your energy is contagious.

My collaborators; the members of the Division of Clinical Pharmacology; Rolf Larsson, Joakim Gullbo, and Sumeer Dhar, you are an amazing team, with you I have never heard that things are impossible; the members of the Department of Medical Nutrition, Joseph Rafter and Pernilla Karlsson, for your enthusiasm and support, and for introducing me to the world of nutrition, which is fascinating in many aspects.

44

Ulrika Huss

The former team of “drug discovery” at Pharmacia, for sharing your knowledge, and also for being so enthusiastic and friendly.

Tina Kunz, thank you for providing a wealth of wisdom during the fi nal hours, I am grate-ful to you for thoroughly reading and discussing my thesis. “Heja, heja” - what else could be necessary to hear during the fi nal weeks?

Magnus Jansson for helping me with the layout of this thesis.

Håkan Tunon, for all your encouragement in a typical “Tunon-manner”, thank you for providing me with nice pictures of plants (last minute is also a minute).

To post-docs and guest reseachers; Laura Segura, (for valuble discussions, and for your lovely Spanish dinners), Adriana Broussalis, Ina Hansen, Hesham El-Seedi and Shaden Kalifa (my “Egyptian family”, thank you for your words of wisdom, and Hesham for dis-cussions and valuable comments on my thesis, and for patiently teaching me the basics in NMR); thank you all for your warm hearts and friendship.

My students; Elisabeth Hola, Martin Molin, Marisa Padoan, Peter Fyhr, Josefi n Larsson, and Jenny Pettersson for skilful technical assistance. Jenny, your help with the thesis in the fi nal hours has been most valuable.

Paul Johnson, for excellent linguistic revision, and for being such an enthusiastic person.

My family; father Haldo and mother Karin, Ann-Katrin and Joakim, Torbjörn and Sal-lyann, grandmother Aili and my late grandfather Arno (for your strong belief in me), and grandmother Lilly; all of you for good advice in all aspects of life, always encouraging me and spreading sunshine in my life. Jacob and Tobias för alla era raka och kloka konstateran-den om livet. Antero and Marianne for your thoughtfulness and support.

The “Melin-family”; Anita and Hans, Anna and Jonas, Barbro and Gösta, and “the Wed-marks”, for your warm hearts, encouragement and all support during these years. I have been lucky to have you in my life.

My friends; Jois, Helena and Anna, for sharing spiritual and non-spiritual thoughts, you are unique. What else can I say? Malin and Sara for always cheering me up, inviting me to dinners, and pushing me to exercise. All of you reminding me about the important things in life.

Anders, my love, for sharing my days, in sunshine, moonlight and in rain. Your love and patience have been immense. Thank you for also spending so many nights with data-sup-port, organizing and proof reading references and providing valuable opinions.

Last but not least, all of you that have encouraged me and made my days sunny in various ways, by dinners, sharing good advice and being there for me. Although not mentioned specifi cally, I will always remember your support.

45


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APPENDIX

General Experimental Procedures –Isolation of fatty acids in Urtica dioica L.

Medium pressure liquid chromatography (MPLC) was performed with SEPARO MPLC equipment (Baeckström Separo AB, Lidingö, Sweden). The columns used were variable-length glass columns with an inner diameter of 1.5 cm. An FMI Lab pump, model QD (Fluid metering Inc., Oyster Bay, NY, USA) was used in the system. Fractions of 5 ml were collected using a Gilson model 201 fraction collec-tor.

Plant material

The aerial parts of Urtica dioica L. were collected in July 1997, in Uppsala, and iden-tifi ed by Dr. Inga Hedberg (Department of Systematic Botany, Uppsala University). A voucher specimen, coded AIP 9707003, has been deposited at the Division of Pharmacognosy, Uppsala.

Extraction and Isolation

The dried and powdered plant material (30.0 g) was successively extracted in a Soxhlet apparatus with 800 ml hexane, CH

2Cl

2, MeCN, and H

2O, respectively. All

extracts were assessed for COX-2 inhibitory effects, and the hexane extract was most active. The extract (0.5 g) was dissolved in a minimum amount of hexane, absorbed on 1.6 g Silica 60 (Mesh 230-400) and applied to a MPLC column (height 1.7 cm), which was connected to a second column packed with Silica gel 60 (height 5.1 cm). A fl ow rate of 20.9 ml/min was used. The columns were eluted with a continu-ous gradient from hexane, over EtOAc to MeOH and H

2O. Initially the mixing

chamber contained 50 mL of hexane, and the reservoir contained the fi rst of the 13 premixed binary (less polar/more polar solvent) gradient mixtures, of 50 ml each, which were successively fed to the reservoir during separation. Every third fraction was spotted on thin layer chromatography (TLC) plates (Silica gel 60, Merck, layer thickness 0.2 mm) eluted in CHCl

3–MeOH–H

2O (9:3:0.5), detected with UV

light (254 and 366 nm) and by spraying with vanillin-sulphuric acid reagent fol-lowed by heating (120ºC). Fractions of similar composition were combined, yield-ing seven fractions, which were tested in the COX-2 assay.

Fraction 3, was found most active and inhibited COX-2 by 92±2.5% and COX-1 by 88±3.6%. Purifi cation of that fraction (43 mg) was performed by column chro-matography over Sephadex LH-20 (CH

2Cl

2), yielding four fractions (A, B, C, and

D). All fractions were assessed for COX-2 inhibition. The most active fractions, C and D, inhibited COX-2 by 89±1% and 90±0.3%, respectively. The COX-2 inhibi-tory effects of these two fractions were related to the presence of unsaturated fatty

55


acids. 1H-NMR spectra were recorded in CDCl3 (270 MHz) with TMS as internal

standard. The 1H-NMR spectra showed the characteristic olefi nic protons at δ 5.36 ppm with typical long chained methylene protons at δ 1.25 and 1.32 ppm, and ter-minal methyl group at δ 0.98 (t, J = 6.6 Hz), in addition to the signal at δ 2.34 for CH

2–COOH.

COX-2 catalysed prostaglandin biosynthesis

Extracts and fractions were assessed for COX-2 inhibitory effect using the COX-2 catalysed prostaglandin biosynthesis in vitro assay as described previously by Noreen et al. (1998). The extract and fractions were dissolved in 20 % DMSO, yielding a fi nal concentration of 4% solvent in the assay, and tested at 100 µg/ml.

General experimental procedures – Murine macrophage experiments

Cell culture and treatment with test compounds

RAW 264.7 cells, a murine macrophage cell line, were purchased from the Euro-pean collection of cell cultures (ECACC, Salisbury, UK) and used between pas-sages 10 and 20 for all experiments. Cells were maintained in Dulbeccos modifi ed essential medium (DMEM) (Sigma), supplied with 2 mM L-glutamin, antibiotic/antimycotic solution, and 10% fetal bovine serum (Gibco BRL), in a humidi-fi ed atmosphere of 95% air and 5% CO

2 at 37°C. In experiments, cells (1 x 106)

were plated in 60 mm dishes and grown for 72 h until reaching 70–80% confl u-ence. Medium was changed 1 h before cells were treated with test agents. Cells were treated with or without lipopolysaccharide (Escheria coli, serotype 0127:B8) (Sigma), in presence or absence of fatty acids, according to the indicated times. The fatty acids were dissolved in DMSO with a fi nal concentration of solvent in the cell cultures of 0.1%.

Cell viability

Cell viability was analysed by measuring the capacity of the macrophages to reduce 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazoliumbromide (MTT, Sigma). Cells were incubated with MTT (fi nal concentration 0.5 mg/ml) for 45 min at room tem-perature. Cell medium was removed and the purple formazan product was dissolved in DMSO. Thereafter, the product formation was detected spectrophotometrically at 570 nm.

RNA isolation and Northern blot analysis

RNA was isolated using a RNeasy Mini kit (Qiagen), according to the manufac-turer’s instructions. Twenty µg total RNA was subjected to electrophoresis in 1.2% agarose/formaldehyde gel. Thereafter, the RNA was transferred to a Hybond™-nylon membrane (Amersham Pharmacia Biotech). The membrane was crosslinked

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Ulrika Huss

before hybridisation. COX-2 cDNA (Cayman Chemical Company) and a plasmid containing the 4 kb murine NOSII cDNA (kindly provided by Dr. Charles J. Lowenstein, John Hopkins University, Baltimore, USA) were radiolabelled with [ α-32P] dCTP using a Megaprime labelling kit (Amersham Pharmacia Biotech). Membranes were prehybridised and hybridised at 65ºC in Church buffer (Church and Gilbert, 1984), modifi ed to contain 0.25 M NaHPO

4, pH 7.2, 7% SDS, and

1 mM EDTA. Hybridisation was done over night with 1.7 x 107 CPM of radiola-belled probe. As a control for equal loading of the samples, the mRNA of the house keeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was measured using radiolabelled GAPDH cDNA (Clontech).

Protein isolation and Western blot analysis

Cytoplasmic proteins were isolated according to Tsai et al., (1999). Briefl y, cells were scraped in 1 ml PBS and centrifuged at 2,000 x g for 30 s. The cell pellet was resuspended in 100 µl lysis buffer (10 mM Hepes, pH 7.6; 10 mM KCl; 0.1 mM EDTA; 1 mM DTT; 0.5 mM phenylmethylsulphonyl fl uoride). The pellet was left on ice for 10 min and was then homogenised. The cells were thereafter centrifuged at 12,000 x g for 20 seconds at 4ºC, and the supernatant containing the cytoplasmic proteins were removed and stored in -80ºC. Protein concentrations of the samples were determined by Lowry´s method (Lowry et al., 1951). Protein (50 µg) from the cell extracts was separated by SDS/PAGE (12%), and transferred to a PVDF transfer membrane, Hybond™-P (Amersham Pharmacia Biotech). The membrane was then incubated with primary anti-bodies against COX-2 (BD Transduction laboratories), 1:250 dilution; anti-iNOS, 1:500 dilution; and anti-Actin, 1:250 dilution (Santa Cruz). The blots were then incubated with the corresponding sec-ondary antibody and conjugated to horseradish peroxidase, and detected with the ECL Western blotting analysis system (Amersham Pharmacia Biotech).

Analysis of PGE2 and nitrite levels

The cell supernatant from RAW 264.7 cells, cultured 20 h for protein determina-tions (as described), was collected and used to measure the release of PGE

2 and

nitrite. To estimate the amount of PGE2 produced, a RIA system was used. The

samples were assessed according to the RIA protocol supplied by Sigma Chemical Company (P5164). Anti-PGE

2 was provided by Sigma Chemical Company, and

[3H]-labelled PGE2 was purchased from Amersham Pharmacia Biotech.

As an indicator of NO production, the nitrite concentration in the cell super-natant was determined using the Griess assay (Sigma). Briefl y, 100 µl of cell supernatant was combined with 150 µl Griess reagent (sulphanilamide/N-(1-naphtyl)ethylenediamine dihydrochloride), in a 96-well plate, and was incubated for 15 min at room temperature. Thereafter, the optical density was measured at 540 nm. Nitrite concentration was determined by comparison with a sodium nitrite standard curve.

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from the Faculty of PharmacyEditor: The Dean of the Faculty of Pharmacy

Distribution:Uppsala University Library

Box 510, SE-751 20 Uppsala, Swedenwww.uu.se, [email protected]

ISSN 0282-7484ISBN 91-554-5739-8

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