Studies on the anti cancer potential of sesquiterpene lactone parthenolide

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CHAPTER 1

CHAPTER 1
INTRODUCTION

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1.1

Parthenolide

1.1.1

Introduction: feverfew and Parthenolide
Feverfew (Tanacetum parthenium) is an aromatic perennial herb grown

originally in Europe. It has been traditionally used as an herbal medicine in treatment
of migraine, fever, arthritis and menstrual problems since ancient times. In the last
few decades, feverfew became especially popular in Britain, France and Canada as a
phytomedicine. Powder of dried leaves has been made into capsules or tablets for oral
consumption. The major pharmacological benefits of feverfew include: (i) prevention
of migraine, (ii) relief of fever, and (iii) treatment of rheumatoid arthritis and
inflammatory disease (Berry, 1984; Johnson et al., 1985).
The main chemical constituents of feverfew include sesquiterpene lactones,
flavonoid glycosides and monoterpenes (Abad et al., 1995). The most predominant
and well studied active component of feverfew is a group of sesquiterpene lactones
such as parthenolide, costunolide and germacranolide, etc. (Knight, 1995). These
sesquiterpene lactones are enriched in the leaves and seeds of the feverfew which are

believed to be produced by superficial leaf glands and act as general insect repellents
for plants. Among feverfew extracts, parthenolide is the principle sesquiterpene
lactone responsible for most of the pharmacological effects of feverfew (Knight,
1995).
1.1.2

Chemical structure, metabolism and bioactivities of parthenolide

1.1.2.1 Chemistry: sesquiterpene lactones and parthenolide
Chemically, parthenolide belongs to the group of sesquiterpene lactone. It was
first isolated from feverfew in 1965 (Berry, 1984). Parthenolide is a 4,5-epoxygermacra-(10), 11(13)-dien-12,6-lactone and its structure is shown in Fig. 1.1.

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CH3

CH2
O
H3C

O
O

Fig. 1.1 Feverfew and chemical structure of parthenolide

The molecular structure contains a terpene compounds with fifteen carbon
atoms and an exocyclic methylene lactone group, an α-methylene-γ-lactone moiety,
which has been shown to process an anti-inflammatory activity. The α-methylene-γlactone group has an exceptional ability to react with nucleophiles by Michael type
addition (Kupchan et al., 1970). Parthenolide has the ability to alkylate intracellular

nucleophiles, such as L-cysteine, glutathione (GSH) and a number of thiol-bearing
cellular proteins, to form adducts (Fig. 1.2) which are believed to be responsible for
its pharmacological effects (Hall et al., 1979; Groenewegen et al., 1986; Knight,
1995). Parthenolide derivative, 11β,13-dihydroparthenolide with saturated exocyclic
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methylene group completely loses its bioactivity suggesting the biological importance
of exocyclic methylene group (Marles et al., 1992). In addition, parthenolide
possesses an epoxide moiety, another functional group with alkylating ability, which
results in an enhanced bioactivity compared to other sesquiterpene lactones. Recently,
it reported that the spatial arrangement of the terpenoid skeleton is more important
than any other functional groups (Neukirch et al., 2003) which are generally believed
to be responsible for the bioactivities of parthenolide.

Fig. 1.2 Formation of parthenolide-thiol adducts

1.1.2.2 Transportation in cell system and bioavailability
After administration of parthenolide, it can be quickly transported and absorbed
by human cells. Khan et al. (2003) has reported that parthenolide is predominately
transported into human intestinal cells (Caco-2) through passive diffusion which can
not be prevented by an inhibitor of multidrug resistance transporter P-glycoprotein
(MRP). This report provides the evidence of the bioavailability of the parthenolide in
cell culture system (Khan et al., 2003). In the in vivo animal model, although the
bioavailability of parthenolide has not been reported, recently work in our laboratory
using UVB-induced skin cancer model suggested the bioavailability of parthenolide
as parthenolide-feeding showed a delayed onset of papilloma incidence and a
significant reduction in papilloma multiplicity (Won et al., 2004).

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1.1.2.3 Bioactivities of parthenolide
Thiol reactivity
As discussed earlier, the chemical reactions of parthenolide involve a covalent
conjugation between α,β-unsaturated carbonyl structure of parthenolide with various
nucleophilic sulphydryl residues (e.g. thiols) resulting in alkylation through Michael
type addition. The binding of parthenolide with sulphydryl groups present in
intracellular proteins may disrupt the normal cellular function of macromolecules.
The three-dimensional structure of both parthenolide and its potential target are the
decisive factors for the steric accessibility of parthenolide to its target. Appropriate
three-dimensional structure is the essential premise for bioactivities of parthenolide
and may provide some extent specificity (Yoshioka et al., 1973). However, the
biological consequences and the importance of parthenolide’s thiol reactivity are
poorly studied.
Anti-inflammatory activity
Inflammation is an important biological process contributing to wound healing
and pathological responses to infection. A complex network of signaling factors
involved in inflammatory response has been evolved (Coussens and Werb, 2002).
Anti-inflammatory activity is one of the most prominent bioactivities of parthenolide.
Feverfew has been traditionally used as a herbal medicine by ancient Greeks and early
Europeans for treatment of inflammatory diseases, such as fever and rheumatoid
arthritis (Berry, 1984). In 1989, a double-blind study carried out in the UK,
demonstrated the effectiveness of feverfew in treatment of the symptoms of
rheumatoid arthritis (Pattrick et al., 1989) which may due to pharmacological
inhibition of pro-inflammatory cytokine prostaglandin (PG) synthesis. The following
in vitro studies further elucidated that feverfew as well as its major bioactive

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component, parthenolide, are potent inhibitors of macrophage production of release of
a group of pro-inflammatory cytokines, including tumor necrosis factor (TNF) and
interleukins (IL) (Hwang et al., 1996) and this inhibitory activity are mainly regulated
via disruption of nuclear factor - kappaB (NF-κB) signaling pathway (Hehner et al.,
1999) and signal transducers and activators of transcription (STAT) pathway (Sobota
et al., 2000).
Anti-cancer activity
It has been suggested that chronic inflammation promotes cancer development
in many types of cancers, such as breast, colorectal, and liver cancer (Coussens and
Werb, 2002). The potent anti-inflammatory activity of parthenolide implies its
potential anti-cancer property. Parthenolide has been reported to interrupt cell cycle
regulation and induce apoptosis in human cancer cells (Wen et al., 2002). In addition,
Patel et al. (2000) and his colleagues also demonstrated that pre-treatment of
parthenolide greatly sensitizes the human breast cancer cells in response to anticancer drug paclitaxel and tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL)-induced apoptosis (Patel et al., 2000; Nakshatri et al., 2004). However, the
anti-cancer potential of parthenolide is still largely unknown. Further studies,
including both in vitro mechanism studies on anti-cancer potential of parthenolide and
in vivo animal study, are needed.
1.1.3

The molecular mechanisms involved in the bioactivities of parthenolide

1.1.3.1 Effects on NF-κB signaling
NF-κB is a highly inducible transcription factor in response to diverse
stimulations such as TNFα, ultraviolet (UV), interleukins, endotoxins etc. NF-κB
activation plays a pivotal role in regulating the inflammatory responses, cell
growth/differentiation and apoptosis (Li and Verma, 2002; Karin and Lin, 2002). In
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the typical NF-κB signaling pathway, NF-κB is a heterodimer formed by RelA (p65)
and p50 subunit which are sequestered as an inactive form in the cytoplasm by NF-κB
inhibitory protein (IκB). Upon stimulation, the IκB protein is phosphorylated by IκB
kinase (IKK), ubiquitinized and degraded by 26s proteosome. This process results in
unmasking the nuclear localization sequence (NLS) of NF-κB protein, NF-κB nuclear
translocation and activation (Li and Verma, 2002). Bork et al. (1997), for the first
time, screened the inhibitory effects of 54 Mexican Indian medicinal plants on
transcription factor NF-κB. Their data suggested that parthenolide, the major
bioactive sesquiterpene lactone extracted from feverfew, shows a potent inhibitory
effect on NF-κB activation at a concentration as low as 5µM (Bork et al., 1997). Later
on, Hehner et al. (1998) reported that parthenolide inhibits several stimulations (TPA,
TNFα, ligation of T cell receptor and hydrogen peroxide)-induced NF-κB activation
by blocking the degradation of phosphorylated IκB while the DNA binding activity of
NF-κB has not been interfered (Hehner et al., 1998). In a subsequent study, they
demonstrated that parthenolide directly inhibits the IKK activity induced by TNFα,
while TNFα-inducible mitogen-activated protein kinase (MAPK) signaling pathways
(p38 and JNK) are not affected by parthenolide (Hehner et al., 1999). Furthermore,
parthenolide is capable of blocking NF-κB activation induced by overexpression of
upstream activators of IKK, such as TNF receptor associated factor 2 (TRAF2) and
mitogen-activated protein kinase 1 (MEKK1), which lead to a conclusion that
parthenolide inhibits TNFα-induced NF-κB activation by targeting the IKK complex
(IKC) (Hehner et al., 1999). At the same time, Rungeler et al. (1999) screened the
inhibitory activity on NF-κB of 28 sesquiterpene lactones including parthenolide.
Using a computer modeling, they proposed a possible molecular mechanism, which
the inhibitory activity of parthenolide may due to the alkylation and cross-linking of

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two cysteine residues (cys 38 and cys 120) located on the p65 subunit of NF-κB
(Rungeler et al., 1999).

Subsequently, two hypotheses emerged to explain the

inhibitory effect of parthenolide on NF-κB activation. First, parthenolide inhibits NFκB activity by direct targeting and inhibiting the IKKβ. Single amino substitution in
the activation loop (C179A) of IKKβ abolished the parthenolide’s IKKβ inhibitory
activity (Kwok et al., 2001). Second, parthenolide suppresses NF-κB activation by
direct modification of p65 at cys38 via alkylation, which in turn prevents NF-κB
DNA binding (Garcia-Pineres et al., 2001). At present, the possible effects of
parthenolide upstream of IKK activation have not been studied.
1.1.3.2 Effects on inflammatory-related molecules
Cytokines
A big family of cytokines plays a pivotal role in inflammatory responses. The
cytokine signaling is highly complicated and the effect of parthenolide seems to be
dependent on specific cell line and cellular contexture. During inflammatory
responses, NF-κB is the most important regulator of the gene expression of proinflammatory cytokines (Tak and Firestein, 2001). Inhibition of NF-κB pathway is
believed to be one of the major mechanisms responsible for anti-inflammatory
activity of parthenolide. Meanwhile, the inflammatory cytokines, such as TNFα and
IL-1 could also activate the NF-κB pathway which in turn promotes the inflammatory
response through a positive feedback control (Lucey et al., 1996). The effects of
parthenolide on expression of several cytokines have been reported. Mazor et al.
(2000) first reported that parthenolide shows a potent inhibitory effect on IL-8
expression in human respiratory epithelium cells (Mazor et al., 2000). In human
macrophages, Kang et al. (2001) observed a similar inhibitory effect of parthenolide
on IL-12 production induced by LPS. The p40 promoter activity of IL-12 which

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contains a NF-κB binding sequence has been greatly suppressed by parthenolide
(5µM) (Kang et al., 2001). As NF-κB regulatory elements have been found in the
promoter sequences of many pro-inflammatory cytokines, the inhibitory effects of
parthenolide on other cytokine secretion have also been demonstrated. In Li-Weber et
al (2002)’s report, IL-4, IL-2 and IFN-γ secretion from normal peripheral blood T
cells are also suppressed by parthenolide (Li-Weber et al., 2002). All these findings
indicate that the parthenolide executes its anti-inflammatory effects by regulating the
secretion of pro-inflammatory cytokines at transcriptional level via inhibition of NFκB.
5-hydroxytryptamine (5-HT) and anti-serotonergic activity
5-HT is a monoamine neurotransmitter secreted by central nervous system and
platelets. It is believed that 5-HT plays a central role in migraine pathophysiology
(Peroutka, 1990). The anti-secretory activity of feverfew extracts, including
parthenolide, was reported in the 1980s (Groenewegen et al., 1986) and parthenolide
has been found to possess a potent inhibitory effect on 5-HT secretion and platelet
aggregation induced by a number of stimulations (Groenewegen and Heptinstall,
1990).

It has also been suggested that parthenolide may act as a low affinity

antagonist at 5-HT receptors (Weber et al., 1997). Recently, the inhibitory effects of
feverfew and parthenolide on 5-HT have been re-examined again (Mittra et al., 2000).
However, the exact mechanisms are still not fully elucidated.
Cell adhesion molecules (CAM)
In the inflammatory process, CAMs, which mediate the interaction between
leukocyte and endothelium cells, play a key role to recruit and accumulate leukocytes
to the site of inflammation (Ulbrich et al., 2003). Many anti-inflammatory drugs
process the inhibitory effect of the CAMs. Since feverfew is a well documented anti-

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inflammatory herbal medicine, the potential effects of its main active component,
parthenolide, on CAMs have also been reported. Piela-Smith and Liu (2001) first
reported that both feverfew extracts and parthenolide greatly inhibited the intracellular
cell adhesion molecule-1 (ICAM-1) expression induced by various cytokines
stimulation, including IL-1, TNFα and IFNγ (Piela-Smith and Liu, 2001). As the
ICAM-1 promoter sequence has a response element potentially regulated by NF-κB,
the suppressed expression ICAM-1 is likely due to the inhibitory effect of
parthenolide on NF-κB (Melotti et al., 2001). Besides the ICAM-1, the effect of
parthenolide on expression of other CAMs has also been addressed. Furthermore,
(VCAM-1)-induced by IL-4 via JAK2-STAT6 signaling was significantly suppressed
by parthenolide (Schnyder et al., 2002). It is interesting to note that this inhibitory
effect seems converge to the suppression of STAT6 nuclear translocation and DNA
binding by an unknown mechanism and the phosphorylation of STAT6 is not affected
by parthenolide (Schnyder et al., 2002). Currently, it is believed that suppression of
CAMs expression which alleviates the inflammatory response is one of the
mechanisms of anti-inflammatory activity of parthenolide.
iNOS and NO production
Nitric oxide (•NO) is one of the important regulator molecules in inflammatory
response. The synthesis and release of the •NO are mediated by an inducible isoform
of nitric oxide synthase (iNOS). Parthenolide has been demonstrated to suppress the
gene expression of iNOS in several cell lines under stimulation of LPS, interferon-γ
(INFγ) or 12-o-tetradecanoylphorbol-13-acetate (TPA) (Fukuda et al., 2000). In rat
aortic smooth muscle cells, parthenolide inhibits the iNOS gene transcription which is
highly correlated with its suppressed activation of NK-κB, one of regulators of iNOS
gene expression (Wong and Menendez, 1999). Similar effect of parthenolide on iNOS

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promoter activity was observed in human monocyte cells in conditions with TPA or
without TPA stimulation and the IC50 is approximately as low as 2µM (Fukuda et al.,
2000). Moreover, in an in vivo rodent’s endotoxic shock model, reduction of iNOS
mRNA and •NO level by parthenolide has also been reported (Sheehan et al., 2002).
This beneficial effect during endotoxic shock also appears to be a direct result of NFκB inhibition by parthenolide. However, the inhibition of LPS-induced iNOS
expression and •NO synthesis in central nervous system seems not related to NF-κB
but rather due to an inhibitory effect on LPS-induced p42/p42 MAPK activation
(Fiebich et al., 2002). These studies imply an extensive role of parthenolide in
regulation of iNOS/NO in response to different stimulations.
COX-2 and prostaglandin production
Prostaglandin (PG) is another group of cytokines responsible for inflammatory
response. During PG synthesis, cyclooxygenase (COX) plays a main regulatory role
since it catalyzes the first rate-limit step in converting the 20-carbon polyunsaturated
fatty acid, such as arachidonic acid, to prostaglandin G2 and H2 (Smith et al., 2000;
Dubois et al., 1998). COX-2 is the highly inducible isoform of COX which plays a
central role in the inflammatory processes as it is induced by pro-inflammatory
cytokines and tumor promoters (Dubois et al., 1998; Williams et al., 1999). As the
COX-2 promoter sequence has NF-κB responsive elements, NF-κB is believed to be
one of the most important regulators of COX-2 expression (Chun and Surh, 2004).
Liang et al. (1999) reported that the inhibitory effect of apigenin and related
flavonoids on the inducible COX-2 and iNOS expression is due to suppressing
lipopolysaccharide (LPS)-induced activation of NF-κB (Liang et al., 1999). More
recently, Kojima et al. (2000) demonstrated further evidence that COX-2 expression
is stimulated by LPS via NF-κB in human colorectal cancer cells (Kojima et al., 2000).

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However, other transcription factor responsive elements are also present on the COX2 promoter sequence including cyclic AMP response element (CRE) and NF-IL-6
element (Kosaka et al., 1994) and it is also reported that COX-2 expression can be upregulated by different simulations through CRE or IL-6 regulatory mechanisms (Xie

et al., 1994; Han et al., 2003).
The effects of parthenolide on prostaglandin synthesis have been demonstrated.
Collier et al. (1980) first showed that feverfew aqueous extracts can inhibit
arachidonic acid metabolism and prostaglandins production without blocking the
cyclooxygenase expression in vitro (Collier et al., 1980). However, subsequent studies
by other researchers found that parthenolide inactivates the prostaglandin synthetasemediated pathway by interacting with sulphydryl amino groups of various enzymes
(Pugh and Sambo, 1988). Further studies demonstrated that feverfew and parthenolide
irreversibly suppress arachidonic acid metabolism via inhibition of cyclooxygenase
and 5-lipoxygenase activities in a leukocyte cell model (Sumner et al., 1992).
Moreover, Hwang et al. (1996) showed that parthenolide inhibits the inducible COX2 in LPS-stimulated murine macrophages cell line (RAW 264.7). Nevertheless, the
molecular mechanisms involved in parthenolide’s inhibitory effect on COX-2 remain
to be further addressed.
1.1.3.3 Effects on Mitogen-activated protein kinase (MAPK) pathway
The inhibition of kinase phosphorylation by parthenolide was first observed by
Hwang et al. (1996) in LPS-induced macrophage (RAW 264.7) cells. Cells exposed to
parthenolide resulted in a non-specific inhibition of MAPKs including extracellular
regulated protein kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) (Hwang et al.,
1996). This observation was further supported by Fiebich et al. (2002) who detected a
similar inhibitory effect on LPS-induced ERK activation by parthenolide. However,

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the phosphorylation of p38 induced by LPS was not affected by parthenolide (Fiebich
et al., 2002). Meanwhile, another study conducted by Uchi et al. showed that
parthenolide suppresses LPS-induced, not TNFα-induced, p38 activation in dendritic
cells which prevents the dendritic cells reaching maturation (Uchi et al., 2002).
Moreover, they also observed that the phosphorylation of upstream kinases of p38
signaling pathway, such as MAPK kinase (MKK) 3 and MKK6, induced by LPS has
also been abolished by parthenolide, indicating the possible upstream targets of

parthenolide in p38 signaling pathway (Uchi et al., 2002).

However, current

knowledge about the potential effects of parthenolide on MAPKs is rather limited.
The exact role of MAPK pathway in the bioactivity of parthenolide remains to be
further investigated.
1.1.3.4 Effects on Janus Kinase (JAK)-Signal Transducers and Activators of
Transcription (STAT) pathway and cytokine signaling
As discussed above, NF-κB-regulated cytokines play a critical role in
inflammatory responses. In addition to NF-κB, the JAK-STAT signaling pathway also
plays an important role in cytokine signaling and inflammatory response (Pfitzner et
al., 2004). The IL-1 type cytokines (IL-1, TNFα etc.) are the central regulators in the
early stage of the inflammatory process. The activation of NF-κB by these cytokines
up-regulates the expression of other cytokines, such as IL-6-type cytokines, which
further participate in the progression of the inflammatory. This process involves the
JAK-STAT pathway (Heinrich et al., 1998; Hanada and Yoshimura, 2002). The
possible effect of parthenolide on JAK-STATs pathway has also been addressed.
Sobota et al. reported that pretreatment with parthenolide not only blocked the
expression of IL-6 secretion but also suppressed the IL-6 signaling through inhibition
of phosphorylation of tyrosine 705 on STAT3 protein (Sobota et al., 2000). They also

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postulated that the conjugation with the sulphydryl (-SH) groups located on JAKs
may be responsible for the mechanism of inhibition. Schnyder et al. (2000)
demonstrated that the nuclear translocation of the STAT6 induced by IL-4 is
prevented by parthenolide. However, in their human endothelial cell model, the
phosphorylation of both upstream JAKs and STAT6 were not affected by

parthenolide (Schnyder et al., 2002). On the other hand, it is interesting to note that
parthenolide can increase the tyrosine phosphorylation of STAT5 protein and upregulate the STAT5 regulated genes, an effect probably due to the negative cross-talk
between NF-κB and STAT5 signaling pathway (Geymayer and Doppler, 2000).
1.1.3.5 Effects on cell proliferation and induction of apoptosis
The effects of parthenolide on cell proliferation and viability are less reported.
Hoffman et al. (1977) first showed the cytotoxicity of parthenolide to human
epidermoid carcinoma cell culture (Hoffmann et al., 1977). Further study was
conducted by Ross and her colleagues. They examined the inhibitory effects of
parthenolide on growth of two tumor cell lines in vitro. The low concentration of
parthenolide can reversibly inhibit the growth of tumor cell lines in a cytostatic
fashion (Ross et al., 1999).
Although the reports of cytotoxity of parthenolide can be traced back to the
1980s, the characteristics and mechanisms of parthenolide-induced cell death have not
been investigated, until recently, when Wen et al. (2002) reported that parthenolide
could trigger apoptosis in invasive sarcomatoid hepatocellular carcinoma cells. Their
study provided evidence of involvement of oxidative stress, caspase activation and
mitochondrial changes in parthenolide-induced apoptosis (Wen et al., 2002). They
also demonstrated that the stress sensitive gene GADD153 expression was induced in
parthenolide-induced apoptosis. On the other hand, parthenolide induced cell death

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was found to be atypical. Pozarowski et al. (2003b) reported, in HL-60 cells,
parthenolide concurrently induces atypical apoptosis and necrosis by unknown
mechanisms (Pozarowski et al., 2003b). Beside the direct apoptosis-inducing activity
of parthenolide, it has also been used in combination with other chemotherapeutic
drugs. In breast cancer cells (MDA-MB-231 and HBL100) which have constitutively
activated NF-κB, parthenolide pretreatment significantly sensitized the breast cancer
cells to chemotherapeutic drug paclitaxel-induced apoptosis (Patel et al., 2000). A

recent report indicated that parthenolide also sensitizes the breast cancer cells to
TRAIL-induced apoptosis via inducing a sustained JNK activation (Nakshatri et al.,
2004). These observations suggest that parthenolide may execute its anti-cancer
property via increasing the sensitivity of cancer chemotherapy. As apoptosis-inducing
ability of parthenolide is closely related to the potential of anti-cancer property, it is
important to further investigate the mechanisms of parthenolide-induced apoptosis
and the molecular targets of parthenolide.
1.1.3.6 Effects on cell cycle regulation
Ross et al. (1999) first reported that low concentration of parthenolide (5µM)
could inhibit cancer cell growth in a cytostatic fashion in vitro, implying certain
effects of parthenolide on cell cycle regulation (Ross et al., 1999). In HL-60 cells,
Pozarowski et al. (2003) observed a transient cell cycle arrest in G2/M phase followed
by an atypical apoptosis under a narrow concentration range (2-10µM) of
parthenolide treatment. This cell cycle arrest was not believed to be related to the
inhibitory effects of parthenolide on NF-κB (Pozarowski et al., 2003a). A similar
G2/M arrest can also be found in hepatoma cells treated with non-cytotoxic
concentration (1 to 3µM) of parthenolide (Wen et al., 2002). All these studies

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indicated that, at sub-lethal concentrations, parthenolide could inhibit cell growth by
inducing cell cycle arrest at G2/M.
1.1.4

in vivo study of parthenolide
The pharmacological effects of parthenolide have also been studied in several in

vivo animal models. In an albino mice model, both parthenolide and feverfew extracts
show an anti-nociceptive effect which significantly increases the pain threshold of

mice. In addition, the anti-inflammatory activity of parthenolide was also observed
(Bejar, 1996). Parthenolide has also been reported to help rodents (rats and mice)
overcome the endotoxin-induced shock. Both pretreatment and post-treatment reduce
the plasma nitrate/nitrite and lung neutrophil infiltration. Moreover, the nitrotyrosine
formation, iNOS mRNA content and apoptosis in thoracic aortas of rats are also
abolished by parthenolide treatment. Accompanied with these effects, parthenolide
suppresses the NF-κB DNA binding in the lung tissue. This study strongly supported
the anti-inflammatory activity of parthenolide in vivo (Sheehan et al., 2002). Another
study from the same group investigated the effects of parthenolide on reperfusioninduced myocardial damage in rats. Intra-peritoneal administration of the parthenolide
has been found to greatly ameliorate the myocardial injury while the activation the
NF-κB activation is concurrently inhibited in vivo (Beranek, 2002). Since
parthenolide shows a potent inhibitory effect on pro-inflammatory cytokines in vitro,
its effects have also been examined in in vivo animal model. Smolinski and Pestka
(2003) investigated inhibitory effects of oral administration of parthenolide on
cytokines release in LPS-stimulated mice. Although parthenolide significantly
suppresses the LPS-induced IL-6 and TNFα secretion in vitro, the IL-6 and TNFα
level in the blood of LPS-stimulated mice are below the detection limit (Smolinski
and Pestka, 2003). A more recent study further addressed this issue. In a

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polymicrobial sepsis rat model, parthenolide treatment significantly reduces the
plasma concentrations of several pro-inflammatory cytokines, including TNFα, IL-6
and IL-10 (Sheehan et al., 2003). In short, the current knowledge of parthenolide’s
biological activities, especially the anti-cancer potential, in in vivo animal model is
rather limited and needs to be further studied.
1.1.5

Toxicity and adverse side effects

Currently, the toxicity and side effects studies only carried out on Feverfew.

Generally, the Feverfew treatment is relatively safe. In animal studies of Feverfew, no
significant toxicity has been observed at doses up to 100 times higher than usual
human dose. No toxic effects on DNA stability or adverse effects to promote
carcinogenesis have been reported by taking Feverfew (Newall et al., 1996). Some
reports have suggested Feverfew may cause allergic response and contact dermatitis.
However, these side effects may due to potential contamination of herbal products
(Paulsen et al., 1998). So far, the side effects of parthenolide have not been reported.
1.2
1.2.1

Oxidative stress, biothiols and intracellular redox balance.
Reactive Oxygen Species

1.2.1.1 Definition
Reactive oxygen species are known to be involved in many biological processes
and in many human diseases. Physically, the atoms are surrounded by multiple orbits
which contain paired electrons spinning in opposite direction. The free radical is
defined as any species capable of independent existence that contains one or more
unpaired electrons. The species with unpaired electrons are highly unstable and can
react with other molecules by means of oxidation or reduction reactions (Halliwell,
1991; Gilbert, 1994). In biological systems, the most important oxygen-based free

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radicals are generally termed as reactive oxygen species (ROS), including superoxide
(O2·-) and hydroxyl radical (·OH) (Bergendi et al., 1999). In addition, other forms of
oxygen-based molecules also functionally act as free radicals in many biological

•−

Fe3+ + O 2 → Fe 2+ + O 2
Fe 2+ + H 2 O 2 →• OH + OH − + Fe3+ (Fenton Reaction)
•−

Net: O 2 + H 2 O 2 ⎯Fe • OH + OH − + O 2 (Haber − Weiss Reaction)
⎯→
Fig. 1.3 Fenton and metal catalyzed Haber-Weiss reaction

oxidative processes, including singlet oxygen (1O2) and hydrogen peroxide (H2O2).
Excessive cellular free radicals and their derivatives may cause oxidative damage due
to their high reactivity towards a variety of molecular targets (Halliwell, 1999;
Marnett, 2000; Cooke et al., 2003).

In the biological systems, most ROS are

produced as O2· - which is instantly converted by superoxide dismutase (SOD) into
H2O2 , a relatively stable form of ROS (Halliwell et al., 2000). However, H2O2 can be
further converted to hydroxyl radical (·OH), the most reactive free radical responsible
for many oxidative damages, via Fenton or Haber-Weiss reaction in vivo (Fig. 1.3)
(Frenkel, 1992; Halliwell et al., 2000). The potent oxidative reactivity of hydroxyl
radical triggers a set of chain reactions resulting in protein oxidation, DNA oxidative
damage (Cooke et al., 2003; Marnett, 2000) and lipid peroxidation (Halliwell and
Chirico, 1993).
1.2.1.2 Sources of ROS
The intracellular ROS are produced via a variety of pathways. Mitochondrion
and endoplasmic reticulum are the major organelles where ROS are produced. In the
aerobic eukaryotic cells, mitochondrion is the central place where the oxygen is

metabolized through the electron transport chain (ETC) to generate adenosine

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triphosphate (ATP). There are five enzymatic complexes, which are arranged by their
oxidative/reductive (Redox) potentials, located in the inner membrane of
mitochondrion. In the aerobic respiration, a series of oxidative/reductive reactions are
catalyzed by using nicotinamide-adenine dinucleotide phosphate (reduced) (NADPH)
and shuttle molecules (such as cytochrome c and coenzyme Q) as substrate to produce
ATP (Forman and Azzi, 1997). Although these enzyme-catalyzed processes are
highly efficient, there are still a fraction of electrons leakage resulting in generation of
O2· - (Yu, 1993). The O2·- is quickly converted by superoxide dismutase (SOD) present
in the mitochondria and converted into H2O2 which could be further converted into
highly reactive ·OH. Another source of intracellular ROS is endoplasmic reticulum
where ROS is generated through NADP/NADPH dependent enzymatic reactions
using both endogenous and exogenous substrates (Trush and Kensler, 1991; Ames et
al., 1993).
1.2.2

Biothiols
In the live organism, ROS are constantly produced during the aerobic

respiration. To avoid potential oxidative damage, a highly regulated antioxidant
defense system, which consists of a series of the enzymatic proteins and nonenzymatic molecules, has been evolved. In this complicated system, the thiol
buffering systems plays a pivotal role in defense of oxidative stress.
1.2.2.1 Definition
Thiols are a group of organic mercaptans (R-SH) that are characterized by the
presence of sulphydryl residues at their active site. In cell system, the biological thiols
(or biothiols) are often referred to biological mercaptans present in the cell. In

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general, biothiols can be classified into low molecular weight free thiols, such as
glutathione (GSH), and high molecular weight protein thiols (Packer, 1995).
1.2.2.2 Biological properties and metabolism
The active sites of most biologically important thiols are located at the
functional cysteinyl residues on the molecular side chain. A thiolates (RS-) is
produced when a thiol (R-SH) loses the H atom from the thiol group or sulphur loses
an electron followed by a proton. Under physiological pH conditions, thiolates are
unstable and may be crosslinked to form a disulphide linkage (-S-S-) between two –
SH group. The dynamic transition of thiol/disulphide depends on cellular
reduction/oxidation status and plays an important role in determination of protein
structure.
All the thiols also act as reducing agents. ROS generated during aerobic
metabolism has a strong tendency to transfer electrons to other molecules and oxidize
the latter. Reducing agents, such as thiols, have negative reductive potentials and thus
act as prompt electron acceptors. A depiction of these reactions is shown in Fig. 1.4.
The ROS can actively interact with thiols and the oxidant-thiol interaction neutralizes
the harmful oxidants into a relatively less toxic by-product and, meanwhile, the
biothiols themselves are oxidized to a disulphide (R-S-S-R).

2R − SH ↔ R − SS − R + 2e − + 2H +
R - SH + R ' - SS - R ' ↔ R - SS - R ' + R − SH
Fig. 1.4 Thiol and disulfides

In biological systems, there are a variety of reductases responsible for recycling
disulphides back to reduced thiols by using cellular reducing equivalents such as
NADPH or NADH (Paget and Buttner, 2003). The general equilibrium reaction

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between thiols and disulphide is depicted in Fig. 1.5. This cycling metabolism plays a
central role in maintaining a favorable oxidative/reductive (Redox) milieu of thiols
containing protein cysteine residues and other small molecules, such as
cysteine/cystine and glutathione (GSH)/glutathione disulphide (GSSG) (Gilbert,
1995).
1.2.3

Anti-oxidant defense system
In order to avoid the oxidative stress-induced biological damage, the aerobic

organisms evolved a highly regulated anti-oxidant defense system. Glutathione is the
most important low molecular biothiol that maintains redox balance within the cell.
GSH is actively synthesized in the cytoplasm by γ-glutamyl cysteine synthestase (γ-

GSH/TRX
Oxidant

Glutathione peroxidase
Thioredoxin reductase

NADPH

Glutathione peroxidase
Thioredoxin peroxidase

GSSG/TRX-SS
Fig. 1.5 Cycling of biothiols

GCS) or GSH synthetase as shown in Fig. 1.6.
Reduced form of glutathione acts as an antioxidant neutralizing the excessive
ROS. And the oxidative form of glutathione (GSSG) will be either transferred back to
the reduced form by glutathione reductase (GRase) using NADPH or exported by
cells to keep an intracellular high ratio of GSH/GSSG (Griffith, 1999). Furthermore,

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GSH is also participating in the synthesis of other small molecular antioxidants such
as ascorbic acid and tocopherols (Meister, 1994).
γ − GCS
L − Glutamate + L − cysteine + MgATP ⎯⎯⎯→
L − γ − Glutamyl − L − cysteine + MgADP + Pi

Fig. 1.6 Glutathione (GSH) synthesis

In addition to GSH, there are a number of protein thiols exist in the cells. The
most important antioxidant non-enzymatic protein thiol system is thioredoxin (TRX).
Thioredoxin is a pleiotropic NADPH-dependent disulphide oxidoreductase which
catalyzes the reduction of protein disulphide bond (-S-S-). The two cysteine residues
of thioredoxin can also be reversibly oxidized into disulphide serving as antioxidant to
remove free radicals and protect cell from oxidative stress. Similar to glutathione, the
oxidized thioredoxin will be recycled through thioredoxin reductase/peroxidase
pathway (Nishinaka et al., 2001; Sen, 2000). Thioredoxin system has also been shown
to be involved in restoring ascorbate and maintaining other forms of antioxidants.
Thioredoxin has been shown to execute its antioxidant capability especially under the
GSH-depleted condition (Iwata et al., 1997).
A group of the anti-oxidant enzymes also plays a critical role in defending

oxidative stress, which includes: (i) SOD which converts superoxide radical to less
harmful H2O2; (ii) catalase which further metabolize H2O2 into oxygen and water; (iii)
glutathione peroxidase which is involved in GSH-GSSG cycling and removal of H2O2
(Michiels et al., 1994).
1.2.4

Redox balance
The intracellular reduction/oxidation status (Redox) is a precise balance

between levels of ROS generation and endogenous thiol buffers exist in the cell

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(Davis, Jr. et al., 2001). Cellular redox status is important in determination of protein
structure, regulation of enzyme activity and control of transcription activity. In the
living cells, aerobic metabolism continually produces a large number of reactive
oxygen species (ROS) which lead to an oxidative stress and a potential harmful
modification of functional cellular proteins. On the other hand a group of low
molecular thiol buffers (GSH) and a variety of reductive enzymatic pathways, such as
ubiquitous disulphide reductase, thioredoxin reductase, contribute to preventing the
oxidation of important proteins thiols by ROS and maintaining a relatively reduced
condition status in living cells (Paget and Buttner, 2003). However, in some situations,
the excessive oxidative stress induced by extracellular stimulations will disrupt the
intracellular redox balance and lead the oxidative damage of many cellular targets.
1.2.5

Biological consequences of redox imbalance

1.2.5.1 Lipid peroxidation

Lipid peroxidation is a free radical-initiated self propagating chain reaction. It is
one of the most well studied biological effects of oxidative damage. Many types of
free radicals can trigger the chain reaction of lipid peroxidation. Firstly, the excessive
free radicals attack the double bond of polyunsaturated fatty acid (PUFA) to generate
a lipid radical L·. The lipid radical L· then directly reacts with O2 which, in turn, form
a lipid peroxy radical LOO· and another series of L· triggering the chain reaction of
lipid peroxidation (Horton and Fairhurst, 1987; Girotti, 1985). As the cell membranes
are enriched with PUFAs, it is believed to be the main target of lipid peroxidation.
The severe oxidation of the phospholipids in the membrane results in a series of
biological effects including re-arrangement of the membrane structure, changing the
permeability of the membrane, disruption of the normal function of membrane-

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binding proteins and simultaneous production of carcinogenic byproducts such as
malondialdehyde (MDA) (Esterbauer et al., 1990).
1.2.5.2 DNA damage
During oxidative stress, DNA can be attacked by many kinds of free radicals
which results in ROS-oxidized DNA modifications, including DNA base
modifications, DNA-protein cross-linking, DNA adducts formation and DNA strand
breaks (Breimer, 1990; Cadet et al., 1999; Dizdaroglu, 1992). Although under
physiological condition, DNA is well protected by nuclear membrane and nuclear
proteins, H2O2 can diffuse freely into nuclei. It can be further concerted to an
extremely reactive ·OH radicals immediately adjacent to the nucleic acid molecules
when iron or copper ions are presented. ·OH radicals can directly attack the DNA
bases and extract hydrogen atoms in genome DNA which result in an OH- form DNA
modification. Among these types of oxidative modifications, 8-hydroxy-2’deoxyguanosine (8-OHdG), which has OH form guanine at C8 position of DNA, is the
most well studied one. The ROS oxidization and modification of DNA has been
suggested to cause gene mutation and is closely related to carcinogenesis (Cooke et al.,

2003; Kamiya et al., 1992).
1.2.5.3 Signal transduction
Signal transduction mainly refers to a series of phosphorylation and
dephosphorylation actions of tyrosine and serine/threonine on the cellular proteins.
These cell signaling proteins are known to be sensitive to redox changes. It has been
demonstrated that the several protein tyrosine kinases can be phosphorylated by
oxidative stress. For instance, the Src-family protein tyrosine kinases have been found
to be activated by ROS (Vasant et al., 2003) and similar phosphorylation /activation

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of PKC protein family members have also been reported (Shibukawa et al., 2003).
The MAPK protein kinase family is also affected by redox changes especially the
stress-activated protein kinase JNK (Inanami et al., 1999). On the other hand, redox
also regulates the protein-DNA binding of transcription factors and then affects gene
transcription. It has been suggested that the cysteine residue in the molecule of NF-κB
is critical for maintaining its spatial structure and DNA binding capability. The
conserved cysteine residue in the RxxRxRxxC motif in the N-terminal of NF-κB Rel
protein is required for optimal DNA-protein interactions and is regulated by redox
changes (Kumar et al., 1992; Toledano et al., 1993; Hayashi et al., 1993).
Furthermore, the unbalanced redox status could also disrupt intracellular calcium
homeostasis and calcium signaling resulting in a variety of cellular function changes
(Donoso et al., 1997).
1.2.5.4 Apoptosis
The involvement of redox in regulation of apoptosis has been extensively
studied (Hampton et al., 1998; Cai and Jones, 1999) which will be discussed in detail
in the following sections.
1.3
1.3.1

Apoptosis and Cancer
Introduction
Apoptosis is a description of a highly regulated and conserved program cell

death in eukaryotic cells which is morphologically and biochemically distinct from
necrotic cell death. It is a genetic controlled process that is involved in embryological
development and maintenance of tissue homeostasis in eukaryotic organisms.
Dysregulation of apoptosis process has been shown to be implicated in tumor
development, chemoresistance, autoimmune syndromes and many other human

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