|The development of resistance by cancer cells to established
chemotherapeutics is a key issue in the clinical treatment of this
devastating disease and has necessitated the generation of therapeutic
agents that utilize novel targeting strategies . Identifying crucial
biochemical alterations between cancer cells and their normal
counterparts is critical in the design of potent chemotherapeutics that
maintain selectivity and overcome drug resistance . Importantly,
the increase in oxidative stress and reactive oxygen species (ROS)
generation observed in cancer cells, in comparison to their normal
counterparts, has been highlighted as a possible selective targeting
strategy . Thus, the use of agents that are able to modulate levels of
intracellular ROS in oxidatively stressed cancer cells forms a potential
|In normal cells, the maintenance of ROS homeostasis is essential
for survival and growth . In fact, ROS are essential in biological
signaling transduction pathways and the regulation of enzyme activity,
such as ribonucleotide reductase (RR) . Importantly, RR is an ironcontaining
enzyme involved in the rate-limiting step of DNA synthesis
that has previously acted as a chemotherapeutic target for hydroxyurea
. While moderate levels of ROS promote cellular proliferation and
differentiation, excessive ROS levels are toxic and can result in oxidative
damage to vital biomolecules, including DNA, proteins and lipids .
Thus, ROS homeostasis is achieved by balancing ROS production and
elimination that occurs through endogenous scavenging systems such
as catalase, thioredoxin and superoxide dismutase [2,3].
|In vitro and in vivo studies and clinical specimens have demonstrated
that neoplastic cells function with increased levels of oxidative stress
compared to normal cells [4-7]. This is evident in the increased levels
of oxidation products, such as lipid peroxides and oxidized DNA bases
in cancer cells [7,8] and is thought to result from factors including
mitochondrial dysregulation and activation of oncogenes [9-11].
Furthermore, levels of ROS-scavenging molecules and enzyme systems
have been previously assessed in primary and malignant tumors (e.g.
colorectal, lung and ovarian cancer) and compared to normal noncancerous
tissue [12-16]. Interestingly, the majority of cancer types
show an imbalance in anti-oxidant enzyme levels in comparison to the
cell of origin . For example, decreased levels of radical-scavenging
systems (e.g. reduced glutathione, glutathione peroxidase 1 and 3,
and glutathione reductase) have been observed in a number of cancer
types (e.g. colorectal cancer, glioblastoma multiforme and transitional
meningioma), in comparison to normal tissue [13,14,16]. In contrast,
ovarian cancers display increased levels of superoxide dismutase and
this increase is believed to be a cellular response to increased ROS levels
. These factors indicate a dys-regulation in ROS homeostasis in
tumors [12-15] and suggest that cancer cells are more susceptible than
normal cells to insult by agents that further disrupt ROS homeostasis.
Thus, exogenous agents that increase intracellular ROS levels, such as
iron chelators that form redox active complexes [17,18], are an exciting
|Interest in thiosemicarbazone iron chelators as anti-cancer
agents has increased in recent years, with the ligand, Triapine®,
entering >20 international clinical trials . Additionally, a number
of thiosemicarbazone analogues, including di-2-pyridylketone
4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and 2-benzoylpyridine
4,4-dimethyl-3-thiosemicarbazone (Bp44mT), overcome resistance
to established chemotherapeutics and have demonstrated potent
and selective anti-cancer activity against a number of human tumor
xenografts in vivo [20,21].
|Importantly, the anti-proliferative activity of thiosemicarbazones
is not only due to the chelation of the essential nutrient, iron, but also
results from the formation of redox-active iron complexes [17,18]. Once
formed, these iron complexes are able to participate in Fenton-like
chemistry to generate ROS and can interact with cellular oxidants and
reductants, establishing a catalytic redox cycling mechanism [1,17,18].
Significantly, Dp44mT and Bp44mT were shown to alter important
thiol-related anti-oxidant systems in vitro, including glutathione,
thioredoxin and glutaredoxin . In fact, these iron chelators
significantly increased the levels of oxidized trimeric thioredoxin and
was most likely due to their ability to inhibit thioredoxin reductase
activity . Moreover, these ligands also resulted in the inhibition of
glutathione reductase activity, which is responsible for the generation
of reduced glutathione, an important cellular anti-oxidant . Such
thiol-containing systems provide reducing equivalents for RR, the
enzyme involved in the rate-limiting step of DNA synthesis . Thus,
targeting these systems also reduce the activity of RR  and these
studies provide further insight into the mechanisms involved in the
anti-cancer activity of thiosemicarbazones .
|Collectively, agents that can modulate levels of intracellular ROS,
either through the increase in ROS generation and/or the inhibition
of ROS-scavenging capacity, form a potential therapeutic avenue to
selectively target cancer . In this light, novel thiosemicarbazone
iron chelators, such as Dp44mT and Bp44mT, have emerged as potent
and selective anti-cancer agents that act via multiple mechanisms,
including: (1) the chelation of iron from biological systems [1,17,18];
(2) the formation of redox active iron complexes [1,17,18]; and (3)
the alteration of thiol-containing anti-oxidant systems to inhibit RR
activity . Significantly, the targeting of multiple cellular systems is an
important strategy in design of selective agents that overcome resistance
to established chemotherapeutics. The ability of these chelators to
target cellular anti-oxidant systems, which are already compromised
in many cancer types, may explain the potent and selective anti-cancer
activity of these ligands observed in vivo [20,21].
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