# Novel Anticancer Platinum(IV) Complexes with Adamantylamine: Their Efficiency and Innovative Chemotherapy Strategies Modifying Lipid Metabolism

Authors: Alois Kozubík; Alena Vaculová; Karel Souček; Jan Vondráček; Jaroslav Turánek; Jiřina Hofmanová
Journal: Metal-Based Drugs
 (2008)

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## Abstract

The impressive impact of cisplatin on cancer on one side and severe side effects, as well as the development of drug resistance during treatment on the other side, were the factors motivating scientists to design and synthesize new more potent analogues lacking disadvantages of cisplatin. Platinum(IV) complexes represent one of the perspective groups of platinum-based drugs. In this review, we summarize recent findings on both in vitro and in vivo effects of platinum(IV) complexes with adamantylamine. Based on a literary overview of the mechanisms of activity of platinum-based cytostatics, we discuss opportunities for modulating the effects of novel platinum complexes through interactions with apoptotic signaling pathways and with cellular lipids, including modulations of the mitochondrial cell death pathway, oxidative stress, signaling of death ligands, lipid metabolism/signaling, or intercellular communication. These approaches might significantly enhance the efficacy of both novel and established platinum-based cytostatics.


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## Body

## 1. Introduction

Platinum-based drugs
are widely used anticancer agents
with a broad range of antitumor activities. Cisplatin [cis-diamminedichloroplatinum(II)] (see Figure 1) has a significant
activity in ovarian, testicular, bladder, head and neck, and lung cancer, where
it is most commonly used in combination with other drugs [1]. Thus, it has become one of the most
successful anticancer drugs used worldwide in almost 50% of solid tumor
chemotherapies. Although the initial response rates can be high with
cisplatin-based regimen, the clinical utility of the drug is often limited by
the onset of acquired or intrinsic resistance [2, 3] and the number of side effects such as kidney
damage, vomiting/nausea, and neurotoxicity [4]. The resistance of tumor cells to cisplatin
remains a major cause of treatment failure in cancer patients, while the high
toxicity of cisplatin limits the dose that can be given to patients. Cancer
cell resistance to platinum chemotherapeutic drugs therefore remains a major
obstacle and demonstrates a need for alternatives. For this reason, vast
efforts are committed to develop novel platinum-based complexes which might
overcome the shortcomings of cisplatin. The ultimate aim is to find and
characterize platinum-based derivatives with higher antitumor activity, which
display a more tolerable toxicologic profile and overcome resistance in many
tumor types. A second alternative approach sought is to develop a more efficient
combination treatment of biologic response modifiers together with
chemotherapeutic platinum drugs.Chemical
structures of cisplatin [cis-diamminedichloroplatinum(II)], LA-9
[(SP-4-3)-(1-adamantylamine)amminedichloroplatinum(II)], and LA-12
[(OC-6-43)-bis(acetato)(1-adamantylamine) amminedichloroplatinum(IV)].
(a)(b)(c)The enormous efforts of a number of research
teams have borne fruit in two derivatives, carboplatin (cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II)) and,
more recently, the so-called “third generation” platinum drug oxaliplatin (trans—R,R.cyclohexane-1,2-diammine)oxalatoplatinum(II)). Both compounds are effective in colon cancer
treatment and are currently clinically approved worldwide [5, 6]. Several other platinum-based drugs have
gained regionally limited approval: Nedaplatin (Japan), SKI2053R (South Korea),
and Lobaplatin (China).Although over 1000 complexes have been studied so far, only 10–20% of
them turned out to be active against cancer cells in preclinical studies [7]. These include, for example, satraplatin which
is currently in the third phase of clinical evaluation [8]. A complementary approach also promises to
employ biologic modifiers such as cytokines, lipids, and other molecules in
order to increase the efficacy of both traditional and novel platinum-based
drugs. Adjuvant biochemotherapy with cytokines, such as interleukin-2 or
interferon-alpha may increase response rates in patients [9], thus improving their efficiency, although
regimen treatments must be selected carefully in order to reduce undesirable
effects. Similar to cytokines, lipids, and their derivatives, drugs interfering
with metabolism of lipid compounds or dietary supplements may provide
additional benefits during the treatment with platinum-based drugs, including
reduced side effects of chemotherapy or increased toxicity towards tumor cells [10–14]. The present review is intended to provide an overview
of some opportunities opened for innovative therapeutic strategies involving both
traditional and novel platinum complexes, with particular emphasis  on the novel class of anticancer platinum(IV)
complexes with adamantylamine, which have been intensively studied in our
laboratories during recent years.


## 2. Development of Platinum(IV) Complexes with Adamantylamine and Their In Vitro/In Vivo Effects

Recently, Žák et al. designed and synthesized a series
of novel platinum(II) and platinum(IV) complexes with amino derivatives of
adamantane as one of its ligands: (SP-4-3)-(1-adamantylamine)amminedichloroplatinum(II),
coded as LA-9, and (OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV),
coded as LA-12 (see Figure1 ). LA-9
represents a four-coordinate(II), and LA-12 an octahedral six-coordinate
platinum complex containing a bulky nonleaving hydrophobic (lipophilic) ligand—adamantylamine [15]. Since the early studies by Rosenberg et al.
[16] it has been known that platinum(IV) as well as
platinum(II) complexes can display antitumor properties. In addition, platinum(IV)
complexes are much more inert to ligand substitution reactions than are their platinum(II)
counterparts [17], and it is generally accepted that reduction
to the lower oxidation state platinum(II) is essential for the anticancer
activity of the platinum(IV) complex [18, 19]. Currently, clinical trials of the platinum(IV)
derivative LA-12 are in progress and some promising results have been shown.
LA-12 has been evaluated in a panel of preclinical studies including in vitro
and in vivo antitumor efficacy and toxicologic and pharmacokinetic studies [20, 21]. In both murine ADJ/PC6 plasmacytoma and a human
A2780 ovarian carcinoma tumor model, higher in vivo antitumor activity of a
single dose as well as of repeated doses was observed, compared to cisplatin
and the other platinum(IV) complex—satraplatin (JM216, the first orally
administered platinum(IV) drug currently evaluated in clinical trials) [21]. Interestingly, the acute toxicity of LA-12 in mice is relatively low
and the effective dose is comparable to that of cisplatin and higher than that
of satraplatin [21].These highly interesting compounds were also
successfully tested in our laboratories. We have demonstrated that LA-9 and
LA-12 display a higher hydrophobicity than cisplatin and exert a higher
cytotoxicity than satraplatin (JM216) in the in vitro tests on a panel
of 14 cancer cell lines of various origin, and a different sensitivity to
cisplatin, including both A2780 (cisplatin-sensitive) and A2780cis (with
acquired cisplatin resistance) ovarian cancer cells, breast carcinoma, colon
cancer, lung carcinoma, and leukemia cells lines, many of which are refractory
to cisplatin treatment [22, 23]. In A2780cis-resistant cells, for example, the
IC50 values were 1.4 and 24.23 μM for LA-12 and cisplatin, respectively, with
LA-12 being effective at the dose approximately equal to cisplatin IC50 in parental A2780 cells,  thus proving
that LA-12 may successfully counter the acquired resistence in vitro [22]. The IC50 value for LA-12 in A2780 was also
approximatelly three times lower than IC50 of LA-12 in nontransformed rat liver
epithelial cells, suggesting that it is not overtly toxic in nontumorigenic
cells [24]. Interestingly, we found strong differences
between the effects of platinum(IV) complex—LA-12 and platinum(II)
derivatives—LA-9 and cisplatin on cytokinetic parameters in SK-OV-3 cells
(with intrinsic cisplatin resistance) [25]. Apparently, both LA-12 and LA-9 are effective
in considerably lower concentrations than cisplatin and can overcome both
acquired and intrinsic resistance to cisplatin. Thus, the mechanisms of the
effects of these compounds should be further investigated, as several recent
studies have demonstrated that LA-12 may have unique effects which could be
responsible for its high efficiency [24, 26].


## 3. A Search for Mechanisms of Action of Novel Platinum Complexes

The activity of the “classical” cisplatin is
generally accepted as being due to its ability to form adducts with DNA (inter-
and intrastrand DNA cross-links) and causing DNA strand breaks in the nucleus [27, 28]. Subsequently, interference with normal
transcription and/or DNA replication mechanisms [29] are its characteristic biological effects,
leading to either repair of the DNA damage and cell survival, or activation of
the irreversible cell death program as a consequence. Indeed, early observations
have shown that cells deficient in DNA repair pathways are hypersensitive to
cisplatin [29, 30], suggesting that cisplatin and other
platinum-based complexes bind DNA.On the other hand, cisplatin may react with
many other cellular components that have nucleophilic sites such as RNA,
proteins, membrane phospholipids,
cytoskeletal filaments, and thiol-containing molecules [31]. Although cisplatin can react with a variety
of cellular macromolecules and there is strong evidence that the most important
target is DNA [27], it is estimated that less than 1% of the
cisplatin molecules that enter the cell actually bind nuclear DNA, with most of
them ending up binding other biomolecules [32]. This indicates that increased attention
should also be focused on the discovery of mechanisms of action of
platinum-based drugs, which are different from those primarily based on DNA
damage. Such mechanisms could substantially interfere with the other
intracellular signaling pathways regulating cell growth and death.


## 4. Unique Properties of Novel Platinum Derivatives

The cytotoxicity of platinum-based anticancer
drugs is therefore a result of many events, beginning with the intracellular
accumulation and proceeding through detoxification by intracellular thiol-containing
molecules, DNA modification, and cellular responses to DNA damage. Modification
of the leaving group(s) in platinum complexes influences both biodistribution and
the general toxicity. On the contrary, modification of the amine ligand(s) will
influence the anticancer properties, since the nonleaving amine ligands are the
reason for structurally different DNA or DNA/protein adducts. The hydrophobicity
(lipophilicity) and bulkiness of the nonleaving amine ligand significantly affect
several factors (permeation through the cell membrane, reactivity towards DNA
and thiols, and recognition of DNA adducts) that are responsible for cytotoxic
potential of platinum complexes on the molecular and cellular levels. In various
platinum(II) complexes, the increase of ligand hydrophobicity is positively
correlated with the increase of the cytotoxic effect [33].It is therefore interesting that a similar
correlation was found within a homologous series of platinum(IV) complexes [23]. The higher hydrophobicity of the platinum
complexes enhanced their penetration through the cell membrane and increased
their accumulation inside the cancer cells. The high intracellular concentrations
of platinum(IV) drugs, together with their reactivity towards DNA, may accelerate
the production of various platinum-DNA or platinum-DNA/protein adducts, which
are more or less endowed with the ability to trigger apoptosis or necrosis in
target cells. The hydrophobic complex LA-12 is a good example of a platinum(IV)
complex with a bulky hydrophobic ligand, enhancing accumulation in cancer cells
and triggering rapid cell death in both cisplatin-sensitive and cisplatin-resistant
cell lines [22, 25]. LA-12 proved to be very toxic against various
cancer cells representing different types of cancer and employing different
mechanisms of resistance towards cisplatin [23].In comparison to cisplatin and satraplatin
(JM-216), LA-12 represents a complex with a very high hydrophobicity due to the
presence of adamantylamine ligand. Moreover, the symmetry of the hydrophobic
adamantyl moiety lightens penetration of the whole complex through biological
membranes. This theoretical presumption has been proven experimentally in cisplatin-sensitive
and cisplatin-resistant cell lines A2780 and A2780/cisR, respectively.  Platinum uptake in sensitive and resistant
A2780 cell lines was more than
one order of a magnitude lower for cisplatin in comparison with LA-12.
Moreover, accumulation of LA-12
in cisplatin-resistant cells was not decreased in the
range having an effect on biological activity [26].


### 4.1. Effects of LA-12 in Cisplatin-Resistant Cells

In all human cancer cell
lines studied so far, it seems apparent that cisplatin resistance is a
multifactorial process, being a result of decreased drug uptake, increased DNA
repair and tolerance to DNA lesions, slow cell proliferation and replicative
bypass, and/or cytosolic drug inactivation. We cannot exclude that LA-12 is a substrate for some of multidrug resistance
proteins (MRP; MDR) and P-glycoprotein (P-gp), but the low cross-resistance
with cisplatin together with high accumulation in cisplatin-resistant cancer
cell line A2780/cisR seem
to imply that MRP/MDR systems are of secondary importance in comparison with
increased ability of hydrophobic LA-12 to penetrate through biological
membranes.Ovarian epithelial cancer cell line A2780 is a well-described
model for studies on cisplatin resistance. The resistant sublines, prepared by
gradual treatment by cisplatin, are available and several mechanisms of resistance
have been described. It seems that the cisplatin-resistant A2780 sublines represent
the type of cisplatin-resistant cells, in which several mechanisms of
resistance are well orchestrated. An organic anion pump that could be involved
in cisplatin resistance is a multidrug resistance protein 2 (MRP2). High
expression of MRP2 was found in A2780/cis70 cell line, together with increased
levels of glutathione [34]. Cisplatin-resistant A2780/cis70 cells
demonstrated enhanced efflux of ciplatin as compared to parent A2780 cells.
Enhanced DNA repair was also observed in A2780/cis70 cells and this mechanism has
been shown to be responsible for cisplatin resistance [35]. Expression of all the above-mentioned
systems gives the cisplatin-resistant A2780 sublines a remarkable
capability to survive the attack of cisplatin. LA-12 demonstrated also very low
cross-resistance in both A2780/cis40 and A2780/cis90 sublines. There was no cisplatin cross-resistance with intermediate
resistant A2780/cis40. In contrast, cross-resistance has been observed for another
new generation platinum(II) complex ZD0473 (formerly known as JM473, AMD473)
in A2780 and A2780/cis40 cells [36]. Interestingly, we have observed a substantial cross-resistance for satraplatin
(JM-216), using  a panel of cisplatin-resistant
cancer cell lines (see Table 1).Table 1
Comparison of the
cytotoxicity of cisplatin, LA-12 and satraplatin (JM-216) against cisplatin-resistant
tumor cell lines. Values of IC50 were
calculated for 24 hour exposure time (adapted from Turánek et al. Anticancer Drugs. 2004,15:537-543).
Cancer cell lineIC50 (μM )CisplatinLA-12SatraplatinK562>80363KG-148263ML-2>80156B16>806>80HT-29N>8012>80HT-2950870HCT116>809>80A42763613HBL100636>80MCF-771870CPRL23/CTR>802556A27807463A2780cis4040340A2790cis90>80763Strong inhibition of DNA polymerization by
LA-12/DNA adducts, enhanced persistence of these adducts due to their less
efficient repair, and DNA-protein cross-linking could be  responsible for the unique cytotoxic activity
of LA-12 [23, 26]. Therefore, increased attention should be paid
to this compound, with the aim to define its mechanisms of action, as well as
to search for alternative approaches improving its anticancer activity. In the
following sections, we summarize the current possibilities of modulating the
effects of cisplatin, which might also be employed for novel platinum-based
drugs, such as platinum(IV) derivatives with adamantylamine. Our attention is primarily
focused on signaling pathways regulating the programmed cell death (apoptosis)
and modulation of cellular lipids. For more information on other drug-resistance
mechanisms, such as MDR, the readers are kindly requested to consult several
recent excellent reviews [37–39].


## 4.1. Effects of LA-12 in Cisplatin-Resistant Cells

In all human cancer cell
lines studied so far, it seems apparent that cisplatin resistance is a
multifactorial process, being a result of decreased drug uptake, increased DNA
repair and tolerance to DNA lesions, slow cell proliferation and replicative
bypass, and/or cytosolic drug inactivation. We cannot exclude that LA-12 is a substrate for some of multidrug resistance
proteins (MRP; MDR) and P-glycoprotein (P-gp), but the low cross-resistance
with cisplatin together with high accumulation in cisplatin-resistant cancer
cell line A2780/cisR seem
to imply that MRP/MDR systems are of secondary importance in comparison with
increased ability of hydrophobic LA-12 to penetrate through biological
membranes.Ovarian epithelial cancer cell line A2780 is a well-described
model for studies on cisplatin resistance. The resistant sublines, prepared by
gradual treatment by cisplatin, are available and several mechanisms of resistance
have been described. It seems that the cisplatin-resistant A2780 sublines represent
the type of cisplatin-resistant cells, in which several mechanisms of
resistance are well orchestrated. An organic anion pump that could be involved
in cisplatin resistance is a multidrug resistance protein 2 (MRP2). High
expression of MRP2 was found in A2780/cis70 cell line, together with increased
levels of glutathione [34]. Cisplatin-resistant A2780/cis70 cells
demonstrated enhanced efflux of ciplatin as compared to parent A2780 cells.
Enhanced DNA repair was also observed in A2780/cis70 cells and this mechanism has
been shown to be responsible for cisplatin resistance [35]. Expression of all the above-mentioned
systems gives the cisplatin-resistant A2780 sublines a remarkable
capability to survive the attack of cisplatin. LA-12 demonstrated also very low
cross-resistance in both A2780/cis40 and A2780/cis90 sublines. There was no cisplatin cross-resistance with intermediate
resistant A2780/cis40. In contrast, cross-resistance has been observed for another
new generation platinum(II) complex ZD0473 (formerly known as JM473, AMD473)
in A2780 and A2780/cis40 cells [36]. Interestingly, we have observed a substantial cross-resistance for satraplatin
(JM-216), using  a panel of cisplatin-resistant
cancer cell lines (see Table 1).Table 1
Comparison of the
cytotoxicity of cisplatin, LA-12 and satraplatin (JM-216) against cisplatin-resistant
tumor cell lines. Values of IC50 were
calculated for 24 hour exposure time (adapted from Turánek et al. Anticancer Drugs. 2004,15:537-543).
Cancer cell lineIC50 (μM )CisplatinLA-12SatraplatinK562>80363KG-148263ML-2>80156B16>806>80HT-29N>8012>80HT-2950870HCT116>809>80A42763613HBL100636>80MCF-771870CPRL23/CTR>802556A27807463A2780cis4040340A2790cis90>80763Strong inhibition of DNA polymerization by
LA-12/DNA adducts, enhanced persistence of these adducts due to their less
efficient repair, and DNA-protein cross-linking could be  responsible for the unique cytotoxic activity
of LA-12 [23, 26]. Therefore, increased attention should be paid
to this compound, with the aim to define its mechanisms of action, as well as
to search for alternative approaches improving its anticancer activity. In the
following sections, we summarize the current possibilities of modulating the
effects of cisplatin, which might also be employed for novel platinum-based
drugs, such as platinum(IV) derivatives with adamantylamine. Our attention is primarily
focused on signaling pathways regulating the programmed cell death (apoptosis)
and modulation of cellular lipids. For more information on other drug-resistance
mechanisms, such as MDR, the readers are kindly requested to consult several
recent excellent reviews [37–39].


## 5. Activation of Signaling Pathways Leading to Cell Death

In general, apoptosis is the most important
form of cell death induced in response of tumor cells to chemotherapeutic
agents, including platinum-based cytostatics. However, in some cell lines,
particularly those with resistance to the drug, platinum-derived
chemotherapeutics may also produce characteristic features of necrotic cell
death. Moreover, in the same population of cisplatin-treated cells, necrotic
and apoptotic cell deaths have been shown to occur simultaneously (for review
see [40]). In addition, other death signaling pathways
may also be stimulated following cisplatin treatment, finally leading to
autophagy [41]. Thus, different types of cell death induced
by the chemotherapeutic drugs have been reported to occur, depending on the
cell type and/or drug concentration.


### 5.1. Apoptotic Pathways Regulated by Anticancer Drugs

Two major distinct apoptotic pathways have been described for mammalian
cells. The intrinsic pathway is characterized by the central role of the
mitochondria in the apoptotic signaling cascade. Bcl-2 family proteins are
known as important regulators at the level of these organelles. Translocation
of proapoptotic proteins from mitochondria to the cytosol results in caspase
activation and execution of apoptosis. This pathway functions in response to
various types of intracellular stress-inducing agents, DNA damage, growth
factor withdrawal, or in response to activation of death receptor-mediated
apoptosis in type II cells. On the other hand, in the extrinsic pathway,
caspase activation is initiated by the death receptors on the surface of the so-called
type I cells and this pathway may proceed independently of mitochondria [42]. Chemotherapeutic agents are known to
induce/modulate apoptosis at the level of both intrinsic and/or extrinsic
pathway.Emerging evidence suggests that defects or
dysregulation of different steps of the apoptotic signaling pathways may be an
important determinant of resistance to anticancer drugs (reviewed in [43, 44]). The mechanisms that inhibit propagation of
the apoptotic machinery induced by cisplatin may include loss of p53 function,
overexpression of Her-2/neu, activation of PI3K/Akt pathway, overexpression of
antiapoptotic Bcl-2 protein, interference in caspase activation, and so forth. The molecular signature defining the
resistant phenotype varies among tumors, and the number of resistance
mechanisms activated in response to selection pressures dictates the overall
extent of cisplatin resistance (for review see [28]).


### 5.2. Interactions of Platinum-Based Drugs and Death Ligands

The onset of resistance or cross-resistance of tumor cells to several
chemotherapeutic drugs is a serious therapeutic complication. Different agents
have been investigated with regard to their ability to induce/modulate
signaling pathways involved in drug-induced apoptosis. Among them, members of
the tumor necrosis factor (TNF) family of apoptotic inductors are of particular
interest. These agents have been shown to be capable of triggering/modulating
mitochondria-dependent and/or mitochondria-independent apoptotic pathways
inside the cell and may contribute to the potentiation of apoptosis, when coadministered
together with chemotherapy [45]. Elucidation of the mechanisms involved in
these effects therefore deserves further attention.A number of studies, including our own
preliminary data, have shown that TNF-related apoptosis-inducing ligand (TRAIL)
(an important member of the TNF family) works synergistically with some
anticancer drugs to kill many types of tumor cells and eliminate resistance to
such drugs. These findings indicate that the combination of TRAIL and
chemotherapeutic drugs is potentially promising in treating refractory cancers.
TRAIL can induce human cancer cell apoptosis through the engagement of its
death receptors (DRs) DR4 and DR5. However, not all cancers are sensitive to
this cytokine. The mechanisms of modulation of the death receptor-mediated
apoptosis (induced by TRAIL) and DR-mediated regulation in colon cancer cells have also
been studied in our laboratory [46, 47]. Recently, it has been shown that low doses of
cytotoxic drugs could restore TRAIL-induced cell death in resistant colon and
prostate cancer cell lines [1, 48]. Ligation of TRAIL to its cognate receptors
results in receptor activation and formation of a death-inducing signaling complex
(DISC), which is responsible for caspase-8 activation and further propagation
of the apoptotic signal [42]. Chemotherapeutic agents have been shown to have a
great impact on these processes.At the level of plasma membrane, upregulation
of the DRs was shown to be responsible for cisplatin-induced sensitization to
DR-mediated apoptosis [49]. Chemotherapeutic agents can induce a
redistribution of CD95, DR4, and DR5 in lipid rafts that accounts for the
synergistic toxicity of chemotherapy and DR ligands in colon carcinoma cells [50]. An increased recruitment of Fas-associated
death domain (FADD) adaptor protein and procaspase-8 to the TRAIL DISC was
demonstrated in colon cancer cells exposed to cisplatin-based drugs [51]. Interestingly, phosphorylated FADD was shown
to play an essential role in the mechanisms of amplification of
chemotherapy-induced apoptosis in prostate cancer cells [52]. Furthermore, cisplatin downregulated the
levels of c-FLIPL (a competitive inhibitor of caspase-8 at the DISC
level) in osteosarcoma cells, sensitizing them to Fas-mediated apoptosis [53].The cisplatin-mediated potentiation of
TRAIL-induced apoptosis has been replaced at the mitochondrial level, as
demonstrated by Bid cleavage, the dissipation of mitochondrial membrane
potential (MMP), and the release of mitochondrial proteins into the cytosol of
the mesotheliomal cells [54]. Exposure of esophageal cancer cells to
sublethal concentrations of cisplatin resulted in profound potentiation of
their susceptibility to TRAIL cytotoxicity, accompanied by significant
activation of caspase-8, -9, and -3. Interestingly, activation of these
caspases was abrogated by overexpression of Bcl-2 or by the selective caspase-9
inhibitor, which pointed to the essential role of mitochondria-dependent
signaling cascade in this process [55]. Similarly, the mitochondria-dependent caspase
activation cascade has been shown to play a significant role in
cisplatin-mediated potentiation of TRAIL-induced apoptosis in thoracic cancer
cells [56]. These results suggest that mitochondria could
be a direct target for the development of more refined strategies to enhance
the therapeutic effect of TRAIL as an anticancer agent.


## 5.1. Apoptotic Pathways Regulated by Anticancer Drugs

Two major distinct apoptotic pathways have been described for mammalian
cells. The intrinsic pathway is characterized by the central role of the
mitochondria in the apoptotic signaling cascade. Bcl-2 family proteins are
known as important regulators at the level of these organelles. Translocation
of proapoptotic proteins from mitochondria to the cytosol results in caspase
activation and execution of apoptosis. This pathway functions in response to
various types of intracellular stress-inducing agents, DNA damage, growth
factor withdrawal, or in response to activation of death receptor-mediated
apoptosis in type II cells. On the other hand, in the extrinsic pathway,
caspase activation is initiated by the death receptors on the surface of the so-called
type I cells and this pathway may proceed independently of mitochondria [42]. Chemotherapeutic agents are known to
induce/modulate apoptosis at the level of both intrinsic and/or extrinsic
pathway.Emerging evidence suggests that defects or
dysregulation of different steps of the apoptotic signaling pathways may be an
important determinant of resistance to anticancer drugs (reviewed in [43, 44]). The mechanisms that inhibit propagation of
the apoptotic machinery induced by cisplatin may include loss of p53 function,
overexpression of Her-2/neu, activation of PI3K/Akt pathway, overexpression of
antiapoptotic Bcl-2 protein, interference in caspase activation, and so forth. The molecular signature defining the
resistant phenotype varies among tumors, and the number of resistance
mechanisms activated in response to selection pressures dictates the overall
extent of cisplatin resistance (for review see [28]).


## 5.2. Interactions of Platinum-Based Drugs and Death Ligands

The onset of resistance or cross-resistance of tumor cells to several
chemotherapeutic drugs is a serious therapeutic complication. Different agents
have been investigated with regard to their ability to induce/modulate
signaling pathways involved in drug-induced apoptosis. Among them, members of
the tumor necrosis factor (TNF) family of apoptotic inductors are of particular
interest. These agents have been shown to be capable of triggering/modulating
mitochondria-dependent and/or mitochondria-independent apoptotic pathways
inside the cell and may contribute to the potentiation of apoptosis, when coadministered
together with chemotherapy [45]. Elucidation of the mechanisms involved in
these effects therefore deserves further attention.A number of studies, including our own
preliminary data, have shown that TNF-related apoptosis-inducing ligand (TRAIL)
(an important member of the TNF family) works synergistically with some
anticancer drugs to kill many types of tumor cells and eliminate resistance to
such drugs. These findings indicate that the combination of TRAIL and
chemotherapeutic drugs is potentially promising in treating refractory cancers.
TRAIL can induce human cancer cell apoptosis through the engagement of its
death receptors (DRs) DR4 and DR5. However, not all cancers are sensitive to
this cytokine. The mechanisms of modulation of the death receptor-mediated
apoptosis (induced by TRAIL) and DR-mediated regulation in colon cancer cells have also
been studied in our laboratory [46, 47]. Recently, it has been shown that low doses of
cytotoxic drugs could restore TRAIL-induced cell death in resistant colon and
prostate cancer cell lines [1, 48]. Ligation of TRAIL to its cognate receptors
results in receptor activation and formation of a death-inducing signaling complex
(DISC), which is responsible for caspase-8 activation and further propagation
of the apoptotic signal [42]. Chemotherapeutic agents have been shown to have a
great impact on these processes.At the level of plasma membrane, upregulation
of the DRs was shown to be responsible for cisplatin-induced sensitization to
DR-mediated apoptosis [49]. Chemotherapeutic agents can induce a
redistribution of CD95, DR4, and DR5 in lipid rafts that accounts for the
synergistic toxicity of chemotherapy and DR ligands in colon carcinoma cells [50]. An increased recruitment of Fas-associated
death domain (FADD) adaptor protein and procaspase-8 to the TRAIL DISC was
demonstrated in colon cancer cells exposed to cisplatin-based drugs [51]. Interestingly, phosphorylated FADD was shown
to play an essential role in the mechanisms of amplification of
chemotherapy-induced apoptosis in prostate cancer cells [52]. Furthermore, cisplatin downregulated the
levels of c-FLIPL (a competitive inhibitor of caspase-8 at the DISC
level) in osteosarcoma cells, sensitizing them to Fas-mediated apoptosis [53].The cisplatin-mediated potentiation of
TRAIL-induced apoptosis has been replaced at the mitochondrial level, as
demonstrated by Bid cleavage, the dissipation of mitochondrial membrane
potential (MMP), and the release of mitochondrial proteins into the cytosol of
the mesotheliomal cells [54]. Exposure of esophageal cancer cells to
sublethal concentrations of cisplatin resulted in profound potentiation of
their susceptibility to TRAIL cytotoxicity, accompanied by significant
activation of caspase-8, -9, and -3. Interestingly, activation of these
caspases was abrogated by overexpression of Bcl-2 or by the selective caspase-9
inhibitor, which pointed to the essential role of mitochondria-dependent
signaling cascade in this process [55]. Similarly, the mitochondria-dependent caspase
activation cascade has been shown to play a significant role in
cisplatin-mediated potentiation of TRAIL-induced apoptosis in thoracic cancer
cells [56]. These results suggest that mitochondria could
be a direct target for the development of more refined strategies to enhance
the therapeutic effect of TRAIL as an anticancer agent.


## 6. Chemotherapy and Mitochondria

Comparison between cisplatin, oxaliplatin, and
carboplatin effects on cancer cells shows that, in addition to the extent of
nuclear DNA platination and fragmentation, impairment of mitochondrial
functions could also be a measure of platinum drug cytotoxicity [57]. Cisplatin has been shown
to accumulate in mitochondria of cultured cells and to have effects on isolated
mitochondria depending on cell type. Cisplatin and its second generation
derivatives produced marked changes of mouse live mitochondrial morphology,
enzyme activity, Ca2+ influx, and surface potential. However, these effects did
not correlate with nephrotoxicity [58]. Cisplatin binds to kidney mitochondrial
proteins in treated mice and inactivates the alpha-ketoglutarate dehydrogenase
complex in cultured LLC-PK1 cells, which is associated with drug bioactivation [59]. Evaluation of mitochondrial oxygen
consumption using various cell lines and tumors from patients showed that
cisplatin does not directly damage the energy-converting mechanism of
mitochondria [60]. In 3T3 and Walker 256 cells, cisplatin had
acute, but reversible, effects on the reorganization of the intermediate filament
component of cytoskeleton as well as mitochondrial function [61]. Using head and neck
squamous cell carcinoma cell lines, it has been shown that cisplatin forms
adducts with mtDNA
more abundantly than those with nuclear DNA and binds preferentially to mitochondrial
membrane proteins [62]. Changes of mitochondria
and their function are associated mainly with induction of intrinsic apoptotic
pathway. Alteration on mitochondria membrane level (dissipation of MMP),
release of mitochondrial proteins into cytosol, and production of reactive oxygen species  are
associated with activities of Bcl-2 family proapoptotic and antiapoptotic
proteins and activation of caspases [62].In
various types of cancer cells, mitochondrial alterations have been shown to be
associated with platinum-drug resistance. The mitochondria of intrinsically
resistant human ovarian cancer cell lines and resistant clones of HeLa cells
showed altered morphology and were fewer in number. In these models, cisplatin
resistance was associated with lower platinum accumulation and mitochondrial
defects [63]. It has been shown that
density of mitochondria in intestinal epithelial cells is the key factor for
the determination of the anticancer activity and side effects of cisplatin [64]. Next, elevation of MMP
in ovarian cancer cells with acquired platinum resistance has been observed,
suggesting that changes at the mitochondrial level would affect the relative
resistance of malignant cells to undergo drug-induced apoptosis [65]. Impairment of
mitochondrial apoptotic signaling pathway is responsible for incomplete
caspase-3 activation and lower sensitivity of gastric cancer cells to cisplatin
[66].The upregulation of the antiapoptotic Bcl-2
protein contributed to the development of cisplatin resistance, which was
reversed by Bcl-2 siRNA [67]. On the other hand, targeted inactivation of
proapoptotic Bax in colon cancer HCT116 cells resulted in a significantly
increased resistance to cisplatin [68]. Failure of caspase-9 activation induced a
higher threshold for apoptosis and cisplatin resistance in testicular cancer [69]. Resistance to apoptosis correlated with the
reduced caspase-3 activation and enhanced the expression of antiapoptotic
proteins in human cervical multidrug-resistant cells [70]. Furthermore, caspase-2 is known to mediate
the recruitment of the mitochondrial apoptotic pathway upon DNA damage induced
by chemotherapy (reviewed in [44]). Cisplatin has been shown to induce
p53-mediated caspase-2 activation resulting in the mitochondrial release of the
apoptosis-inducing factor (AIF) and subsequent apoptosis in renal tubular
epithelial cells [71]. From the information described above, it
follows that the knowledge of platinum drug effects on mitochondria may help to
design strategies for increase of the effectiveness of tumor cell treatment. Importantly,
similar approaches might be potentially used to improve efficacy of
both currenly used platinum derivatives and novel compounds, such as LA-12.


## 7. Chemotherapy and Oxidative Metabolism

Studies in a variety of cell types have
suggested that chemotherapeutic drugs induce cancer cell apoptosis at least in
part by inducing formation of ROS. In several cancer
cell models it has been demonstrated that cisplatin promotes increased
production of ROS, which can activate nuclear factor-kappaB (NF-κB).
This can lead to increased expression of proinflammatory mediators and
intensify the cytotoxic effects of cisplatin [72]. A positive correlation has been reported between
lipid peroxidation and the declined survival rate of murine melanoma cells
after treatment with novel platinum(II) complexes [73]. On the other hand, in B lymphoma cells the
cisplatin-induced apoptosis occurs via a mechanism not involving oxidants [74]. Similarly, in mouse fibrosarcoma and human
astrocytoma cells, cisplatin caused oxidative stress-independent apoptotic cell
death. Importantly, in the same cell systems, overproduction of ROS was
detected after treatment with the novel platinum(IV) complex with a higher
cytotoxicity compared to cisplatin, which induced primarily necrosis [75].Nevertheless, the role of oxidative metabolism
associated with the action of metal-based cytostatics is still rather controversial.
Production of ROS and generation of lipid peroxides (LP), and thus induction of
oxidative stress, are considered to be some of the main causes of metal-based
drug organ toxicity, especially nephrotoxicity and hepatotoxicity [76]. Antioxidants can reduce or prevent many of
these side effects. In this context, the beneficial effects of compounds with
antioxidative properties like resveratrol or green tea have been demonstrated [77].It seems evident that the production and role
of ROS and LP in the effects of platinum drugs may depend on the structure and
concentration of the drug, the cellular system used, the capacity of the antioxidant
system, and can be further modulated by the presence of other factors like TNF,
which may alter oxidative/antioxidative balance [78].


## 8. Interactions of Platinum-Based Drugs with Cell Membranes and with Lipid Signaling/Metabolism

### 8.1. Impact of Polyunsaturated Fatty Acids (PUFAs) on Anticancer Drug Effects

There is increasing interest in the new
therapeutic approaches based on the knowledge of structure, function, and
alteration of membrane lipids [79]. Experimental data in vitro and in vivo and also several preclinical studies have
shown that certain PUFAs, especially of omega-3 type (found in marine plankton,
fish oil, and some plant oils), possess anticarcinogenic activities including
suppression of neoplastic transformation, cell growth inhibition, enhanced
apoptosis, and antiangiogenicity especially in colon, breast, and prostate
tissues. This topic is behind the scope of our review and many reviews have
been published until now [80–82]. Therefore, we pay
attention mostly to the mechanisms, which could be considered in possible anticancer
strategy using combined treatment with cytostatics and PUFAs.Several molecular mechanisms whereby omega-3
PUFAs may modify carcinogenic process have been proposed. These include
suppression of biosynthesis of arachidonic acid-derived eicosanoids (especially due to
decreased  COX-2 expression and
activity), modulation of membrane properties and signal transduction pathways,
effects on transcription factor activity, gene expression, alteration of
estrogen metabolism and insulin sensitivity, and increased or decreased
production of reactive oxygen and nitrogen radicals [83, 84] (for more details see below). Supplementation
of tumors with long-chained omega-3 PUFAs results in enrichment of tumor
phospholipid fractions with these PUFAs. Such cells then possess membranes with
increased fluidity, an elevated unsaturation index, enhanced transport
capabilities that result in accumulation of selective anticancer agents,
increased activity of selected drug-activating enzymes, and alteration of
signaling pathways important for cancer progression. These changes may enhance
tumor responsiveness to antineoplastic agents such as doxorubicin, mitomycin C,
or 5-fluorouracil [10, 85, 86], especially in tumor lines that are resistant
to chemotherapy. Studies of associations between the levels of fatty acids,
especially docosahexaenoic acid (DHA) stored in breast adipose tissue and the
tumor response to various chemotherapeutic agents in breast carcinoma patients
suggest that increased DHA content may improve therapeutic effects [87]. There are several reports demonstrating
possibilities of enhancement of cisplatin cytotoxicity and modulation of
cisplatin resistance by PUFAs in ovarian A2780, breast MCF-7, and small cell
lung GLC4-sensitive and GLC4-resistant cancer cell lines [11, 12, 88]. The anticancer activity of cisplatin has been
shown to be effectively supported by fish-oil diet under a conditioned balance
of oxidation and antioxidation adjusted by vitamins E and C [89]. Therefore, it appears that dietary administration
of omega-3 PUFAs might be a relatively nontoxic form of cancer-supporting
therapy increasing the effects of various anticancer agents including those
based on platinum. In addition, certain PUFAs may also prevent or reduce some
of the side effects of these therapies and tumor cachexia [90]. Therefore, it is important to study the
mechanisms of the effects of PUFAs and other lipid molecules and to look for their
links with cytostatic drug effects.


### 8.2. Cell Membrane Properties and Drug Effects

It has been shown that the levels of lipids in
cell membranes of cancer patients and in cancer cells resistant to chemotherapy
are altered [91, 92]. Nevertheless, the importance of cell membrane
composition/properties and their modulation for the effects of platinum drugs is
not clear. It has been found that cisplatin-resistant epidermal cells have a higher
membrane potential and more fluid membranes than cisplatin-sensitive cells [93]. On the other hand, no differences have been
found in major phospholipids (PLs) or in free cholesterol content between cisplatin-resistant
and cisplatin-sensitive human ovarian carcinoma cells. The decreased cisplatin accumulation
in resistant cells is probably not due to membrane changes, which might lead to
retardation of passive diffusion of the drug into the cells [94]. It has been demonstrated that cisplatin may  interact
and form complexes with negatively charged PLs like phosphatidylserine and
could change fluidity of the membranes [95, 96]. Modulation
of membrane lipid composition and metabolism may be important for cell behaviour,
especially in tumor cells, as their cell membranes are characterized by changes
in PL constituents and membrane fluidity, namely, by a decreased content of
PUFAs [97]. All above studies seem to suggest that modulation
of cell membrane composition and functions might modulate the effects of
platinum-based drugs.


### 8.3. Mechanisms of PUFAs Effects

The mammalian
organism is not able to synthesize PUFAs and thus they have to be supplied in
the diet. These essential PUFAs play an important role in transducing signals
from the extracellular space and function as inter- and intracellular mediators
and modulators of the cellular signaling network [98]. Their effects on various levels of cell organization
and their interaction with other endogenous or exogenous factors can finally significantly
influence cell proliferation, differentiation, and apoptosis [99].The mechanisms of action of PUFAs are not yet
fully understood and are likely to be very complex. The main mechanisms at the cellular
level might include:(1)
incorporation of PUFAs into cancer cell
membranes, thus altering their physical and functional properties and modulating
signaling pathways involved in the regulation of cytokinetics; changes in
membrane composition further determine the ability of associated proteins, such
as receptors, ion channels, and so forth, to move and interact;(2)
alteration of cell oxidative metabolism due
to lipid peroxidation and production of ROS or reactive nitrogen species (RNS);(3)
conversion to their reactive and biologically
active metabolites (e.g., eicosanoids);(4)
activation of specific nuclear receptors
and transcription factors leading to altered expression of genes involved in
proliferation, differentiation, and apoptosis (cytokinetics);(5)
interaction with other signal transduction
pathways.The most important of the above mechanisms are
discussed below.


### 8.4. PUFAs and Lipid Effects on Cell Membrane Level

As components of membrane PLs, PUFAs cause changes of membrane fluidity
and remodeling of membrane structure, which may influence receptor-ligand
interactions, signal transduction properties, and various aspects of membrane-mediated
cellular functions [100].PUFAs, particularly DHA as the longest and most
unsaturated acyl chain commonly found in membranes, are thought to be essential
components of lipid rafts which represent small dynamic plasma membrane
microdomains of tightly packed proteins and lipids enriched with cholesterol
and sphingolipids [101, 102]. These structures are functionally implicated
in the compartmentalization, modulation, and integration of cell signaling,
thus modulating important processes including cell growth, survival, and
adhesion [103]. Sphingolipids constitute a class of lipids
that may function as second messengers in different cellular processes as cell
differentiation, growth, and death. In particular, ceramides have been implied
in intracellular signal transduction systems regulating cellular
differentiation, survival, and apoptosis, and thus appear capable of changing
the lifestyle of virtually any cell type. Their production by sphingomyelinases
(SMase) can play a pivotal signaling role through direct interaction with
signaling proteins or through facilitating the formation and trafficking of
signal transduction complexes [104]. As components of lipid rafts, EPA and DHA might
increase the activity of SMase and the production of ceramide in breast cancer
cells simultaneously decreasing the level of EGF receptors in lipid rafts, thus
altering EGFR signaling and inhibiting cancer cell growth [105]. Apoptotic stimuli, such as death ligands or
chemotherapeutic agents, can also activate SMases and generate ceramide [106]. Based on these findings, PUFAs might modulate
function of proteins bound to membrane lipid domains including the transporter
function of proteins like P-glycoprotein (P-gp). The lipid phase of the plasma
membrane plays an important role with respect to MDR and P-gp, whose activity is highly sensitive
to lipid environment. P-gp may also be involved in lipid trafficking and
metabolism. PUFAs play a specific structural function in membranes and are
capable of positioning a double bond into the inner leaflet of a membrane. There is not much information about direct effects of PUFAs on
P-glycoprotein expression and activity. The available data suggest that DHA
enhanced bioavailability of CYP3A substrates in the rat intestine, but did not
inhibit P-gp [107]. In mouse leukemia cell line P388 GLA and DHA pretreatment improved
doxorubicin  (DOX)  cytotoxicity due to enhanced
DOX accumulation and changes of antioxidant enzyme activities. However, no
changes of P-gp expression were observed [108]. Promising results were achieved using fatty acid diesters, which are
more potent in reversing MDR in vitro due to their direct P-gp binding
properties [109].


### 8.5. PUFAs and Mitochondria

Apart from the
cytoplasmic membrane, PUFAs also physically interact with mitochondrial
membranes and alter their permeability by opening the permeability transition
pores and decreasing MMP [110]. Apoptotic signals depend on redox catalytic
interactions of cytochrome c with main mitochondrial PLs: cardiolipin and phosphatidylserine
[111]. It has been demonstrated that in colonocytes,
DHA is preferentially incorporated into cardiolipin, which coincides with
increasing unsaturation, induction of oxidative stress, release of cytochrome c,
and apoptosis [112]. PUFAs have also been shown to significantly
modulate the level of several Bcl-2 family proteins (e.g., Bid, Bcl-2) interacting
with mitochondrial membrane lipids which regulate apoptosis; cardiolipin has
been proposed to be a “lipid receptor” of Bid [113].


### 8.6. PUFAs and Oxidative Metabolism

Antiproliferative
and apoptotic effects of PUFAs were shown to be associated with changes on
mitochondria and induction of oxidative stress. Susceptibility of their double
bonds to oxidation is the reason for production of various types of
biologically active metabolites like eicosanoids, ROS and RNS, and lipid
peroxides (LPs) [114]. We together with many others have shown that
supplementation of cultured cells with PUFAs dose-dependently enhanced ROS
production and lipid peroxidation [115, 116]. Many studies have documented that PUFAs
augmented free radical generation and formation of LPs selectively in the tumor
cells compared to normal cells despite the fact that the uptake of fatty acids
in the tumor cells was lower [117]. There is a close correlation between the rate
of lipid peroxidation and the degree of malignancy of tumor cells,
and the susceptibility to ROS-induced cytotoxicity. Dividing cells show low
levels of lipid peroxidation as compared to slowly dividing or nondividing
cells. Resistance to lipid peroxidation appears to occur at the premalignant
stage of the carcinogenic process. ROS also serve as common messengers
downstream of various stimulus-specific pathways leading to NF-κB activation, including TNF-α, PUFAs, or cyclooxygenases (COXs), and are
involved in the regulation of immune response, proliferation, and apoptosis [118, 119].Taken together, ROS and LPs can function as a “double-edged
sword.” Their excess may damage normal cell populations, while they may have
antiproliferative and apoptotic effects in cancer cell population [97]. These events may interfere with the
modulation of oxidative metabolism induced by platinum-based anticancer drugs,
as described earlier.


### 8.7. Effects of PUFAs on Signal Transduction and Gene Expression

PUFAs and their metabolites are involved in
many intracellular signaling pathways activating or inhibiting various
components of this machinery and modulate gene expression [120]. They may alter the levels and signaling
pathways of various mediators, such as eicosanoids and cytokines [46, 115, 121].The increase of phosphatidylserine by DHA
resulted in a modification of the PI3K/Akt pathway, which is a major signaling
pathway mediating proliferative
and differentiation signals in many cell types in vitro and in vivo.ω-3 PUFAs
decrease cell proliferation and induce apoptosis possibly by decreasing signal
transduction through the Akt/NF-κB
cell survival pathway. In addition, PUFA-mediated modulation of other signaling
pathways, such as those involving protein kinase C, extracellular
signal-regulated kinase, and p38 mitogen-activated protein kinase, has been
reported [122]. Fatty acids and their metabolites are
generally considered as agonists of peroxisome proliferator-activated receptors
(PPARs). Furthermore, PUFAs can also interact with other transcription
mediators including NF-κB [120]. PPAR and NF-κB activation can be further
associated with the regulation of expression of COX-2, an enzyme playing an important
role in many pathological conditions [123].Several studies using in vitro cell cultures have confirmed that
certain PUFAs have a selective cytotoxic or antiproliferative effect on tumor
cells and a minimal or no effect on normal cells. However, often cells from
different species and different tissues were compared. The effects of PUFAs also
depend on time of treatment, concentration, and culture conditions such as
growth rate, cell confluency, type of medium, and amount of serum [124]. Nevertheless, it has been shown that the fatty acid composition of tumor tissues differs from normal ones and that also activities of
fatty acid metabolism enzymes and oxidative defence were distinct. Tumor cells
are characterized by the loss or decreased activity of desaturases and thus a decreased
metabolism of precursor omega-6 linoleic acid and omega-3 α-linolenic acid to
longer chain fatty acids such as AA or EPA and DHA. They also have a low level
of cytochrome P450 enzymes which can initiate and propagate lipid peroxides. Moreover, malignant cells have markedly enhanced levels of antioxidants and low
levels of superoxid dismutase, glutathione peroxidase, and catalase enzymes. These properties may be prerequisite for tumor-selective augmentation of free
radical generation and formation of lipid peroxides compared to normal cells after
exogenous addition of PUFAs. These products of PUFA oxidative metabolism are
powerful inducers of p53 activity, influence the expression and activity of
members of the Bcl-2 family, activate caspases, and shorten telomeres, thus
inducing apoptosis of cancer cells. They were also shown to play a role in the
metastatic process and angiogenesis [97]. Recently developed animal models can closely mimic
the clinical course of carcinogenesis and showed that it is especially the
ratio of omega-3 to omega-6 PUFAs which plays an important role in cancer
development [125]. The data from multiorgan carcinogenesis model in
male rats treated with various types of PUFAs provide evidence of their
organotropic effects on carcinogenesis, which correlates with reduction of
tissue AA levels in the target organs [126]. It was reported that PUFAs are potent inhibitors of
hepatic glycolysis and de novo lipogenesis and promote a shift from fatty acid
synthesis and storage to oxidation inhibiting lipogenic genes through specific
transcription factors SREBP-1 and ChREBP [127]. Thus, further studies evaluating especially the
effects of PUFAs on tumor versus normal cells and tissues using in vivo systems
are necessary before copper-bottomed therapeutic use of omega-3 PUFAs for
cancer patients. The future promising approaches include use of new PUFA
analogues [109] and the development of specific conjugates of PUFAs
with cytostatic drugs. In tumor-bearing mice, it has been demonstrated that
conjugates of DHA with taxanes are stable in plasma for a long time, are less
toxic than taxanes alone, and posses significantly increased antitumor activity
[128, 129]. Recently, the new conjugates of second generation of
taxoids with DHA exhibited strong activity against drug-resistant colon cancer
and drug-sensitive ovarian cancer xenografts in mice [130] .


## 8.1. Impact of Polyunsaturated Fatty Acids (PUFAs) on Anticancer Drug Effects

There is increasing interest in the new
therapeutic approaches based on the knowledge of structure, function, and
alteration of membrane lipids [79]. Experimental data in vitro and in vivo and also several preclinical studies have
shown that certain PUFAs, especially of omega-3 type (found in marine plankton,
fish oil, and some plant oils), possess anticarcinogenic activities including
suppression of neoplastic transformation, cell growth inhibition, enhanced
apoptosis, and antiangiogenicity especially in colon, breast, and prostate
tissues. This topic is behind the scope of our review and many reviews have
been published until now [80–82]. Therefore, we pay
attention mostly to the mechanisms, which could be considered in possible anticancer
strategy using combined treatment with cytostatics and PUFAs.Several molecular mechanisms whereby omega-3
PUFAs may modify carcinogenic process have been proposed. These include
suppression of biosynthesis of arachidonic acid-derived eicosanoids (especially due to
decreased  COX-2 expression and
activity), modulation of membrane properties and signal transduction pathways,
effects on transcription factor activity, gene expression, alteration of
estrogen metabolism and insulin sensitivity, and increased or decreased
production of reactive oxygen and nitrogen radicals [83, 84] (for more details see below). Supplementation
of tumors with long-chained omega-3 PUFAs results in enrichment of tumor
phospholipid fractions with these PUFAs. Such cells then possess membranes with
increased fluidity, an elevated unsaturation index, enhanced transport
capabilities that result in accumulation of selective anticancer agents,
increased activity of selected drug-activating enzymes, and alteration of
signaling pathways important for cancer progression. These changes may enhance
tumor responsiveness to antineoplastic agents such as doxorubicin, mitomycin C,
or 5-fluorouracil [10, 85, 86], especially in tumor lines that are resistant
to chemotherapy. Studies of associations between the levels of fatty acids,
especially docosahexaenoic acid (DHA) stored in breast adipose tissue and the
tumor response to various chemotherapeutic agents in breast carcinoma patients
suggest that increased DHA content may improve therapeutic effects [87]. There are several reports demonstrating
possibilities of enhancement of cisplatin cytotoxicity and modulation of
cisplatin resistance by PUFAs in ovarian A2780, breast MCF-7, and small cell
lung GLC4-sensitive and GLC4-resistant cancer cell lines [11, 12, 88]. The anticancer activity of cisplatin has been
shown to be effectively supported by fish-oil diet under a conditioned balance
of oxidation and antioxidation adjusted by vitamins E and C [89]. Therefore, it appears that dietary administration
of omega-3 PUFAs might be a relatively nontoxic form of cancer-supporting
therapy increasing the effects of various anticancer agents including those
based on platinum. In addition, certain PUFAs may also prevent or reduce some
of the side effects of these therapies and tumor cachexia [90]. Therefore, it is important to study the
mechanisms of the effects of PUFAs and other lipid molecules and to look for their
links with cytostatic drug effects.


## 8.2. Cell Membrane Properties and Drug Effects

It has been shown that the levels of lipids in
cell membranes of cancer patients and in cancer cells resistant to chemotherapy
are altered [91, 92]. Nevertheless, the importance of cell membrane
composition/properties and their modulation for the effects of platinum drugs is
not clear. It has been found that cisplatin-resistant epidermal cells have a higher
membrane potential and more fluid membranes than cisplatin-sensitive cells [93]. On the other hand, no differences have been
found in major phospholipids (PLs) or in free cholesterol content between cisplatin-resistant
and cisplatin-sensitive human ovarian carcinoma cells. The decreased cisplatin accumulation
in resistant cells is probably not due to membrane changes, which might lead to
retardation of passive diffusion of the drug into the cells [94]. It has been demonstrated that cisplatin may  interact
and form complexes with negatively charged PLs like phosphatidylserine and
could change fluidity of the membranes [95, 96]. Modulation
of membrane lipid composition and metabolism may be important for cell behaviour,
especially in tumor cells, as their cell membranes are characterized by changes
in PL constituents and membrane fluidity, namely, by a decreased content of
PUFAs [97]. All above studies seem to suggest that modulation
of cell membrane composition and functions might modulate the effects of
platinum-based drugs.


## 8.3. Mechanisms of PUFAs Effects

The mammalian
organism is not able to synthesize PUFAs and thus they have to be supplied in
the diet. These essential PUFAs play an important role in transducing signals
from the extracellular space and function as inter- and intracellular mediators
and modulators of the cellular signaling network [98]. Their effects on various levels of cell organization
and their interaction with other endogenous or exogenous factors can finally significantly
influence cell proliferation, differentiation, and apoptosis [99].The mechanisms of action of PUFAs are not yet
fully understood and are likely to be very complex. The main mechanisms at the cellular
level might include:(1)
incorporation of PUFAs into cancer cell
membranes, thus altering their physical and functional properties and modulating
signaling pathways involved in the regulation of cytokinetics; changes in
membrane composition further determine the ability of associated proteins, such
as receptors, ion channels, and so forth, to move and interact;(2)
alteration of cell oxidative metabolism due
to lipid peroxidation and production of ROS or reactive nitrogen species (RNS);(3)
conversion to their reactive and biologically
active metabolites (e.g., eicosanoids);(4)
activation of specific nuclear receptors
and transcription factors leading to altered expression of genes involved in
proliferation, differentiation, and apoptosis (cytokinetics);(5)
interaction with other signal transduction
pathways.The most important of the above mechanisms are
discussed below.


## 8.4. PUFAs and Lipid Effects on Cell Membrane Level

As components of membrane PLs, PUFAs cause changes of membrane fluidity
and remodeling of membrane structure, which may influence receptor-ligand
interactions, signal transduction properties, and various aspects of membrane-mediated
cellular functions [100].PUFAs, particularly DHA as the longest and most
unsaturated acyl chain commonly found in membranes, are thought to be essential
components of lipid rafts which represent small dynamic plasma membrane
microdomains of tightly packed proteins and lipids enriched with cholesterol
and sphingolipids [101, 102]. These structures are functionally implicated
in the compartmentalization, modulation, and integration of cell signaling,
thus modulating important processes including cell growth, survival, and
adhesion [103]. Sphingolipids constitute a class of lipids
that may function as second messengers in different cellular processes as cell
differentiation, growth, and death. In particular, ceramides have been implied
in intracellular signal transduction systems regulating cellular
differentiation, survival, and apoptosis, and thus appear capable of changing
the lifestyle of virtually any cell type. Their production by sphingomyelinases
(SMase) can play a pivotal signaling role through direct interaction with
signaling proteins or through facilitating the formation and trafficking of
signal transduction complexes [104]. As components of lipid rafts, EPA and DHA might
increase the activity of SMase and the production of ceramide in breast cancer
cells simultaneously decreasing the level of EGF receptors in lipid rafts, thus
altering EGFR signaling and inhibiting cancer cell growth [105]. Apoptotic stimuli, such as death ligands or
chemotherapeutic agents, can also activate SMases and generate ceramide [106]. Based on these findings, PUFAs might modulate
function of proteins bound to membrane lipid domains including the transporter
function of proteins like P-glycoprotein (P-gp). The lipid phase of the plasma
membrane plays an important role with respect to MDR and P-gp, whose activity is highly sensitive
to lipid environment. P-gp may also be involved in lipid trafficking and
metabolism. PUFAs play a specific structural function in membranes and are
capable of positioning a double bond into the inner leaflet of a membrane. There is not much information about direct effects of PUFAs on
P-glycoprotein expression and activity. The available data suggest that DHA
enhanced bioavailability of CYP3A substrates in the rat intestine, but did not
inhibit P-gp [107]. In mouse leukemia cell line P388 GLA and DHA pretreatment improved
doxorubicin  (DOX)  cytotoxicity due to enhanced
DOX accumulation and changes of antioxidant enzyme activities. However, no
changes of P-gp expression were observed [108]. Promising results were achieved using fatty acid diesters, which are
more potent in reversing MDR in vitro due to their direct P-gp binding
properties [109].


## 8.5. PUFAs and Mitochondria

Apart from the
cytoplasmic membrane, PUFAs also physically interact with mitochondrial
membranes and alter their permeability by opening the permeability transition
pores and decreasing MMP [110]. Apoptotic signals depend on redox catalytic
interactions of cytochrome c with main mitochondrial PLs: cardiolipin and phosphatidylserine
[111]. It has been demonstrated that in colonocytes,
DHA is preferentially incorporated into cardiolipin, which coincides with
increasing unsaturation, induction of oxidative stress, release of cytochrome c,
and apoptosis [112]. PUFAs have also been shown to significantly
modulate the level of several Bcl-2 family proteins (e.g., Bid, Bcl-2) interacting
with mitochondrial membrane lipids which regulate apoptosis; cardiolipin has
been proposed to be a “lipid receptor” of Bid [113].


## 8.6. PUFAs and Oxidative Metabolism

Antiproliferative
and apoptotic effects of PUFAs were shown to be associated with changes on
mitochondria and induction of oxidative stress. Susceptibility of their double
bonds to oxidation is the reason for production of various types of
biologically active metabolites like eicosanoids, ROS and RNS, and lipid
peroxides (LPs) [114]. We together with many others have shown that
supplementation of cultured cells with PUFAs dose-dependently enhanced ROS
production and lipid peroxidation [115, 116]. Many studies have documented that PUFAs
augmented free radical generation and formation of LPs selectively in the tumor
cells compared to normal cells despite the fact that the uptake of fatty acids
in the tumor cells was lower [117]. There is a close correlation between the rate
of lipid peroxidation and the degree of malignancy of tumor cells,
and the susceptibility to ROS-induced cytotoxicity. Dividing cells show low
levels of lipid peroxidation as compared to slowly dividing or nondividing
cells. Resistance to lipid peroxidation appears to occur at the premalignant
stage of the carcinogenic process. ROS also serve as common messengers
downstream of various stimulus-specific pathways leading to NF-κB activation, including TNF-α, PUFAs, or cyclooxygenases (COXs), and are
involved in the regulation of immune response, proliferation, and apoptosis [118, 119].Taken together, ROS and LPs can function as a “double-edged
sword.” Their excess may damage normal cell populations, while they may have
antiproliferative and apoptotic effects in cancer cell population [97]. These events may interfere with the
modulation of oxidative metabolism induced by platinum-based anticancer drugs,
as described earlier.


## 8.7. Effects of PUFAs on Signal Transduction and Gene Expression

PUFAs and their metabolites are involved in
many intracellular signaling pathways activating or inhibiting various
components of this machinery and modulate gene expression [120]. They may alter the levels and signaling
pathways of various mediators, such as eicosanoids and cytokines [46, 115, 121].The increase of phosphatidylserine by DHA
resulted in a modification of the PI3K/Akt pathway, which is a major signaling
pathway mediating proliferative
and differentiation signals in many cell types in vitro and in vivo.ω-3 PUFAs
decrease cell proliferation and induce apoptosis possibly by decreasing signal
transduction through the Akt/NF-κB
cell survival pathway. In addition, PUFA-mediated modulation of other signaling
pathways, such as those involving protein kinase C, extracellular
signal-regulated kinase, and p38 mitogen-activated protein kinase, has been
reported [122]. Fatty acids and their metabolites are
generally considered as agonists of peroxisome proliferator-activated receptors
(PPARs). Furthermore, PUFAs can also interact with other transcription
mediators including NF-κB [120]. PPAR and NF-κB activation can be further
associated with the regulation of expression of COX-2, an enzyme playing an important
role in many pathological conditions [123].Several studies using in vitro cell cultures have confirmed that
certain PUFAs have a selective cytotoxic or antiproliferative effect on tumor
cells and a minimal or no effect on normal cells. However, often cells from
different species and different tissues were compared. The effects of PUFAs also
depend on time of treatment, concentration, and culture conditions such as
growth rate, cell confluency, type of medium, and amount of serum [124]. Nevertheless, it has been shown that the fatty acid composition of tumor tissues differs from normal ones and that also activities of
fatty acid metabolism enzymes and oxidative defence were distinct. Tumor cells
are characterized by the loss or decreased activity of desaturases and thus a decreased
metabolism of precursor omega-6 linoleic acid and omega-3 α-linolenic acid to
longer chain fatty acids such as AA or EPA and DHA. They also have a low level
of cytochrome P450 enzymes which can initiate and propagate lipid peroxides. Moreover, malignant cells have markedly enhanced levels of antioxidants and low
levels of superoxid dismutase, glutathione peroxidase, and catalase enzymes. These properties may be prerequisite for tumor-selective augmentation of free
radical generation and formation of lipid peroxides compared to normal cells after
exogenous addition of PUFAs. These products of PUFA oxidative metabolism are
powerful inducers of p53 activity, influence the expression and activity of
members of the Bcl-2 family, activate caspases, and shorten telomeres, thus
inducing apoptosis of cancer cells. They were also shown to play a role in the
metastatic process and angiogenesis [97]. Recently developed animal models can closely mimic
the clinical course of carcinogenesis and showed that it is especially the
ratio of omega-3 to omega-6 PUFAs which plays an important role in cancer
development [125]. The data from multiorgan carcinogenesis model in
male rats treated with various types of PUFAs provide evidence of their
organotropic effects on carcinogenesis, which correlates with reduction of
tissue AA levels in the target organs [126]. It was reported that PUFAs are potent inhibitors of
hepatic glycolysis and de novo lipogenesis and promote a shift from fatty acid
synthesis and storage to oxidation inhibiting lipogenic genes through specific
transcription factors SREBP-1 and ChREBP [127]. Thus, further studies evaluating especially the
effects of PUFAs on tumor versus normal cells and tissues using in vivo systems
are necessary before copper-bottomed therapeutic use of omega-3 PUFAs for
cancer patients. The future promising approaches include use of new PUFA
analogues [109] and the development of specific conjugates of PUFAs
with cytostatic drugs. In tumor-bearing mice, it has been demonstrated that
conjugates of DHA with taxanes are stable in plasma for a long time, are less
toxic than taxanes alone, and posses significantly increased antitumor activity
[128, 129]. Recently, the new conjugates of second generation of
taxoids with DHA exhibited strong activity against drug-resistant colon cancer
and drug-sensitive ovarian cancer xenografts in mice [130] .


## 9. Targeting of Cisplatin-Based Drugs

New strategy of development of PUFA-anticancer
drug conjugates might be based on the fact that some PUFAs are taken more
rapidly by tumor cells than by normal cells. The new DHA-taxoid conjugates
exhibit a strong activity against drug-resistant colon cancer and
drug-sensitive ovarian cancer xenografts in mice and a reduced systemic
toxicity as compared to taxoids alone [130]. It might be speculated that agents with
higher lipophilicity, such as LA-12, would be more effective in suppression of
proliferation and induction of cell death in the conditions of modulated
phospholipid metabolism. The above-mentioned effects should be taken into
account when developing new strategies targeting platinum-based drugs.
Encapsulation in liposomes and linking of cytotoxic platinum drugs to
macromolecular carriers are strategies for targeting to solid tumors. Effective
accumulation of drug carriers in the tumor requires a long half-time in
circulation as well as an appropriate size of carriers (liposomes or polymers),
which should be below 120 nm to penetrate into the tumor tissue through
fenestrations in the vasculature of capillary vessels nourishing the tumor. The
extracellular matrix represents another hindrance in the penetration of drug
carriers deep into the tumor tissue [131]. A liposomal cisplatin preparation SPI77 has
been studied in both pediatric and adult patients, but the liposomes used were
too stable and cisplatin was insufficiently released from them [132]. An improved liposomal cisplatin termed
lipoplatin [133] is about to enter phase III studies, and
liposomal oxaliplatin is expected to enter the clinical stage of development in
the near future [134].Several cisplatin derivatives with long alkyl
chains were prepared to improve their direct incorporation into the liposomal
membrane. Preliminary evidence of antitumor activity of L-NDDP (liposomal
formulation ofcis-bis(neodecanoato)(trans-R,r-cyclohexane-1,2-diamine)platinum(II))
was reported from a phase II trial in refractory metastatic colorectal cancer [135]. Nevertheless, clinical development has been
halted in order to improve drug formulation.


## 10. Other Mechanisms

Although this review is dedicated principally
to the role of lipids and possibilities of modulating the effects of novel
platinum derivatives, we would also like to briefly touch the issues of
inhibition of intercellular communication and possibilities of modulation of
multidrug resistance.


### 10.1. Modulation of Gap-Junctional Intercellular Communication by Platinum Derivatives

Intracellular communication through gap
junction (GJIC) has been shown to be crucial for both tissue and cellular
homeostasis. Disruption of GJIC through defects in expression, posttranslational
modification, and localization of connexins and/or mutation of genes coding for
connexins seems to be one of the mechanisms involved in carcinogenesis [136]. Many chemopreventive and antitumor agents upregulate
GJIC, which can be a significant mechanism of their effects [137]. On the other hand, tumor promoters (e.g., phorbol
esters, dithiothreitol) and also some anticancer drugs such as doxorubicin,
taxol, and also cisplatin decrease GJIC [138–140]. Our own data indicate that disturbance of
tissue homeostasis owing to inhibition of GJIC may be linked to a direct
cytotoxic effect of platinum drugs, rather than an indirect effect found for
some carcinogens (e.g., TPA) or polyaromatic tumor promoters [24]. It has been
shown that the increased level of connexin 43 may significantly improve the efficiency
of anticancer therapy [141–143]; the level of connexin 43 expression
correlates with the sensitivity to anticancer therapy and the quality of
regeneration after the therapy [141, 144]. These studies strongly support the importance
of “bystander” cells and functional GJIC in cancer therapy and recovery [145, 146]. Interestingly, it has been shown that
cisplatin can induce cell-interdependent cell death through Ku/DNA-dependent
protein kinase signaling in mouse embryonic fibroblasts [147]. Inhibition of GJIC might thus significantly
limit its efficiency in target cells, suggesting that restoration of GJIC in
tumor cells or prevention of inhibitory effects of anticancer drugs on GJIC
might increase the effectiveness of both established and novel platinum
derivatives. This area of research presents a challenge for the development of
new drugs and/or treatment protocols, which would lead to elimination of these side
effects.


### 10.2. Role of Resistance in Response to Platinum Derivatives

Resistance is a major problem in the application
of platinum-based therapy. The postulated mechanisms of cisplatin resistance
include reduced drug accumulation by changing the profile of uptake/efflux,
inactivation of cisplatin by increased levels of intracellular thiols such as
glutathione, metallothionein, or other sulfur-containing molecules, increased
repair of cisplatin adducts, increased tolerance to cisplatin adducts, and
failure of apoptotic response. Reduced intracellular drug accumulation in
resistant cells might be ascribed to an inhibition of drug uptake, an increase
in drug efflux, or both. Resistance to cisplatin can be acquired through
chronic drug exposure or it can present itself as an intrinsic phenomenon. The
results from our laboratory showed a great potential of LA-12 to overcome both
acquired [22] and intrinsic resistance in ovarian cancer
cells [25]. The mechanisms enabling LA-12 to defeat
platinum resistance are currently unknown. However, they might be related to a
number of events, including enhanced accumulation of the drug, strong
inhibition of DNA polymerization, decreased DNA repair, and formation of
DNA-protein cross-links as compared to cisplatin [26].The development of alternative platinum analogues
or their application, in combination with drugs acting through different
mechanisms, seems to be an effective strategy, which would help to solve problems
of cisplatin resistance. However, this strategy usually fails in cases when the
resistance of the cells is due to their inability to die by apoptosis, which is
not directly drug-dependent [148]. Interestingly, LA-12 is able to induce not
only apoptosis, but also a caspase-independent type of cell death [25]. This could be an advantage when using this novel
platinum(IV) agent for the treatment of tumors with defects in the classical
apoptotic pathway.Another strategy to address the problem of
cisplatin resistance is drug resistance reversal therapy [149]. This therapy is simply focused on restoration
of sensitivity to platinum-based drugs through targeting of known mechanisms of
resistance using specific inhibitors of influx, efflux channels and
transporters, glutathione-S-transferase, DNA repair, p53 pathway, and so forth. It is
currently not known whether such strategies would also increase the efficiency
of LA-12 and related compounds.


## 10.1. Modulation of Gap-Junctional Intercellular Communication by Platinum Derivatives

Intracellular communication through gap
junction (GJIC) has been shown to be crucial for both tissue and cellular
homeostasis. Disruption of GJIC through defects in expression, posttranslational
modification, and localization of connexins and/or mutation of genes coding for
connexins seems to be one of the mechanisms involved in carcinogenesis [136]. Many chemopreventive and antitumor agents upregulate
GJIC, which can be a significant mechanism of their effects [137]. On the other hand, tumor promoters (e.g., phorbol
esters, dithiothreitol) and also some anticancer drugs such as doxorubicin,
taxol, and also cisplatin decrease GJIC [138–140]. Our own data indicate that disturbance of
tissue homeostasis owing to inhibition of GJIC may be linked to a direct
cytotoxic effect of platinum drugs, rather than an indirect effect found for
some carcinogens (e.g., TPA) or polyaromatic tumor promoters [24]. It has been
shown that the increased level of connexin 43 may significantly improve the efficiency
of anticancer therapy [141–143]; the level of connexin 43 expression
correlates with the sensitivity to anticancer therapy and the quality of
regeneration after the therapy [141, 144]. These studies strongly support the importance
of “bystander” cells and functional GJIC in cancer therapy and recovery [145, 146]. Interestingly, it has been shown that
cisplatin can induce cell-interdependent cell death through Ku/DNA-dependent
protein kinase signaling in mouse embryonic fibroblasts [147]. Inhibition of GJIC might thus significantly
limit its efficiency in target cells, suggesting that restoration of GJIC in
tumor cells or prevention of inhibitory effects of anticancer drugs on GJIC
might increase the effectiveness of both established and novel platinum
derivatives. This area of research presents a challenge for the development of
new drugs and/or treatment protocols, which would lead to elimination of these side
effects.


## 10.2. Role of Resistance in Response to Platinum Derivatives

Resistance is a major problem in the application
of platinum-based therapy. The postulated mechanisms of cisplatin resistance
include reduced drug accumulation by changing the profile of uptake/efflux,
inactivation of cisplatin by increased levels of intracellular thiols such as
glutathione, metallothionein, or other sulfur-containing molecules, increased
repair of cisplatin adducts, increased tolerance to cisplatin adducts, and
failure of apoptotic response. Reduced intracellular drug accumulation in
resistant cells might be ascribed to an inhibition of drug uptake, an increase
in drug efflux, or both. Resistance to cisplatin can be acquired through
chronic drug exposure or it can present itself as an intrinsic phenomenon. The
results from our laboratory showed a great potential of LA-12 to overcome both
acquired [22] and intrinsic resistance in ovarian cancer
cells [25]. The mechanisms enabling LA-12 to defeat
platinum resistance are currently unknown. However, they might be related to a
number of events, including enhanced accumulation of the drug, strong
inhibition of DNA polymerization, decreased DNA repair, and formation of
DNA-protein cross-links as compared to cisplatin [26].The development of alternative platinum analogues
or their application, in combination with drugs acting through different
mechanisms, seems to be an effective strategy, which would help to solve problems
of cisplatin resistance. However, this strategy usually fails in cases when the
resistance of the cells is due to their inability to die by apoptosis, which is
not directly drug-dependent [148]. Interestingly, LA-12 is able to induce not
only apoptosis, but also a caspase-independent type of cell death [25]. This could be an advantage when using this novel
platinum(IV) agent for the treatment of tumors with defects in the classical
apoptotic pathway.Another strategy to address the problem of
cisplatin resistance is drug resistance reversal therapy [149]. This therapy is simply focused on restoration
of sensitivity to platinum-based drugs through targeting of known mechanisms of
resistance using specific inhibitors of influx, efflux channels and
transporters, glutathione-S-transferase, DNA repair, p53 pathway, and so forth. It is
currently not known whether such strategies would also increase the efficiency
of LA-12 and related compounds.


## 11. Conclusion

More than thirty years of experimental and clinical studies on platinum-derived
compounds have led to the development of potent anticancer drugs. However, despite
the progress within this field, treatment of generally more resistant types of
cancer, such as colon or prostate cancer, by more efficient cisplatin compounds
still remains elusive. Recently developed platinum(IV) complexes with
adamantylamine have the potential for a significantly improved therapy of many types
of tumors. Based on the findings discussed above, we propose that several innovative
approaches might be used to modulate the anticancer effects of platinum(IV)
complexes with adamantylamine. These may include, for example, combined
application with PUFAs and/or death ligands, or modulation of intercellular
communication. Although sufficient data on the role of endogenous regulators in
the mechanism of action of platinum-based drugs in the regulation of mammalian
cell kinetics (proliferation, differentiation, and apoptosis) are currently limited,
the role of PUFAs in modulating these processes should be emphasized, as
summarized in Figure2. A possible potentiation of platinum-based
drug effects by relatively cheap PUFAs from the diet and/or lipid preparations
would allow to decrease the doses of expensive antineoplastic drugs, thus
preventing side effects and allowing to use them also in low-sensitivity tumor types.
Further studies are needed to address these questions and to understand the
mechanisms involved.Figure 2
A diagram depicting potential
innovative chemotherapy strategies which might be employed to increase efficacy
of novel platinum(IV) complexes.



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*Source: 417897-2008-04-08.xml*