# Proteomic Approaches in Understanding Action Mechanisms of

Authors: Ying Wang; Jen-Fu Chiu
Journal: Metal-Based Drugs
 (2008)

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## Abstract

Medicinal inorganic chemistry has been stimulating largely by the success of the anticancer drug, cisplatin. Various metal complexes are currently used as therapeutic agents (e.g., Pt, Au, and Ru) in the treatment of malignant diseases, including several types of cancers. Understanding the mechanism of action of these metal-based drugs is for the design of more effective drugs. Proteomic approaches combined with other biochemical methods can provide comprehensive understanding of responses that are involved in metal-based anticancer drugs-induced cell death, including insights into cytotoxic effects of metal-based anticancer drugs, correlation of protein alterations to drug targets, and prediction of drug resistance and toxicity. This information, when coupled with clinical data, can provide rational basses for the future design and modification of present used metal-based anticancer drugs.


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## Body

## 1. Metal-Based Anticancer Drugs

Medicinal applications of metal complexes as therapeutic drugs have a more than 5000-year
history [1]. Since the discovery of the
anticancer activity of cisplatin by Shimizu and Rosenberg 35 years ago, there has been
a rapid expansion in research to find new,
more effective metal-based anticancer drugs [2]. The major classes of
metal-based anticancer drugs include platinum (II), gold (I) and gold (III),
metalloporphyrin, ruthenium (II) and ruthenium (III), bismuth (III), rhenium (I),
and copper (II)  compounds.


### 1.1. Platinum(II) Anticancer Drugs

Cisplatin represents one of the most potent drugs available in the cancer chemotherapy
for several solid tumors, such as testicular, ovarian, bladder, and neck
cancers [3]. It is generally believed
that cisplatin exhibited its anticancer effects through preferentially binding
to quinine N-7 of DNA, and then cause DNA damage specifically in cancer cells, subsequently
leading to cell death [4]. After successes achieved
with platinum complexes, there is a tremendous increase in the search for
platinum complexes with different ligands that might produce more specific
anticancer effects. Some of these platinum-based drugs have been approved by
the Food and Drug Administration (FDA), including carboplatin for the treatment
of ovarian cancer [5, 6], oxaliplatin for metastatic
colorectal cancer [7, 8], satraplatin for
hormone-refractory prostate cancer [9], and picoplatin for
small-cell lung cancer [10].Transplatinum compounds follow different patterns of cell killing in comparison to
cisplatinum, thus giving a reason for optimism in their development as a new
class of platinum-based anticancer drugs [11]. The initial report of
anticancer properties of a dinuclear platinum complex in 1988 started a new
paradigm in platinum-based chemotherapy. Several multinuclear platinum
complexes have entered clinical trials in recent years, with varying results [12, 13]. The major limitations of cisplatin
and other platinum anticancer drugs are related to drug resistance and their
side effects, including nephrotoxicity, neurotoxicity, and emetogensis [14]. Resistance to cisplatin is
multifactorial, most cases consist of mechanisms limiting the formation of DNA
adducts or operating downstream of the cisplatin-DNA interaction to promote
cell survival [15].


### 1.2. Gold (I) and Gold (III) Anticancer Complexes

Gold (I) complexes had been used for the treatment of arthritis some decades ago, but most of them
disappeared from the drug market because of intolerable side effects, such as
gastrointestinal adverse reactions, nephrotoxicity, and haematological
reactions. However, the design and testing of gold complexes, especially gold (III)
complexes with anticancer activity begin to be intensively pursued in the past
few years. The potential use of gold (III) complexes as anticancer drugs were
based on three rationales [16–18]: (a) analogies between square
planar complexes of both platinum (II) and gold (III) are d8 ions;
(b) analogy to the immunomodulatory effects of gold (I) antiarthritic agents;
and (c) complexation of gold (I) and gold (III) with known anticancer agents to
form new compounds with enhanced activity. Buckley et al. first reported some organogold (III) complexes endowed with
significant cytotoxic and anticancer properties [19]. During the past decades,
various gold (III) complexes with sufficient stability in the physiological
environment have been synthesized and evaluated for in vitro anticancer properties. Some of these gold (III)
complexes turned out to exhibit relevant cytotoxic effects in vitro and were the subject of
further biochemical and pharmacological investigations [20–33]. Our previous findings showed
that gold (III) mesotetraarylporphyrin
1a was stable against demetallation in physiological conditions and exhibited
higher cytotoxicity than cisplatin against a panel of human cancer cell lines [34–37]. The major limitation of gold
(III) complexes is that few exhibit good stability under physiological
conditions, due to the reduction of gold (III) to gold (I) [38]. However, low cisplatin cross
resistance has been observed in gold complexes [39]. There is therefore
considerable interest in the development of tumor-selective and stable gold
anticancer drugs.


### 1.3. Metalloporphyrin Drugs

Metalloporphyrin drugs are new class of antioxidant enzyme mimetics with novel structure: a
metal in the center of porphyrin ligand. Metalloporphyrins (e.g., MnTBAP) have previously
been used to inhibit age-related oxidative damage in myocardium of mice that
are lacking mitochondrial enzyme manganese superoxide dismutase [40]. Afterwards, metalloporphyrin
drugs began to be used as photodynamic therapy agents for certain solid tumors [41]. Photodynamic therapy is
based on the concept that porphyrins are known to be rapidly and preferentially
taken up by the tumor cells with higher intakes of lipoproteins [42, 43]. When such photosensitizers
are irradiated with an appropriate wavelength of visible or near infrared (NIR)
light, the excited molecules can transfer their energy to molecular oxygen in
the surroundings, which is normally in its triplet ground state. This results
in the formation of cytotoxic reactive oxygen species (ROSs), particularly
singlet oxygen [44]. ROSs are responsible for
oxidizing various cellular compartments including plasma, mitochondrial,
lysosomal, and nuclear membranes, resulting in irreversible damage of tumor [34, 44]. Therefore, under appropriate
conditions, photodynamic therapy offers the advantage of an effective and
selective method of destroying diseased tissues without damaging adjacent
healthy cells [42, 43].Since the approval
of Photofrin by FDA for chemotherapy [45], porphyrin derivatives with
different metal in the center of the molecule have been widely used as
photosensitizers for photodynamic therapy in the treatment of cancer, including
chlorophyllin copper complex as superoxide dismutase mimics [46, 47], FeTBAP and MnTBAP [48, 49], ZnTBAP [50], motexafin gadolinium (MGd) [51]. Different modes of actions have been
suggested for different kinds
of metalloporphyrins. For example, MGd has been shown to inhibit heme
oxygenase-1 (HO-1) activity that results in inactivation of the antiapoptotic properties of the
products of HO-1 [51]. While FeTBAP and MnTBAP have
been reported to be superoxide anion scavengers [48]. MGd is also an active
inhibitor of cytochrome P450 enzymes, although with a lower potency than that
exhibited for inhibition of HO-1.


### 1.4. Other Metal-Based Anticancer Drugs

In recent years, other approaches in the search for new, metal-based anticancer agents are to
examine complexes that contain other transition metals. In the design of these
new drugs, octahedral ruthenium (II) and ruthenium (III) complexes have shown
antineoplastic properties on a number of experimental tumors. Tetraammine-,
pentaammine-, heterocycle-, and dimethylsulfoxide-coordinated ruthenium
complexes have been synthesized and shown high affinity for nitrogen donor
ligands in vitro and as a result exhibit anticancer action in
vivo [52–54]. Other transition metals have
been used as anticancer drugs, including bismuth (III) labeled antibodies for
systemic radioimmunotherapy [55, 56], rhenium (I) complexes as
DNA-binding agents [57], (MTR)2Zn2+ complex that induces cancer cell death by binding to chromatin [58], and Cu2+ compound
chlorophyllin initiated apoptosis in human colon cancer cells through caspase-8
and apoptosis-inducing factor (AIF) activation in a cytochrome c-independent
manner [46].


## 1.1. Platinum(II) Anticancer Drugs

Cisplatin represents one of the most potent drugs available in the cancer chemotherapy
for several solid tumors, such as testicular, ovarian, bladder, and neck
cancers [3]. It is generally believed
that cisplatin exhibited its anticancer effects through preferentially binding
to quinine N-7 of DNA, and then cause DNA damage specifically in cancer cells, subsequently
leading to cell death [4]. After successes achieved
with platinum complexes, there is a tremendous increase in the search for
platinum complexes with different ligands that might produce more specific
anticancer effects. Some of these platinum-based drugs have been approved by
the Food and Drug Administration (FDA), including carboplatin for the treatment
of ovarian cancer [5, 6], oxaliplatin for metastatic
colorectal cancer [7, 8], satraplatin for
hormone-refractory prostate cancer [9], and picoplatin for
small-cell lung cancer [10].Transplatinum compounds follow different patterns of cell killing in comparison to
cisplatinum, thus giving a reason for optimism in their development as a new
class of platinum-based anticancer drugs [11]. The initial report of
anticancer properties of a dinuclear platinum complex in 1988 started a new
paradigm in platinum-based chemotherapy. Several multinuclear platinum
complexes have entered clinical trials in recent years, with varying results [12, 13]. The major limitations of cisplatin
and other platinum anticancer drugs are related to drug resistance and their
side effects, including nephrotoxicity, neurotoxicity, and emetogensis [14]. Resistance to cisplatin is
multifactorial, most cases consist of mechanisms limiting the formation of DNA
adducts or operating downstream of the cisplatin-DNA interaction to promote
cell survival [15].


## 1.2. Gold (I) and Gold (III) Anticancer Complexes

Gold (I) complexes had been used for the treatment of arthritis some decades ago, but most of them
disappeared from the drug market because of intolerable side effects, such as
gastrointestinal adverse reactions, nephrotoxicity, and haematological
reactions. However, the design and testing of gold complexes, especially gold (III)
complexes with anticancer activity begin to be intensively pursued in the past
few years. The potential use of gold (III) complexes as anticancer drugs were
based on three rationales [16–18]: (a) analogies between square
planar complexes of both platinum (II) and gold (III) are d8 ions;
(b) analogy to the immunomodulatory effects of gold (I) antiarthritic agents;
and (c) complexation of gold (I) and gold (III) with known anticancer agents to
form new compounds with enhanced activity. Buckley et al. first reported some organogold (III) complexes endowed with
significant cytotoxic and anticancer properties [19]. During the past decades,
various gold (III) complexes with sufficient stability in the physiological
environment have been synthesized and evaluated for in vitro anticancer properties. Some of these gold (III)
complexes turned out to exhibit relevant cytotoxic effects in vitro and were the subject of
further biochemical and pharmacological investigations [20–33]. Our previous findings showed
that gold (III) mesotetraarylporphyrin
1a was stable against demetallation in physiological conditions and exhibited
higher cytotoxicity than cisplatin against a panel of human cancer cell lines [34–37]. The major limitation of gold
(III) complexes is that few exhibit good stability under physiological
conditions, due to the reduction of gold (III) to gold (I) [38]. However, low cisplatin cross
resistance has been observed in gold complexes [39]. There is therefore
considerable interest in the development of tumor-selective and stable gold
anticancer drugs.


## 1.3. Metalloporphyrin Drugs

Metalloporphyrin drugs are new class of antioxidant enzyme mimetics with novel structure: a
metal in the center of porphyrin ligand. Metalloporphyrins (e.g., MnTBAP) have previously
been used to inhibit age-related oxidative damage in myocardium of mice that
are lacking mitochondrial enzyme manganese superoxide dismutase [40]. Afterwards, metalloporphyrin
drugs began to be used as photodynamic therapy agents for certain solid tumors [41]. Photodynamic therapy is
based on the concept that porphyrins are known to be rapidly and preferentially
taken up by the tumor cells with higher intakes of lipoproteins [42, 43]. When such photosensitizers
are irradiated with an appropriate wavelength of visible or near infrared (NIR)
light, the excited molecules can transfer their energy to molecular oxygen in
the surroundings, which is normally in its triplet ground state. This results
in the formation of cytotoxic reactive oxygen species (ROSs), particularly
singlet oxygen [44]. ROSs are responsible for
oxidizing various cellular compartments including plasma, mitochondrial,
lysosomal, and nuclear membranes, resulting in irreversible damage of tumor [34, 44]. Therefore, under appropriate
conditions, photodynamic therapy offers the advantage of an effective and
selective method of destroying diseased tissues without damaging adjacent
healthy cells [42, 43].Since the approval
of Photofrin by FDA for chemotherapy [45], porphyrin derivatives with
different metal in the center of the molecule have been widely used as
photosensitizers for photodynamic therapy in the treatment of cancer, including
chlorophyllin copper complex as superoxide dismutase mimics [46, 47], FeTBAP and MnTBAP [48, 49], ZnTBAP [50], motexafin gadolinium (MGd) [51]. Different modes of actions have been
suggested for different kinds
of metalloporphyrins. For example, MGd has been shown to inhibit heme
oxygenase-1 (HO-1) activity that results in inactivation of the antiapoptotic properties of the
products of HO-1 [51]. While FeTBAP and MnTBAP have
been reported to be superoxide anion scavengers [48]. MGd is also an active
inhibitor of cytochrome P450 enzymes, although with a lower potency than that
exhibited for inhibition of HO-1.


## 1.4. Other Metal-Based Anticancer Drugs

In recent years, other approaches in the search for new, metal-based anticancer agents are to
examine complexes that contain other transition metals. In the design of these
new drugs, octahedral ruthenium (II) and ruthenium (III) complexes have shown
antineoplastic properties on a number of experimental tumors. Tetraammine-,
pentaammine-, heterocycle-, and dimethylsulfoxide-coordinated ruthenium
complexes have been synthesized and shown high affinity for nitrogen donor
ligands in vitro and as a result exhibit anticancer action in
vivo [52–54]. Other transition metals have
been used as anticancer drugs, including bismuth (III) labeled antibodies for
systemic radioimmunotherapy [55, 56], rhenium (I) complexes as
DNA-binding agents [57], (MTR)2Zn2+ complex that induces cancer cell death by binding to chromatin [58], and Cu2+ compound
chlorophyllin initiated apoptosis in human colon cancer cells through caspase-8
and apoptosis-inducing factor (AIF) activation in a cytochrome c-independent
manner [46].


## 2. Proteomics

### 2.1. Introduction to Proteomics

The proteome is defined as all expressed protein complement of a cell, organ or organism, and
it includes all isoforms and posttranslational variants. Proteomic technology,
first coined in 1995 [59], attempts to separate,
identify, and characterize a global set of proteins in an effort to provide
information about protein abundance, location, modification, and
protein-protein interaction in the proteome of a given biological system [59, 60]. This postgenomic technology
provides a direct measurement of the presence and relative abundance of
proteins, and reveals the consequence of protein functioning in establishing
the biological phenotype of organisms in different states. By studying
interrelationships of protein expressions and modifications in health and
disease or drug treatment, proteomics contributes important insights into
determining the pathophysiological basis of disease [61], validating drug targets [62], and illustrating drug action
[63], toxicity, and side effects [64].


### 2.2. Technological Platforms

In the field of proteomics, several
well-established methods persist as means to resolve and analyze complex mixtures
of proteins derived from cells and tissues. Currently, the most commonly used
proteomic platforms include two-dimensional gel electrophoresis (2DE) and
protein chip arrays, isotope-coded affinity tages (ICATs), and immobilized metal affinity
chromatography (IMAC) (Table1). These technological platforms are most often incorporated
with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) [65], surface enhanced laser
desorption ionization time of flight (SELDI-TOF) [66], electrospray ionization
(ESI) [67], and/or tandem mass
spectrometry (MS/MS). In addition, inductively coupled plasma (ICP) mass spectrometry
has also been applied in proteomic-based research of drug discovery [68].Table 1
Major technological platforms in proteomics.
Technique2DE (Two-dimensional gel electrophoresis)SELDI-TOF MSICAT (Isotope-coded affinity tags)IMAC (Immobilized metal affinity chromatography)Principles2DE separates protein mixtures by
their isoelectric points and molecular weights, proteins can be identified by
MALDI-TOF MS through enzyme digestionThis technique employs protein
chip separates protein mixtures by different surface binding affinity and
molecular weights, and roughly identifies proteins through SELDI-TOF MSICAT separates proteins by
chemical labeling and relative abundance, and then obtains protein identification
through ESI MS/MSIMAC is a powerful protein
fractionation method used to enrich metal-associated proteins and peptides,
proteins and peptides can be determined by ESI MS/MSRemarksSuitable for whole proteome or
specific pre-fractioned proteomes, detect large quantity of proteins in a single run,
not suitable for low abundant proteins, affected by posttranslational
modificationsSimple preparation procedures, sensitive detection limit,
small sample requirement, significant results,
wide detection range in molecular weight, modified by different surface
affinitiesBiotinylated tags labeling before analysis;
suitable for low abundant proteins; not suitable for
post translational modified proteins;
more automatedEnrich metal-associated proteins
and peptides, easy regeneration, longevity and stability to proteolytic degradation,
have to be facilitated by other separation methods, suitable for posttranslational
modificationPotential applicationsStudy drug-induced cellular
signaling pathway in a global scale [35, 69]Identification of drug targets [70]Identification of drug targets [70]Screen drug candidates [72, 73]Receptome profiling [75, 76]Mapping of phosphoproteomes [77, 78] and metalloproteomes [79, 80]Study of drug toxicity and side
effects [71]Study of protein-drug
interactions [74]


## 2.1. Introduction to Proteomics

The proteome is defined as all expressed protein complement of a cell, organ or organism, and
it includes all isoforms and posttranslational variants. Proteomic technology,
first coined in 1995 [59], attempts to separate,
identify, and characterize a global set of proteins in an effort to provide
information about protein abundance, location, modification, and
protein-protein interaction in the proteome of a given biological system [59, 60]. This postgenomic technology
provides a direct measurement of the presence and relative abundance of
proteins, and reveals the consequence of protein functioning in establishing
the biological phenotype of organisms in different states. By studying
interrelationships of protein expressions and modifications in health and
disease or drug treatment, proteomics contributes important insights into
determining the pathophysiological basis of disease [61], validating drug targets [62], and illustrating drug action
[63], toxicity, and side effects [64].


## 2.2. Technological Platforms

In the field of proteomics, several
well-established methods persist as means to resolve and analyze complex mixtures
of proteins derived from cells and tissues. Currently, the most commonly used
proteomic platforms include two-dimensional gel electrophoresis (2DE) and
protein chip arrays, isotope-coded affinity tages (ICATs), and immobilized metal affinity
chromatography (IMAC) (Table1). These technological platforms are most often incorporated
with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) [65], surface enhanced laser
desorption ionization time of flight (SELDI-TOF) [66], electrospray ionization
(ESI) [67], and/or tandem mass
spectrometry (MS/MS). In addition, inductively coupled plasma (ICP) mass spectrometry
has also been applied in proteomic-based research of drug discovery [68].Table 1
Major technological platforms in proteomics.
Technique2DE (Two-dimensional gel electrophoresis)SELDI-TOF MSICAT (Isotope-coded affinity tags)IMAC (Immobilized metal affinity chromatography)Principles2DE separates protein mixtures by
their isoelectric points and molecular weights, proteins can be identified by
MALDI-TOF MS through enzyme digestionThis technique employs protein
chip separates protein mixtures by different surface binding affinity and
molecular weights, and roughly identifies proteins through SELDI-TOF MSICAT separates proteins by
chemical labeling and relative abundance, and then obtains protein identification
through ESI MS/MSIMAC is a powerful protein
fractionation method used to enrich metal-associated proteins and peptides,
proteins and peptides can be determined by ESI MS/MSRemarksSuitable for whole proteome or
specific pre-fractioned proteomes, detect large quantity of proteins in a single run,
not suitable for low abundant proteins, affected by posttranslational
modificationsSimple preparation procedures, sensitive detection limit,
small sample requirement, significant results,
wide detection range in molecular weight, modified by different surface
affinitiesBiotinylated tags labeling before analysis;
suitable for low abundant proteins; not suitable for
post translational modified proteins;
more automatedEnrich metal-associated proteins
and peptides, easy regeneration, longevity and stability to proteolytic degradation,
have to be facilitated by other separation methods, suitable for posttranslational
modificationPotential applicationsStudy drug-induced cellular
signaling pathway in a global scale [35, 69]Identification of drug targets [70]Identification of drug targets [70]Screen drug candidates [72, 73]Receptome profiling [75, 76]Mapping of phosphoproteomes [77, 78] and metalloproteomes [79, 80]Study of drug toxicity and side
effects [71]Study of protein-drug
interactions [74]


## 3. Potential Applications of Proteomics in Illustrating Metal-Based Drug  Development and Discovery

Ever since the initial discovery of the anticancer activity of cisplatin, major efforts have
been devoted to elucidate the biochemical mechanisms of anticancer activities
of metal-based drugs to facilitate rational design of novel metal-based drugs
with better pharmacological profiles. A comprehensive understanding of the
molecular action mechanisms, which are triggered by metal-based drugs to kill
cancer cells can lead to the design of more effective anticancer drugs, as well
as to provide new therapeutic strategies based on the molecular activity of
metal-based drug activity.


### 3.1. Target Discovery and Validation

Target discovery, which involves the identification and early validation of disease-associated
targets, is an essential first step in the drug discovery pipeline [81]. Indeed, the drive to
determine protein function has been stimulated, both in industry and academia,
by the human genome and proteome projects in progress. Proteomics, the study of
cellular protein expression, is an evolving technology platform that has the
potential to identify novel proteins involved in key biological processes in
cells. These proteins may serve as potential drug targets. Proteomics thus
holds great promise as a powerful technique for drug target discovery. It must
be pointed out, however, that numerous drug-targeted proteins are
membrane-bound proteins, for example, receptors and ion channels. These
proteins may not be amenable for study by proteomics due to their poor
solubility and low abundance, and thus they are disproportionally represented
in proteome profiles [82]. Up to date, only a fraction
of putative drug targets has been identified by proteomic approaches, including
the volume-sensitive organic osmolyte/anion channel as key elements of tumor
development, migration, and invasiveness [83], and integrin alpha-4 as a
molecular target of oxidative stress [84].Studying protein expression profiling of drug-treatment leads to the identification of a number
of drug-specific targets both in vivo and in vitro. Using
HPLC-MALDI-TOF  MS, Hasinoff et al. have identified topoisomerase IIα con- tained at least five free cysteins (170,
216, 300, 392, and 405) and two disulfide-bonded cyteine pairs (427-455 and
997-1008) [85]. Cisplatin was found to antagonize
the formation of a fluorescence adduct between topoisomeraser IIα and the sulfhydryl-reactive maleimide reagent
10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-naphtho[2,1-b]pyran-2-carboxylic
acid methyl ester (ThioGlo-1). Based on these results, the authors suggested
that topoisomeraser IIα cysteines may be possible sites responsible for the
inhibition of the catalytic activity of topoisomeraser IIα observed in the
presence of cisplatin, and topoisomeraser IIα cysteins and DNA as targets
responsible for cisplatin-induced inhibition of topoisomeraser IIα [85]. Besides, mitochondrial
proteins, especially ATP synthase-beta subunit, have been reported to be key
proteins that serve as primary target of manganese porphyrin [MnTnHex-2-PyP(5+)]
treatment during renal ischemia/reperfusion injury by proteomic study [86].ICP MS coupled with capillary electrophoresis (CE) has been used in the identification,
characterization, and determination of different chemical species of an element
in complex biological systems, that is, the impetus of biochemical speciation
analysis [87, 88]. Polec-Pawlak et al. have used this approach to
study the platinum group metallodrug-protein binding aiming to characterize the
interactions between cancer-inhibiting metal complexes and serum transport
proteins [89]. Such binding does not only
regulate the uptake and accumulation of the drug in tumor tissue but also
determines its overall distribution and exertion and differences in efficacy, activity, and
toxicity [90, 91]. Their study provides clear
evidence that a ruthenium (III) complex
[trans-tetrachlorobis(1H-indazole)ruthenate (III)] (KP1019) preferentially
binds toward albumin whose adduct is a dominating protein-bound species of ruthenium
[89].


### 3.2. Validation of Drug Toxicity and Resistance

Insights into toxic responses are an asset for the interpretation of adverse drug effects and
contribution to accurate risk assessment for humans. Proteomics in combination
with combinatorial chemistry and high-throughput screening can help to bring
forward validating toxicity and resistance of an unprecedented number of
potential lead compounds [92]. Benefits can be expected in
optimized clinical trials based on the availability of biologically relevant
markers of drug efficacy and safety. Proteomics has demonstrated
proof-of-concept in toxicology as shown by a number of successful applications
in mechanistic toxicology and lead selection. Proteomic studies of liver
toxicity have been carried out with thioacetamide [93] and ethanol [64]. Other researches have
studied nephrotoxicity of cyclosporine A by proteomic approaches [94, 95] and reported a profound downregulation
of the calcium binding protein calbindin D28 responsible for cyclosporine
A-induced kidney toxicity. However, this technology-driven acceleration in drug
discovery moves the bottlenecks in drug development to the downstream, which is
the improvement in the selection of patient populations for clinical trials.Proteomic approaches have been used to identify the underlying mechanisms for cisplatin
resistance [96]. In this study, the authors
used cervix squamous cell carcinoma cell line A431 and its cisplatin-resistant
subline, A431/Pt as model system. The identified differentially expressed
proteins can be classified into several groups, including molecular chaperones
(e.g., heat-shock protein HSP60, HSP90, and HSC71), Ca2+-binding
proteins (e.g., calmodulin and calumenin), proteins involved in drug
detoxification (e.g., peroxiredoxin 2 and 6, and glutathione-S-transferase),
antiapoptotic proteins (e.g., 14-3-3 switched on in cisplatin-exposed cells)
and ion channels (e.g., voltage-dependent anion channel 1, voltage-dependent
anion-selective channel). Besides this, proteome profile of cisplatin sensitive
ovarian cell line IGROV1 and its cisplatin-resistant counterpart IGROV1-R10
have been compared aiming to find any protein markers or to establish new
therapeutic strategies [97, 98]. Increased expression of
cytokeratin 8 and cytokeratin 18 was considered to play a role in acquired chemoresistance of
IGROV1-R10 cancer cell line to cisplatin [97, 98]. Cytokeratin 8 and cytokeratin
18 have been implicated in resistance to TNF-α-induced apoptosis by binding the
cytoplasmic domain of tumor necrosis factor receptor 1 [69, 99]. Moreover, human
nasopharyngeal carcinoma cells deficient for cytokeratin 8 were more sensitive
to cisplatin-induced apoptosis [100].In addition, acquired and intrinsic cellular drug resistances are multifactorial processes,
involving induction of drug detoxifying mechanisms, quantitative and
qualitative modification of drug targets, cell cycle arrest, regulation of DNA
replication or reparation mechanisms, modulation of apoptosis, and other mechanisms
[101, 102]. Global examination of the glycoproteomes of
the cisplatin-resistant ovarian cancer cell line IGROV-1/CP using shotgun
glycopeptide capture approach coupled with MS has been used to study cisplatin resistance [103]. In this approach,
glycopeptides derived from glycoproteins are enriched by selective capture onto
a solid support using hydrazide chemistry followed by enzymatic release of the
peptides and subsequent analysis by MS/MS. This method improves solubility of
large membrane proteins and exposes all of the glycosylation sites to ensure
equal accessibility to capture reagents. Stewart et al. also used isotope-coded affinity tags (ICATs) integrated
with mRNA expression levels to study cisplatin resistance in ovarian cancer
cells [104]. Their study identified three
pathways in Panther database (http://www.pantherdb.org/) that were significantly (p<.05) upregulated
in cisplatin-sensitive cells, including glycolysis, interleukin signaling
pathway, and PI 3-kinase pathway [104].


### 3.3. Mapping Drug Action Mechanisms

An understanding of protein function within the context of complex cellular networks is required
to facilitate the discovery of novel drug targets and, subsequently, new
therapies directed against them. Proteomics offers comprehensive monitoring of
protein alterations at molecular level upon drug treatments. Being the basic
biochemical mode of drug activities, drug action mechanism should be better
understood to provide valuable insights into drug modification and new drug
development [34, 105]. Successful examples in drug
mechanism study using proteomics include the illustration of insulin-like
growth factor-binding protein-6-induced sublethal hydrogen peroxide stress in
human diploid fibroblasts cells [106]. By using ESI MS/MS, Kanski et al. have applied proteomic
analysis of protein nitration in aging skeletal muscle and identified
nitrotyrosine-containing sequences in vivo [107].In our previous study, we have used 2DE-based proteomic technology to compare the protein
profile of human nasopharyngeal carcinoma SUNE1 cell line treated with gold (III)
porphyrin 1a, and a number of differentially expressed proteins were identified
[35]. These proteins can be
classified into several categories based on their major biological functions,
including cellular structural proteins, stress-related and chaperone proteins,
proteins involved in ROS, enzyme proteins and translation factors, proteins
that mediate cell death and survival signaling, and proteins that participate
in the internal degradation system [35]. Among these proteins, one of
the significant increased proteins is voltage-dependent anion channel 1 (VDAC
1). VDAC 1 is a mitochondrial outer membrane channel protein, which functions
as the pathway for the movement of various substances in and out of the
mitochondria [108]. It is considered to be a
component of the permeability transition pore oligoprotein complex that plays a
role in the permeability transition [109, 110]. VDAC 1 also plays an
essential role in Bax/Bak-induced apoptotic mitochondrial changes in the
process of mammalian cell death [111–113]. In this process, the
proapoptotic proteins Bax and Bak bind to VDAC 1, and enhance its permeability
so that cytochrome c passes through the channel and releases to cytoplasm [111–113]. Our data on VDAC 1
upregulation [35] and Bax overexpression [37] suggest that gold (III)
porphyrin 1a may induce cell death via the mitochondria-mediated apoptosis
pathway. Further functional studies revealed that gold (III) porphyrin 1a
caused depletion of mitochondrial transmembrane potential (ΔΨm) soon
after uptake with suppression of Bcl-2, and activation of caspase 9 and caspase
3 [34]. Taken together, these
results suggested that mitochondria are the primary target of gold (III)
porphyrin 1a.Quantitative proteomic analysis on other metal-based anticancer drugs has also been pursuit.
Schmidt et al. have used nano-LC coupled offline MALDI-TOF/TOF-MS
to study cisplatin-induced apoptosis in Jurkat T cells [114]. Their results showed that
this method is more accurate than the commonly used online LC-ESI-MS.


## 3.1. Target Discovery and Validation

Target discovery, which involves the identification and early validation of disease-associated
targets, is an essential first step in the drug discovery pipeline [81]. Indeed, the drive to
determine protein function has been stimulated, both in industry and academia,
by the human genome and proteome projects in progress. Proteomics, the study of
cellular protein expression, is an evolving technology platform that has the
potential to identify novel proteins involved in key biological processes in
cells. These proteins may serve as potential drug targets. Proteomics thus
holds great promise as a powerful technique for drug target discovery. It must
be pointed out, however, that numerous drug-targeted proteins are
membrane-bound proteins, for example, receptors and ion channels. These
proteins may not be amenable for study by proteomics due to their poor
solubility and low abundance, and thus they are disproportionally represented
in proteome profiles [82]. Up to date, only a fraction
of putative drug targets has been identified by proteomic approaches, including
the volume-sensitive organic osmolyte/anion channel as key elements of tumor
development, migration, and invasiveness [83], and integrin alpha-4 as a
molecular target of oxidative stress [84].Studying protein expression profiling of drug-treatment leads to the identification of a number
of drug-specific targets both in vivo and in vitro. Using
HPLC-MALDI-TOF  MS, Hasinoff et al. have identified topoisomerase IIα con- tained at least five free cysteins (170,
216, 300, 392, and 405) and two disulfide-bonded cyteine pairs (427-455 and
997-1008) [85]. Cisplatin was found to antagonize
the formation of a fluorescence adduct between topoisomeraser IIα and the sulfhydryl-reactive maleimide reagent
10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-naphtho[2,1-b]pyran-2-carboxylic
acid methyl ester (ThioGlo-1). Based on these results, the authors suggested
that topoisomeraser IIα cysteines may be possible sites responsible for the
inhibition of the catalytic activity of topoisomeraser IIα observed in the
presence of cisplatin, and topoisomeraser IIα cysteins and DNA as targets
responsible for cisplatin-induced inhibition of topoisomeraser IIα [85]. Besides, mitochondrial
proteins, especially ATP synthase-beta subunit, have been reported to be key
proteins that serve as primary target of manganese porphyrin [MnTnHex-2-PyP(5+)]
treatment during renal ischemia/reperfusion injury by proteomic study [86].ICP MS coupled with capillary electrophoresis (CE) has been used in the identification,
characterization, and determination of different chemical species of an element
in complex biological systems, that is, the impetus of biochemical speciation
analysis [87, 88]. Polec-Pawlak et al. have used this approach to
study the platinum group metallodrug-protein binding aiming to characterize the
interactions between cancer-inhibiting metal complexes and serum transport
proteins [89]. Such binding does not only
regulate the uptake and accumulation of the drug in tumor tissue but also
determines its overall distribution and exertion and differences in efficacy, activity, and
toxicity [90, 91]. Their study provides clear
evidence that a ruthenium (III) complex
[trans-tetrachlorobis(1H-indazole)ruthenate (III)] (KP1019) preferentially
binds toward albumin whose adduct is a dominating protein-bound species of ruthenium
[89].


## 3.2. Validation of Drug Toxicity and Resistance

Insights into toxic responses are an asset for the interpretation of adverse drug effects and
contribution to accurate risk assessment for humans. Proteomics in combination
with combinatorial chemistry and high-throughput screening can help to bring
forward validating toxicity and resistance of an unprecedented number of
potential lead compounds [92]. Benefits can be expected in
optimized clinical trials based on the availability of biologically relevant
markers of drug efficacy and safety. Proteomics has demonstrated
proof-of-concept in toxicology as shown by a number of successful applications
in mechanistic toxicology and lead selection. Proteomic studies of liver
toxicity have been carried out with thioacetamide [93] and ethanol [64]. Other researches have
studied nephrotoxicity of cyclosporine A by proteomic approaches [94, 95] and reported a profound downregulation
of the calcium binding protein calbindin D28 responsible for cyclosporine
A-induced kidney toxicity. However, this technology-driven acceleration in drug
discovery moves the bottlenecks in drug development to the downstream, which is
the improvement in the selection of patient populations for clinical trials.Proteomic approaches have been used to identify the underlying mechanisms for cisplatin
resistance [96]. In this study, the authors
used cervix squamous cell carcinoma cell line A431 and its cisplatin-resistant
subline, A431/Pt as model system. The identified differentially expressed
proteins can be classified into several groups, including molecular chaperones
(e.g., heat-shock protein HSP60, HSP90, and HSC71), Ca2+-binding
proteins (e.g., calmodulin and calumenin), proteins involved in drug
detoxification (e.g., peroxiredoxin 2 and 6, and glutathione-S-transferase),
antiapoptotic proteins (e.g., 14-3-3 switched on in cisplatin-exposed cells)
and ion channels (e.g., voltage-dependent anion channel 1, voltage-dependent
anion-selective channel). Besides this, proteome profile of cisplatin sensitive
ovarian cell line IGROV1 and its cisplatin-resistant counterpart IGROV1-R10
have been compared aiming to find any protein markers or to establish new
therapeutic strategies [97, 98]. Increased expression of
cytokeratin 8 and cytokeratin 18 was considered to play a role in acquired chemoresistance of
IGROV1-R10 cancer cell line to cisplatin [97, 98]. Cytokeratin 8 and cytokeratin
18 have been implicated in resistance to TNF-α-induced apoptosis by binding the
cytoplasmic domain of tumor necrosis factor receptor 1 [69, 99]. Moreover, human
nasopharyngeal carcinoma cells deficient for cytokeratin 8 were more sensitive
to cisplatin-induced apoptosis [100].In addition, acquired and intrinsic cellular drug resistances are multifactorial processes,
involving induction of drug detoxifying mechanisms, quantitative and
qualitative modification of drug targets, cell cycle arrest, regulation of DNA
replication or reparation mechanisms, modulation of apoptosis, and other mechanisms
[101, 102]. Global examination of the glycoproteomes of
the cisplatin-resistant ovarian cancer cell line IGROV-1/CP using shotgun
glycopeptide capture approach coupled with MS has been used to study cisplatin resistance [103]. In this approach,
glycopeptides derived from glycoproteins are enriched by selective capture onto
a solid support using hydrazide chemistry followed by enzymatic release of the
peptides and subsequent analysis by MS/MS. This method improves solubility of
large membrane proteins and exposes all of the glycosylation sites to ensure
equal accessibility to capture reagents. Stewart et al. also used isotope-coded affinity tags (ICATs) integrated
with mRNA expression levels to study cisplatin resistance in ovarian cancer
cells [104]. Their study identified three
pathways in Panther database (http://www.pantherdb.org/) that were significantly (p<.05) upregulated
in cisplatin-sensitive cells, including glycolysis, interleukin signaling
pathway, and PI 3-kinase pathway [104].


## 3.3. Mapping Drug Action Mechanisms

An understanding of protein function within the context of complex cellular networks is required
to facilitate the discovery of novel drug targets and, subsequently, new
therapies directed against them. Proteomics offers comprehensive monitoring of
protein alterations at molecular level upon drug treatments. Being the basic
biochemical mode of drug activities, drug action mechanism should be better
understood to provide valuable insights into drug modification and new drug
development [34, 105]. Successful examples in drug
mechanism study using proteomics include the illustration of insulin-like
growth factor-binding protein-6-induced sublethal hydrogen peroxide stress in
human diploid fibroblasts cells [106]. By using ESI MS/MS, Kanski et al. have applied proteomic
analysis of protein nitration in aging skeletal muscle and identified
nitrotyrosine-containing sequences in vivo [107].In our previous study, we have used 2DE-based proteomic technology to compare the protein
profile of human nasopharyngeal carcinoma SUNE1 cell line treated with gold (III)
porphyrin 1a, and a number of differentially expressed proteins were identified
[35]. These proteins can be
classified into several categories based on their major biological functions,
including cellular structural proteins, stress-related and chaperone proteins,
proteins involved in ROS, enzyme proteins and translation factors, proteins
that mediate cell death and survival signaling, and proteins that participate
in the internal degradation system [35]. Among these proteins, one of
the significant increased proteins is voltage-dependent anion channel 1 (VDAC
1). VDAC 1 is a mitochondrial outer membrane channel protein, which functions
as the pathway for the movement of various substances in and out of the
mitochondria [108]. It is considered to be a
component of the permeability transition pore oligoprotein complex that plays a
role in the permeability transition [109, 110]. VDAC 1 also plays an
essential role in Bax/Bak-induced apoptotic mitochondrial changes in the
process of mammalian cell death [111–113]. In this process, the
proapoptotic proteins Bax and Bak bind to VDAC 1, and enhance its permeability
so that cytochrome c passes through the channel and releases to cytoplasm [111–113]. Our data on VDAC 1
upregulation [35] and Bax overexpression [37] suggest that gold (III)
porphyrin 1a may induce cell death via the mitochondria-mediated apoptosis
pathway. Further functional studies revealed that gold (III) porphyrin 1a
caused depletion of mitochondrial transmembrane potential (ΔΨm) soon
after uptake with suppression of Bcl-2, and activation of caspase 9 and caspase
3 [34]. Taken together, these
results suggested that mitochondria are the primary target of gold (III)
porphyrin 1a.Quantitative proteomic analysis on other metal-based anticancer drugs has also been pursuit.
Schmidt et al. have used nano-LC coupled offline MALDI-TOF/TOF-MS
to study cisplatin-induced apoptosis in Jurkat T cells [114]. Their results showed that
this method is more accurate than the commonly used online LC-ESI-MS.


## 4. Conclusion and Future Prospects

The potential value of proteomics in metal-based drug development, especially in
mapping drug action mechanisms, has been demonstrated in many successful
examples. Proteomic approaches have been recognized as promising techniques
that can facilitate the systematic characterization of a drug targets’
physiology, thereby helping to reduce the typically high attrition rates in
discovery projects, and improving the overall efficiency of pharmaceutical
research processes. However, at present stage, the bottleneck for taking full
advantage of this new experimental technology is the rapidly growing volumes of
automatically produced biological data, and technical challenges with regards
to sampling, tumor heterogeneity, and lack of standardized methodologies. In
addition, to complement the limitation of current proteomic technology, systematic
biological and pharmaceutical studies should be integrated with proteomics to
better serve the purpose of illustrating the action mechanism of drugs and thus
contribute to the success in metal-based drug development.



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*Source: 716329-2008-07-22.xml*