Download Chapter 1 Introduction 1.1 An overview Carcinogenesis is an

Chapter 1
Introduction
1.1 An overview
Carcinogenesis is an intricate multistep and multifactorial process in which
environment-gene interaction plays an important role (Godderis et al.,
2012). It involves sequential genetic mutation resulting in uncontrolled cell
proliferation and different homeostatic dysregulation in normal cells.
Mechanism of cancer progression is still partially known, which had made
it as leading cause of death in the world (Jemal et al., 2011). The concepts
of gene-environment interactions and human cancer risk were generated by
the synthesis of chemical during the industrialisation in early 20th century.
Global
epidemiologic
studies
have
identified
environmental
and
occupational exposure of chemicals as a potent carcinogen (Loeb & Harris,
2008). Evidence from epidemiological, occupational and migration studies
has consistently pointed to environmental factors as the major cause for
cancer (Luch, 2005). In, this regard involvement of ploy aromatic
hydrocarbons becomes very important as we are routinely exposed to these
arrays of compounds.
1.2 Poly Aromatic Hydrocarbon (PAHs) and its carcinogenicity:
Poly Aromatic Hydrocarbon (PAHs) produced naturally by forest fires and
volcanoes. Industrialization and change in human life style e.g. burning of coal,
wood, petroleum products, tires, polypropylene and motor vehicle exhaust, the
level of PAHs has increased in our environment (Cherng et al., 1996).There are
approximately 100 different known PAHs in air, soil, foodstuffs, water and
diesel exhaust which contains significant amounts of PAHs (Zedeck,1980).
Polycyclic aromatic hydrocarbons (PAHs) refer to a ubiquitous group of
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several hundred chemical which are environmentally persistent organic
compounds of various structures and varied toxicity. Most of them are formed
by a process of thermal breakdown and recombination of organic
molecules. Often, PAHs consist of three or more fused benzene rings
containing only carbon and hydrogen. PAH-induced carcinogenesis can result
in formation of bulky PAH-DNA adduct which is critical for the regulation of
cell differentiation or growth. If this DNA aberration remains unrepaired
during the cell replication process, it will result in gene mutation which leads to
cell transformation or carcinogenesis. The carcinogenicity of certain PAHs is
well established in laboratory animals. Researchers have reported increased
incidences of skin, lung, bladder, liver, and stomach cancers. Animal studies
show that certain PAHs can affect different biological systems likehematopoietic, immune systems, reproductive, neurologic, and developmental
system (Dasgupta and Lahiri, 1992; Szczeklik et al., 1994; Zhao, 1990). MCA
alkylated derivative of benz[a]anthracene and being widely used all around the
world for in vitro cell transformation assay (Peterson et al., 1981; Sakiyama et
al., 1986;Miller and Hall,1991)
1.3 Cell Transformation Assay (CTAs)
In vitro cell transformation assay has provided powerful tool to study the
cellular and molecular mechanisms of chemical carcinogenesis (Fernandez et
al., 1980).
Two stage cell transformation assay measures the phenotypic
conversion of cell from normal to malignant type. CTAs not only provide the
powerful tool to study the carcinogenic potential of any chemical compound
but it also gives us the molecular mechanism of cancer. When cells are exposed
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Introduction
to sub-threshold dose of carcinogen (a tumor initiator) and subsequently with a
tumor promoter (a typical non-genotoxic carcinogen), results in the formation
of transformed foci (Sakai, 2007). Two-stage cell transformation has been
performed in many cell systems is regarded as a important method for the
screening of chemical as well as valuable approach for the mechanistic study
in carcinogenesis. Thus, two-stage cell transformation assay mimics in vivo
multistage carcinogenesis (Sakai, 2007). Contact inhibition and anchorage
independence are the important characteristic of two-stage cell transformation
(Urani et al.,2009 ;Fang et al.,2001).In recent years, cDNA and oligonucleotide
microarray technology has allowed researchers to study the effects of chemical
induce carcinogenesis in experimental animal and human. Toxicogenomics
tools have also been utilized to investigate the chemical-gene interaction to
enhance the information on toxicological properties of a particular chemical.
Chemical-Gene expression signatures can be used to determine mechanism of
unknown test compound. In cancer toxicology this technique can be readily
used to identify dysregulated pathways/genes involved in chemical-Gene
interaction (Godderis et al., 2012; Luch, 2005). In CTA and as well as in the in
vivo carcinogenesis, normal and cancerous cells will be growing in constant
communication with surrounding invasive and non invasive cell colonies and
are undergoing concomitantly complementary changes in gene expression
profile (Priya et al., 2013).
1.4 Redox regulation by GSH in cancer
Glutathione is a tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) which has
important functions in eukaryotic cells. It is an antioxidant which carry active
thiol group. It protects cell by interacting with free radical, reactive oxygen
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Chapter 1
Introduction
species (ROS), and reactive nitrogen species (RNS) because of its reactivity,
high intracellular concentration (10mM in the liver cell and malignant cells)
and high redox potential. GSH is synthesis is a two step process catalyzied by
L-glumate: L-cysteine γ-ligase and glutathione synthase (Estrela et al., 2006).
Glutathione in cancer cells is particularly relevant in the regulation
of carcinogenic mechanisms; sensitivity against cytotoxic drugs, ionizing
radiations, some
cytokines, DNA
synthesis, cell proliferation and death
(Ortega et al., 2011). The intra and extra cellular GSH levels are responsible for
the cell homeostasis. It can be determined by the balance between its
production, consumption and transportation. Imbalance of GSH is observed in
a wide range of pathologies, including cancer, neurodegenerative disorders,
cystic fibrosis (CF), HIV, and aging. The elevated level of GSH inside a cell,
disturb the cell homeostasis which causes cell transformation. Maintaining
proper GSH levels and oxidation state are important for cell function and their
disruptions are observed in many human diseases. GSH deficiency leads to an
increased susceptibility to oxidative stress and, thus, progression of many
disease states. On the other hand, elevated GSH levels increase antioxidant
capacity and resistance to oxidative stress and this is observed in many types of
cancer (Abdalla, 2011).
While GSH is important in the detoxification of
carcinogens, Increase in GSH level is already reported in different human
carcinoma cell lines e.g. A549 cell (human lung carcinoma) and HepG2 cell
(heptocellular carcinoma) which may increase resistance or alters the
cytotoxicity of many chemotherapy drugs or radiation (Vojislav et al., 2001;
Balendiran et al., 2004).This increase in GSH may be an important factor in
chemo- or radiotherapy resistance seen in these cells (Goodwin and Baylin,
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Chapter 1
Introduction
1982; Carney et al., 1983; Huang et al., 2000Yu and Brown, 1984; Guichard et
al., 1986).
Therefore, GSH depletion is a common strategy used by the
pharmacologist as a possible target for cancer prevention. Manipulation of
intracellular GSH using chemicals such as L-buthionine-(S,R)-sulfoximine
(BSO), Diethyl maleate(DEM), Phorone(PHO) and t-butyl hydro peroxide have
been found to reduce GSH level (Griffith et al., 1979,Anderson,1998).These
chemicals has been used to increase the sensitivity of different tumour cell
lines to therapy and showed that selective differential chemotherapy responses
of normal versus tumour cells is possible (Griffith et al., 1979; Williamson et
al., 1982). However, it’s important to note that different cells respond
differently to oxidative stress inducing therapies (Mattson et al., 2009).The
relationship between GSH depletion, chemotherapy, and/or the radiation
response has been examined in many tumor cells after treatment with
different
drugs,
including BSO,
diethylmaleate,
2-oxothiazolidine-4-
carboxylate, and different radio sensitizing agents (Rappa et al 1997;Mistry and
Harrap,1991;Estrela
et
al.,2006;Bump
and
Brown,1990;Meister,1991).
Moreover, GSH depletion only appears to be therapeutically effective when
very low levels of this tripeptide can be achieved within the cancer cells
(Estrela et al., 2006). Thus, achievement of selective tumour GSH depletion
under in vivo or in vitro conditions appears as a remarkable pharmacological
challenge. In fact, since the molecular background is firmly established,
the potential benefits of GSH depletion for cancer therapy have remained in
the shadow for two decades. Nevertheless, recently, new research has offered
some light on how to make GSH depletion a useful tool in cancer therapy
(Ortega et al., 2011).
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The strategy of GSH depletion as a chemo-therapeutic tool for cancer
prevention is already used by other researchers but the genomic profile
responsible for the GSH depletion, using a GSH depletor, is still unknown and
never been evaluated earlier. In light of these facts, we proposed following
aims and objectives of our study.
1.5
Aims and objective

Study regulatory significance of cellular GSH depletion in
experimental carcinogenesis.

Elucidate its mechanism using microarray approach and validate
altered gene expression using qPCR.

Identify functional relevance of critically altered genes/pathway
for their regulatory role in toxicity-carcinogenesis.
1.6 Study plan
1.6.1 Chemical induced Cell Transformation Assay (CTAs) in Fibroblast
cell lines:
We determined the cell transforming dose of MCA and transformed
C3H10T1/2 cells using MCA (initiator) +TPA (promoter), in two-stage cell
transformation assay. Cell transformation was characterized by soft agar assay,
which measures acquisition of the anchorage independent growth. In addition
to Giemsa staining, CytoselectTM based method was used to quantify colonies
formed in the soft agar. Altered genomic profile of transformed cell was
investigated using microarray approach and validation of altered gene
expression was done by qPCR. Results of this study (C3H10T1/2 cells) were
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also validated in MCA +TPA treated BALB/c cells using RT-PCR (Chapter
3).
1.6.2 DEM exposure to MCA+TPA transformed cell lines
We determined the non-cytotoxic dose of DEM (Diethyl malate) in
MC+TPA transformed C3H10T1/2 cells was identified. GSH content, ROS
generation, Cell cycle, TUNEL assay was performed to measure the changes
due to DEM exposure in both the transformed cells (C3H10T1/2 and BALB/c).
Altered genomic profile of DEM treated transformed cell was investigated
using microarray approach and validation of altered gene expression was
quantified by quantitative PCR (qRT-PCR). Result of this study (C3H10T1/2
cells) were also validated in DEM treated transformed BALB/c cells using RTPCR. CytoselectTM based method was used to quantify colonies formed in the
soft agar by DEM exposure (Chapter 4).
1.6.3 PHO exposure to MCA+TPA transformed cell lines
In this plan, we have investigated the alteration in global gene
expression profile of MCA+TPA transformed C3H10T1/2 after GSH depletion
by exposure to PHO; and the similar set of investigations were conducted in
BALB/c 3T3 cell line and validated using qPCR (Chapter 5).
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