Download - P-Glycoprotein Significance in Cancer
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
FACULTY OF SCIENCEMODULAR DEGREE SCHEME
BSc HONOURS DEGREE
IN
PHARMACOLOGY
Kudakwashe Emmanuel Mupamhanga
K0433939
The Biological and Clinical Significance of
P-Glycoprotein in cancer
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ABSTRACT & BACKGROUND
CANCER
- BIOLOGY OF CANCER
- CANCER CAUSES
- CANCER TYPES
- GENES IN CANCER
- TREATMENT SCHEMES
P-GLYCOPROTEIN
- HISTORY
- STRUCTURE
- LOCALISATION OF Pgp IN NORMAL TISSUES
Pgp SIGNIFICANCE ON THE THERAPY OF CANCER
- DRUG RESISTENCE
- P-GP EXPRESSION IN CANCER CELLS
- PHARMACOLOGICAL TARGETING OF Pgp
- 1ST TO 3RD GENERATION Pgp INHIBITORS
- STEM CELLS AND CANCER GENE THERAPY
CONCLUSION
REFERENCES
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCERABSTRACT
Cancer is a leading cause of death worldwide. It is characterized by rapid and
unregulated growth of the body’s cells as a result of a mutation in a proto-oncogene
or a tumor suppressor gene or both. It is however resultant of multiple mechanism
leading to its development ranging from environmental factors to hereditary
influence. The membrane bound MDR1 gene product P-glycoprotein (Pgp) has been
reported to be associated with drug resistance incidence in cancer to chemotherapy.
Pgp is thought to have an evolutionary role as a protective mechanism against toxins
ingested or inhaled from the environment. Pharmacological and gene therapy
research has strived to modulate the effects of Pgp or more recently make use of this
drug resistant characteristic for chemoprotection respectively.
Background
According to the World Health Organisation (WHO), cancer is a leading cause of
death worldwide and accounts for an estimated 7.4 million deaths (2004 statistics),
13% of all deaths worldwide. Cancer can affect any part of the body and there are
approximately 200 different types of cancer. (Cancer Research UK)
In the U.K alone there are approximately 285,000 new cases of cancer diagnosed
each year and it is estimated that 1 in 3 people will develop some form of cancer in
their lifetime. It is non-discriminate of age however it is most prevalent in older people
with 75% of cases developing in people at the age of 60 and above. Cancers in
children, teenagers and young adults account for approximately 1% of all cases.
Although cancer incidence has remained relatively stable over the last decade there
has been an overall increase in incidence rates in the U.K constituting a rise of one
quarter since 1975.
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Table 1: Main types of cancer leading to mortality each year
Cancer type Number of
deaths per
year
Lung 1.3 million
Stomach 803 000
Colorectal 639 000
Liver 610 000
Breast 519 000
WHO FACT SHEET No. 297
The WHO projects deaths from cancer worldwide to continue rising with an estimated
12 million deaths worldwide in 2030. Both men and women are affected however
variations exist between genders for the most frequent types of cancer.
Among men lung cancer is the most prevalent whereas breast cancer dominates
cancer incidences among women (Table 1). In the U.K the overall cancer death rates
have fallen by about 10% however a staggering 150,000 deaths, 1 in 4 of all deaths
are as a result of cancer (Figure 1). Despite declines in death caused by uterine,
oesophageal and male skin cancer 1 in 5 cancer fatalities are attributed to lung
cancer.
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Figure 1: All deaths caused by cancer 2006
Cancer Research UK (2007)
These figures and statistics give insight into the global burden of cancer. The
projected increases (12 million by 2030) place an ever mounting challenge on the
scientific community to understand the process of carcinogenesis and to find
effective treatment strategies to combat the disease.
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Biology of Cancer
“Tumors destroy man in a unique and appalling way, as flesh of his own flesh which
has somehow been rendered proliferative, rampant, predatory, and ungovernable . . .
Yet, despite more than 70 years of experimental study, they remain the least
understood. . . What can be the why for these happenings?”
Peyton Rous, in his acceptance lecture for the Nobel Prize in Physiology or Medicine
(1966)
Cancer refers to group of diseases that develop across time and are predominantly
characterised by uncontrolled division of the body’s cells. In the case of normal cells
external growth factors are required to instruct the cell to divide. Normal cell
regulation inhibits these growth factors accordingly and halts further division. Cancer
cells operate independently of these positive growth factors and thus divide in their
presence or absence. National Institutes for Health (1999)
Cancerous cells begin to dictate their own agenda for proliferation. Not only does this
ancestral cell display inappropriate proliferation all of its subsequent progeny operate
in this manner a mass of cells formed of these abnormal cells are referred to as a
tumour which can either stay in the tissue it originated in ( in situ cancer) or it may
begin to invade nearby tissue (invasive cancer). Invasive tumors are said to be
malignant. National Institutes for Health., (1999)., Normal cells can divide to fill in a
gap but a soon as there are a sufficient number of cells to fill the gap they cease to
divide. Cancer cells show no contact inhibition and continue to divide after they touch
other cells and consequentially form this large mass of cells -tumour. Blackburn et
al.,
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Cells have a lifespan and this entails ageing and death via apoptosis which is the
normal, regulated programmed death of cells. Their ability to replicate their DNA is
limited to approximately 50 times due to the fact that each time a chromosome
replicates the (ends) shorten. Growing cells utilise telomerase enzymes to replace
lost cells whereas maturet cells lack this enzyme resulting in their limited replications.
Cancerous cells have the ability to activate telomerase in adult cells and this allows
for an unlimited number of cell divisions. Columbia Encyclopaedia 6th edition.,
(2007) ; National Institutes for Health., (1999); Blackburn et al.,
In addition when DNA is damaged or replicates abnormally, normal cells cease to
divide and since cancerous cells divide regardless of DNA damage or abnormal cell
replication they manage to accumulate increasing amounts of damaged DNA.
Blackburn et al.,
Figure 2: Stages of Tumour development
National Cancer Institutes (1999)
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Malignant tissue can cause secondary effects whereby expanding abnormal growth
puts pressure on surrounding organs and tissue or cancer cells metastasize and
invade other organs by shedding into the blood or the lymph.
It is believed that tumour development and growth is preceded a mutation within a
cell(s) leading to hyperplasia (figure 2) mentioned above which creates an
environment conducive to further mutation. This applies to virtually all of the body’s
tissues as all of them are susceptible to mutations. As a result of these mutations
cancerous cells attain a selective advantage over normal proliferating cells and thus
multiply much more rapidly.
The general consensus within modern science is that cancer is a disease of
molecules and genes. It is a multistep process developing across time as long
succession of genetic changes. Through these changes precancerous cells manage
to acquire the traits together and manifest into a malignant growth of cells. National
Institutes for Health., (1999) The main functioning genes are subdivided into three
categories. Proto-oncogenes produce proteins that enhance cell growth and division.
When mutated these genes are referred to as oncogenes. The second group are the
tumour suppressor genes and these create proteins that terminate cell division and
induce apoptosis. The third group are genes coding for DNA repair mechanisms
which help repair DNA from molecular changes that lead to cancer. National
Institutes for Health., (1999)
Mutations in any three of the groups of genes potentiate the development of cancer
(Table 2).
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Table 2: Gene mutations and their implications
Mutation Present Effects potentiating Cancer
Proto-oncogenes Results in oncogenes-
accelerated and
uncontrolled growth
Tumour suppressor genes Limited ability of signalling
inhibitory messages
DNA repair genes Loss of ability to repair
alterations in DNA
.
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Genes in Cancer
Modern science views on cancer centre around it being a multistep process
developing across time as long succession of genetic changes. Through these
changes precancerous cells manage to acquire the traits together and manifest into
a malignant growth of cells. National Institutes for Health., (1999)
Mutations to proto-oncogenes forms oncogenes and these genes stimulate
excessive division whilst mutations in tumour suppressor genes (figure 4), results in
their inactivation. Consequentially the ability to inhibit excessive growth is lost and
collectively mutations in these groups of cancers account for most human cancers.
Proto-oncogenes code for proteins that play a part in pathways that process and
receive growth signals (figure 3) from other cells within a particular tissue. When
growth factors
Figure 3: Control of gene expression, stimulatory signals
Blackburn et al,.
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are produced they move into the gaps between cells attaching to specific receptors
on their membranes. Upon activation these receptors transmit stimulatory signals to
proteins within the cell cytoplasm. These proteins in turn convey these messages
stimulating other proteins all the way to the nucleus and activate genes that facilitate
the movement of the cell through its growth cycle. The mutated form of these genes
(oncogenes) cause proteins involved in growth signalling pathways to become
overactive and as a result cell proliferation progresses at much faster rate than in
comparison to the rate before the mutation. Oncogenes also produce deviating
signals to receptor proteins leading to the release of stimulatory signals in the
presence or absence of growth factors. This continuous disruption of the signalling
cascade results in the nucleus receiving constant stimulatory signals instructing it to
divide. Blackburn et al,.
Figure 4: Some Genes implicated in Cancer development
Blackburn et al,.
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When DNA repair mechanisms involved in maintenance of the chromosome are
damaged errors in the DNA go unattended. Without DNA mechanisms mutations are
allowed to accumulate within the cell. These mutations increase the cancerous
changes within a cell. Blackburn et al,.
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CANCER CAUSES
There maybe multiple mechanisms leading to the development of cancer (figure 5).
Although disputed the prevailing model centres around mutations occurring within
tumour suppressor and oncogenes which result in cancer. Alternative models
indicate mutations occurring in “master genes” which control the cell cycle and that
once a mutation occurs in these genes inappropriate gene dosing occurs. As a result
of this cells produce too much or too little of particular proteins required for proper
cell growth and an imbalance of this sort leads to cancer. Blackburn et al,.; National
Institutes for Health (1999)
Viruses that affect humans may also promote cancer; this is the case with viruses
which insert their DNA into the chromosome at the same point in which proto-
oncogenes are located thus inactivating them (converting them into oncogenes).
Virus DNA located close to genes involved in the regulation of cell growth may take
over host machinery resulting in increased transcription of those genes
(inappropriately) which provides the possibility of a cancer occurring. The table below
shows a few viruses that have been implicated in cancer. Blackburn et al,.
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Table 3: Viruses that can potentiate cancer
Viruses associated with increased risk of
cancer
Cancer type caused
Human papillomavirus Genital Carcinomas
Hepatitis B Liver Carcinoma
Epstein-Barr virus Burkitts lymphoma
Human T-cell leukaemia virus T-cell lymphoma
Herpes Virus Kaposi’s sarcoma, B
cell lymphoma
Cancer research over the years had lead to knowledge that environmental factors
contribute to an individual’s chances of getting cancer. In 1775 Percival Pott found
that there was an unusually high incidence of scrotal cancer amongst men who
worked as chimney sweep boys. Hawes et al (1775) When there is a significant
correlation between exposure to an environmental factor and occurrence of a specific
cancer the factor is referred to as a carcinogenic agent. Carcinogenic agents range
from X-rays, UV light, Tobacco smoke, industrial solvents. Some cancers associated
with these factors are not associated with cancer genes furthermore some are
preventable. Blackburn et al,.
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Figure 5:The Genesis of cancer 4 Theories
Padilla-Nash Hesed M and Reid Thomas (2003)
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CANCER TYPES
Cancers are classified according to the type of tumour that develops and this is
based on where the original alteration occurred (See Table 4 below). Blackburn et
al,.
Table 4: Origin of mutation and cancer
Type Origin
Carcinoma Epithelial cells (most common)
Sarcoma Muscle, bone, fat and
connective tissue
leukaemia White blood cells
Lymphoma Bone marrow (lymphatic
system)
Myeloma Specialised anti-body
producing white blood cells
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Pgp SIGNIFICANCE IN THE THERAPY OF CANCER
The ideal cancer therapy aims at being highly efficacious with high tumour specificity
and maintaining minimum levels of toxicity. Roylance (2007)
TREATMENT SCHEMES
The basis of medical cancer therapy falls under the categories of chemotherapy,
radiotherapy, endocrine therapy and biological therapy. Although their mechanisms
may differ, treatment strategies tend to involve a combination of separate therapies.
Table 5: Mainline Therapies in cancer
Therapy type Treatment strategy
Chemotherapy The use of cytotoxic agents
Radiotherapy Ionising gamma radiation
Endocrine Blocking hormonal action
Biological Monoclonal antibodies
Surgery -ectomy
Chemotherapy is one of the mainline treatments in cancer and involves the use of
alkylating agents, platinum compounds, anthracyclines, antimicrotubuleagnets,
antimetabolites and topoisomerase II inhibitors. These drugs target rapidly dividing
cells consistent with tumour growth with a 90% efficacy on 10% of all cancers
Roylance 2007. They are however non-specific to tumour cells thus normal cells my
affected if they exhibit similar characteristics of division.
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Figure 6: Chemotherapeutic effects on the cell cycle
Roylance 2007
Combination chemotherapy involves using more than 1 class of chemotherapeutic
agent at optimal dose and schedule. This enables synergistic action of the drugs as
different drug classes will affect different points within the cell cycle (figure 6).
Radiotherapy much like chemotherapy is non-specific to cancer cells thus normal
cells are damaged in the process. Unlike chemotherapy these cells have the ability to
repair themselves after. Cancer Research UK
It involves the use of ionising radiation in measured doses (X-rays). This radiation
damages cells hindering their growth and division. It is usually given before and after
surgery to reduce tumour size and after to improve treatment results. Palliative
treatment of cancer by radiotherapy buys times for other treatment strategies to be
implemented. Cancer Research UK
Hormonal therapy is usually associated with breast and prostate cancer and it is a
relatively specific form of treatment with minimal toxicity. Beaston 1896 postulated a
link existed between the ovaries and the proliferation of breast cells after removing
the ovaries of a woman with advanced metastatic breast cancer, she responded
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drastically. When tamoxifen a selective oestrogen modulator was developed as a
contraceptive its uses in breast cancer soon superseded its relatively low efficacy as
a contraceptive (it even induced ovulation in some cases). In 1973 it was licensed for
use in breast cancer and its mechanism of action (Jordan 1974) involved the
blockade of oestradiol to oestrogen receptor (OER) in human breast and rat
mammary tumour. Hormonal therapy currently includes anti-oestrogen agent
(fulvesant) and aromatase inhibitors both steroidal (exemestane) and non-steroidal
(arimidex). Roylance (2007).
Biological therapy involves the use of monoclonal antibodies and small molecule
inhibitors. This strategy is efficacious however the mechanism of action is not fully
understood. The treatment is specific and of minimal toxicity (e.g. herceptin). This is
a relatively novel treatment strategy in cancer thus many of the treatment regimes
are not implemented unless conventional treatment has failed. Roylance (2007)
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P-GLYCOPROTEIN
P-glycoprotein (Pgp) is the most characterised member of the human ATP Binding
Cassette (ABC)-transporter family. It is the gene product of the MDR1 gene and
serves as an integral efflux membrane protein. Pgp and members of this superfamily
are characterised by the ATP driven active transport of substances out of the cell. It
is believed to have evolved as a protective mechanism against harmful toxins
Gottesman et al (1995). Evidence of this is based on the highly conserved protein
regions that can be observed in prokaryotes, archea and eukaryotes.Seelig and
Landwojtowicz (2000)
The ABC efflux transporters are primarily located in plasma membranes; here they
extrude a variety of structurally diverse drugs, drug conjugates and metabolites
Schinkel and Jonker (2002). The human genome codes approximately 48 ABC
proteins, which are subdivided into subfamilies via sequence alignments (From A to
G). For a transporter to be considered functional it must contain at least 2 ABC
subunits. These proteins are membrane bound consisting of various domains and
specialised structures. (Table 6)
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Table 6: ABC transporters
Szakacs et al 2008
Pgp transports a variety of chemically diverse substances across the cell membrane
thus protecting the cell from passively transported drugs. It plays a role in the
bioavailability of substances that enter the cell (including drugs, metabolites, and
xenobiotics). This wide substrate specificity limits compounds crossing tissues with a
protective barrier function which include; blood-brain barrier, liver, intestines, kidney
and testis/placenta (Figure 4). Most Pgp substrates are hydrophobic and many of
them contain aromatic ring structures Endicott and Ling (1989); Gottesman and
Pastan (1993). Its substrate specificity extends to non cytotoxic compounds as well;
calcium channel blockers, immunosuppressants, steroid hormones and neuroplectic
drugs.
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P-gp History
Juliano and Ling (1976) first characterised P-gp while working with Chinese hamster
ovary (CHO) clonal cell lines where they reported the expression of a protein (P-gp)
in colchicine resistance. This followed previous work by Ling and Thompson in
(1974) (figure7) in which they isolated a series of related colchicine resistant CHO
clonal cell lines using single step selections. They also found that increasing
colchicine resistance correlated to increased resistance to other drugs and reduced
uptake.
figure 7: Development of Pgp understanding
Mcdevitt and Callaghan (2007)
The isolation of clones using Chinese hamster ovary (CHO) cells (Juliano and Ling
1976) was useful as a method of investigating somatic cell mutations and the
phenotypic expression in cultured mammalian cells. Isolation also aided in identifying
environmental mutations.
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Researchers attempted to investigate genetically linked mutations and specialised
only in genuine mutations (that is heritable alterations in particular genes). Heritable
mutations are valuable as they can also serve as genetic markers. Single step
selection methods were implemented and analyses were undertaken to determine
whether the appearance of variants was consistent with spontaneous mutation rates.
In 1970 while assessing the resistance of murine leukaemia sublines (L5178Y and
L5178Y/D) to actinomycin D Kessel and Bosmann (1970) found that administration of
50µg/kg actinomycin D inhibited uridine incorporation into RNA in L5178Y but not in
L5178Y/D. Following enzymatic studies (using galactosyl transferases, fucosyl
transferases, glucosyl transferases) they managed to identify an altered cell surface
glycoprotein. These alterations to the membrane composition where found to have
attributed to the changes in actinomycin D permeability.
These findings concurred with those of other researchers at the time and during the
isolation of colchicine-resistant CHO cells in which Juliano and Thompson (1974)
found that the colchicine-resistant cells characterised had pleiotropic cross-
resistance to other drugs (daunomycin and puromycin) furthermore reduced uptake
of colchicine was proportionate to the degree of drug resistance. This led to the
conclusion that colchicine resistance was bought about by alterations to membrane
permeability.
Juliano and Ling (1976) attributed the multi-drug resistant characteristic of tumor
cells as being brought about by the overexpression of this surface glycoprotein and
named it Plasma glycoprotein.
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STRUCTURE OF P-GP
The structure of Pgp like other functional entities of ABC transporters consists of 4
domains.2 membrane domains (MDs) and 2 nucleotide binding domains (NBDs also
referred to as the ATP-binding cassettes). The NBDs are responsible for the
generation of motional force while the MDs provide a translocation pathway for
substrates bound to the protein. Pgp shares a number of conserved sequence motifs
with other ABC transporters (Walker A, Walker B and the ABC signature) due to ATP
their common substrate. MDs however are more diverse and this diversity is
reflected by the large diversity of substrates that are transported. Seeger and W. van
Veen ( 2008)
Figure 8a: P glycoprotein structure
Schinkel and Jonker 2003
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MDs (Figure 8a) consist of 6 putative transmembrane segments with heavy
glycosylation occurring on the first loop. The NBDs or similarly the ATP binding
cassettes are located intracellularly (Figures 8a and 8b). In vitro studies on Pgp
structure Schinkel et al (1993) showed that N-glycosylation was not necessary for
basic transport function. Up to 3 sites present in the mdra1 protein, they also deleted
a stretch of 20 amino acids containing 2 out of the 3 glycosylation sites following
transfection into drug-sensitive cells the effects of the mutations were analysed. It
was found that the absence of N-glycosylation did not alter the level or pattern of
cross resistance however drastically reduced the efficiency in which drug-resistant
clones where generated. As a result of these findings it was postulated that the
Glycosylation of the transmembrane loops contributed to the stability of Pgp (within
the plasma membrane) but not to drug transportation.
Figure 8b: Pgp structure showing mechanism of action
Modified from Gottesman et al,.
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Photoaffinity labelling experiments Cornwall et al (1986); Bruggeman et (1992)
show that following the passive diffusion of chemical agents into the cell
cytoplasm they are bond by Pgp and exported out into the extracellular space
ATP hydrolysis or GTP in certain scenarios Ambudkar et al 1992
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LOCALISATION OF Pgp IN NORMAL TISSUES
Figure 9: localisation of Pgp as detected by MRK16 a monoclonal antibody
Theibaut et al 1987
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Thiebaut et al ‘s findings support the hypothesis of the MDR1 gene product (P-gp) as
having a role as a pump against physiological metabolites and chemotherapeutic
agents.
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DRUG RESISTANCE
“The first successful chemotherapy of human cancer led soon to the realization that
drug resistance was going to be a major impediment to cure or long-term palliation”
Gottesman and Ling (2006)
In the 1940s Sidney Farber administered aminopterin in patients who where
diagnosed with leukaemia in what was the beginnings of modern age treatment to
caner. This also symbolised the beginnings of the understandings of drug resistance
and its clinical consequences as the treated children experienced an initial response
to therapy followed by remissions that were resistant to further treatment.
Drug resistance is a phenomenon associated with the chemotherapy of metastatic
cancers. Conventional chemotherapy of most common cancers (see table 1) can be
expected to reduce tumour size in 50% of all cases. In almost all cases drug
resistance develops and is the major cause of fatalities Baird and Kaye (2003).
Resistance falls into 2 categories; intrinsic or acquired. Chemotherapy has limited
efficacy in patients with intrinsic resistance which is present at the time of diagnosis
whereas acquired resistance is born of tumours which are initially responsive to
chemotherapy however reoccurrence of the tumour expresses a completely different
phenotype which is non-responsive to previous therapy. In some cases “multidrug
resistance”(MDR) occurs and this refers to pleiotropic cross-resistance to a range of
structurally unrelated compounds as a result of increased expression of the
transporter protein.
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Drug resistance is either as a result of alterations in the cancer cells or insufficient
drug exposure. Insufficient dosing, low bioavailabiulity, increased metabolism,
excretion are some of the factors that lead to drug resistance. Alterations in cancer
cells can lead to increased drug efflux (ABC transporters; Pgp), decreased drug
influx or activation of detoxification systems. Biard and Kaye (2003)
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Pgp EXPRESSION IN CANCER CELLS
In vitro studies of multidrug resistance reveal that application of a single
chemotherapeutic agent, to a selected cancerous cell line can in fact confer
resistance, to other unrelated compounds. Salmon et al (1989) while investigating
multiple myeloma found that the frequency of Pgp expression increased after
chemotherapy (Fig 6 ).
(See Clinical Studies table 7).
The MDR1 gene is reported to be overexpressed in up to 50% of clinical tumour
specimens Goldstein et al (1989): Gottesman et al (1995) and the overexpression of
MDR1 transcripts, is associated with lack of treatment efficacy in a number of
cancers Baldini et al 1996; Chan et al 1990. Furthermore studies carried out by
Campos et al 1992; Pirker et al in the same year identified Pgp expression to be an
independent risk factor for treatment failure.
Despite the extensive cataloguing of Pgp expression in many cancer types a clear
relationship between Pgp detection and its implications on prognosis and response
to therapy is still debated. Poor design of clinical trials and a general lack of
consensus on detection methods, low patient numbers significantly hampered
progress Pgp understanding. In 1996 Beck et al attempted to standardise the
detection of Pgp expression making recommendations for future studies which
included:
(a) Although detection of Pgp and MDR1 is at present likely to be more reliable in
leukemias and lymphomas than in solid tumors, accurate measurement of low levels
of Pgp expression under most conditions remains an elusive goal;
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(b) Tissue-specific controls, antibody controls, and standardized MDR cell lines are
essential for calibrating any detection method and for subsequent analyses of clinical
samples;
(c) Use of two or more vendor-standardized anti-Pgp antibody reagents that
recognize different epitope improves the reliability of immunological detection of Pgp;
(d) Sample fixation and antigen preservation must be carefully controlled;
(e) Multiparameter analysis is useful in clinical assays of MDR1/Pgp expression
(f) Immunostaining data are best reported as staining intensity and the percentage of
positive cells; and
(g) Arbitrary minimal cut-off points for analysis compromise the reliability of
conclusions
Beck et al (1996)
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Table 6: clinical studies implicating Pgp expression in cancer.
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Figure 9: Cancers reprted to have high MDR1 expression post-chemotherapy
Gotetesman and Pastan et al 1991
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PHARMACOLOGICAL TARGETING OF Pgp
Drug resistance presents the greatest challenge to cancer treatment by
chemotherapy and is the main reason for failure of treatment Aouali et al (2005). One
of the first compounds to be identified as being able to circumvent resistance
(vincristine in particular) was the calcium channel blocker Verapamil . This was
followed up in 1986 by Slater et al in which cyclosporine was found to have
modulating ability on resistance. Early clinical trials Salmon et al (1991) on verapamil
assessed it clinical feasibility as a modulator. Salmon et al (1991) first evaluated the
resistance patterns from bone marrows from 59 myeloma patients and found that
verapamil was capable of sensitising myeloma cells exhibiting resistance to
doxorubicin and vincristine in vitro but did not enhance sensitivity of cells that were
drug sensitive (P>.001). Clinical trials were then conducted on 22 patients with
myeloma refractory to vincristine-adriamycin-dexamethasone (VAD) which was
administered with i.v verapamil. The clinical efficacy of the trials prompted the
supposition that clinical reversal of MDR could be achieved by verapamil. Cardiac
toxicity however was observed in some patients (fig 9), an unacceptable routine for
cancer treatment. Licht et al (1998)
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Figure 9: Toxicities of VAD Plus High-Dose IV Verapamil in 22 Patients
Salmon et al 1991
The use of verapamil, cyclosporine Yahanda et al (1992) and other 1st generation
Pgp inhibitors was plagued by an inability to reach sufficient plasma concentrations
to block Pgp activity and by clinically significant toxicity profiles. These findings
prompted research using available in vitro assays to investigate any common
pharmacophoric elements on substrates of
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCERPgp. In 2003 Wang et al utilised quantitative structure activity relationships (QSAR)
to describe criteria that must be fulfilled in order for a substance to be a substrate or
modulator for Pgp. Their findings proposed that a Pgp modulator candidate should
have
i. a log P value of at least 2.92
ii. 18-atom-long molecular axis and
iii. a high Ehomo value
iv. at least 1 tertiary base nitrogen atom
(P version 4.0 QSAR software and HyperChem version 5.0 program) (Wang et al
2003)
Figure 10: 1st -3rd Generation Pgp Modulators
Mcdevitt and Callaghan et al 2007
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
The low binding affinities of the 1st generation Pgp inhibitors necessitated the use of
high doses subsequently leading to unacceptable toxicity. Despite the works of
Zamora et al 1988 and Wang et al 2003 the pharmacophoric parameters lacked the
needed stringency to facilitate drug development significantly. Moreover 1st
generation modulators had shown efficacy and thus they formed the template of
further drug development. 2nd generation Pgp modulators (Fig 10)
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
1ST -3RD GENERATION Pgp MODULATORS
For a compound to eligible as a modulator of Pgp it must fulfil at least one of the
following criteria:
i. Increase the potency of cytotoxic drugs in resistant cells
ii. Increase the accumulation of cytotoxic drugs intracellularly
iii. Interfere with photo affinity labelling of Pgp
As mentioned before (pharmacological targeting of Pgp) 1st generation modulators
resulted in high levels of toxicity and a major contributing factor to this was the low
binding affinities observed which prompted higher doses. The clinical application of
cyclosporine has been extensively investigated Yahanda et al (1992); Sonneveld et
al (1992).
Manetta et al 1993 conducted phase I trials in which the potential clinical use of
cyclosporine A modulation of cisplatin was investigated, they also set out to identify a
tolerable dose of Cyclosporin A when combined with a standard dose of cisplatin
(75mg/m2 ). Their sample size consisted of 20 patients with refractory gynaecologic
cancer received 81 courses of therapy.
20% of patients developed nephrotoxicity with 25% of the patients being partial (3
patients) and complete (2 patients) responders. Although there was evidence of
chemosensitising of MDR the overall results showed cyclosporine A achieves this
with considerable toxicity levels.
The toxicity of 1st generation chemosensitising agents prompted the development of
new compounds. Analogues of these agents where developed and these included
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
dexverapamil, dexniguldipine, PSC 833 and VX-710. Wilson et al 1995 reported 10-
fold decrease in cardiac toxicity at equal chemosensitising levels. The most
characterised of these compounds is PSC 833 a derivative of cyclosporine that was
reported Boekhorst et al 1992; Twentyman and Bleehen 1991 to inhibit Pgp by 10-
20fold greater activity. Many studies show it to have high reversing potency Boesch
et al 1991; Gaveriaux et al 1991. While findings by Coley et al 2000 on fresh tumour
material from patients with soft tissue sarcoma recorded modest on accumulation of
ancthracycline (20%) when given at 1nM. Animal models of 2nd Pgp generation
modulators show significant results Watanabe et al 1996 however clinical trials show
evidence of limited success. VX710 like PSC 833 has been extensively studied. It
directly interferes with the efflux of substances due to its affinity to the Pgp pump as
well as other related ABC transporters (namely MRP1). The coadministration of this
compound with chemotherapeutic agents has had limited success in the treatment of
refractory cancers. Gandhi et al 2007 conducted a phase II trial in order to evaluate
the efficacy of VX710 when co administered with doxorubicin and vincristine patients.
The patients suffered from inoperable, local advanced or metastatic sarcoma of the
soft tissue. In addition the patients had anthracycline-resistance. The study was on
36 patients who enrolled over a two year period and of varying demographic. VX710
was reported as no significantly enhancing anti-tumour activity or survival.
Neutropenia was also found to be the major toxicity occurring in 26/30 patients in
trials. There was however partial responders 7/36 patients as detected by radiology
however accurate calculation of response duration was hindered by censoring of the
results and a halt to the trials. Similarly earlier studies conducted by Bramwell et al
2002 revealed objective responses for the drug with disease stabilisation in partial
responders lasting duration of only 3-4 months.
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Ghandi et al 2007
2nd generation inhibitors have a better pharmacologic profile than 1st generation
inhibitors however they also posses similar characteristics which limits their chemical
profile. By limiting the metabolism and clearance of chemotherapeutic agents they
facilitate the incidence of unacceptable toxicity that necessitates the reduction in
doses administered within the trials.
Cytochrome P450 enzymes are also induced along with ABC transporters leading to
suppositions that regulatory elements of these genes overlap Lum and Gosland
1995. The P450 3A4 isoenyme shares many substrates with Pgp thus substances
affected by MDR development are also liable to metabolism by P450 3A4 (PSC 833
and VX710) and thus results in many of the reported unpredictable pharmacokinetic
interactions. These agents inhibit the P450 3A4 mediated metabolism and is the
main cause of toxicity associated with modulators off Pgp. Dose reductions had to be
implemented by researchers due to safety however achieving therapy at a safer
dosing regime limits the efficacy of many of these 2nd generation modulators.
Furthermore intrinsic activity of some 2nd generation modulators (VX710, PSC 833)
suggests they have affinities for other ABC transporters as well as Pgp and this
inhibitory activity of non-target transporters may contribute to the limited efficacy and
raised toxicity of these agents Yanagisawa et al 1999;Rowinsky et al 1998.
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
3rd generation Pgp modulators (fig 6 pharmacological targeting of Pgp) specifically
and potently inhibit Pgp function. Their development occurred as a result of structure
activity relationships and combinational chemistry Thomas and Coley 2003. At
relevant concentrations they do not show activity at P450 3A4 enzymes Dantzig et al
1999 where the selectivity of LY335979’s selectivity for Pgp was evaluated as well as
its effects of P450 activities. It was found to have a significantly lower affinity for
CYP3A than for Pgp. Furthermore it was characterised as a potent modulator of Pgp
and not other members within the ABC transporter family (namely MPR1 and MPR2).
Other 3rd generation modulators; XR9576, R101933,ONT-093,GF120918 are of
varying chemical structures but are common in their level of potency on Pgp. This
has also been observed in clinical trials as well the most promising agent of 3rd
generation inhibitors is XR9576 which is believed to achieve it modulation by binding
to the ATP binding sites of Pgp. Martin et al 1996; roe et al 1999 findings identified
XR9576 as a potent inhibitor of Pgp in vivo and in vivo. Martin et al 1999 used drug
resistant CHO ovary cell lines and demonstrated XR9576 showing greater selectivity,
duration of inhibition and potency of interaction at Pgp than with any other reported
modulators.
Furthermore it was shown to increase accumulation of [H3]-Vinblastine and [3H]-
paclitaxel transport as high as in cell lines that were not overexpressing Pgp
(EC50=487+50 nM). By binding to the ATP sites means that Xr9576 like most 3rd
generation modulators are not substrates themselves. This induces conformational
changes which prevent hydrolysis of ATP a requirement in the efflux mechanism
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
MODULATION OF Pgp; A GENETIC APPROACH
Molecular strategies directed at modulating Pgp activity have focused on interfering
with the synthesis of Pgp. This has brought into use antisense oligonuleotides,
ribozymes and protein that regulate differentiation of cancer cells Corrias et al 1992;
Efferth et al 1993. These strategies focus on cleavage of MDR1 mRNA and have the
ability to restore chemosensitivity in MDR cells Kobayashi et al 1999. They
developed 2 anti-MDR1 hammerhead ribozymes driven by the beta-lactin promoter.
They transduced these ribozymes into MDR1 expressing cells which were designed
to target specific predetermined sites (fig11) in addition a retroviral vector containing
polymerase III promoter was used to improve ribozyme activity.
Figure 11: MDR1 gene and anti-MDR1 hammerhead ribozymes
Kobayashi et al 1999
Human leukaemia cell lines (MOLT-3 where MDR subline used was
MOLT-3/TMQ800) where used and these were co-cultured with virus producer cells.
Ribozyme efficacy was then determined by G418 selection and ribozyme transduced
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
cells became vincristine-sensitive. Original MOLT-3/TMQ800 cells(conrol) were
reported to be 600-fold more resistant to vincristine whereas stably transduced cells
showed 92 to 296-fold resistance. Partial restoration of vincristine sensitivity and
doxorubicin activity was accompanied by decreased expression MDR1, decrease in
Pgp amount and function (fig 10). Kobayashi et al (1999)
iMDR1-sRz one of the ribozymes used targeted the translation-initiation site and
pooled transformants that occurred as a resulted of its introduction to original MOLT-
3/TMQ800 cells exhibited 10% of MDR1 mRNA compared to controls.
Further studies by Scanlon et al (1993) complimented these findings and revealed
that in addition to MDR1-directed ribozymes, c-fos interacting ribozymes could also
decrease expression of Pgp. Hammerhead ribozymes designed to cleave c-fos
mRNA cloned into pMAMneo were transfected into drug sensitive human ovarian
carcinoma (A2780) cell lines. Cells exhibiting MDR also had elevated levels of the c-
fos furthermore cells transfected with c-fos also exhibited MDR and the anti-fos
ribozyme reversed the MDR phenotype in A2780AD cells. These studies reveal
potential pharmacological targets in particular Fos which was previously believed to
play a role in resistance to agents not within the MDR family such as cisplatin, AZT
and 5-Flourouracil Scanlon et al (1993). Transcription factors are involved in the
regulation of expression in drug resistance genes and thus appear to be suitable
targets for strategies to circumvent drug resistance.
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
STEM CELLS AND MDR1 EXPRESSION
The prospect of transferring drug resistance genes to chemosensitive cells like stem
cells has been explored to protect them from the adverse effects of chemotherapy.
The drug resistance genes may serve as selectable markers in vivo facilitating gene
expression in transduced cells after exposure to drugs.
Licht et al (1998) transferred MDR1 cDNA to mouse bone marrow cells that lacked
expression of lineage –specific antigens nor MHC II antigen Ia. These cell had high
expression of Ly6A/E. Isolated cells were expanded ex vivo using growth factors.
Gene transfer was achieved via coculture containing retrovirus. Functional Pgp was
detected in 60% of expanded cells. The recipient animals expressed Pgp in high
proportion compared to those observed in MDR cancers in the clinic.
In 1994 Ward et al conducted research using human CD34+ progenitor cells a
reflection of the feasibility of gene transfer to stem cells as demonstrated in previous
animal models. In normal and also in tumour tissue MDR expression varies greatly
whereas in bone marrow cells it remains consistently low. This renders stem cell
particularly sensitive to many chemotherapeutic agents; taxol, anthracylcines,
etoposide, vinca alkaloids all of which are effect against many cancer forms.
They reported the successful transfer and expression of the human MDR1into
human bone marrow cells (via amphotropic retroviral supernatant) and the resistance
to taxol exposure gained thereafter. PCR analysis of transduced, colchicine resistant,
amphotoric cells indicated 17/20 clones possessing th MDR cDNA
The main MDR producer A12M1 had the highest titer (5 x 1014 viral particles/ mL).
FCAS analysis showed a 1to 2 log increase in MDR expression compared t
untransduced cells.
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Taxol was introduced to MDR-transduced cells and their level of MDR expression
compared to non-transduced cells after exposure to 1 x 10-6 mol/L on day 10 post
transduction. Analysis took place on day 12 and 12.5% of cells showed increase in
Pgp compared to non-transduced cells.
The levels of taxol resistance achieved by MDR-transduced cells and non-
transduced cells and preferential survival was observed in transduced cells at doses
of 5 x 10-8 mol/L. This ability to select for progenitor cells resistant to taxol bears
significant perspective on its potential use in providing drug resistant marrow cells for
patients undergoing cancer therapy. Alternatively it could be useful in enriching
marrow populations of cells with the MDR gene and a non-selectable gene (e.g beta-
globin) Ward et al (1994).
Be it promising the first published clinical trials conducted on MDR1 levels reported
to be disappointingly low. Hanania et al (1996). It is believed that low and unstable
expression of these transferred genes is the major cause for failure of gene therapy
Licht et al (1998). As a result trials conducting high-dose chemotherapy with
autologous hematopoietic stem cell transplantation report insufficient function of
reconstituted bone marrow limiting the efficacy of transplantation chemotherapy.
Efforts by Takahashi et al 2007 (Fig 12) to increase transduction efficiency of MDR1
gene transfer yielded limited success.
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Figure 12: MDR gene therapy
Takahashi et al 2007
Peripheral blood was stimulated using 5 cytokines; SCF, TPO, IL-6, FL-Ligand & sIL-
6r. They achieved transduction efficiencies of 8-17% by Pgp expression.
Transplantation of induced peripheral blood stem cells (PBSC) (1/3) and untreated
PBSC (2/3) yielded 3-5% increases in the ratio of peripheral white blood cells which
corresponded to the ratio of induced MDR1-tranduced CD34+ cells. However despite
this after 6 months Pgp expressing cells decreased to an undetectable level. These
limited successes in clinical scenarios may indicate low survival chances of MDR1-
transduced stem cells in the bone marrow or may lie in transduction strategies which
still seem to lack sufficient efficiency.
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Discussion/ Conclusion
Extensive studies have been carried out on the MDR gene and it gene product P-
glycoprotein. It has been characterised as a ATP-dependant efflux protein whose
main role is the expulsion of toxic substances that enter the cells of the body.
The use of radiolabelled dyes monoclonal antibodies have elucidated its distribution
within the body’s organs leading to researchers suggesting evolutionary significance
pertaining to its protective characteristics. This is further substantiated by it
occurrence in many different species.
More than 50% of cancer relapses show an overexpression of MDR1 gene as a
result of conventional chemotherapy. Furthermore the increased expression of Pgp
has been associated with multidrug resistance to a number of unrelated
chemotherapeutic agents.
This greater understanding of Pgp gave rise to many pharmacological studies aimed
at modulating Pgp function as a means of increasing drug accumulation of
chemotherapeutic agents. As a result drug development on Pgp inhibitors advanced
rapidly. Many agents proved promising however clinical trials yielded limited
successes. A significant trend in these studies was that although Pgp modulators
have the potential to increase drug accumulation of cytotoxic agents a significant
number of studies resulted in toxicities associated with this increase accumulation of
drug. Alterations of doses and more potent drugs were designed slightly increasing
the number of responders to therapy.
Research has generally shifted to stem cell biology and gene therapy and significant
progress has been made since the early days of introducing foreign genes into
murine cells via retroviral vectors. Gene therapy directed at transferring resistance to
stem cell as a means of protection from chemotherapy was anticipated to
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
revolutionize cancer treatment drastically however optimism in these ambitions have
rapidly declined. This largely due to the limited positive results obtained in clinical
trails to a variety of gene therapy approaches in which low efficiency was reported.
P-glycoprotein’s significance in cancer relates to multidrug resistance and its
overexpression leads to the failure of many conventional chemotherapeutic
strategies. Despite advances in pharmacotherapy and stem cell research many
challenges lie ahead in the translation of recent advances into reproducible clinical
benefit.
A major factor to be considered is that resistance to chemotherapeutics is
multicausative. A number of genes have been identified that are associated with
chemoresistance and despite strategies having being developed to circumvent Pgp
mediated resistance in cancer various other approaches involving other genes are
still at a preclinical stage.
Any major advances in Pgp modulation or application will have to encompass other
related ABC transporters and other factors contributing to the MDR phenomenon this
project recognises the feasibility of pharmacological Pgp modulation and its
application in gene therapy which is still to be validated as a means of cancer
therapy.
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THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
REFERENCES
Ambudkar S V, Lelong I H and Zhang J P. (1992). Partial purification
and reconstitution of tthe human multidrug-resistance pump-
Characterisation of drug stimulable ATP hydrolysis. Proc Natl Acad Sci
USA; 89: 8472-8476
Alan Milburn. (2000). The NHS cancer plan: A plan for investment A
plan for reform. www.doh.gov.uk/cancer
Aouali Nassera, Eddebra Lahcen, Macadre Jerome and Morjani Hamid.
(2005). Immunosuppressors and reversion of multidrug-resistance.
Critical Reviews in Oncology/Hematology; 56: 61-70
Baird R D and Kaye S B. (2003). Drug resistance reversal-are we
getting closer? European Journal of Cancer; 39: 2450-2461
Baldini Nicola, Scotlandi Katia, Barbanti-Brodano, Manara Maria
Cristina, Maurici Daniela, Bacci Gaetano, Bertoni Franco, Picci Piero,
Sottili Sandra, Campanacci Mario and Massimo Serra. (1995).
Expression of P-Glycoprotein in High-Grade Osteosarcomas in
Relation to Clinical Outcome. The New England Journal Of Medicine;
333: 1380-1385
Bates Susan E, Mickley Lyn A, Chen Yi-Nan, Richert Nancy, Rudick
Jonathan, Biedler June L and Fojo Antonio T. (1989). Expression of
Drug Resistance Gene in Human Neuroblastoma Cell Lines:
Modulation by Retinoic Acid-Induced Differentiation. Molecular and
Cellualr Biology; 9: 4337-4344
Page
51
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Bech-Hansen N T, Till J E and Ling V.(1975). Pleiotropic Phenotype of
Colchicine-Resistant CHO Cells Cross-Resistance and Collateral
Sensitivity. J. Cell, Pysiol; 88: 23-32
Beck W T, Grogan T M, William C L, Cordon-Cardo C, Parham D M
and Kuttesch J F. (1996). Methods to detect P-glycoprotein-associated
multidrug resistance in patients’s tumors: Consensus
recommendations. Cancer Research; 56: 3010-3020
Bertino Joseph R.( 2008).Transfer of drug resistance into
Hematopoietic Stem Cells for Marrow Protection. The
Oncologist;13:1036-1042
Blackburn Elizabeth, Druker Brian, Hartwell Leland, King Mary-Claire,
Weinberg Robert. (2009). Rediscovering Biology: Cell Biology and
Cancer. www.learner.org/courses/biology/index.html
Bodine DM, seidel NE, Gale MS, Nienhuis AW and Orlic D.( 1994).
Efficient retrovirus transduction of mouse pluripotent hematopoietic
stem cells mobilized into peripheral blood by treatment with granulocyte
colony-stimulating factor and stem cell factor. Blood; 84:1482-1491
Bramwell Vivien H C, Morris Donald, Ernst Scott D, Hings Ingrid,
Blackstein Martin, Venner Peter M, Ette Ene I, Harding Matthew W,
Waxman Allison and Demetri George D. (2002). Safety and Efficacy of
the Multidrug-Resistance Inhibitor Biricodar (VX-710) with Concurrent
Doxorubicin in Patients with Anthracycline-resistant Advanced Soft
Tissue Sarcoma. Clinical Cancer Research; 8: 383-393
Page
52
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Bruggemann E P, Currier S J, Gottesman M M, Pastan I. (1992).
Characterisation of the azidopine and vinblastine binding site of P-
glycoprotein. J Biol Chem; 264: 15483-15488
Campos L, Guyotat D and Achimbaud E. (1992). Clinical significance of
multidrug resistance P-glycoprotein expression on acute
nonlymphoblastic leukemia cells at diagnosis. Blood; 79: 473-476
Cancer Research UK. (2009). Cancer Stats Key Facts, All cancers.
info.cancerresearchuk.org/cancerstats/incidence/
http://www.cancerhelp.org.uk/help/default.asp?page=91
Chaudhary P M, Mechetner E B and Roninson I B. (1992) Expression
and activity of the multidrug resistance P-glycoprotein in human
peripheral blood lymphocytes. Blood Journal; 80: 2735-2739
Chan H S L, Thorner P S and Haddad G. (1990). Immunohistochemical
detection of P-glycoprotein. Prognostic correlation in soft tissue
sarcoma of childhood. Journal of Clinical Oncology; 8: 689-704
Coley H M, Verrill M W, Gregson S E, Odell D E, Fisher C and Judson I
R. (2000). Incidence of P-glycoprotein overexpression and multidrug
resistance (MDR) reversal in adult soft tissue sarcoma. European
Journal of Cancer; 36: 881-888
Page
53
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Cordon-Cardo C, O’brien J P, Boccia J, Casals D, Bertino J R and
Melamed M R. (1990). Expression of the Multidrug resistance Gene
Product (P-Glycoprotein) in Human Normal and Tumor Tissues. The
Journal of Histochemistry and Cytochemistry; 38: 1277-1287
Cordon-Cardo Carlos, O’brien James P, Casals Dolors, Rittman-
Grauer Lana, Biedler June L, Melamed Myron R and Bertino Joseph R.
(1989). Multidrug-resistance gene (P-glycoprotein) is expressed by
endothelial cells at blood-brain barrier sites. Medical Sciences; 86: 695-
698
Corrias M V and Tonini G P. (1992). An oligomer complimentary to the
5’ region of the MDR1 gene decreases resistance to doxorubicin of
human ardenocrcinoma multidrug resistance cells. Anticancer
Research; 12: 1431-1438
Cornwell M M, Safa A R and Felsted R L. (1986). Membrane vesicle
from multidrug resistant human cancer cells contain a specific 150-170
kDa protein detected by photoaffinity labelling. Proc Natl Acad Sci USA;
83: 3847-3890
Dantzig A H, Shepard R L, Law K L, Tabas L, Pratt S, Gillespies J S,
Binkley S N, Kuhfel M T, Starling J J and Wrighton S A. (1999) .
Selectivity of the Multidrug Resistance Modulator, LY335979, for P-
Glycoprotein and Effect on Cytochrome P-450 Activities. The journal of
Pharmacology and Experimental Theraputics; 290: 554-562
Page
54
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Debenham Paul G, Kartner Norboert, Siminovitch Louis, Riordan John
R and Ling Victor. (1982). DNA-Mediated Transfer of Multiple Drug
Resistance and Plasma Membrane Glycoprotein Expression. Molecular
and Cell Biology; 2: 881-889
Efferth T and Volm M. (1993). Modulation of P-glycoprotein mediated
multidrug resistance by monoclonal antibodies, immunotoxins or
antisense oligodeoxynucleotides in kidney carcinoma and normal
kidney cells. Oncology; 50: 303-308
Endicott Jane A and Ling Victor.(1989) The Biochemistry of P-
Glycoprotein-Mediated Multidrug Resistance. Annual Reviews
Biochemistry; 58: 137-171
Fenneteau Frederique, Turgeon Jacques, Couture Lucie, Michaud
Veronique, Li Jun, Nekka Fahima. (2009). Assesing drug distribution in
tissues expressing P-glycoprotein through physiologically based
pharmacokinetics modelling: model structure and parameters
determination. Theoretical Biology and Medical Modelling; 6: 1742-
4682
Page
55
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Fromm Martin F. (2002). Genetically determined differences in P-
glycoprotein function: implications for disease risk. Toxicology;181-
182:299-303
Frredenberg William R, Salzaman Sherry A, Phan Sonja M and
Burmester James K. (1999). Transforming growth factor-β and
multidrug resistance in chronic lymphocytic leukemia. Medical
Oncology;16:110-118
Ghandhi Leena, Harding Matthew W, Neubauer Marcus, Langer Corey
L, Moore Melvin, Ross Helen J, Johnson Bruce E and Lynch Thomas J.
(2007). A Phase II Study of the Safety and Efficacy of the Multidrug
Resistant Inhibitor VX-710 Combined With Doxorubicin and Vincristine
in Patients With Recurrent Small Cell Lung Cancer. American Cancer
Society; 109: 5
Gibbs Wayt W. (2003). Untangling the roots of cancer. Scientific
American.
Gottesman Michael M. (1993). How Cancer Cells Evade
Chemotherapy: Sixteenth Richard and Hinda Rosenthal Foundation
Award Lecture. Cancer Research; 53: 747-754
Gottesman Michael M. (2009). Learning some new tricks from a
multidrug transporter. National Health Institute;
Page
56
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Gottesman Michael M and Ling Victor. (2006). The molecular basis of
multidrug resistance in cancer: The early years of P-glycoprotein
research. FEBS Letters; 580: 998-1009
Goldstein L, Galsi H and Fojo A. (1989). Expression of multidrug
resistance gene in human cancers. J Natl Cancer Inst; 81: 116-124
Hanania E G, Giles R E, Kavanagh J, Ellerson D, Zu Z, Wang T , Su Y,
Kudelka A, Rahman Z, Holmes F, Hortobagyi G, Claxton D, Bachier P,
Thall P, Cheng S, Hester J, Ostrove J M, Bird R E, Chang A Korbling
M, Seong D, Cote R, Holzmayer T, Merchetner E, Heimfeld S,
Berenson R, Burtness B, Edwards C, Bast R, Andreeff M, Champlin R
and Deisseroth A B. (1996). Results of MDR-1 vector modification trial
indicate that granulocyte/macrophage colony-forming unit cells do not
contribute to post transplant hematopoietic recovery following intensive
systemic therapy. Medical Sciences; 93: 15346-15351
Israeli David, Ziaei Simin, Gonin Patrick and Garcia Luis. (2005). A
proposal for the physiological significance of mdr1 and bcrp1/abcg2
gene expression in normal tissue regeneration and after cancer
therapy. Journal of Theoretical Biology; 232: 41-45
Jones M E, Thomas S M and Rogers A. (1993). Luria-Delbruck
Fluctuation Experiments: Design and Analysis. Genetics Society of
America*
Page
57
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Jones Craig V. (2006). Tamoxifen (ICI46,474) as a targeted therapy to
treat and prevent breast cancer. British Journal of Pharmacology; 147:
269-276
Juliano R L, Ling V. (1976). A suface glycoprotein modulating drug
permeability in Chinese hamster ovary cell mutants. Biochim Biophys
Acta; 455: 152-162
Juliano R L. (1978). Adhesion and Detachment Characteristics of
Chinese Hamster Cell Membrane Mutants. Journal of Cell Biology; 76:
43-49
Kang Hyumin, Fisher Michael, Xu Dong, Miyamoto Yuko J, Marchand
Arnaud, Van Aerchot Arthur, Herdewijn Piet and Juliano Rudolph L.
(2004). Inhibition of MDR1 gene expression by chimeric HNA antisense
oligonucleotides. Nucleic Acids Research; 32: 441-4419
Kessel David and Bosmann Bruce. (1970). On the Characteristics of
Actinomycin D Resistance in L5178Y Cells. Cancer Research; 30:
2695-2701
Kobayashi Hiroyuki, Takemura Yuzuru, Wang Fu-Sheng, Oka Tetsuo,
Ohnuma Takao.(1999). Retrovirus-mediated transfer of anti-MDR1
hammerhead ribozymes into multidrug-resistant human leukemia cells:
screening for effective target sites. Int. J. Cancer; 81: 944-950
Page
58
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Kuroda Yukiaki. (1974). Mutagenesis in cultured human diploid cells I.
Effects of some mutagens and a selective agent on cell survival. Japan
Journal Of Genetics; 49: 379-387
Kuwazuru Yasuo, Yoshimura Akihiko, Hanada Shuichi, tsunomiyaAtae
U, Makino Torahiko, Ishibashi Kazuaki, Kodama Masahiko, Iwahashi
Masato, Arima Terukatsu, and Akiyama Shin-Ichi. Expression of the
Multidrug Transporter P-glycoprotein, in Acute Leukemia Cells and
Correlation to Clinical Drug Resistance. Cancer; 66: 868-873
Kuo Macus Tien, Bednarski Patrick, Kohno Kimitoshi, Salerno Milena,
Tarasuik Jolanta and Zunino Franco. (2009). Redox Regulation of
Multidrug Resistance in Cancer Chemotherapy. Antioxid Redox Signal;
1: 99-134
Lagas Jurjen S, Sparidans Rolf W, van Waterschoot Robert A B,
Wagenaar Els, Beijen Jos H and Schinkel Alfred H. (2008). P-
Glycoprotein Limits Oral Availabity, Brain Penetration and Toxicity of an
Anionic Drug, the Antibiotic Salinomycin. Antimicrobial Angents and
Chemotherapy; 52: 1034-1039
Lee Kyung-Yung, Prak Jae-Gahb, Gazdar Adi F, Goldstein Lori J,
Hwang Ee-Sook and Kim Jin-Pok. (1990). Correlation between MDR1
Gene Expression and In Vitro Drug Sensitivity Testing (DST) of Human
Cancer Cell Lines. Journal of Korean Cancer Association;
Page
59
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Li Yan-Hong, Wang Yong-Hua, Li Yan, Yang Ling. (2006). Gene
Polymorphisms and Clinical Relevance. Acta Genetica Sinca; 33: 93-
104
Li Qing-Quan, Wang Wen-Juan, Xu Jing-Da, Cao Xi-Xi, Chen Qi, Yang
Jin-Ming and Xu Zu-De. (2007). Involvement of CD147 in regulation of
multidrug resistance to Pgp substrate drugs and in vitro invasion in
breast cancer cells. Cancer Sci; 98: 1064-1069
Licht Thomas, Gottesman Michael M, Pastan Ira. (1998). Clinical
applications of gene therapy in cancer: modification of sensitivity to
therapeutic agents. Stem cell biology and gene therapy
Ling Victor and Thompson Larry H. (1974). Reduced permeability in
CHO cells as a mechanism of resistance to colchicine. J. Cell. Physiol;
83: 103-116
Loscher Wolfgang and Potschka Heidrum. (2005). Progress in
Neurobiology; 76: 22-76
Lown Kenneth S, Mayo Robert R, Leichtman Alan B, Hsiao Hsiu-ling,
Turgeon Kim, Schmiedlin Phyllissa, Brown Morton B, Guo Wesheng
and Rossi Stephan J. (1997). Role of intestinal P-glycoprotein (mdr1) in
interpatient variation in the oral bioavailability of cyclosporine. Clinical
Pharmacology Therapy; 62: 248-260
Lum Bert L, Fisher George A, Brophy Nathalie A, Yahanda Anne M,
Adler Kent M, Kaubisch Sonja, Halsey Joanne R N and Sikic Branimir I.
Page
60
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
(1993). Clinical Trials of Modulation of Multidrug Resistance. Cancer
Supplement; 72: 11
Martin Catherine, Berridge Georgina, Mistry Prakash, Higgins
Chistopher, Charlton Peter, Callaghan Richard. (1999). The molecular
interaction of the high affinity reversal agent XR9576 with P-
glycoprotein. British Journal of Pharmacology; 128: 403-411
Manetta Alberto, Boyle Julie, Berman Michael L, Disaia Philip J, Lentz
Samuel, Yuan Lia Shu, Mutch David, Slater Lewis. Cyclosporin
Enhancement of Cisplatin Chemotherapy in Patients with Refractory
Gynecologic Cancer. Cancer; 73: 1
Marcus Menashe, Fainsod Abraham and Diamond Gill. (1985). The
Genetic Analysis of Mammalian Cell-Cycle Mutants. Annual Review of
Genetics; 19: 389-421
McDevitt Christopher A and Callaghan Richard. (2007). How can we
best use structural information on P-glycoprotein to design inhibitors?
Pharmacology & Therapeutics; 113: 429-441
Moscow Jeffrey A and Cowan Kenneth H. (1988). Multidrug
Resistance. Journal of the National Cancer Institute; 80: 14-20
National Institutes of Health. (1999). NIH Publication No. 99-4646
Pastan Ira and Gottesman. (1991) Multidrug Resistance. Annual
Reviews in Medicine; 42: 277-86
Page
61
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Pirker R, Wallner J and Gotzl M. (1992). MDR1 RNA expression is an
independent prognostic factor in acute myeloid leukemia. Blood; 80:
557-559
Riehm Hansjorg and Biedler June L. (1971). Cellular Resistance to
Daunomycin in Chinese Hamster Cells in Vitro. Cancer Research; 31:
409-412
Roe Michael, Folkes Adrian, Ashworth Philip, Brumwell Julie, Chima
Lal, Hunjan Sukhjit, Pretswell Ian, Dangerfield Wendy, Ryder Hamish
and Charlton Peter. (1999).Reversal of P-Glycoprotein mediated
Multidrug Resistance by Novel Anthranilamide Derivatives. Bioorganic
& Medicinal Chemistry Letters; 9: 595-600
Roylance Rebecca. (2007) Basis of medical cancer therapy.
www.smd.qmul.ac.uk/morbidanatomy/medica~1.ppt
Saad Maha, Garbuzenko Olga B and Minko Tamara. (2008). Co-
delivery of siRNA and an Anticancer Drug for Treatment of Multidrug-
resistant Cancer. Nanomedicine; 6: 761-776
Salmon S E, Dalton W S, Grogan T M, Plezia P, Lenhert M, Roe D J
and Miller T P. (1991). Multidrug-resistant myeloma: laboratory and
clinical effects of verapamil as a chemosensitizer. Blood Journal; 78:
44-50
Scanlon Kevin J, Ishida Hironori, Kashani-Sabet Mohammed.(1994).
Ribozyme-mediated reversal of the multidrug-resistant phenotype.
Biochemistry; 91: 11123-11127
Page
62
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Schinkel Alfred H and Jonker Johan W. (2003). Mammalian drug efflux
traansporters of the ATP binding cassette (ABC) family: an overview.
Advanced Drug Delivery Reviews; 55: 3-29
Schinkel Alfred H, Wagenaar Els, van Deemter Liesbeth, Mol Carla A A
M and Borst Piet. (1995). Absence of the mdr1a P-Glycoprotein in Mice
Affects Tissue Distribution and Pharmacokinetics of Dexamethasone,
Digoxin and Cyclosporin A. Journal of Clinical Investigations; 96: 1698-
1705
Schinkel Alfred H, Kemp Stephan, Dolle Martijn, Rudenko Gabrielle
and Wagnaar Els. (1993). N-glycosilation and Deletion Mutants of the
Human MDR1 P-glycoprotein. The Journal of Biological Chemistry;
268: 7474-7481
Scripture Charity D and Figg William D. (2006). Drug Interactions in
Cancer Therapy. Nat Rev Cancer; 7:546-558
Seeger Markus A and van Veen Hendrik. (2008). Molecular basis of multidrug
transport by ABC transporters. Biochimica et Biophysica Acta; uncorrected
version
Sheps Jonathan A and Ling Victor. (2007). Perface: the concept and
consequences of multidrug resistance. European Journal of
Physiology; 453: 545-553
Sonneveld P, Suciu S, Weijermans P, Beksac R, Neuwirtova G
Lokhorst H, Van Der Lelie J, Dohner H, Gerhartz H, Sergeren M,
Page
63
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Willemze R and Lowenberg B. (2001). Cyclosporin A combined with
vincristine, doxorubicin and dexamethasone (VAD) compared with VAD
alone in payients with advanced refractory multiple myeloma: an
EORTC-HOVON randomised phase III study (06914). British Journal of
Haematology; 115: 985-902
Sotomayor Maria G, Yu Hua, Antonia Scott, Sotomayor Eduardo M and
Pardoll Drew M.( 2002). Advances in Gene Therapy for Malignant
Melanoma. Cancer Control; 9: 1
Stein U, Shoemaker R H and Schlag P M. (1996). MDR1 Gene
Expression: Evaluation of its Use as a Molecular Marker for Prognosis
and Chemotherapy of Bone and Soft Tissue Sarcomas. European
Journal of Cancer; 32: 86-92
Styczynnski Jan and Wysocki Mariusz. (2007). Cyclosporine increases
in vitro sensitivity to prednisolone in childhood acute lymphoblastic
leukemia. Cancer Therapy; 5: 213-218
Szakacs Gergely, Varadi Andras, Ozvegy-Laczka and Sarkadi Balazs.
(2008). The role of ABC transporters in drug absorption, distribution,
metabolism, excretion and toxicity (ADME-Tox). Drug Discovery Today;
13: 1359-6446
Takahashi Shunji, Aiba Keisuke, Ito Yoshinori, Hatake Kiyohiko,
Nakane Minoru, Kakayashi Takayuuki, Minowa Sayuri, Shibata Harumi,
Mitsuhashi Junko, Tsukahara Satomi, Ishikawa Estuko, Suzuki Rieko,
Tsuruo Takashi and Sugimoto Yoshikazu. (2007). Pilot study of MDR1
Page
64
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
gene transfer into hematopoietic stem cells and chemoprotection in
metastatic breast cancer patients. Cancer Sci;98:1069-1616
Thiebaut Franz, Tsuruo Takashi, Hamada Hirofumi, Gottesman Michael
M, Pastan Ira and Willingham Mark C. (1987). Cellular localization of
the multidrug-resistance gene product P-glycoprotein in normal human
tissues. Medical Sciences; 84: 7735-7738
Thomas Hilary and Coley Helen M.(2003). Overcoming Multidrug
resistance in Cancer: An Update on the Cinical Strategy of Inhibiting P-
Glycoprotein. Cancer Control; 10: 2
Trivedi Meghana, Budihardjo Imawati, Loureiro Kirsten, Reid Tony R
and Ma Joseph D. (2008). Epothilones: A Novel Class of Microtuble-
Stabalizing Drugs for the Treatment of Cancer. Future Oncology; 4:
483-500
Visvader Jane E and Lindeman Geoffrey J. (2008). Cancer Stem Cells
in Solid Tumours: Accumulating Evidence and Unresolved Questions.
Nature Review Cancer; 10: 755-768
Wang R B, Kuo C L, Lien L L and Lien E J. (2003). Structure-activity
relationships: analyses of p-glycoprotein substrates and inhibitors.
Journal of Clinical Pharmacy and Therapeutics; 28: 203-228
Ward M, Richardson C, Pioli P, Smith L, Podda S, Goff S, Hesdorffer C
and Bank A. (1994).Transfer and expression of the human multiple
drug resistance gene in human CD34+ cells. Blood Journal; 84: 1408-
1414
Page
65
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Watanabe Toru, Naito Mikihiko, Oh-hara Tomoko, Itoh Yohjiro, Cohen
Dalia and Tsuruo Takashi. (1996). Modulation of Multidrug Resistance
by SDZ PSC 833 in leukemic and Solid-tumor-bearing Mouse Models.
Jpn. J. Cancer Res; 87: 184-193
World Health Organisation. (2009). Cancer. Factsheet No.297.
www.who.int/mediacentre/factsheets/fs297/en.hmtl
Yahanda A M, Adler K M and Fisher G A. (1992). Phase I trial of
etopiside with cyclosporine as a modulator of multidrugresistance.
Journal Clinical Oncology; 10: 1624-1634
Page
66
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Acknowledgements
I would like to thank Dr. Helmout Modjtahedi For all his assistance. He
managed to create a positive environment conducive to successful
completion of this poroject. Many thanks.