THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER FACULTY OF SCIENCE MODULAR DEGREE SCHEME BSc HONOURS DEGREE IN PHARMACOLOGY Kudakwashe Emmanuel Mupamhanga K0433939 The Biological and Clinical Significance of P-Glycoprotein in cancer
Cancer is a leading cause of death worldwide with WHO global estimates projecting a 12 million death rate by 2015 and in many of these cancer p-glycoprotein is implicated this project reviews some of the steps taken in the battle against cancer with P-glycoprotein under particular focus.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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
Page
2
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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.
Page
4
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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.
Page
5
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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.
Page
6
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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.,
Page
7
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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)
Page
8
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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).
Page
9
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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
.
Page
10
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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,.
Page
11
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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,.
Page
12
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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,.
Page
13
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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,.
Page
14
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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,.
Page
15
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Figure 5:The Genesis of cancer 4 Theories
Padilla-Nash Hesed M and Reid Thomas (2003)
Page
16
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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
Page
17
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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
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.
Page
18
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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
Page
19
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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)
Page
20
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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)
Page
21
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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.
Page
22
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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.
Page
23
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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.
Page
24
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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
Page
25
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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,.
Page
26
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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
Page
27
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
LOCALISATION OF Pgp IN NORMAL TISSUES
Figure 9: localisation of Pgp as detected by MRK16 a monoclonal antibody
Theibaut et al 1987
Page
28
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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.
Page
29
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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.
Page
30
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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)
Page
31
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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;
Page
32
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
(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)
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Table 6: clinical studies implicating Pgp expression in cancer.
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
Figure 9: Cancers reprted to have high MDR1 expression post-chemotherapy
Gotetesman and Pastan et al 1991
Page
35
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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)
Page
36
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER
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
Page
38
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)
Page
39
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
Page
40
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.
Page
41
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.
Page
42
THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER