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

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 CANCER

ABSTRACT 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.

Table 1: Main types of cancer leading to mortality each year

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

Cancer type

Number of deaths per

Lung Stomach Colorectal Liver Breast

year 1.3 million 803 000 639 000 610 000 519 000

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.

Figure 1: All deaths caused by cancer 2006

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

Cancer Research UK (2007)

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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 tofind effective treatment strategies to combat the disease.

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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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., 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)

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).

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 oncogenesaccelerated and uncontrolled growth

Tumour suppressor genes

Limited ability of signalling inhibitory messages

DNA repair genes

Loss of ability to repair alterations in DNA

.

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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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,. 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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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,.

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,.

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 protooncogenes 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,.

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

Table 3: Viruses that can potentiate cancer Viruses associated with increased risk of cancer Human papillomavirus Hepatitis B Epstein-Barr virus Human T-cell leukaemia virus Herpes Virus Cancer type caused Genital Carcinomas Liver Carcinoma Burkitts lymphoma T-cell lymphoma 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,.

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)

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 Carcinoma Sarcoma Origin Epithelial cells (most common) Muscle, bone, fat and connective tissue leukaemia Lymphoma Myeloma White blood cells Bone marrow (lymphatic system) Specialised anti-body producing white blood cells

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 BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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 Chemotherapy Radiotherapy Endocrine Biological Surgery Treatment strategy The use of cytotoxic agents Ionising gamma radiation Blocking hormonal action Monoclonal antibodies -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.

Figure 6: Chemotherapeutic effects on the cell cycle
PHASE NON-SPECIFIC Alkylating agents Cisplatin Nitrosoureas Antibiotics VINCA ALKALOIDS TAXANES

M G0 G2 G1

S
METHOTREXATE HYDOXYUREA CYTOSINE ARABINOSIDE ANTHRACYCLINES

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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 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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

a relatively novel treatment strategy in cancer thus many of the treatment regimes are not implemented unless conventional treatment has failed. Roylance (2007)

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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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)

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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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.

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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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.

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 crossresistance 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.

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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.

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)

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

Figure 8a: P glycoprotein structure

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

Schinkel and Jonker 2003
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,.

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

LOCALISATION OF Pgp IN NORMAL TISSUES

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

Figure 9: localisation of Pgp as detected by MRK16 a monoclonal antibody

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

Theibaut et al 1987 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.

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.

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)

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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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; (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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

(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

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

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

PHARMACOLOGICAL TARGETING OF Pgp

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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)

Figure 9: Toxicities of VAD Plus High-Dose IV Verapamil in 22 Patients

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

Salmon et al 1991
The use of verapamil, cyclosporine Yahanda et al (1992) and other 1stgeneration 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 CANCER

Pgp. 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. ii.
iii.

a log P value of at least 2.92 18-atom-long molecular axis and a high Ehomo value at least 1 tertiary base nitrogen atom

iv.

(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

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)

1ST -3RD GENERATION Pgp MODULATORS

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

For a compound to eligible as a modulator of Pgp it must fulfil at least one of the following criteria: i. ii. iii. Increase the potency of cytotoxic drugs in resistant cells Increase the accumulation of cytotoxic drugs intracellularly 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 dexverapamil, dexniguldipine, PSC 833 and VX-710. Wilson et al 1995 reported 10fold decrease in cardiac toxicity at equal chemosensitising levels. The most characterised of these compounds is PSC 833 a derivative of cyclosporine that was

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

reported Boekhorst et al 1992; Twentyman and Bleehen 1991 to inhibit Pgp by 1020fold 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.

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.

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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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 3rdgeneration 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

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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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 cells became vincristine-sensitive. Original MOLT-3/TMQ800 cells(conrol) were reported to be 600fold more resistant to vincristine whereas stably transduced cells showed 92 to 296fold 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 MOLT3/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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

mRNA cloned into pMAMneo were transfected into drug sensitive human ovarian carcinoma (A2780) cell lines. Cells exhibiting MDR also had elevated levels of the cfos 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.

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

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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.

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 nontransduced 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 betaglobin) 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.

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

Efforts by Takahashi et al 2007 (Fig 12) to increase transduction efficiency of MDR1 gene transfer yielded limited success.

Figure 12: MDR gene therapy

Takahashi et al 2007
Peripheral blood was stimulated using 5 cytokines; SCF, TPO, IL-6, FL-Ligand & sIL6r. 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 MDR1transduced stem cells in the bone marrow or may lie in transduction strategies which still seem to lack sufficient efficiency.

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 Pglycoprotein. 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

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.

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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Acknowledgements

THE BIOLOGICAL AND CLINICAL SIGNIFICANCE OF P-GLYCOPROTEIN IN CANCER

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.