Introduction

Multidrug resistance (MDR) is defined as the resistance of tumour cells to the cytotoxic actions

of multiple, structurally divergent drugs commonly used in cancer chemotherapy.

Multidrug resistance is the principal mechanism by which many cancers develop resistance to

chemotherapy drugs. It is a major factor in the failure of many forms of chemotherapy. It affects

patients with a variety of blood cancers and solid tumours, including breast, ovarian, lung and

lower gastrointestinal tract cancers. Tumours usually consist of mixed population of malignant

cell, some of which are drug sensitive while others are drug naive or drug resistant cells.

Chemotherapy kills all drug resistant cells but leaves behind sometimes a smaller proportion of

the drug naïve cells. At first a noticeable and rather impressive remission in the population of

active cancer cells is seen as a result of the killing of the predominant drug sensitive cells in

which the tumour shrinks to an undetectable size. Yet the remaining drug resistant cells, all of

whose progeny are also drug resistant, continue to multiply; they eventually dominate the cell

population of the tumour, which might grow to fatalistic size resulting ultimately in the death of

the patient.

What then could be accounted for this phenomenon?

Resistance to chemotherapy has been correlated to the presence of at least two molecular pumps

in tumour cell membrane that actively expel chemotherapy drugs from the interior. This allows

tumour cells to avoid the toxic effect of the drug or molecular processes within the nucleus or the

cytoplasm. The two pumps commonly found to confer chemoresistance in cancer are P-

glycoprotein (Pgp) and the so-called Multidrug Resistance Associated Protein (MRP).

Historical perspective

That cells have mechanisms to transport a variety of molecules out of the cytoplasm has been

known for decades. For example organic cation transporters were some of the earliest such

mechanisms identified and the kidney capability in this affair was first demonstrated 1947. The

first correlation between cell membrane transporters or pumps and drug resistance phenotype

were made in Chinese hamster ovary cell lines in the mid-1970s. ; When it was shown that a

glycoprotein of 170kD, called P-glycoprotein correlated with the degree of drug resistance in

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several cell lines. A variety of cells were found that were resistant to colchicines, vinblastine,

doxorubicin, vinca alkaloids, etoposide, paclitaxel and other small molecules used in cancer

chemotherapy.

P-glycoprotein was purified in 1979, and strong evidence in support of its role in pleitropic drug

resistance came in 1982, when it was shown that DNA from resistant cell lines that were

transferred to non-resistant cells was able to confer resistance to the latter that correlated with the

expression of the protein. The gene encoding P-glycoprotein called MDR1 was cloned in 1985

and the protein’s putative function as an energy dependent pump that expels molecule from cell

interior was postulated on the basis of sequence homologies on the bacterial hemolysin transport

proteins and on other studies.

Work on a lung cancer cell lines that was resistant to doxorubicin and other chemotherapeutic

agents showed that this cell lines did not over-express P-glycoprotein, but did express another

protein, namely MRP (Multidrug Resistant Protein), cloned in the 1992. MRP was also found to

be a pump, specifically a member of the ATP-binding cassette transmembrane transporter

superfamily.

Types of Multidrug Resistance

There are generally two types or forms of MDR in tumour cells:

Intrinsic MDR → As the name suggests, this is a form of multidrug resistance which is

already present in tumour cells at the time of first treatment with a chemotherapeutic

agent. An example of this type of MDR that readily come to mind is the resistance

obtained in brain tumours as a result of the protection put up by the blood-brain barrier

which is known to be only selectively permeable to micronutrients.

Acquired MDR→ A tumour cell resistance to multiple drug chemotherapy is described

as acquired when the disease becomes insensitive to treatment upon relapse, and it’s

achieved either by adaptation or selection. An example is breast cancer.

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Proposed Mechanisms of Multidrug Resistance in Cancer Cells

1. Modulation of The Influx/Efflux of Intracellular Concentration of Drugs

This is the most documented and so far the most important pathway of multidrug

resistance in tumour cells. Cancer cells have been implicated to possess mechanism that

modulate decrease influx and increase efflux of chemotherapeutic agents thereby leading

to low cellular concentration of the drug which is under-sufficient o elicit its cytotoxic

effect. However since data concerning alterations of drug uptake by cells are scarce,

therefore the pathway leading to the increase efflux of drugs is more favoured. The

culprit that has been identified as responsible for the mediation of ATP-dependent

unidirectional efflux of drug substrates is P-glycoprotein (Pgp), a member of the MDR

family. Multidrug resistance associated protein (MRP), another member of the MDR

superfamily has also been implicated in the transport of drugs outward of their target

cell(s).

Other Mechanisms of Multidrug Resistance

2. Alterations of Cellular Metabolism of Drugs

This is perhaps the second most significant pathway by which tumour cells achieve

phenotypic resistance to miscellaneous chemotherapy through either decreased activation

or increased deactivation. An example of this metabolic pathway of tumour cell

multidrug resistance is the glutathione conjugation prior to transport out of cell.

3. Alterations of Cellular Target(s) of The Drug

Examples of this are the mutations which have been shown to render topoisomerse

resistant to messenger amsacrine and which are presumed to occur in tubuline, thereby

preventing the binding of paclitaxel or vinca alkaloids to the tubulin.

4. DNA Damage Repair

A fourth mechanism which a cell may employ to become drug resistant is through

enhancement of repair, as for the repair of DNA damage due to alkylating agents.

5. Finally a fifth pathway through which a cell may attain self-defense against cancer

multidrug therapy, though not sufficiently investigated as yet, is the proposed cell

survival pathways. These may involve growth receptor pathway, signal transduction

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pathway and the alterations of the genes and proteins involved in the control of apoptosis

e.g. upregulation/downregulation of p53 and Bcl-2.

Table 1 show the summary of the various mechanisms of Drug resistance

Drug Transport

-Impaired Entry

-Enhanced Efflux

Altered Drug Metabolism

-Impaired Activation

-Enhanced Inactivation

Altered Target

-Increased/Decreased Expression

-Mutation

Altered Repair -Enhanced DNA Repair

Alterations in Cell Survival Pathways

-Oncogenes

-Apoptosis

-Growth Factors/Receptors

Multidrug Resistance Mediated By P-Glycoprotein (Pgp)

Human Pgp is coded by the gene MDR1 which is a member of the MDR family. The

gene is located on chromosome 7(7q21.1).

P-glycoprotein, the gene product of MDR1, is a large transmembrane glycoprotein with

molecular weight of approximately 170kD consisting of two similar halves each

including six hydrophobic transmembrane sequence. This suggests that Pgp molecule

span the membrane twelve times. The Pgp molecule has two ATP binding domains

showing that Pgp function is energy dependent. One of the most popular hypothesis hold

that the drug molecule binds to a specific site of Pgp within the lipid bilayer of the cell

plasma membrane and by means of the energy of ATP hydrolysis is transported out of the

cell. The Pgp molecule in this case acting as a molecular “pump” or an energy dependent

channel for the extrusion of drug molecules.

P-glycoprotein belongs to the ATP binding cassette (ABC) superfamily of transporters.

More than a hundred transport proteins are included in the ABC family and they have

been found in different species ranging from E.coli to humans. The proteins of this family

transport very different substrates ranging from inorganic ions to polysaccharides and

proteins. These diverse transporters are united by a common domain organization - the

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presence of transmembrane and ATP-binding domains which are also named ABC

domains.

Figure 1 is the schematic structure of P-glycoprotein as revealed by X-ray

crystallography

\Figure 1ic structural organization of P-glycoprotein

Schematic structural organization of P-glycoprotein. Each half contains a highly hydrophobic domain with six transmembrane α-

helices involved in chemotherapeutic drug efflux, and a hydrophilic domain located at the cytoplasmic face of the membrane,

nucleotide binding domain 1(NBD1) or NMD 2, containing an ATP-binding site with cheracteristic Walker motifs A and B and

the S signature of ABC transporters. The two half molecules are separated by a highly charged "linker region which is

phosphorylated at several sites by protein kinase C and the first extracellular loop is heavily N –glycosylated.

Functions of ABC transporters

Although the physiologic functions of ABC transporters are not well known, they are expressed

constitutively in not only tumor cells but also normal cells in the digestive system including the

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small intestine, large intestine, liver, and pancreas; epithelial cells in the kidneys, adrenals, brain,

and testes; and endothelial cells . From the aspect of the tissue distribution, ABC transporters are

thought to participate in the absorption and secretion of endogenous and exogenous substances.

Endogenous and exogenous substrates for ABC transporters reported so far are summarized in

Table 2. Especially, the ABC transporters have shown to function as an efflux pump for lipid,

multiple drugs, natural products and peptides. It is proposed to operate as a hydrophobic vacuum

cleaner, expelling non-polar compounds from the membrane bilayer to the exterior, driven by the

energy of ATP hydrolysis. ATP-dependent trans-bilayer lipid transporters are classified into

cytofacially-directed flippases and exofaciallydirected floppases. Floppase activity has been

associated with the ABC transporters although not all ABC transporters are floppases .

Endogenous substrates for Pgp include corticosterone, beta-estradiol 17beta-D-glucuronide, an

endogenous cholestatic metabolite of estradiol, 1-O alkyl-2-acetyl-sn-glycero-3- phosphocholine

(generically platelet-activating factor, PAF), glutamate and endorphin . It was also recently

reported that Pgp has the function of removing beta-amyloid, which was reported as the causal

substance of Alzheimer's disease. MRP1 effluxes various conjugated substrates such as

leukotriene C4 conjugates steroid conjugates and the GSH conjugate of aflatoxin B1, which is a

mycotoxin . Cells can, upon hypoxic demand, use BCRP to reduce heme or porphyrin

accumulation, which can be detrimental to cells. When cancer originates not only from cells

normally expressing efflux pump but also cells having genes but not expressing, gene expression

is initiated due to the exposure to anticancer drugs, resulting in resistance to anticancer drugs,

eventually interfering with chemotherapy.

Multidrug Resistance Mediated By MRP Protein

Multidrug resistance associated protein (MRP) was discovered in 1992, thus the results

elucidating its role in the evolution of malignant tumours are not as documented as data obtained

in the studies of P-glycoprotein. This protein, with molecular weight 190kD contributes

resistance to tumour to almost the same anti-tumour drugs as Pgp. It functions as an energy

dependent (ATP-dependent) pump extruding toxic substances from the cell. MRP like Pgp

belongs to the family of ABC transporters. The role of MRP in drug resistance is shown by

means of the introduction of MRP gene into target cells in vivo. Though MRP and Pgp are

known to have similar drug substrate line, they however differ in their functionality in that, for

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MRP to efficiently function as a drug effluxing pump, it needs cellular glutathione. The protein

is therefore one of the transporters of glutathione conjugates, so called GS-X pumps. The

substrate specificity for MRP is not dissimilar to Pgp; however the patterns of cross-resistance

have some differences.

Multidrug Resistance Mediated By LRP Protein

The multidrug resistant protein LRP (Lung Resistance Related Protein), molecular weight

110kD, discovered in 1993, is found not on the cell membrane (as it’s the case of Pgp and MRP)

but in the cytoplasm. It is expressed by the cells of normal epithelium and cells of tissues

exposed to toxic substances. LRP is a major protein of ribonucleoprotein cell particles named

“vaults”. The function of these organelles is not yet known, but it was suggested that they

participate in the transport of substrates from the nucleus and lysosomes; this fact connects its

function with drug transport and sequestration into vesicles. Probably, after this a drug can be

excluded from the cell by exocytosis. LRP mediated MDR has been implicated in cases of

ovarian cancer and acute myeloid leukemia.

Drug Resistance Mediated By Detoxification of the Drug in the Cell

The cellular glutathione (GSH) system is a critical component of detoxification of cytotoxins

(cytostatics) in the cell. Glutathione, a non-protein thiol, can interact via its thiol with the

reactive site, resulting in the conjugation of the drug glutathione. The conjugated product is less

active and more water soluble and it is exited from the cell with the participation of a transport

protein named GS-X (including MRP). Increased levels of glutathione were found in cell lines

resistant to alkylating agents, thereby increasing the rate of drug detoxification in the target

cell(s). Therefore, the upregulation of these enzymes can cause cellular drug resistance.

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Figure 2

Reaction of the binding of alkylating drug (chlorambucil) with the glutathione molecule and conjugate (monoadduct) formation.

The SH group of glutathione binds the reactive group of chlorambucil. GSTα and GSTλ are some glutathione S-transferases

catalyzing the reaction.

Drug Resistance Mediated By Alterations of Drug Targets or By Enhancement of Target

Repair.

Some cancer drugs are inhibitors of topoisomerase (topoisomerase 1&2). These drugs stabilize

the DNA topoisomerase complex which in normal circumstances is easily decomposed. In cell

lines selected for the resistance to topoisomerase 2 inhibiting drugs, the activity or the quantity of

this enzyme is reduced due to mutations in the topoisomerase 2 gene. This type of drug

resistance arises due for adriamycin, daunorubicine, mitoxantrone, etoposide, e.t.c. Aso

enhanced DNA repair is probably implicated in drug resistance to the drugs interacting with

DNA, for example nitrosomethylene or platinum derivatives.

The Role of Key Genes Controlling Apoptosis in Drug resistance of tumour Cells

Normal p53 protein is activated in response to different injuries and its alterations result in cell

cycle arrest or apoptosis. Thus injured cells either are eliminated from the cell population or

damaged DNA is repaired. Alterations of p53 are very frequent in tumours, and these alterations

result in impaired p53 function; the cell become unable to die by apoptosis or to stop in cell cycle

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checkpoints after damage. Thus alterations of p53 function can a priori result in changes of

tumour cell sensitivity to drugs, in particularly in MDR.

Different influences activate p53: various DNA damaging agent, altered ribonucleotide pools,

changes in redox potential, disruption of the mitotic spindle e.tc. Many anticancer drugs kill the

cell using some of these mechanisms; thus it is evident that most cytostatics have to be p53

activators. Cells with mutant p53 more often were resistant to a drug with different mechanisms

of action (for example, to Cisplatin and 5-Fluorouracil) than cells with wild type p53. These data

stress the importance of p53 in tumour sensitivity to chemotherapy

.

Two Ancient Pathways of Cancer Formation and Their Significance

The signaling pathways that govern proliferation, survival and oncogenesis are prime interest in

the biology of cancer. The nuclear factor kB (NF-kB), transcription factors (p50/p105(NF-kB1),

p52/p100(NF-kB2),RelA(p65),RelaB, c-Rel) and the pathways that control Nf-kB activation are

best known for their role in inflammatory responses, but also critical to many physiological and

pathophysiological responses, including cell differentiation, adhesion, survival and apoptosis. In

most resting cells, NF-kB is sequestered in the cytoplasm by binding to the inhibitory IkB

proteins. NF-kB is activated by a variety of stimuli such as carcinogens, inflammatory agents

including phorbol esters, bacterial endotoxins, and tumour necrosis factor (TNF). These stimuli

promote dissociation of IkB through phosphorylation, ubiquitination and proteasome mediated

degradation. This process unmask the nuclear localization sequences of NF-kB, which then

accumulates in the nucleus, binds to kB regulatory elements and activates target genes, whose

expression in the nucleus impede the process of apoptosis. Chemopreventive measures such as

curcumin and capsaicin are know to block the NF-kB activation process.

Wnts are an evolutionary conserved family of growth factors that are essential for a wide array of

developmental and physiological processes. The activity of the Wnt/β-catenin signaling pathway

is dependent upon the amount of β-catenin in the cytoplasm. In the absence of β catenin ligands,

the cytoplasmic β-catenin level is through continous ubiquitin-proteosme-mediated degradation,

by a multiprotein destruction complex containing axin, adenomatous polypopsis coli (APC) and

glycogen synthase kinase 3 (GSK3). Upo binding to the receptor complex Frizzled (Fz) and

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LDLD receptor –related proteins (LRP), a signal is transduced to activate the cytoplasmic

phosphoprotein Dishevelled (Dvl). Activated Dvl inhibits GSK3, thus preventing

phosphorylation of β-catenin . Mutation of APC gene occurs the majority of sporadic coleratal

cancers, as well as familiar adenomatous polypopsis. Some chemopreventive therapies are

known to block the Wnt/β-catenin signaling pathway. Therefore NF-kB and Wnt pathways are

important molecular targets of cancer prevention by natural compounds.

OVERCOMING MULTI-DRUG RESISTANCE (The use of synthetic Drugs)

Small Molecules Inhibitors

First- and second-generation MDR drugs

The relative promiscuity of drug transport by P-gp and other MDR-associated transporters

inspired a wide search for compounds that would not be cytotoxic themselves but would inhibit

MDR transporters. The initial demonstration of verapamil as a P-gp inhibitor was followed by

many additional compounds reported to inhibit drug transport and thus sensitize MDR cells to

chemotherapeutic drugs. Variously called chemosensitizers, MDR reversal agents, modulators or

convertors, these 'first-generation' MDR drugs included compounds of diverse structure and

function such as calcium channel blockers (eg, verapamil), immunosuppressants (e.g.,

cyclosporin A), antibiotics (e.g., erythromycin), antimalarials (eg, quinine), psychotropic

phenothiazines and indole alkaloids (eg, fluphenazine and reserpine), steroid hormones and anti-

steroids (e.g., progesterone and tamoxifen) and detergents (eg, cremophor EL). Although the

structure of these compounds is very different, many are amphipathic molecules with ternary

nitrogen and a planar ring or ring system. First-generation MDR drugs were not specifically

developed for inhibiting MDR. They often had other pharmacological activities, as well as a

relatively low affinity for MDR transporters, and thus were limited in application. Clinical trials

with first-generation MDR inhibitors failed for various reasons, often due to side effects resulting

from adverse reactions to the MDR drug itself. Second-generation drugs were based on the first

generation, but were specifically selected or designed to reduce the side effects of the latter by

eliminating their non-MDR pharmacological actions. For example, the R-enantiomers of

verapamil (R-verapamil) and the dihydropyridine niguldipine (dexniguldipine) were much

weaker calcium channel blockers but nearly equally effective as the L-enantiomers in blocking

P-gp. Unfortunately, these compounds did not fare any better as MDR drugs in clinical studies,

most likely because their affinity towards Pgp still fell short of producing significant inhibition

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of MDR in vivo at tolerable doses. However, the early clinical trials were useful in that they

indicated the complexity of clinical drug resistance compared with in vitro MDR models and

highlighted conceptual problems and study design issues that must be addressed in future studies.

Among the more important lessons to be learnt was the need to determine which MDR protein is

upregulated in the patient population (eg, P-gp) and to utilize an anticancer drug that would most

clearly benefit from inhibition of that protein (eg, a taxane). Another lesson was that the plasma

concentrations of the tested MDR drug must be monitored in order to verify that an effective

inhibitory concentration was in fact achieved in vivo. Finally, and perhaps most important, is the

need to avoid pharmacokinetic interactions between the MDR drug and the anticancer drug(s)

used in the study; co-administration of an MDR drug may significantly elevate plasma

concentrations of an anticancer drug by interfering with its clearance (e.g., via biliary

elimination) or metabolism (eg, viathe cytochrome P450 system). This would result in an

increase in the area under the curve (AUC) leading to unacceptable side effects, necessitating

dose reductions down to pharmacologically ineffective levels. Because of these problems, van

Zuylen et al stated that "…administration of MDRconvertors is unlikely to improve the

therapeutic index of anticancer drugs unless such agents lack significant pharmacokinetic

interactions". Some of the design issues and recommendations for clinical studies with MDR

drugs are summarized in Box 1.

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Third-generation MDR drugs

Third-generation MDR drugs are characterized by high affinity to P-gp (and/or other MDR

transporters) enabling inhibition at low nanomolar concentrations in in vitro models of MDR.

Several such compounds originating from drug development programs are currently undergoing

clinical trials in specific forms of advanced cancer. Results from previously completed clinical

trials were reviewed recently. The first of these drugs to be studied was PSC-833 (Valspodar;

Novartis AG; Figure 2), a non-immunosuppressive cyclosporin D derivative. While early trials

with PSC-833 were encouraging, further work revealed potentially significant pharmacokinetic

interactions with several anticancer drugs and, furthermore, possible inhibition of non MDR-

related transporters. Novartis subsequently discontinued development of this compound,

although it is currently being examined in additional phase III studies in acute myeloid leukemia

(AML), multiple myeloma and myelodysplastic syndrome sponsored by the National Cancer

Institute (NCI). MS-209 is a quinolone derivative under development by Schering AG (formerly

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Mitsui Pharmaceuticals). Currently, MS-209 is in phase III trials in Japan for the treatment of

breast cancer and phase I trials in breast and lung cancer in Europe.

All other prospective MDR drugs are still in phase II or phase I trials. XR-9576 (tariquidar;

Xenova Group plc/QLT Inc;) has successfully concluded phase II studies with paclitaxel and

vinorelbine in ovarian cancer. US FDA approval for the initiation of phase III trials has been

granted, and the trials are expected to commence in mid- 2002 [22]. By April 2002, the NCI was

enrolling a total of 24 patients for a phase I trial and pharmacokinetic study of XR- 9576 in

children with refractory solid tumors including brain tumors [23]. VX-710 (biricodar, Incel;

Vertex Pharmaceuticals Inc; Figure 2), a high-affinity P-gp and MRP inhibitor, appears to have

no pharmacokinetic interactions with doxorubicin and is currently undergoing phase II trials in

solid tumors. R-101933 (Janssen Pharmaceutica NV) exhibits desirable pharmacokinetic

characteristics with respect to taxols and is also now undergoing a European Organisation for

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Research and Treatment of Cancer (EORTC)/NCI. sponsored phase II study in metastatic breast

cancer in combination with these anticancer drugs. Another drug that is not typically third-

generation is mitotane, long utilized for treatment of adrenocortical carcinoma and recently

found to inhibit P-gp, is now similarly being studied in combination with anticancer drugs in an

NCI sponsored phase II study. Additional candidate MDR drugs in phase II studies are listed in

Table 1. Many more compounds are undoubtedly in phase I and preclinical development.

Noteworthy among those is GF-120918 (elacridar; GlaxoSmithKline plc), initially characterized

as a P-gp inhibitor but now known to inhibit also BCRP, which has completed phase I studies

and shows no pharmacokinetic interactions with doxorubicin. Annamycin (Antigenics Inc) is an

exception, as itis not a P-gp inhibitor but an anthracycline that is not transported by P-gp (see

below).

NOVEL APPROACHES TO MDR THERAPY

The discussion so far has focused on direct inhibitors of MDR transporters, mainly P-gp

inhibitors. The difficulties encountered in applying these drugs in the clinic and the emerging

complexity of the MDR phenotype has engendered several alternative approaches to MDR

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therapy, designed either to inhibit MDR in novel ways or to cleverly circumvent MDR

mechanisms altogether.

Inhibiting MDR mechanisms

Downregulation of MDR transporters by antisense oligonucleotides has been suggested as an

alternative and more specific way to overcome MDR than the use of conventional small

molecule pharmacological inhibitors. Recent advances in antisense oligonucelotide technologies

are reflected in patents such as those of Isis Pharmaceuticals and Hybridon, claiming methods to

suppress P-gp expression using antisense oligonucleotides with improved stability and cellular

permeability. Another proposed approach is to exploit or target physiological mechanisms

involved in regulation of MDR proteins. Induction of MDR1 gene expression in tumor cells

occurs upon treatment with cytotoxic drugs, whereas this response is inhibited by

pharmacological inhibitors of calcium-dependent signaling in what could be a novel therapeutic

strategy, as proposed in a recent patent by the University of Illinois. MDR1 and cytochrome p450

3A4 (CYP3A4) gene expression can also be stimulated by anticancer drugs such as taxol via its

direct interaction with and activation of the nuclear steroid and xenobiotic receptor (SXR),

leading to increased drug resistance and faster drug clearance. Hence, antagonists of SXR such

as ET-743 (PharmaMar SA/Ortho Biotech Inc; Figure 3) may be utilized in conjunction with

anticancer drugs to counteract the induction of MDR1 and CYP3A4.

Figure

Acquisition of the MDR phenotype is often associated with upregulation of glucosylceramide

(GlcCer), which results from elevated GlcCer synthase activity. Overexpression of recombinant

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GlcCer synthase confers resistance to adriamycin and to ceramide in human breast cancer cells,

suggesting that drug resistance in GlcCer synthase-transfected cells is related to stimulation of

glucosylation of ceramide and the resultant inhibition of drug-induced apoptotic signaling. The

role of GlcCer synthase in drug resistance was demonstrated directly by antisense suppression of

GlcCer synthase expression in MDR cell. These results are consistent with the hypothesis that

GlcCer synthase contributes to drug resistance in MDR cells by attenuating drug-induced

formation of apoptotic ceramide and indicate that GlcCer synthase may represent a novel drug

target in cancer MDR. A recent US patent application from Shayman et al describes novel

amino-ceramide analogs that inhibit GlcCer synthase and thereby may elevate ceramide

production inn MDR cells, enhancing drug-induced apoptosis.

CIRCUMVENTING MDR MECHANISMS

MDR mechanisms reflect the innate adaptive potential of living cells and may thus prove to be

intractable. Therefore, researchers have looked for various ways to circumvent rather than

directly inhibit MDR mechanisms. One approach has focused on developing anticancer drugs

that are poor substrates for MDR transporters. Examples include anthracyclines such as

idarubicin and annamycin. The latter has reached phase II development with Antigenics, which

is currently evaluating its development program. Another anticancer drug with such properties is

an olivacine derivative, S16020-2 (Servier; Figure 4), that is similarly able to bypass Pgp-

mediated resistance possibly because of its rapid uptake kinetics compared with standard

anticancer agents

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Figure 4

Tumours require an adequate blood supply in order to grow and are capable of inducing the

formation of new blood vessels that provide them with oxygen and nutrients, a phenomenon

called angiogenesis. The angiogenic response requires proliferation of vascular endothelial cells

which depends on angiogenic factors, and it can be inhibited by anti-angiogenic factors. Recent

work has shown that the latter can effectively inhibit tumor growth and hence anti-angiogenic

therapy may become an important anticancer treatment modality. As anti-angiogenic factors do

not target the tumor cells themselves but rather the endothelial cells, anti-angiogenic therapy

should, in principle, be equally effective toward non-MDR and MDR tumors. A possible proof

of feasibility for this therapeutic strategy is provided by recent clinical studies showing the

effectiveness of thalidomide (Celgene Corp; Figure 5), an antiangiogenic drug, in treating

patients with refractory multiple myeloma. However, the action of thalidomide may additionally

reflect modalities other than anti-angiogenesis, since it also induces apoptosis in drug resistant

multiple myeloma cells in vitro.

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Figure 5

A novel procedure for circumventing MDR mechanisms, involving immunization with an

autologous tumor cell vaccine, was proposed by Shtil et al. This work shows that vaccination

with irradiated myeloma cells engineered to express GM-CSF, elicits a strong cytotoxic T-

lymphocyte response leading to > 90% graft rejection. Significantly, similar response rates were

obtained with drug resistant myeloma cells, indicating that cell killing bypasses the resistant

apoptotic pathway(s). Additional studies suggest that the T-lymphocyte cytotoxicity occurs by

perforin-induced necrosis, although granzyme B-induced apoptosis may also be involved. This

strategy holds great promise but the road to clinical implementation is still long and arduous. A

conceptually related approach involves the use of rituximab (Genentech Inc/IDEC

Pharmaceuticals Corp), an apoptosis inducing monoclonal antibody directed against the

CD20receptor. Rituximab induces apoptosis in drug sensitive cells and may modulate the

threshold for drug-induced apoptosis in MDR cells. Recent clinical studies show that rituximab

improves the efficacy of chemotherapy.

Finally, a recent patent outlines an elegant way to overcome MDR simply by elevating the

anticancer drug dosage. The problem is to achieve this without complete eradication of bone

marrow stem cells, which is considered to be a major dose-limiting toxicity factor. Gottesman et

al thus suggest upregulating drug resistance of autologous bone marrow stem cells by

transfection with vectors carrying the MDR1 cDNA. This would result in multidrug resistant

bone marrow cells which may be employed to reconstitute a lethally irradiated endogenous

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hemopoietic system, providing protection against a chemotherapeutic regimen at otherwise

unacceptable doses, and thus overcoming MDR

Overcoming Drug Resistance (The use of Plant Phytochemicals)

Tea

Tea (Camellia sinensis) consumption has been shown to reduce the risk of tumor formation at

different organ sites including the skin, oral cavity, esophagus, stomach, intestine, lung, liver,

pancreas, mammary gland, urinary bladder, and prostate. Epigallocatechin gallate (EGCG), a

major water-extractable constituent of tea, has been presumed to be the active compound for the

cancer preventive effects. A cup of green tea (2.5 g of dried green tea leaves brewed in 200mL of

water) may contain 90 mg of EGCG, and similar or a slightly smaller amount of

epigallocatechin, about 20 mg each of epicatechin gallate and epicatechin. Nomura et al.

reported the inhibitory activity of EGCG on NF-kB pathway in mouse epidermal cells. In the

JB6 mouse epidermal cells, a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)

markedly induced NF-kB activation. EGCG blocked TPA-induced phosphorylation of IkB

resulted in the inhibition of NF-kB activity. It was also reported that EGCG inhibits ultraviolet

(UV)-induced phosphorylation of IkB and activation of NF-kB in human epidermal

keratinocytes. Dashwood and colleagues have reported the inhibition of heterocyclic amine 2-

amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced formation of intestinal polyps

in Apcmin mouse by tea. They subsequently revealed the inhibition of Wnt/β-catenin signaling

by tea constituents using β-catenin/TCF reporter construct. β-Catenin/TCF reporter activity in

human embryonic kidney (HEK293) cells was inhibited by 25μM EGCG. Kim et al. reported the

inhibition of Wnt/β-catenin signaling by EGCG in human invasive breast cancer cells. EGCG

induced the HBP1 transcriptional repressor, a suppressor of Wnt signaling, thus reducing both

tumorigenic proliferation and invasiveness of breast cancer cells.

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In addition to the anti-carcinogenic effects of EGCG, we have revealed the inhibitory effects of

EGCG and other tea catechins on the function of P-glycoprotein using human carcinoma KB-C2

cells and fluorescent P-glycoprotein substrates daunorubicin and rhodamine 123. KB-C2 is a

multidrug-resistant human epidermal carcinoma cell line that over-expresses P-glycoprotein.

Daunorubicin and rhodamine have often been used in studies of P-glycoprotein-mediated

transport.

Turmeric

Curcumin is a major component of the culinary spice turmeric (Curcuma longa) and it gives

specific flavor and a yellow color to curry. Curcumin has strong anti-oxidant and anti-

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inflammatory properties and is reported to prevent the initiation, promotion, or progression of

cancer. Curcumin is one of the most extensively investigated chemopreventive phytochemicals.

It is also reported that curcumin is pharmacologically safe. Human clinical trials indicated no

dose-limiting toxicity when administered at doses of up to 10 g/day. Curcumin is reported to

suppress NF-kB activation induced by TNF, phorbor esters, and hydrogen peroxide through

suppression of IkB degradation. In 1995, Singh and Aggarwal firstly reported that curcumin is a

potent inhibitor of NF-kB signaling. They used human myelomonoblastic leukemia cell line ML-

1a and electrophoretic mobility shift assay (EMSA) to show the activation of NF-kB in ML-1a

cells by TNF, TPA and hydrogen peroxide. Curcumin blocked the TNF-induced phosphorylation

and degradation of IkB, and translocation of NF-kB to the nucleus. The activation of NF-kB by

TNF, TPA and hydrogen peroxide were inhibited by curcumin. Chun et al. also reported that

curcumin inhibits the TPA-induced expression of cyclooxygenase-2 in mouse skin through the

suppression of NF-kB activation. The Wnt signaling pathway is also an important target of the

cancer preventive activity of curcumin. Jaiswal et al. showed that β-catenin-induced c-Myc

expression is important in curcumin-induced growth arrest and apoptosis in human colon cancer

HCT-116 cells. Park et al. have reported the inhibitory mechanism of curcumin using β-

catenin/TCF reporter construct. They suggested that the inhibitory mechanism of curcumin is

related to the reduced amount of nuclear β-catenin and TCF-4 proteins. The mammalian target of

rapamycin (mTOR), a serine/threonine kinase, is downstream in the phosphatidylinositol 3-

kinase (PI3K)/Akt (protein kinase B) cascade. mTOR integrates signals regarding nutrient and

energy availability, mitogen activation, and thus regulates the cell growth and survival. Recent

findings show that curcumin can inhibit mTOR signaling in cancer cells. Yu et al. have reported

curcumin inhibits mTOR signaling in human prostate cancer PC-3 cells through a protein

phosphatase-dependent dephosphorylation mechanism.

The inhibitory effect of curcumin on human P-glycoprotein was investigated using P-

glycoprotein-overexpressing human carcinoma KB-C2 cells and fluorescent P-glycoprotein

substrates. Curcumin increased the accumulation of daunorubicin in KB-C2 cells in a

concentration-dependent manner. The cellular accumulation of rhodamine 123 was also

increased in the presence of curcumin. Since curcumin inhibits the efflux of P-glycoprotein

substrates, the elevation of substrate accumulation seems to be induced by the inhibition of the

efflux transporter. The efflux of rhodamine 123 from KB-C2 cells was decreased by curcumin.

21

Taken together, these findings indicate that curcumin has an inhibitory effect on P-glycoprotein

function, and may have chemosensitizing potency, in addition to its own chemopreventive

properties.

Chili Pepper and Ginger

Capsaicin is a pungent component of hot and red chili pepper (Capsicum annuum). In addition to

alleviating pain and itching in humans, capsaicin has exhibited chemopreventive effects,

suppressing carcinogenesis of the skin, colon, lung, tongue, and prostate. Singh et al. have

reported the inhibition of TNF-induced NF-kB activation in human myeloid ML-1a cells by

capsaicin. Capsaicin blocked the degradation of IkB, and nuclear translocation of NF-kB. Han et

al. also showed that capsaicin inhibits the TPA-induced activation of NF-kB in mouse skin.

Gingerol is a phenolic substance responsible for the spicy taste of ginger (Zingiber officinale).

Gingerol has also been linked with prevention of cancer. It has been reported that gingerol

inhibits the proliferation of cancer cells including prostate, gastric, and breast. Kim et al.

reported that gingerol suppresses TPA-induced degradation of IkB and translocation of NF-kB to

the nucleus, consequently inhibits the activation of NF-kB. Lee et al. recently reported that

gingerol downregulates β-catenin-depenent cyclin D1 levels and induce apoptosis in human

colon cancer cells.

Capsaicin and gingerol have also shown to have inhibitory effects on human P-glycoprotein.

Capsaicin and gingerol increased the intracellular concentration of P-glycoprotein substrates by

inhibiting this anticancer drug efflux transporter. In the presence of 50μM capsaicin or gingerol,

multidrug-resistant carcinoma KB-C2 cells were more susceptible to the cytotoxicity of

vinblastine, a P-glycoprotein substrate, as compared with vinblastine alone. This demonstrates

that capsaicin and gingerol can partially reverse multidrug resistance in cells that express P-

glycoprotein.

Rosemary

The leaves of rosemary (Rosmarinus officinalis) are commonly used as a spice in cooking.

However, because of the presence of phenolic diterpenes and triterpenes with strong

antioxidative activity, interest has grown in using rosemary as a natural antioxidant in foods.

Extract of rosemary leaves have been used to prevent lipid autooxidation and to inhibit the

oxidation of edible oils or fats. The antioxidative activity of rosemary extract is comparable with

that of known antioxidants, such as butylated hydroxytoluene (BHT) and butylated

22

hydroxyanisole (BHA), without the cytotoxic and carcinogenic risk. Among the constituents of

rosemary extracts, 90% of the total antioxidative activity was derived from carnosic acid and

carnosol. Rosemary also yields substantial quantities of the polyphenolic antioxidant rosmarinic

acid. Rosemary extract is regarded as safe, and a relatively high concentration, 0.02 to 0.05%

(w/w), is used for food production. A typical commercial rosemary extract contains 20% (w/w)

carnosic acid. Therefore, 100 g of meat could contain 30μmol of carnosic acid.

In addition to antioxidative activities, rosemary phytochemicals are reported to have

antimicrobial, anti-inflammatory, and anticancer properties. Topical application of carnosol and

ursolic acid to mouse skin inhibited TPA-induced inflammation and tumorigenesis. Carnosol

restricts the invasion of B16/F10 mouse melanoma cells by reducing metalloproteinase-9 activity

through inhibition of NF-kB [50]. Carnosol is shown to block the lipopolysaccharide (LPS)-

induced phosphorylation and degradation of IkB, and nuclear translocation of NF-kB . Ursolic

acid inhibits NF-kB activation induced by various carcinogens. Carnosol is also reported to

prevent β-catenin/APC-associated intestinal carcinogenesis. Therefore, rosemary

phytochemicals, carnosic acid, carnosol, rosmarinic acid, and ursolic acid, are regarded as strong

natural antioxidants and promising dietary chemopreventive agents.

The effects of rosemary phytochemicals on the function of human P-glycoprotein has been

examined. Carnosic acid, carnosol, and ursolic acid increased the cellular accumulation of

daunorubicin or rhodamine 123 in multidrug-resistant KB-C2 cells. On the other hand,

rosmarinic acid had no effect on the accumulation of daunorubicin. We have previously

investigated the effects of flavonoids on P-glycoprotein function. The inhibitory effects of

flavonoids were in the order of kaempferol > quercetin, baicalein > myricetin > fisetin, morin.

Quercetin-3-glycoside and rutin had no effects. These results indicate that the hydrophobicity of

phytochemicals is important for their inhibitory effects on the efflux of substrates by P-

glycoprotein. Rosmarinic acid, a hydrophilic antioxidant, had no effect on P-glycoprotein. In

contrast, ursolic acid, a steroid-like triterpene that has little or no antioxidative activity, inhibited

P-glycoprotein function. Therefore, the hydrophobicity, not the antioxidative activity, of

phytochemicals could be important for their inhibitory effects on P-glycoprotein activity. We

have also examined the effects of rosemary phytochemical on the resistance to vinblastine

cytotoxicity. In the presence of as little as 10μM concentration of carnosic acid, KB-C2 cells

were more susceptible to the cytotoxicity of vinblastine, a P-glycoprotein substrate, as compared

23

with vinblastine alone. This demonstrates that rosemary phytochemical carnosic acid has a

chemosensitizing effect, reversing P-glycoprotein-mediated multidrug resistance by increasing

the intracellular accumulation of anticancer drug.

Citrus Fruits

The ingestion of citrus fruit has been reported to be beneficial for the reduction of certain types

of human cancer. Citrus phytochemicals, including monoterpenes, limonoids, flavonoids, and

coumarins are demonstrated to have anti-carcinogenic effects . Auraptene (7

geranyloxycoumarin), a coumarin-related compound occurring widely in citrus fruit (e.g.,

grapefruit), markedly inhibited 4-nitroquinoline 1-oxide-induced oral carcinogenesis in rats and

suppressed the growth of human prostate carcinoma cells. Auraptene also showed suppression of

TPA- and LPS- induced inflammatory responses . Nobiletin (5,6,7,8,3',4'-hexamethoxyflavone),

a polymethoxyflavonoid in citrus fruit (e.g., mandarins and oranges), is an inhibitor of the

generation of both NO and O2- in leukocytes and showed strong inhibitory effects on TPA-

induced skin inflammation, oxidative stress, and tumor promotion in mice and the growth of

human prostate carcinoma cells. Therefore, citrus phytochemicals, auraptene and nobiletin, are

considered promising chemopreventive agents. The concentration of auraptene is 6.03 μM in

grapefruit juice, and the concentration of nobiletin is 6.71 to 11.43μM in Valencia orange juice.

It is reported that these concentrations could be sufficient to inhibit the function of P-

glycoprotein . However, further studies of the absorption, distribution, metabolism, and excretion

of citrus phytochemicals in the human body are needed to clarify the inhibitory effects of these

compounds at the site of drug action, in the tumor tissues and cancer cells. It is reported that ATP

hydrolysis and substrate transport are tightly coupled, and most compounds that are known to be

transported by the ABC transporter stimulate ATPase activity. To explore the inhibitory

mechanism of citrus phytochemicals on the function of P-glycoprotein, effects on ATPase

activity of P-glycoprotein was measured using human P-glycoprotein membranes from

baculovirus infected insect cells. The ATPase activity of P-glycoprotein was stimulated by

verapamil, a known substrate of P-glycoprotein. Auraptene or nobiletin alone also stimulated the

basal ATPase activity of P-glycoprotein. Auraptene and nobiletin further enhanced the

verapamil-stimulated ATPase activity of P-glycoprotein. These results suggest that auraptene

and nobiletin could be substrates of P-glycoprotein, and competitively interact at drug-binding

site of P-glycoprotein.

24

Future Perspectives

Although P-glycoprotein and MRP1 are key determinants of drug sensitivity, other anticancer

drug efflux transporters, such as breast cancer resistance protein (BCRP, ABCG2), can cause

multidrug resistance [3,4]. At present, interactions between natural compounds and anticancer

drug efflux transporters, other than P-glycoprotein and MRP1, have not been well investigated.

Therefore, it is important to study the inhibitory effects of phytochemicals on BCRP and other

ABC transporters.

In the studies using cancer cell lines, relatively higher doses of phytochemicals are often used.

Even though the natural compounds are regarded as safe, a low amount is favorable for future in

vivo studies. It is noteworthy to consider that the level of P-glycoprotein expression in the cell

line is much greater than that in human tissues. Thus, lower concentrations of phytochemicals

would be effective in modulating in vivo P-glycoprotein activities. In addition, synergistic effects

with other dietary phytochemicals on ABC transporter function should be considered.

Combinatorial therapy with several natural compounds and conventional anticancer drugs must

be developed for the future cancer chemotherapy.

As described, cellular signaling pathways, such as NF-kB and Wnt, are important molecular

targets of cancer prevention by natural compounds. Recent studies have revealed that P-

glycoprotein is also regulated by these pathways. β-Catenin/TCF binding sites in the MDR1

promoter were described previously and recently it has been shown that the activation of Wnt/β-

catenin signaling increases P-glycoprotein expression. In contrast, suppression of Wnt/β-catenin

signaling decreased P-glycoprotein expression. Natural compounds that inhibit both of P-

glycoprotein function and expression can be useful for overcoming multidrug resistance in

human cancer. Therefore, I am now trying to find such compounds by using both P-glycoprotein-

lacking anticancer drug sensitive cells and P-glycoprotein-overexpressing multidrug-resistant

cells It is also very important to investigate the absorption, distribution, metabolism, and

excretion of chemopreventive phytochemicals in the human body, to clarify the interaction with

anticancer drugs at the site of drug action, in the tumor tissues and cancer cells.

Conclusions

1. So far, the complexity and versatility of cellular MDR mechanisms have hindered the

search for effective and clinically applicable MDR therapies. Yet, in the last two decades

25

we have learnt much about the required properties of putative MDR drugs and how best

to evaluate them. Afew good candidates have emerged that may soon make it into the

clinic. Novel inhibitors of MDR transporters are continually being discovered, including

many natural products derived from rare plants and marine fauna (e.g., WO-00149279

[107] and US-06087370). In addition, novel approaches are being devised in order to

bypass rather than block MDR mechanisms. With these advances in sight there is ground

for cautious optimism that improvements in the efficacy of cancer chemotherapy maybe

expected in the not too distant future.

2. In addition to the beneficial effects of natural compounds, the inhibitory effects of

phytochemicals on the anticancer drug efflux transporter P-glycoprotein and MRP1 have

been revealed, which can reverse the multidrug resistance. Therefore, dietary

chemopreventive phytochemicals, such as nobiletin, guggulsterone and glycyrrhetinic

acid, can be considered promising lead compounds for the design of more efficacious and

less toxic chemosensitizing agents to enhance the efficacy of chemotherapy in cancer

patients.

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29

MULTI-DRUG RESISTANCE IN CANCER CELLS

By:

Akinade Kehinde Adetola

Matriculation Number 152981

A seminar topic presented to:

The Department of Biochemistry, Faculty of Basic Medical

Sciences, College of Medicine University of Ibadan.

In partial fulfillment for the award of Master’s degree (MSc)

in Biochemistry.

February; 2011

Table of Content

30

1.0 Introduction

2.0 Historical Perspective

3.0 Types of Multi-drug Resistance

3.1 Intrinsic Multi-drug Resistance

3.2 Acquired Multi-drug Resistance

4.0 Mechanisms of Multi-drug Resistance

4.1 Modulation of Influx/Efflux Intracellular Concentration of Drugs

4.2 Alterations of Cellular Metabolism of Drugs

4.3 Alterations of Cellular Targets of Drug

4.4 DNA Damage Repair

5.0 Functions of ABC Transporters

6.0 Ancient Pathways of Cancer Formation

7.0 Overcoming Multi-drug Resistance

7.1 Small Molecule Inhibitors

7.1.1 First and Second Generation MDR Drugs

7.1.2 Second Generation MDR Drugs

7.2 Novel Approaches

7.2.1 Proposed Clinical Methods

7.2.2 The Use of Plant Phytochemicals

8.0 Conclusion

9.0 References

31