Multidrug Resistance Essay

8/8/2019 Multidrug Resistance Essay

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Multidrug Resistance Essay

Terms & Definitions and General Perspectives

Drug resistance:The ability of a cell or microorganism to withstand theeffects of a drug that is lethal to most members of theirequivalents.

Malignant neoplasmsvary in their response to cytotoxic drugs: some aresensitive, others resistant. Understanding of the biochemical basis of this resistance might lead

to the development of markers that would correlate with the clinical response to the drugs - or

even lead to ways of overcoming the resistance.

Multidrug resistance:

1 The resistance ofTumor cells to more than onechemotherapeutic agent. Resistance may be

attributed to a P-glycoprotein transmembrane pumpthat lowers the concentration of drugs in the cell.

2 The resistance of bacteria, especially

Mycobacterium tuberculosis, against more than two ofthe antibiotics that were once effective.

Multidrug resistance"multiple drug resistance in some

malignant cell lines"= Resistance to many structurally

unrelated chemotherapy agents in cells that were discovered

to have developed natural resistance to a single cytotoxic

compound.


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Discovery

Multidrug resistance was first described in 1970 afterselection of Chinese hamster ovarian cancer cellsexposed to increasing concentrations of actinomycin

D.1 Though the cells had been selected by a singleagent, they proved to be resistant to a range ofclinically important anticancer drugs, including the

Anthracyclines (doxorubicin and daunomycin), theVinca alkaloids (vincristine, vinblastine, and

vindesine), Etoposide, and Colchicine.

Criteria of multidrug resistant cells

Riordan and Ling went on to show that the multidrug resistant cells had

lower concentrations of the drug

a drug accumulation deficit - and

that a membrane glycoprotein of 170 kDa was responsible.2

At first the deficit was thought to be due to a fault in Permeationand therefore theglycoprotein was named P glycoprotein (P-gp).3

Genetics

The gene for this, mdr-1, has been cloned andsequenced, and the amino acid has beensequenced and the amino acid sequence

derived.

4

P glycoprotein has1280

amino acids,and its structure suggests that it arose from thefusion of two closely related genes; it uses ATPas a source of energy.

The precise molecular mechanism whereby Pglycoprotein can transport such a wide range of


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structurally diverse drugs is uncertain, andseveral models have been proposed.

P glycoprotein& multidrugresistance associated protein

(MRP).

P glycoprotein belongs to a superfamily of ATP bindingcassette transporters, whose members include the cystic

fibrosis transmembrane regulator, the chloroquine

transporter of Plasmodium falciparum, and a yeast

transporter.8

Recently a new memberof this family was cloned from a

human small cell lung cancer cell line, H69AR.

Though it was resistant to doxorubicin and vincristine, it did

not express P glycoprotein.9 This new protein has 1522 amino

acids and has been termed multidrug resistance associated

protein (MRP).

Cells with this type of resistance may not have accumulation

deficits of doxorubicin. One possible explanation is that

multidrug resistance associated protein facilitates the

sequestration of cytotoxic drugs into intracellular organelles.9

Whether this protein binds to the drugs against which it

confers resistance is not known, but the emergence of a

potential new target for modulation, if it is widely expressed inhuman tumours, is clearly an exciting development.

Several probes have been developed that can detect the P glycoprotein

gene, its mRNA, and its protein product.10,11

Studies have shown that the

gene expressed in normal gastrointestinal mucosal cells, renal tubular

cells, biliary canalicular cells, and adrenocortical cells .11


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Tumours that are initially sensitive to chemotherapy but resistant on relapse commonly show

increases in expression of P glycoprotein.

Leukaemias with high concentrations of P glycoprotein tend to be resistant to inductive

treatment with chemotherapy based on anthracycline.12

Expression of P glycoprotein is an adverse factor in multivariate prognostic models for

childhood sarcoma and neuroblastoma.13,14

BEFORE LONG IT MAY BE PRACTICABLE TO TISSUE TYPE TUMOURS ACCORDING TO

EXPRESSION OF P GLYCOPROTEIN AND, PERHAPS, TO AVOID THE TOXICITY AND REDUCED

QUALITY OF LIFE ASSOCIATED WITH TREATMENT WITH INEFFECTIVE ANTICANCER DRUGS.

After an initial observation by Tsuruo et al,15

several groups

have shown that transport defect mediated by P glycoprotein

can be blocked by many non-cytotoxic drugs, including

nifedipine, verapamil, quinine, chloroquine, progestogens,tamoxifen, cyclosporin A and its analogues, reserpine and

tricyclic anti-depressants.16 Clinical trials have assessed the

combination of conventional chemotherapy with modulators

of P glycoprotein; in one study of relapsed non-Hodgkin's

lymphoma 13 of18 patients responded to infusional

chemotherapy with high doses of verapamil.17

Unfortunately,

the amounts of verapamil needed to reverse drug resistance

produced congestive cardiac failure and heart block.Research

groups are currently looking for potent, non-toxic modulators

of P glycoprotein to combine with conventional

chemotherapy. Cyclosporin A has been reported to reverse

clinical multidrug resistance in myeloma.18 The combination

of quinidine with the antineoplastic agent epirubicin was

recently assessed in a prospective, placebo controlled

randomised study in patients with advanced breast

cancer.19Tumour response rates and survival were similar in

the two arms of the trial, a finding that probably reflects the

low potency of quinidine to bind P glycoprotein.7

Another possible application of the genetics of P glycoprotein is in gene therapy. Transgenic

mice that express human P glycoprotein in their bone marrow are resistant to chemotherapy,20

so in theory if human bone marrow could be transfected with the gene that might protect it

against myelosuppression from anticancer chemotherapy.8

Novel, improved modulators of P glycoprotein seem likely to be developed in parallel with mechanistic

research on the function of the gene, and thus should lead to the rational design of inhibitor. Future


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phase II clinical trials with new modulators of P glycoprotein must include pharmacokinetic studies since

interactions can occur with cytotoxic drugs - for example, Etoposide's activity is increased by80% when it is given in combination with cyclosporin A.21 These trials should focus onthose cancers that are known to express high concentrations of P glycoprotein (including

renal and colorectal cancers and sarcomas).

Since P glycoprotein is expressed in normal tissues there is

some concern that its modulators might alter the cellular

transport of physiological metabolites such as bilirubin and

thereby delay the clearance of cytotoxic drugs that undergo

hepatobiliary excretion. These clinical trials should therefore

be performed in units used to handling the toxicity associated

with intensive chemotherapy.

Oxford Journals

y Volume95, Issue4

y Pp. 255-257.

Multidrug Resistance: Can New Drugs Help

Chemotherapy Score Against Cancer?

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Redundant Mechanisms

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Nanoparticles may play a role in inhibiting the multidrug resistance in

chemotherapy

(Nanowerk Spotlight) Multidrug resistance, the principal mechanism by which many

cancers develop resistance to chemotherapy drugs, is a major factor in the failure of

many forms of chemotherapy. New research by Chinese scientists suggests that

nanoparticle surface chemistry and size as well as the unique properties of the

magnetic nanoparticles themselves may contribute to a synergistic enhanced effectof drug uptake of targeted cancer cells. These findings could result in promising

biomedical applications for cancer therapy.

ProfessorXuemei Wang from the the State Key Laboratory of Bioelectronics (Chien -

Shiung Wu Laboratory) in Nanjing, PR China, together with several of her col leagues

from Southeast University, recently published a paper titled "Synergistic

enhancement effect of magnetic nanoparticles on anticancer drug accumulation in

cancer cells" in the June 26, 2006 online issue of Nanotechnology.

In it, the researchers describe their investigation of the synergistic effect of threekinds of magnetic nanoparticles, nano Fe 3O4, Ni and Fe2O3, on the drug uptake of

anticancer drug daunorubicin in leukemia K562 cells.

They show how Fe3O4 nanoparticles could remarkably enhance the uptake or

diffusion efficiency of anticancer drugs into target cancer cells (especially drug

resistance cancer cells). If Fe3O4 nanoparticles, which are biocompatible and very

stable, are fixed at the ailing area by using external magnetic field during the tumor

treatment, the chemotherapy effect could be considerably enhanced by combination

of the application of the new magnetic nanoparticles in drug delivery systems for

achieving the targeting and controlled drug release.


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Microscopy images (200 m 200 m) of Fe3O4 incubated leukemia cells in the

absence and presence of a magnetic field. The cells numbered in the images

illustrate the movement of the cells, while squared cells in the left image wereobserved to move out of vision after the external magnetic field was applied, and the

cycled cells in the right image appeared upon application of the external m agnetic

field. The arrows indicate the direction of the magnetic field and the picture was

captured after the magnetic field was applied for 5 s. (Reprinted with permission from

IOP Publishing)

"Our results illustrate that the presence of magnetic nanopar ticles could facilitate the

drug accumulation of daunorubicin inside leukemia cells and the enhancement effect

of nano Fe3O4 is much stronger than that of the other two magnetic nanoparticles"

Wang explains the findings to Nanowerk. "These observations are consistent with the

results of our recent biological experimental studies, which indicates that the

presence of Fe3O4 nanoparticles could apparently inhibit the growth of the respective

leukemia cells (Interestingly, the Fe 3O4 nanoparticle itself could al so inhibit the cell

growth somehow); especially, when treated the target cells by anticancer drug

daunorubicin together with Fe 3O4 nanoparticles, the growth of leukemia cells could

be much more remarkably inhibited than that with only daunorubicin or other

nanoparticles. Since these three kinds of nanoparticles were all capped with the

tetraheptylammonium, our observations suggest that both the size and the unique

properties of magnetic nanoparticles themselves may contribute to the synergistic

enhanced effect of the drug uptake of targeted cancer cells."

The magnetic targeting offers a unique opportunity to treat tumors without systemic

toxicity. It is known that the cure efficiency of cancer chemotherapy depends not only

on the anticancer drug itself but also on how it is delivered to its targets. As already

reported in some literature, it has been observed that the magnetic particles can be

targeted and concentrated in some tumor tissue at significantly high level.


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"Our observations indicate that magne tic nanoparticles with different size and surface

chemistry have different ability to enter target cells and thus the relative efficiency of

the drug delivery systems by the conjugation of drugs with nanoparticles will be

critically dependent upon nanopart icle surface chemistry and size of the

functionalized nanoparticles" says Wang.

"Based on these observations, our future research with regard to cancer therapy may

focus on the relative mechanisms of new magnetic nanoparticles" Wang describes a

possible direction for her group's future research. "Magnetic nanomaterials are

especially promising for the early diagnosis of some cancers and for efficiently

targeting chemotherapy.

Multidrug resistance

Mechanism and function of multidrug transporters in pro- and eukaryoticcells

The development of resistance to multiple drugs is a major problem in the treatment of

cancer cells and infections by pathogenic microorganisms. One of the mechanisms by

which human cells and bacteria can acquire multidrug resistance involves the expression of

membrane proteins which mediate the active extrusion of drugs from the cell. Current

research focuses on the molecular properties of four distinct multidrug transport proteins.

Multidrug transporter LmrP

InLactococcuslactisLmrP mediates drug resistance by extruding amphiphilic, organic

compounds from the inner leaflet of the cytoplasmic membrane.LmrP is a secondary

multidrug transporter which is typical for prokaryotes. The antibiotic specificity ofLmrP is

exceptionally broad and includes tetracyclins, quinolones, aminoglucosides, lincosamides,

macrolides, streptogramins and others. To facilitate functional and structural studies,

histidine-tagged LmrPprotein was overexpressed inL. lactis, solubilized with

dodecylmaltoside, purified to homogeneity by nickel chelate affinity chromatography, and

functionally reconstituted in proteoliposomes. Interestingly, LmrP is able to transport

detergents such as Triton X-100. Therefore, the choice of the detergent used for

solubilization of the protein was critical.

Multidrug transporter LmrA

A second protein in L. lactis, LmrA, mediates antibiotic resistance via a similar mechanism,

and with a similar specificity as the secondary multidrug transporter LmrP. Unlike other

known bacterial multidrug resistance proteins, LmrA is an ATP-binding cassette (ABC)

transporter.LmrA is homologous to the human multidrug resistance P-glycoprotein,

encoded by the MDR1 gene, overexpression of which is one of the major causes of

resistance of human cancers to chemotherapy. In collaboration with Prof. C.F. Higgins in

Oxford, LmrA was expressed in human lung fibroblast cells to compare the

pharmacological properties ofLmrA with those ofP-glycoprotein. Surprisingly, LmrA was


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targeted to the plasma membrane and conferred typical multidrug resistance on these

human cells. The pharmacological characteristics ofLmrA and P-glycoprotein- expressing

lung fibroblasts were very similar, and the affinities of both proteins for vinblastine and

magnesium-ATP indistinguishable. Blockers ofP-glycoprotein-mediated multidrug

resistance also inhibited LmrA-dependent drug resistance. Kinetic analysis of drug

dissociation from LmrA, expressed in plasma membranes of insect cells, revealed the

presence of two allosterically-linked drug binding sites indistinguishable from those ofP-

glycoprotein. These findings have implications for the reversal of antibiotic resistance in

pathogenic microorganisms. Taken together, these observations demonstrate that

bacterial LmrA and human P-glycoprotein are functionally interchangeable and that this

type of multidrug resistance efflux pump is conserved from bacteria to man.LmrA has

been overexpressed in L. lactis up to a level of 35% of total membrane protein. This

protein is functional, and has been purified and reconstituted into proteoliposomes to

facilitate detailed structure-function analyses. Currently, mg quantities of reconstituted

LmrA can be obtained rather easily.

Multidrug transporter HorA

LactococcalLmrA is homologous to other prokaryotic ABC transporters such as the hop-

resistance protein HorA in the beer-spoilage bacterium Lactobacillus brevis. To study HorA

in more detail, the protein was functionally expressed in L. lactis. Studies in whole cells

and membrane vesicles derived thereof demonstrate that HorA is a multidrug transporter

able to transport hop compounds (iso-a-acids), with a substrate specificity similar to that

ofLmrA. Recently, the protein has been purified and reconstituted into proteoliposomes.

Cholatetransporter

Micro-organisms able to colonize the gastrointestinal tract in mammals must tolerate high

levels of bile salts, powerful detergents that disrupt biological membranes.Physiological

evidence has been obtained for the presence of an ATP-dependent cholate transporter in L.

lactis. Interestingly, this transport activity may be related to the transport activities for

fluorescent anionic dyes BCECF and FTUG in this organism. Current work aims at the

isolation of the gene(s) encoding the cholate transporter.

Multidrug resistance-associated protein

Multidrug resistance in humans is caused by the overexpression of two multidrug

transporters: P-glycoprotein and the Multidrug Resistance-Associated Protein (MRP1).

Although MRP1 contains two nucleotide binding domains (NBDs), it is not known whether

both NBDs can function as an active ATPase, and whether drug-protein interactions inthese domains play a role in the drug-stimulated ATPase activity of MRP1. The NBDs of

MRP1 were expressed in fusion with glutathione S-transferase, purified by affinity

chromatography, cleaved from the fusion partner by thrombine, and purified to

homogeneity by gel filtration. Both NBDs hydrolyze ATP, with a Km of 340 mM Mg-ATP and

Vmax of 6.0 nmol Pi/mg/min for NBD1, and a Km of 910 mM Mg-ATP and Vmax of 7.5

nmol Pi/mg/min for NBD2. The properties of NBD1 were further studied. Surprisingly, the

Vmax of the NBD1 ATPase reaction was stimulated more than 3-fold by the MRP substrate


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decyl glutathione but not by decylmaltoside or decanol, whereas the Km of the reaction

was not affected. The interaction between S-alkyl glutathione conjugates and NBD1 was

analyzed by using the intrinsic tryptophan fluorescence of NBD1 as a reporter. The

tryptophan fluorescence was quenched in a concentration-dependent manner by S-alkyl

glutathione conjugates, but not by alkyl maltosides or alkanols. The apparent KD of NBD1

for S-alkyl glutathione conjugates decreased with an increasing length of the alkyl chain.

Thus, NBD1 is suggested to have a high-affinity binding site for S-alkyl glutathione

conjugates.


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Multidrug Resistance Essay

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