Review

Why chemotherapy can fail?

M. Król, K.M. Pawłowski, K. Majchrzak, K. Szyszko, T. Motyl

Department of Physiological Sciences, Faculty of Veterinary Medicine,Warsaw University of Life Sciences – SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland

Abstract

There are many reasons that lead to failure of cancer chemotherapy. Cancer has the ability tobecome resistant to many different types of drugs. Increased efflux of drug, enhanced repair/in-creased tolerance to DNA damage, high antiapoptotic potential, decreased permeability and enzy-matic deactivation allow cancer cell survive the chemotherapy. Treatment can lead to the death ofmost tumor cells (drug-sensitive), but some of them (drug-resistant) survive and grow again. Thesetumor cells may arise from stem cells.

There are many studies describing human experiments with multidrug resistance, especially inbreast cancer. Unfortunately, studies of canine or feline ABC super family members are not asextensive as in human or mice and they are limited to several papers describing PGP in mammarycancer, cutaneous mast cell tumors and lymphoma.Multidrug resistance is one of the most significant problems in oncology today. The involvement ofmany different, not fully recognized, mechanisms in multi drug resistance of cancer cells makes thedevelopment of effective methods of therapy very difficult. Understanding the mechanisms of drugresistance in cancer cells may improve the results of treatment. This review article provides a synopsisof all aspects that refer to cancer cell resistance to antitumor drugs.

Key words: chemotherapy, multidrug resistance, ATP binding cassette, cancer, apoptosis

Introduction

Treatment of cancer is complicated due to myriadmechanisms by which tumor cells are/become drug re-sistant and patient’s ability to absorb or activate drugs.

Although gene therapy or immunotherapy in can-cer treatment are increasingly in use, chemotherapy isstill the most important method. Unfortunately, sometumors are naturally resistant to anticancer drugs (inthat cases chemotherapy fails), however other tumorsmay respond well to drugs at first, but eventually thetreatment fails because the cancer acquires drug resis-tance (Selby 1984). If the disease is refractory tochemotherapy from the beginning, then multidrugresistance is called “intrinsic”, but if the cancer be-

Correspondence to: Tomasz Motyl, tel/fax: +48 22 847 24 52, e-mail: tomasz-motyl@sggw.pl

comes insensitive during the treatment then it iscalled “acquired”. Understanding the mechanisms ofdrug resistance in cancer cells may improve the resultsof treatment.

Several factors (physiological and pharmacologi-cal) can contribute to the development of anticancerdrug resistance. The main physiological factors are:

1. Increased efflux of drug, mediated by: P-glycop-rotein, multidrug resistance protein (MRP), breastcancer resistance protein (BCRP) and a few otherproteins.

2. Mechanisms that precede “apoptotic decisionpoint”: enhanced repair/increased tolerance to DNAdamage; and high antiapoptotic potential.

Polish Journal of Veterinary Sciences Vol. 13, No. 2 (2010), 399-406

3. Decreased permeability: drugs cannot enter thecell due to: compromised angiogenesis in solid tu-mors, increased intra-tumor fluid pressure, andnon-cycling cells.

4. Enzymatic deactivation (e.g. glutathione conju-gation); altered binding-sites of chemotherapeuticagents or alternate metabolic pathways (the cancercompensates for the effect of the drug).

Increased efflux of the drug

Solid tumors consist of very heterogeneous cellpopulations. The cancer stem cell hypothesis statesthat all cancers contain a minority population of stemcells (Raguz et al. 2008). Some of proliferating anddifferentiating cancer cells arise from stem cells thatare responsible for sustaining the tumor. Cancer stemcells retain the important mechanism of self-protec-tion through the activity of multiple drug resistancetransporters (“efflux-pumps”; Fig. 1). The multidrugresistance is frequently associated with the overex-pression of two or more membrane pumps that effluxthe anticancer drugs from the cytoplasm. This pro-tects the tumor cells against the drug effects and itscorrelated molecular processes within the nucleus orthe cytoplasm. If the cells that build the mass of thetumor are sensitive to chemotherapy, but the tumorstem cells are not, the tumor will initially respond todrug, but eventually recur (Borst et al. 2007).

Fig. 1. The role of the efflux pump and its inhibitor in a drug-resistant cancer cell.

But not only stem cells or tumors that arise fromstem cells do express membrane pumps. It is worthremembering, that the normal physiological functionof efflux pumps is very important in all healthy organ-isms. It is recognized as an important component ofthe blood-brain barrier, which limits entry and accu-mulation of many substances into the brain (Lotsch etal. 2002, Dagenais et al. 2004) and protects the func-tion of the central nervous system (Mizuno et al.2003). Another important blood-tissue barrier inwhich these proteins are involved, is the maternal-fe-tal interface (Fromm et al. 2004). It is also expressedin the intestine (function is not clearly defined, but isbelieved to decrease the drugs bio-availability), liverand kidney (Sun et al. 2004). The enlightening study,performed by Roulet and coworkers, documentedthat increased ivermectin sensitivity of several herdingbreeds (e.g. Collies, Bobtails, Australian Shepherds) iscaused by polymorphism of one of the efflux pumps(called multidrug resistance protein), a 4-bp deletionmutation (Roulet et al. 2003). It can also lead to toxiceffect of other drugs given in normal, tolerable (forother breeds) doses, like: opioids, antiparasitic agents,antimicrobial drugs, immunosupresants and alsochemotherapy. It means that anticancer drugs in thesebreeds should be given with caution.

Tumors derived from tissues which physiologicalrole requires high expression of transporter proteinshave intrinsic multidrug resistance to cytostatic agentseven before chemotherapy is initiated. Expression

400 M. Król et al.

of efflux pumps is usually higher in tumors that orig-inate from tissues that normally express these pro-teins. But, expression of efflux pumps is always higherin tumors than in normal cells (Ullah 2008).

Because increased efflux is such a significant con-tributor to multidrug resistance in cancer cells, cur-rent research is aimed at blocking this specific mech-anism.

A brief history

In 1976 Juliano and Ling described the glycop-rotein (called: P-glycoprotein) which was supposed tocorrelate with the drug resistance in several cell lines.The molecule contains trans-membrane domains(TMDs), and also a large cytoplasmic domain whichcontains ATP-binding site (Viguie 1998). The strongevidence of it’s role in multidrug resistance (MDR)became apparent in 1982, when DNA from resistantcell lines was transfected to nonresistant cells devel-oping their drug resistance phenotype (Debenham etal. 1982). The PGP is nowadays the best examinedplasma membrane protein of the ATP-binding cas-sette (ABC) super family of energy-dependent effluxprotein pumps (Sun et al. 2004).

P-glycoprotein is also called: PGP, P-gp, Pgp,ABCB1, ATP-binding cassette sub-family B member(Ueda et al. 1987). P-glycoprotein is encoded by theMDR genes: MDR1 and MDR3 in humans, andMDR1a, MDR1b and MDR2 genes in mice (Arboixet al. 1997).

Until 1992 PGP was thought to be the only mem-brane protein responsible for the cytotoxic drugs ef-flux. Experiments with a lung cancer cell line, whichwas resistant to doxorubicin, surprisingly showed thatthis cell line did not overexpress P-glycoprotein. Coleand coworkers (1992) showed that this cell line ex-pressed another protein called MRP (multidrug resis-tance protein). MRP was also found to be an effluxpump, which belongs to the ATP-binding cassettetrans-membrane transporter super family. Since 1992both MRP and P-glycoprotein are targets for antican-cer treatment (Doyle et al. 1998). Although both pro-teins belong to the same ABC super family, they arequite different (Gottesman et al. 1993). Now, manydifferent efflux pumps are known and about 48 ABCproteins are encoded in the human genome, but onlyabout 10 are responsible for chemotherapy resistance(Kuo 2007). Most of them are different isoforms ofPGP or MRP, however it is important to note thatthere are some proteins, like breast cancer resistanceprotein (BCRP) or lung cancer resistance protein(LCRP), with a different formation. Over the next fewyears, due to the extensive molecular experiments andstudies on multi-drug resistance of cancer cells, it isprobable that more MDR proteins will be discovered.

Efflux pumps in canine tumors

There are many studies describing human experi-ments on multidrug resistance, especially in breastcancer. Unfortunately, studies of canine or felineABC super family members are not as extensive as inhuman or mice and they are limited to several papersdescribing PGP in mammary cancer, cutaneous mastcell tumors and lymphoma (Myioshi et al. 2002; Pet-terino et al. 2006). Miyoshi and coworkers examinedPGP and MRP expression in canine cutaneous mastcell tumors of different grade of malignancy (Myioshiet al. 2002). Interestingly, they found that PGP andMRP were expressed mostly in established differenti-ated tumors of grade I malignancy. It would thereforesuggest that less differentiated and more malignanttumors should be more susceptible to chemotherapy.In fact, it is not as evident in clinical environment. Leeand coworkers examined canine lymphoma and foundthat 67% of them did not show any PGP expression(Lee et al. 1996). They also found that PGP express-ion was higher after relapse, in comparison to pret-reatment case.

Multidrug resistance in caninemammary cancer

Experiments which assess the drug resistance ofcanine mammary cancer are particularly compelling,as in dogs the risk of this type of tumor is three timeshigher than in human. The treatment of choice for thecanine patients with mammary cancer is surgical mas-tectomy. Unfortunately, it is often unsatisfactory be-cause at the time of surgery the micrometastases arepresent. Although chemotherapy has been shown tobe effective in individual dogs with mammary glandtumors, there are still no large studies that prove thebenefit of chemotherapy in dogs which are necessaryto help establish chemotherapy treatment protocols(Karayannopoulou et al. 2009). In some veterinary cli-nics, the adapted protocols of human chemotherapyare used (usually doxorubicin and cyclophosphamide).Unfortunately, these methods of treatment can oftenfail, as there is minimal information about drug resis-tance of canine mammary cancer and the effective-ness of each individual chemotherapeutic agent.Moreover, mammary cancer sarcomas are quite fre-quent tumors in dogs, whereas there are just severalreports describing this kind of breast tumor.

The first manuscript describing PGP expression incanine mammary gland tissue samples was publishedin May 2006 (Petterino et al. 2006). The authorsfound that neoplastic cells in benign mammary tumorsshowed diffuse cytoplasmic staining pattern for PGP,in contrast to malignant tumors that showed mainlythe membranous staining pattern. These results pro-

Why chemotherapy can fail? 401

vide the first indication that routine evaluation ofPGP expression in canine mammary gland tumorsmay be useful for selecting cases for chemotherapy.Three years later, Honscha and co-workersdocumented (by real-time PCR) seven differentmultidrug resistance proteins in canine mammarycancer cells (MRP1, MRP3, MRP5, MRP6, MRP7,BCRP, and PGP): more than half of the examinedtumors expressed all seven of them (Honscha et al.2009). In this experiment all examined tumorsshowed expression of BCRP. In other study, Nowaket al. (2009) histopathologically verified BCRP-1 ex-pression in canine mammary adenocarcinomas andadenomas. They have demonstrated that 85% of ad-enocarcinomas show BCRP-1 expression. Authorsshowed that the expression level correlates withgrade of malignancy. Only 28% of canine mammaryadenomas express BCRP.

In our microarray studies (Król et al. 2010a) weshowed higher expression of PGP in canine mam-mary cancer cell lines with high metastatic potentialand their metastases to the lungs than in other ca-nine mammary cancer cell lines with low metastaticpotential. There is no information in the literaturethat PGP takes part in the metastatic process ormetastatic ability of cancer cells, but it is commonlyknown that cancers that metastasize are more resis-tant to chemotherapy (Król et al. 2010a).

Each protein is thought to be responsible for theresistance to a specific cytotoxic drug, but the func-tion of different ABC-transporters to pump out vari-ous substances overlaps. For example overexpressionof PGP is linked to resistance to vinca alkaloids, an-thracyclines, epipodophyllotoxins and also tubulinpolymerizing drugs (Litman et al. 2001) whereasMRP1 is responsible for resistance to vincristine andvinblastine, methothrexate, anthracyclines, etopo-side, paclitaxel and irinotecan (Deeley et al. 2006).The specific substrates for each efflux “pump” havenot been assessed in animals yet. In 2001 Ma andco-workers found that MRP1 in dogs protects cancercells against vinca alkaloids, epipodophyllotoxins,and anthracyclines. Dr. Honscha and co-workers(2009) functionally analyzed BCRP in Madin-Derbycanine kidney cells (cells were stable transfected withcBCRP). They found that transfected cells showedmore than 5 times higher resistance to doxorubicinbut in the presence of methotrexate cell survival wasnot affected by cBCRP. In face of information that85-100% of canine mammary tumors express BCRP(Honscha et al. 2009, Nowak et al. 2009) authorssuggest that application of doxorubicin (as it is some-times used according to the adapted human chemo-therapy protocols) for treatment of this kind of tu-mor is inappropriate.

Investigators all over the World try to find drugsthat could be PGP inhibitors. Several of them have

been proposed with limited success, such as antican-cer/chemotherapy co-agents, Ca2+ channel blockers(e.g. verapamil), steroids, opioids, antibiotics (e.g.Cyclosporine A), lipid-lowering agents, and hista-mine H1 receptor antagonists (Fromm et al. 2004).Some of the multidrug resistance proteins inhibitors(called response modifiers: RM) are widely de-scribed in many in vitro experiments as effective inhuman and mice (Colombo et al. 1994, Arboix et al.1997). Since these compounds have already been ap-proved for other purposes, it seems natural to exam-ine their effects on the treatment of cancer withchemotherapeutic agents. Uozurmi and coworkers(2005) described strong verapamil inhibition of PGPfunction in a canine B cell lymphoma cell line thatwas cultured in medium containing gradually in-creasing levels of doxorubicin. Two years later rever-sal effect of verapamil in drug-resistant mast cell tu-mor cell lines originated from oral-mucosal tumorand gastrointestinal tumor was described (Nakaichiet al. 2007). But, verapamil has been reported to beineffective in restoring drug sensitivity ofMRP1-overexpressing human cells. In these cells itstimulates glutathione transport by MRP1 and trig-gers apoptosis of resistant cells through stimulationof MRP1-mediated glutathione efflux (Perrotton etal. 2007). Raguz et al. (2008) described some clinicaltrials that indicated that commonly usedefflux-pumps antagonists were ineffective or toxic atthe doses required to attenuate P-glycoprotein func-tion. Moreover, they described also some clinicaltrials indicating that the modulation of P-glycop-rotein function can be achieved. It shows that mech-anism of efflux pumps modulation is very compli-cated and only partially explored.

Canine mammary cancer cell lines appear to rep-resent a good model for further investigation ofMDR and the resistance breakage. In the face of ourprevious results showing that in vitro experiments us-ing cell lines can clearly reflect interactions in vivo(Pawłowski et al. 2009), the experiments withover-expressed PGP cell lines may be especially sig-nificant. It can expedite evaluation of strategiesmodulating and preventing drug resistance in bothcanines and humans.

The novel genetic approaches, such as genemodification, seem to give hope in multidrug resis-tance studies. For example, Marthinet et al. (2000)described that introduction of antisense nucleotidesequence of the promoter of the human MDR1 genewas effective in reversing the drug resistance ofleukemia cells. Woodahl et al. (2004) examined cellstransfected with mutant and wild type PGP. Theyshowed significant differences in resistance of thesecell lines to vinca alkaloids and also differences inrhodamine-123 efflux.

402 M. Król et al.

Mechanisms that precede “apoptoticdecision point”

Chemotherapeutic agents, as alkylating agents(cisplatin, carboplatin, melphalan), inhibitors ofDNA topoisomerase II (anthracyclines, etoposide,teniposide), inhibitors of topoisomerase I and anti-metabolites induce DNA double strands breaks(DSBs).

Cancer cells have the ability of increased DNAdamage repair via different mechanism, for examplealterations in methylguanine DNA methyl transfer-ase or changes in topoisomerase II activity (Lis-covitch et al. 2002). The lack of chemotherapy suc-cess is also linked with the cancer cells tolerance tothe DNA damage. Maintaining chromosomal integ-rity is crucially dependent on cell cycle checkpoints.A failure of the G2/M checkpoint increases the likeli-hood that cancer cells will enter mitosis withdamaged chromosomes. A majority of cancer cellshave dysfunctional cell cycle checkpoints. It meansthat cancer cells enter mitosis with DNA damagewhereas in normal cells the apoptotic pathway wouldbe induced (Fojo 2001).

Fig. 2. Cumulative graph showing the number of apoptotic cells (%) in different canine mammary cancer cell lines treated with0.3 µg/ml of campthotecin (CPT) for 1, 3, 6 and 12 hours. The number of apoptotic cells and mean fluorescence related toBcl-2 expression was measured in Skan^R screening station. Cells were stained with anti-Bcl-2 antibodies (Dako). Two celllines (simple carcinoma and adenocarcinoma) showed the highest Bcl-2 expression and the lowest number of apoptotic cells.

The other observed pleiotropic resistance of can-cer cells to many different apoptosis-inducing agents(eg. oxidative stress, radiation, tumor necrosis factor)indicates that defects in apoptotic pathways (eg.through overexpression of antiapoptotic proteinssuch as Bcl-2) are also efficient drug resistancemechanisms (Liscovitch 2002). Some other investiga-tors also suggest that cells with high antiapoptoticpotential may be more resistant to chemotherapeuticdrugs. Brown and Wouters performed deep analysisof susceptibility to anticancer drugs in different tu-mors (with mutant p53 or overexpression of Bcl-2family proteins) (Brown et al. 1999). They found thatapoptosis and its controlling genes (like p53 or Bcl-2)play little or no role in the insensitivity of these cellsfor killing effects by anticancer drugs and radiation.A group guided by Prof. Piet Borst from the Nether-lands Cancer Institute examined doxorubicin-resis-tant mammary tumors (which did not show MDR1over-expression) and did not find any alteration inthe expression of genes known to be involved in cel-lular apoptosis/senescence in tumors (Borst et al.2007). They conclude that resistance due to blocks inapoptosis or senescence is not an available option for

Why chemotherapy can fail? 403

examined tumors. Although other authors found thatoverexpression of Bcl-2 resulted in a high drug resis-tance of the tumor cells (Schmitt et al. 2001, Schmittet al. 2002) they, on the contrary, suggested thatBcl-2 can act as a potent multidrug resistance gene.

Our own results indicate that canine mammarycancer cells with high expression of Bcl-2 can showless susceptibility to campthotecin (CPT). It wasmanifested by a small number of apoptotic cells(Król et al. 2010a). These cell lines after 12 hoursincubation in a medium containing CPT (0.3 µg/ml)showed low number of apoptotic cells (Fig. 2). Thisfinding supports the hypothesis that Bcl-2 proteinguarantee the immortality of cancer cells.

Decreased permeability

Decreased permeability may be caused by poorlystructured vasculature. Tumors induce blood vesselgrowth (angiogenesis) by secreting various growthfactors, which can induce capillary growth into thetumor. Angiogenesis is required to transition froma small cluster of cells to a large tumor. However, insome types of tumors angiogenesis is compromised(Jain 1987) resulting in poor tumor vasculature. Thisalters the accessibility of the drug to the cancer cellsand limits cytotoxicity. The microenvironment of thetumor is totally different from that of normal tissue.Moreover, lack of nutrition and hypoxia due to poorvasculature can additionally increase resistance tocancer cells to drugs that act on actively dividing cells(mostly in the mitosis phase of cell cycle) or the cel-lular drug uptake (Ueda et al. 1987, Demant et al.1990).

Enzymatic deactivation

Other mechanisms of multidrug resistance in-volve enzyme systems that decrease drug activity butdo not influence the drug concentration in cytop-lasm. The most important enzyme of drugs metab-olism is glutathione-S-transferase (GST), which fa-cilitate excretion. Moreover, GST increases biotran-sformation of anticancer drugs and its function hasbeen described in many different cancer cells (Batistet al. 1986, Hao et al. 1994).

It is still unresolved how drug resistance arisesand whether the capacity to develop drug resistancearises before or after tumorigenic transformation.Important question is also: what is the minimumnumber of altered pathways required to permit thisevent. Dimri et al. (2005) described model of trans-formed fibroblast cells and three possibilities afterthe cancer treatment: apoptosis, senescence or ac-

quired drug resistance. In their studies the ability toacquire drug resistance is intrinsic to the early stepsin the tumorigenic pathway necessary for transform-ation. In their opinion acquired drug resistance ariseearlier than the full malignant transformation. Thisphenomena require deeper investigation.

There are many aspects of the multidrug resis-tance that still remain questionable. The involvementof many different mechanisms in cancer cells drugresistance makes the development of effectivemethods of therapy very difficult. The most import-ant problem is the understanding the up-regulationmechanisms of ABC transporter genes in cancers, aswell as development of new reversal agents.

Acknowledgements

The authors would like to thank: Ms. VictoriaBrennan for her contribution in preparation of themanuscript.

Some author’s experiments cited in this paperwere supported by the grant no N308230536 fromthe Ministry of Science and Higher Education.

References

Arboix M, Gonzales Paz O, Colombo T, D’IncalciM (1997) Multidrug resistance-reversing agents in-crease vinblastine distribution in normal tissues express-ing the P-glycoprotein but do not enhance drug penetra-tion in brain and testis. J Pharmacol Exp Ther281: 1226-1230.

Batist G, Tulpule A, Sinha BK, Katki AG, Myers CE,Cowan KH (1986) Overexpression of a novel anionicglutathione transferase in multidrug-resistant humanbreast cancer cells. J Biol Chem 261: 15544-15549.

Borst P, Jonkers J, Rottenberg S (2007) What makes tu-mors multidrug resistant? Cell Cycle 6: 2782-2787.

Brown JM, Wouters BG (1999) Apoptosis, p53, and tumorcell sensitivity to anticancer agents. Cancer Res59: 1391-1399.

Cole SP, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE,Almquist KC, Stewart AJ, Kurz EU, Duncan AM,Deeley RG (1992) Overexpression of a transporter genein a multidrug-resistant human lung cancer cell line.Science 258: 1650-1654.

Colombo T, Zucchetti M, D’Incalci M (1994) CyclosporinA markedly changes the distribution of doxorubicin inmice and rats. J Pharmacol Exp Ther 269: 22-27.

Dagenais C, Graff CL, Pollack GM (2004) Variable modu-lation of opioid brain uptake by P-glycoprotein in mice.Biochem Pharmacol 67: 269-276.

Debenham PG, Kartner N, Siminovitch L, Riordan JR,Ling V (1982) DNA-mediated transfer of multiple drugresistance and plasma membrane glycoprotein express-ion. Mol Cell Biol 2: 881-889.

Deeley RG, Cole SPC (2006) Substrate recognition andtransport by multidrug resistance protein 1 (ABCC1).FEBS Lett 580: 1103-1111.

404 M. Król et al.

Demant EJ, Sehested M, Jensen PB (1990) A model forcomputer simulation of P-glycoprotein and transmem-brane delta pH-mediated anthracycline transport inmultidrug-resistant tumor cells. Biochim Biophys Acta1055: 117-125.

Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y,Rishi AK, Ross DD (1998) A multidrug resistancetransporter from human MCF-7 breast cancer cells.Proc Natl Acad Sci USA 95: 15665-15670.

Fojo T (2001) Cancer, DNA repair mechanisms, andresistance to chemotherapy. J Natl Cancer Inst93: 1434-1436.

Fromm MF (2004) Importance of P-glycoproteinat blood-tissue barriers. Trends in Pharmacol Sci25: 423-429.

Gottesman MM, Pastan I (1993) Biochemistry of multidrugresistance mediated by the multidrug transporter. AnnuRev Biochem 62: 385-427.

Hao XY, Widersten M, Ridderstrom M, Hellman U, Man-nervik B (1994) Co-variation of glutathione transferaseexpression and cytostatic drug resistance in HeLa cells:establishment of class M glutathione transferase M3-3as the dominating isoenzyme. Biochem J 297: 59-67.

Honscha KU, Schirmer A, Reischauer A, Schoon HA, Ein-spanier A, Gabel G (2009) Expression of ABC-trans-port proteins in canine mammary cancer: consequencesfor chemotherapy. Reprod Domest Anim 44: 218-223.

Jain RK (1987) Transport of molecules in the tumour inter-stitium: a review. Cancer Res 47: 3039-3051.

Juliano RL, Ling V (1975) A surface glycoprotein modula-ting drug permeability in Chinese hamster ovary cellmutants. Biochim Biophys Acta 445: 152-162.

Karayannopoulou M , Kaldrymidou E, Constantinidis TC,Dessiris A (2001) Adjuvant Post-operative Chemother-apy in Bitches with Mammary Cancer. TransboundEmerg Dis 48: 85-96.

Liscovitch M, Lavie Y (2002) Cancer multidrug resistance:a review of recent drug discovery research. IDrugs5: 349-355.

Król M, Pawłowski KM, Skierski J, Rao NA, Hellmen E,Mol JA, Motyl T (2009) Transcriptomic profile of twocanine mammary cancer cell lines with different prolif-erative and antiapoptotic potential. J Physiol Pharmacol60: 95-106.

Król M, Pawłowski KM, Skierski J, Turowski P, MajewskaA, Polańska J, Ugorski M, Morty RE, Motyl T (2010)Transcriptomic “portraits” of canine mammary cancercell lines with various phenotype. J Appl Genet 51, inpress. (a)

Król M, Polańska J, Pawłowski KM, Skierski J, Turowski P.,Majewska A, Ugorski M, Morty RE, Motyl T (2010)Molecular signature of cell lines isolated from mammaryadenocarcinoma metastases to lungs. J Appl Genet51: 37-50. (b)

Kuo MT (2007) Roles of Multidrug Resistance Genes inBreast Cancer Chemoresistance. In: Oncology in BreastCancer Sensitivity, Landes Biosciences, USA.

Lee JJ, Hughes CS, Fine RL, Page RL (1996) P-glycop-rotein expression in canine lymphoma: a relevant, inter-mediate model of multidrug resistance. Cancer77: 1892-1898.

Linardi RL, Natalini CC (2006) Multi-drug resistance(MDR1) gene and P-glycoprotein influence on phar-

macokinetic and pharmacodymanic of therapeuticdrugs. Ciknc Rural, 36: 336-341.

Litman T, Druley TE, Stein WD, Bates SE (2001) FromMDR to MXR: new understanding of multidrug resis-tance systems, their properties and clinical significance.Cell Mol Life Sci 58: 931-959.

Lotsch J, Skarke C, Tegeder I, Geisslinger G (2002) Druginteractions with patient-controlled analgesia. J ClinPharmacokined 41: 31-57.

Ma L, Pratt SE, Cao J, Dantzig AH, Moore RE, Slapak CA(2002) Identification and characterization of the caninemultidrug resistance – associated protein. Mol CancTher 1: 1335-1342.

Marthinet E, Divita G, Bernaud J, Rigal D, Baggetto LG(2000) Modulation of the typical multidrug resistancephenotype by targeting the MED-1 region of humanMDR1 promoter. Gene Ther 7: 1224-1233.

Miyoshi N, Tojo E, Oishi A, Fujiki M, Misumi K, SakamotoH, Kameyama K, Shimizu T, Yasuda N (2002) Im-munohistochemical detection of P-glycoprotein (PGP)and multidrug resistance-associated protein (MRP) incanine cutaneous mast cell tumors. J Vet Med Sci64: 531-533.

Mizuno N, Niwa T, Yotsumoto Y, Sugiyama Y (2003) Im-pact of drug transporter studies on drug discovery anddevelopment. Pharmacol Rev 55: 425-461.

Nakaichi M, Takeshita Y, Okuda M, Nakamoto Y, ItamotoK, Une S, Sasaki N, Kadosawa T, Takahasi T, TauraY (2007) Expression of the MDR1 gene and P-glycop-rotein in canine mast cell tumor cell lines. J Vet Med Sci69: 111-115.

Nowak M, Madej JA, Dziegiel P (2009) Expression ofbreast cancer resistance protein (BCRP-1) in caninemam mary adenocarcinomas and adenomas. In Vivo23: 705-709.

Pawłowski KM, Król M, Majewska A, Badowska--Kozakiewicz A, Mol JA, Malicka E, Motyl T (2009)Comparison of cellular and tissue transcriptional pro-files in canine mammary tumor. J Physiol Pharmacol60: 85-94.

Petterino C, Rossetti E, Bertoncello D, Martini M, Zap-pulli V, Bargelloni L, Castagnaro M (2006) Immunohis-tochemical detection of P-glycoprotein (clone C494) incanine mammary gland tumours. J Vet Med A PhysiolPathol Clin Med 53: 174-178.

Perrotton T, Trompir D, Chang XB, Di Pietro A,Baubichon-Cortay H (2007) (R)- and (S)-verapamil dif-ferentially modulate the multidrug-resistant proteinMRP1. J Biol Chem 282: 31542-31548.

Raguz S, Yague E (2008) Review to chemotherapy: newtreatments and novel insights into old problem. BritJ Canc 99: 387-391.

Roulet A, Puel O, Gesta S, Lepage JF, Drag M, Soll M,Alvinerie M., Pineau T (2003) MDR1-deficient geno-type in Collie dogs hypersensitive to the P-glycoproteinsubstrate ivermectin. Eur J Pharmacol 460: 85-91.

Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoff-man RM, Lowe SW (2002) A senescence program con-trolled by p53 and p16INK4a contributes to the out-come of cancer therapy. Cell 109: 335-346.

Schmitt CA, Lowe SW (2001) Apoptosis is critical for drugresponse in vivo. Drug Resist Updat 4: 132-134.

Selby P (1984) Acquired resistance to cancer chemother-

Why chemotherapy can fail? 405

apy. Brt Med J 288: 1251-1253.Steingold SF, Sharp NJ, McGahan MC, Hughes CS, Dunn

SE, Page RL (1998) Characterization of canine MDR1mRNA: its abundance in drug resistant cell lines and invivo. Anticancer Res 18: 393-400.

Sun J, He ZG, Cheng G, Wang SJ, Hao XH, Zou MJ(2004) Multidrug resistance P-glycoprotein: crucial sig-nificance in drug disposition and interaction. Med SciMonit 10: RA5-14.

Turker MS, Duffin KZ, Smith AC, Martin GM, MartinAW, DiMartino DL, Kersey DS (1991) Multidrug resis-tance phenotype associated with selection of aminop-terin resistant dog kidney cell line. Pharmacogenetics11: 149-460.

Ueda K, Clark DP, Chen CJ, Roninson IB, GottesmanMM, Pastan I (1987) The human multidrug resistance

(mdr1) gene. cDNA cloning and transcription initiation.J Biol Chem 262: 505-508.

Ullah MF (2008) Cancer multidrug resistance (MDR):a major impediment to effective chemotherapy. AsianPac J Cancer Prev 9: 1-6.

Uozurmi K, Nakaichi M, Yamamoto Y, Une S, TauraY (2005) Development of multidrug resistance in a ca-nine lymphoma cell line. Res Vet Sci 78: 217-224.

Woodahl EL, Yang Z, Bui T, Shen DD, Ho RJY (2004)Multidrug resistance gene G1199A polymorphism altersefflux transport activity of P-glycoprotein. J PharmacolExp Ther 310: 1199-1207.

Viguie F (1998) ABCB1. Atlas of Genetics and Cytogeneticsin Oncology and Haematology, http://atlasgeneticsoncol-ogy.org//Genes/PGY1ID105.html.

406 M. Król et al.