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Bast RC Jr, Kufe DW, Pollock RE, et al., editors. Holland-Frei Cancer Medicine. 5th edition. Hamilton (ON): BC Decker; 2000.

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Holland-Frei Cancer Medicine. 5th edition.

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Chapter 39Drug Resistance and Its Clinical Circumvention

, MD, PhD and , MD, PhD.

Systemic therapy with cytotoxic drugs is the basis for most effective treatments of disseminated cancers. Additionally, adjuvant chemotherapy can offer a significant survival advantage to selected patients, following the treatment of localized disease with surgery or radiotherapy, presumably by eliminating undetected minimal or microscopic residual tumor. However, the responses of tumors to chemotherapeutic regimens vary, and failures are frequent owing to the emergence of drug resistance. Patterns of treatment response and tumor sensitivity are conveniently divided into three groups. First, with modern treatments, prompt cytoreduction and cures are common for some intrinsically drug-sensitive tumors, such as childhood acute lymphoblastic leukemia (ALL), Hodgkin’s disease, some non–Hodgkin’s lymphomas, and testicular cancer. A second group comprises tumors such as breast carcinomas, small cell lung cancers, and ovarian carcinomas which are also usually highly responsive to initial treatments but more often become refractory to further therapy. Relapses in either group of tumors, particularly during or shortly after the completion of therapy, generally herald the emergence of tumor cells which are resistant to the antineoplastic agents used initially and often to drugs to which the patient was never exposed. Therefore, success with conventional salvage chemotherapies has been limited. Finally, a third common pattern of drug sensitivity is found in tumors which are intrinsically resistant to most chemotherapeutic agents. This group is represented by malignancies such as non–small cell lung cancers, malignant melanoma, and colon cancer. For these tumors, the number of active antineoplastic agents is low, and significant chemotherapeutic responses are effected only in a minority of cases.

The phenomenon of clinical drug resistance has prompted studies to clarify mechanisms of drug action and identify mechanisms of antineoplastic resistance. It is expected that through such information, drug resistance may be circumvented by rational design of new non–cross-resistant agents, by novel delivery or combinations of known drugs and by the development of other treatments which may augment the activity of or reverse resistance to known antineoplastics. Multiple mechanisms of antineoplastic failure have been identified using in vitro (tissue culture) and in vivo (animal and xenograft) models of antineoplastic resistance. A list of these general mechanisms of drug resistance are categorized in Table 39.1. Considered here are mechanisms involving anatomic, pharmacologic, and host-drug-tumor interactions which are uniquely pertinent to patients and to in vivo models of drug resistance, as well as cellular mechanisms which can be described at the molecular level. These mechanisms are frequently interrelated as, for example, altered gene expression must ultimately underlie most of the cellular and biochemical mechanisms listed in Table 39.1. Furthermore, multiple independent mechanisms of antineoplastic resistance may coexist in a population of tumor cells.

Table 39.1. General Mechanisms of Drug Resistance.

Table 39.1

General Mechanisms of Drug Resistance.

While mechanisms of drug resistance have been largely determined in experimental systems, many have been implicated in at least some examples of clinical chemotherapeutic failure. Evidence which bears upon these mechanisms of resistance as well as strategies to circumvent them are discussed below. First, we discuss the general mechanisms of cellular drug resistance and then some specific examples in the sections that follow. Additionally, the important concept of resistance to multiple antineoplastic agents, resistance to specific classes of drugs, and resistance mechanisms unique to in vivo situations are discussed.

General Mechanisms of Drug Resistance

Experimental selection of drug resistance by repeated exposure to single antineoplastic agents will generally result in cross-resistance to some related agents of the same drug class. This phenomenon is explained on the basis of shared drug transport carriers, drug metabolizing pathways, and intracellular cytotoxic targets of these structurally and biochemically similar compounds. Generally, the resistant cells retain sensitivity to drugs of different classes with alternative mechanisms of cytotoxic action.1,2 Thus, cells selected for resistance to alkylating agents or antifolates will usually remain sensitive to unrelated drugs, such as anthracyclines. Exceptions include emergence of cross-resistance to multiple, apparently structurally and functionally unrelated drugs, to which the patient or cancer cells were never exposed during the initial drug treatment. Despite apparent differences in the families of drugs associated with multi-drug resistance (MDR) phenotypes, when the mechanisms underlying these phenotypes are identified, we frequently discover that the involved antineoplastic agents share common metabolic pathways, efflux transport systems, or sites of cytotoxic action. Conceptually then, the targets of MDR mechanisms are similar to the targets of single-agent resistance mechanisms.

In this section, we describe broadly defined processes related to drug resistance and a few specific examples. A more comprehensive discussion follows in the sections on resistance to specific classes of drugs.

Decreased Drug Accumulation

Decreased intracellular levels of cytotoxic agents is one of the most common mechanisms of drug resistance. This may result from decreased drug influx due to a defective carrier-mediated transport system. Decreased influx via a high affinity folate-transport system3 as well as via a reduced folate carrier4 is a well-described cause of methotrexate resistance.5,6 A deficient membrane transport system has similarly been identified in cells resistant to nitrogen mustard.7 Enhanced drug efflux may also lower intracellular steady state levels of drugs. Cells which have multiple resistance to antineoplastic drugs due to overexpression of the P-glycoprotein drug efflux pump (classic MDR) are important examples of this mechanism of resistance.8,9

Altered Drug Metabolism

Modified drug activation, drug inactivation, or cofactors can confer resistance to selected antineoplastic agents. For example, many antimetabolites and some alkylating agents (e.g., cyclophosphamide) are administered as prodrugs, which must be activated to their cytotoxic forms by the targeted tumor or by other tissues. Resistance to some nucleobase drugs has been associated with decreased conversion of these analogues to their cytotoxic nucleoside and nucleotide derivatives by kinases and phosphoribosyl transferase salvage enzymes.10,11 Similarly, cellular sensitivity to the cytotoxicity of the topoisomerase I poison, CPT-11, is in part governed by the level of carboxyesterase—an activity necessary to convert CPT-11 to its active metabolite, SN38.12,13 Furthermore, enhanced inactivation of pyrimidine and purine analogues by elevated deaminases has been linked to resistance toward these agents.14,15 Finally, cofactor levels may modify drug toxicity. For example, optimal formation of inhibitory complexes between 5-fluorodeoxyuridine monophosphate (FdUMP) and its target enzyme, thymidylate synthase, require the cofactor 5,10-methylene tetrahydrofolate.16

Increased Repair or Cellular Tolerance or Drug-Induced Damage

Cells contain multiple complex systems involved in the repair of membrane and DNA damage. Because such damage may occur as a direct or secondary consequence of cytotoxic drug action, altered intrinsic repair mechanisms can influence drug sensitivity. For example, resistance to cisplatin, a drug whose cytotoxic action is thought to involve intrastrand DNA cross-linkages (see below), has been associated with altered activities presumed to reflect increased DNA repair. Conversely, defects in mismatch repair are associated with tolerance to cisplatin-induced DNA damage.17 It is hypothesized that in this form of platinum resistance, the repair system is unable to recognize platinum-DNA adducts, and that this defect leads to the failure to activate the normal, appropriate programmed cell death (apoptotic) response.

Different classes of anticancer drugs initiate their cytotoxicities through a variety of primary molecular targets. However, increasingly, the view is held that most, if not all, cancer drugs ultimately effect cell death via common downstream signalling pathways associated with programmed cell death or apoptosis.18–22 Drug insults may lead to several alternative cellular responses, including cell cycle arrest and activation of repair processes, or active cell suicide by apoptosis. Mutations or alterated expression levels of the key genes regulating these alternative responses to drug-induced stress—genes that include p53, p21cip/WAF, and bcl2 family genes—can profoundly influence cellular sensitivity or resistance to cancer drugs.

Altered Drug Targets

The mechanisms of cell kill of several antineoplastic drugs involve interactions between the drug and an essential intracellular enzyme. These interactions result in alteration or inhibition of normal functions. Quantitative or qualitative changes in these enzyme targets of antineoplastic drugs can compromise drug efficacy. These changes have been demonstrated in several enzymes associated with drug resistance cells, including dihydrofolate reductase,23,24 thymidylate synthase,25 and topoisomerases I and II.26–32

Altered Gene Expression

The cellular mechanisms of drug resistance outlined above depend upon altered levels or function of key gene products. These alterations may result from changes which occur at any point along the pathways of gene expression and regulation. Indeed, multiple molecular processes have been shown to be involved in examples of drug resistance, including DNA mutation, deletion or amplification; altered transcriptional or post-transcriptional control of RNA levels; and altered post-translational modifications of proteins. The prevalence of these changes reflects the phenotypic and genetic instability of cancer cells under the selective, and perhaps mutagenic, pressures of xenobiotic toxin and drug exposure.

Resistance to Multiple Drugs

De novo and acquired cross-resistance to multiple antineoplastic agents can result from several alternative factors and processes. Accordingly, we have grouped the major patterns of crossresistance into several categories, on the basis of their presumed underlying mechanisms (Table 39.2). First, MDR patterns of cross-resistance are frequently associated with decreased drug accumulation, usually due to increased drug efflux. Classic MDR associated with resistance to drugs listed in Table 39.3 is mediated by P-glycoprotein (MDR1, P-170). More recently, a similar but distinct MDR phenotype has been attributed to the energy-dependent drug efflux activities of multidrug resistance protein (MRP) family members.33–36 Another overlapping but discrete resistance MDR phenotype is associated with increased expression of the recently isolated putative efflux transporter, breast cancer resistance protein (BCRP).37,38 MDR has also been described in association with overexpression of the lung resistance protein (LRP). The mechanism of LRP-associated resistance is unclear, and whether LRP alone is sufficient to confer resistance is unknown. It is speculated that as a major vault protein, LRP is involved in nucleocytoplasmic transport and cytoplasmic sequestration of drugs.39,40 Drug resistance defined by alterations in topoisomerases represents a third major category of MDR.26–32 Additionally, more speculative mechanisms of MDR mediated by nonspecific xenobiotic metabolizing enzymes, and cell-to-cell transfer of genetic information are discussed separately. As discussed below, there can be overlap among some of these mechanisms—for example, high level resistance to some drugs may depend on expression of both the phase II drug conjugating glutathione/glutathione S-transferase system and the MRP1 glutathione conjugate transporter.41

Table 39.2. Mechanisms of Multidrug Resistance (MDR).

Table 39.2

Mechanisms of Multidrug Resistance (MDR).

Table 39.3. Cross-Resistance Pattern of Classic (P-glycoprotein–mediated) MDR.

Table 39.3

Cross-Resistance Pattern of Classic (P-glycoprotein–mediated) MDR.

Classic (P-glycoprotein–dependent) MDR

An in vitro model of MDR was described by Biedler and co-workers three decades ago.42 In these studies, cultured cells selected for resistance by exposure to actinomycin D developed cross-resistance to a surprising array of structurally diverse compounds, including Vinca alkaloids, puromycin, daunomycin, and mitomycin C. Subsequently, induction of this pattern of cross-resistance has been observed by numerous investigators, who have selected cells in the presence of the same and other drugs. Generally, exposure of cells to any of the drugs (many of which are listed in Table 39.3) related to this MDR phenotype can result in cross-resistance to all other members of the phenotype.8,9 Drug transport studies using parental and MDR cells have demonstrated that the reduced cytotoxicity of these drugs is the result of decreased drug accumulation secondary to enhanced drug efflux.43,44 Furthermore, the emergence of MDR has been associated with increased levels of a membrane-bound glycoprotein, P-glycoprotein (P-170 or MDR1 protein).

Although it is widely accepted that P-glycoprotein mediates an energy-dependent decrease in drug accumulation, there is considerable debate on the precise mechanism(s) involved. Drugs associated with the classic MDR phenotype are generally freely permeable to the plasma membrane. Upon entry into the cytosol, they may be recognized by P-glycoprotein and exported back across the plasma membrane in association with adenosine triphosphate (ATP) hydrolysis.9 Another proposal, termed the “hydrophobic vacuum cleaner” model, suggests that the lipid-soluble drugs may be recognized by P-glycoprotein within the plasma membrane and expelled without ever entering the cytoplasm.9,45 Numerous other mechanisms of P-glycoprotein–dependent drug transport have been proposed including the lipid “flippase” model,46 or processes in which drug efflux is indirectly influenced by P-glycoprotein–mediated changes in membrane potential or chloride channels.47–50 Finally, it has been noted that if the freely diffusible, lipid-soluble drugs are the substrates of P-glycoprotein, then huge expenditures of energy would be required in order to maintain reduced drug accumulation in cells continually exposed to extracellular drug. To obviate this thermodynamic obstacle, it has been suggested that the true substrates of P-glycoprotein–mediated efflux may not be the parent drugs but rather the previously unidentified amphiphilic and membrane-impermeable drug conjugates formed within the cell.51

Regardless of the mechanistic details, the consensus view that P-glycoprotein is the energy-dependent drug efflux pump responsible for MDR is supported by pharmacologic, genetic, and biochemical data. First, the expression of P-glycoprotein is associated with concomitant increases in drug efflux and resistance which are sensitive to metabolic poisons. Furthermore, gene transfer experiments have shown that the expression of P-glycoprotein genes is sufficient to confer drug resistance.52,53 P-glycoproteins are encoded by members of a multi-gene family. Analyses of these mdr genes has revealed a striking sequence homology between P-glycoproteins and several bacterial transport proteins.54,55 The deduced amino acid sequences of P-glycoproteins predict the presence of two pairs of six transmembrane domains and two ATP-binding sites (Fig. 39.1). Photoaffinity labeling experiments have demonstrated direct binding of drugs to P-glycoprotein.56 Finally, the distribution of P-glycoprotein on the luminal surfaces of normal tissues including renal tubules, colon, small intestine, and bile canaliculi is consistent with its proposed role in excretory transport.57 Thus, P-glycoprotein appears to fulfill the requirements predicted of a membrane-bound energy-dependent drug exporter.

Figure 39.1. Models of P-glycoprotein and MRP1.

Figure 39.1

Models of P-glycoprotein and MRP1.

P-glycoprotein associated MDR is subject to significant phenotypic heterogeneity. The relative degree of cross-resistance to the drugs listed in Table 39.3 will vary depending on the cell line and the selecting drug. While the level of drug resistance is roughly correlated with the level of P-glycoprotein expression, protein and RNA levels may be disproportionately higher or lower than expected for the level of resistance observed. Similarly, the magnitude of the drug accumulation defect may appear insufficient to account for the degree of resistance. The phenotypic variability may result from the concomitant expression of alternative resistance mechanisms. Although, there are two human mdr genes, only mdr1 has been shown to confer drug resistance.8,9 Mutations in the coding region of the mdr1 gene have been reported to alter the relative resistance patterns of cells.58 Post-translational modifications of P-glycoprotein may also alter pump function. For example, P-glycoprotein can be phosphorylated by protein kinase C59,60 and by a novel membrane associated protein kinase.61 Specific sites of protein kinase C–mediated phosphorylation are clustered in the linker region between the two halves of P-glycoprotein.60 Transport studies on MDR cells treated with protein kinase C activators and inhibitors as well as with inhibitors of protein phosphatases show that increased phosphorylation of P-glycoprotein is associated with decreased vinblastine accumulation.60,62,63 These results indicate that P-glycoprotein phosphorylation status, as determined by the relative levels of opposing protein kinase and protein phosphatase activities, may influence drug efflux pump function, drug resistance, and MDR phenotypic diversity. Other cofactors involved in the augmentation of P-glycoprotein function have been proposed but not yet identified.51,64 Lastly, other mechanisms of drug resistance may coexist with classic MDR.

A thorough understanding of the regulation of P-glycoprotein production and the means to suppress its expression might significantly influence future cancer treatment strategies. Studies addressing this issue have shown that high levels of P-glycoprotein expression in vitro are often associated with mdr gene amplification and transcriptional activation.8,9 Increased expression of P-glycoprotein can also be stimulated by heat shock,65 heavy metals, cytotoxic drugs,66–68 regenerating liver,66,67 differentiating agents,69–71 and by repeated exposure to ionizing radiation.72 However, the responses to these treatments appear to vary between species and are cell line specific. Thus, predictable modulation of mdr gene expression is not yet possible. Under certain conditions in some cells, the mdr1 promoter activity can be regulated by altered expression of oncogenes (raf and ras) and the tumor suppressor gene, p53.73–77

A considerable literature has accumulated which concerns the importance of P-glycoprotein in human cancer. P-glycoprotein RNA or protein has been detected in tumor specimens derived from patients with acute and chronic leukemias,78–80 ovarian cancer,81 multiple myeloma,82 breast cancer,83,84 neuroblastoma,85 soft tissue sarcomas,86 renal cell carcinoma,87 and others.88 Although the numbers of patients with particular tumors in these studies were small, the results have tended to link P-glycoprotein expression with a history of prior therapy (usually with MDR-associated drugs) or toxin exposure, emergence of intrinsic or acquired drug resistance, and treatment outcome. Ma et al.78 reported that in two patients with acute nonlymphoblastic leukemia (ANLL), disease progression with treatment (including an anthracycline) was associated with increasing P-glycoprotein levels in leukemic blasts. In a study of 15 additional patients with ANLL, Sato et al79 found that P-glycoprotein was commonly present in leukemic blasts but more prevalent in blasts derived from patients of poor prognostic groups including those with a history of prior toxin exposure. More recently, three prospective studies have shown that increased P-glycoprotein in patients with acute myelogenous leukemia (AML) is associated with decreased complete remission rates and reduced remission duration with use of conventional chemotherapy.89–91 Although P-glycoprotein was frequently present in tumor specimens from both treated and untreated patients with neuroblastoma, P-glycoprotein RNA tended to be higher in patients treated with regimens that included doxorubicin than in untreated patients.85 Moreover, in patients with advanced neuroblastoma, P-glycoprotein expression has been strongly associated with aggressive biologic behavior, poor treatment response, and poor outcome.92 The impressive correlations between P-glycoprotein expression and aggressive neuroblastoma persisted even when the data were corrected, by multivariant analyses, for other confounding prognostic features. However, the significance of mdr1 expression in neuroblastomas is controversial as other data have suggested the opposite—that increased mdr1 expression is associated with more favorable clinical variables in patients with neuroblastoma.93 In tumor specimens obtained from patients with childhood ALL80 and soft tissue sarcomas,86 the presence of P-glycoprotein was associated with anthracycline pretreatment, increased rate of remission induction failure, and increased frequency of relapse. Over 400 tumor specimens were tested for P-glycoprotein RNA levels in a large study.88 Increased levels of P-glycoprotein RNA were more prevalent in tumors which tend to be intrinsically resistant to therapy (colon, renal, adrenal, hepatic, and pancreatic cancers) compared with intrinsically sensitive tumors. Furthermore, P-glycoprotein RNA was often increased in tumors at relapse (acute leukemias, breast cancer, neuroblastoma, pheochromocytoma, and nodular poorly differentiated lymphoma).

In general, the relationship between increased P-glycoprotein and adverse outcome in human cancers is strongest in hematologic malignancies. This correlation is particularly demonstrated in adult multiple myeloma, AML, and lymphoma, as well as in pediatric ALL.94–96 Moreover, efforts to reverse clinical resistance to chemotherapy using P-glycoprotein inhibitors (see below) have similarly been most promising in the treatment of selected hematologic malignancies.95,97–99 Among solid tumors, the relationship between P-glycoprotein expresssion and response to therapy is less convincing,100 although as noted, significant correlations between P-glycoprotein and adverse outcome in pediatric rhabdomyosarcoma and neuroblastoma have been reported.94

Additional and prospective studies will be required to fully evaluate the clinical significance of P-glycoprotein in human cancer. However, the available results indicate that P-glycoprotein overexpression is associated with clinical evidence of drug resistance and treatment failure in a significant number of patients—especially, selected groups with hematologic malignancies. P-glycoprotein determinations in clinical specimens need to be carefully standardized and the correlated outcomes and end points clearly defined.101 Such studies will help establish for which cancers the determination of P-glycoprotein levels in patients at diagnosis or relapse may have an important role in the design of treatment protocols.

Multidrug Resistance Protein Family

Similar phenotypes of multiple resistance to antineoplastic agents have been described that are associated with the expression of other membrane proteins. In many of these examples resistance occurs independently of P-glycoprotein expression.102–106 A distinct gene, mrp1 (multidrug resistance protein 1 or multidrug resistance-associated protein 1), was isolated from a doxorubicin-selected MDR lung cancer cell line.107 Except for the absence of P-glycoprotein expression, the phenotype of this cell line, which includes the property of reduced drug accumulation, was similar to classic MDR. The mrp1 gene encodes a 190 kDa transmembrane protein, whose structure is strikingly homologous to P-glycoprotein/MDR1 and other members of the ATP-binding cassette (ABC) transmembrane transporter proteins.107,108 Primary sequence analysis predicts the transmembrane structure shown in Figure 39.1. The structure, supported by immunochemical data, includes 11 plus 4 (or alternatively 11 plus 6) transmembrane domains with 2 cytosolic ATP-binding sites.109 Increased MRP1 expression is associated with MDR, and decreased MRP1 expression is associated with reversion to drug sensitivity. Gene transfer experiments have established that MRP1 can confer MDR to a variety of drugs including anthracyclines, epipodophyllotoxins, and Vinca alkaloids.110–112 Transport studies have indicated that MRP1 is involved in ATP-dependent efflux of some native natural product anticancer drugs. Additionally, MRP1 is an ATP-dependent transporter of a variety of anionic conjugates of drugs and other xenobiotics—conjugates that include glutathione conjugates, glucuronides, and sulfates.113–118 Thus, MRP1 is an important xenobiotic-conjugate transport pump that is involved in efflux detoxification of a wide range of cellular poisons, including anticancer drugs and their conjugates. The significance of these conjugate substrates is further discussed in a following section. In contrast to P-glycoprotein, whose substrates are generally lipophilic neutral or cationic compounds, MRP1 preferentially recognizes amphiphilic organic anions including the conjugates described above. While neutral, hydrophobic compounds such as vincristine are also substrates of MRP1, reduced glutathione is required for their transport.116,119 Although no covalent linkage between glutathione and vincristine is observed, it is believed that both glutathione and the neutral drug must be simultaneously present to effect efflux, and that they both may be co-transported by MRP1.

MRP1 is ubiquitously expressed in tumor and normal tissues.120–122 The importance of MRP1 overexpression in clinical drug resistance is unknown. However, because levels of MRP1 expression vary widely in tumor cells, MRP1 may be a significant mediator of drug resistance in human cancer.

There are at least five other human MRP isoforms identified.123,124 Among them, MRP2 (cMOAT) and MRP3 are also capable of supporting efflux detoxification of cancer drugs, including epipodophyllotoxins (MRP2 and 3), doxorubicin and cisplatin (MRP2).125,126 Recent results indicate that MRP1 and MRP2 are also able to confer resistance to the polyglutamatable antifolate, methotrexate.126,127 Unlike MRP1, which is expressed on the basolateral plasma membrane surface of polarized cells, MRP2 is normally targeted to the apical membrane surface of bile canalicular and renal tubular epithelium.128–130 MRP3 is localized to the basolateral surface in various tissues including the colon, liver, and pancreas.123,131,132 The roles of MRP2 and 3, as well as those of the less characterized isoforms MRP4, MRP5, and MRP6, in clinical drug resistance are presently speculative.

MDR Associated with Topoisomerase Poisons

Topoisomerases are nuclear enzymes which catalyze the formation of transient single- or double-stranded DNA breaks, facilitate the passage of DNA strands through these breaks, and promote rejoining of the DNA stands.133,134 As a consequence of these activities, topoisomerases are thought to be critical for DNA replication, transcription, and recombination. The cytotoxicity of many drugs which target topoisomerases, a class of drugs here termed topoisomerase poisons (Table 39.4), is thought to depend on the DNA cleavage activities of topoisomerases. There are two classes of mammalian enzymes, topoisomerases I and II. Topoisomerase I catalyzes the formation of single-stranded DNA breaks, while topoisomerases II (α and ß isoforms) catalyze both single- and double-stranded breaks. During the cleavage reactions reversible DNA-topoisomerase complexes (cleavable complexes) can be stabilized by interactions with topoisomerase poisons. The formation of these stabilized DNA-topoisomerase-drug complexes is thought to initiate the production of lethal DNA strand breaks. Of the chemotherapeutic drugs that affect topoisomerase activities, the topoisomerase II poisons have been the most important clinically. A partial list of these agents, which include DNA intercalating and nonintercalating drugs appears in Table 39.3. A growing list of useful topoisomerase I poisons are now available, including topotecan, CPT-11 (irinotecan), and SN38.

Table 39.4. Topoisomerase II Poisons.

Table 39.4

Topoisomerase II Poisons.

Several laboratories have described an MDR pattern characterized by resistance of cells to several or all of the drugs listed in Table 39.3.135,136 It is readily apparent that many of these topoisomerase II–targeting drugs are also members of the classic MDR phenotype (see Table 39.2). Hence, decreased drug accumulation via increased expression of P-glycoprotein or MRP1 represents a potential mechanism of resistance to these topoisomerase II poisons. However, a distinct pattern of the topoisomerase II–related MDR has been described that differs from the pattern of P-glycoprotein–associated MDR in several important ways. First, resistance to these drugs is not usually associated with reduced drug accumulation or P-glycoprotein expression. Exceptions may reflect the presence of multiple simultaneous mechanisms of resistance. Additionally, cells that display this topoisomerase II–related resistance phenotype are usually sensitive to antimicrotubule drugs associated with classic MDR, including Vinca alkaloids and colchicine, unless a concomitant drug transport or microtubule alteration exists. The mechanism of resistance to topoisomerase II poisons is thought to involve altered topoisomerase II activity. Both qualitative and quantitative changes in enzyme activity have been demonstrated in resistant cell lines. Reduced levels of topoisomerase activity have been associated with decreased drug-induced DNA strand breaks as well as reduced drug cytotoxicity.137,138 Other studies have implicated intrinsic changes in drug-induced catalytic properties or associated cofactors as the basis of drug resistance in some cells.28,139–141 The nature of the topoisomerase II alterations may influence the cross-resistance patterns observed. For example, cells which develop alterations in topoisomerase II following exposure to m-AMSA (amsacrine) may show cross-resistance to other intercalating topoisomerase II poisons but not to epipodophyllotoxins.140 Collectively, these data indicate that reduced topoisomerase protein levels or selectively altered enzyme activities influencing drug-enzyme interactions may render cells relatively more resistant to drugs by interfering with the formation of stable cleavable complexes and hence cytotoxic DNA strand breaks. Indeed, the normal downregulation of topoisomerase II in nondividing cells133 may explain the relative insensitivity to topoisomerase II poisons of some solid tumors containing a large proportion of quiescent cells. Finally, there are two mammalian isozymes of topoisomerase II, a 170 kDa form (topoisomerase IIα) and a 180 kDa form (topoisomerase IIß).142–144 These isozymes differ with respect to their regulation during the cell cycle145 and their relative sensitivities to topoisomerase II poisons.142,143 Hence, the relative levels of the specific topoisomerase II isozymes as well as the total topoisomerase II activity may be significant determinants of the sensitivity of tumor cells to topoisomerase II drugs.

The molecular bases of drug resistance associated with qualitatively altered topoisomerase II have been suggested in several reports.146 Point mutations leading to amino acid substitutions in topoisomerase IIα isolated from cells selected for resistance to topoisomerase II drugs have been described. These mutations are clustered within the conserved ATP-binding consensus sequences31,147–150 or near the Tyr 804 residue involved in covalent attachment of topoisomerase IIα to DNA.149,151 Although these topoisomerase IIa mutations are associated with drug resistance in intact cells and, in some cases, with altered enzymatic activities in vitro, the exact mechanism(s) of drug resistance and the relationship of these mutations to a specifically altered enzymatic property are incompletely understood. Moreover, the relevance for clinical drug resistance of these topoisomerase IIα mutations identified in experimentally drug-selected resistant cell lines is unknown. Indeed, one study of topoisomerase IIα derived from leukemic blasts of 15 relapsed patients failed to identify mutations in either of the above two regions implicated in experimental drug resistance.149 Other qualitative alterations in topoisomerase II activity and structure have been described in cell lines selected for resistance to topoisomerase II poisons. These include a selective decrease in nuclear matrix–associated topoisomerase II152 and a truncated form of topoisomerase IIα.29 In some resistant cell lines, cytoplasmic or membrane components may be responsible for the altered topoisomerase II activity implicated in the emergence of drug resistance.153 Alternatively, altered subcellular localization of topoisomerase II isoforms146,154,155 or altered post-translational phosphorylation146,156 have been reported in association with some etoposide-resistant cell lines.

The cytotoxicity of topoisomerase II poisons is believed to depend on the formation of DNA strand breaks secondary to stabilization of the reversible enzyme-DNA cleavable complex.133 It is thought that a collision between the complex and the DNA replication fork is necessary to generate the cytotoxic lesions. If DNA replication is delayed or altered until after the drug is cleared, the cleavable complex can be reversed and the cytotoxic lesion does not form.146 Thus, altered DNA replication or repair timing could also mediate topoisomerase II poison resistance.

A new family of drugs targeting topoisomerase II function have emerged that include fostriecin, merbarone, aclarubicin, and bis (2,6-dioxopiperazine) derivatives (e.g., ICRF193 and ICRF 187). In contrast to the topoisomerase II poisons that stablize cleavable complexes (above and Table 39.4), this new family of drugs target the catalytic cycle of topoisomerase II activity in which DNA strands are intact. As the toxicity of these “catalytic inhibitors” is independent of cleavable complex stablization, cross-resistance with the topoisomerse II poisons is less likely.146,157,158

The cytotoxic agent, camptothecin has been shown to enhance topoisomerase I–mediated strand breaks. Earlier, host toxicity prohibited the clinical use of such topoisomerase I poisons. However, the prospect of less toxic analogues of this drug that maintain a high level of activity against topoisomerase I–rich human cancer cells has renewed interest in the clinical application of this class of compounds.159 Consequently, the emergence of resistance to these agents may become an increasingly important consideration. There are reports of topoisomerase I mutations derived from cell lines selected for resistance to camptothecin or its derivative, CPT-11.30,160,161 In two of these resistant cell lines, the mutant enzyme has altered topoisomerase I activity with a reduced capacity to mediate camptothecin-induced DNA strand breaks.160–162

MDR Associated with Altered Expression of Drug Metabolizing Enzymes and Drug-Conjugate Export Pumps

The manner in which cells metabolize cancer drugs and other xenobiotics is often described as three phases of detoxifications (Fig. 39.2).163 While none of these phases are obligatory steps in the metabolism of every drug, the concept illustrated in Fig. 39.2 represents a useful framework with which to view cellular detoxification mechanisms. Alterations in any of these three phases can influence the sensitivity or resistance to a particular drug or xenobiotic toxin. Phase I metabolism is mediated by cytochome P450 mixed function oxidases. Generally, the drug or xenobiotic is rendered a more electrophilic, reactive intermediate—a process that may enhance toxicity. These metabolites, or the unmodified drug, may then be converted to a less reactive, presumably less toxic, form in phase II reactions. Phase II detoxifications include the formation of drug/xenobiotic conjugations with glutathione (GSH), glucuronic acid, or sulfate—reactions that are catalyzed by multiple isozymes each of glutathione S-transferase (GST), UDP-glucuronosyl transferase, and sulfatase, respectively.164–168 Phase III detoxification consists of export of the parent drug/xenobiotic or its metabolites by energy-dependent transmembrane efflux pumps including P-glycoprotein, MRP family members, and BCRP, described above.

Figure 39.2. Phase I, II, and III drug detoxification.

Figure 39.2

Phase I, II, and III drug detoxification.

Frequently, in cellular and animal models of drug or xenobiotic resistance, a coordinated downregulation of phase I drugactivating enzymes and an upregulation of specific phase II drug-conjugating enzymes is observed.66,67,169,170 Such a programmed cellular stress response offers a versatile, generalized protective mechanism against exposure to a variety of exogenous toxins.

Of the phase II enzymes, the GSTs have been the most extensively studied.

GSTs164,165 comprise multiple soluble and membraneassociated isozymes, which catalyze the conjugation of electrophilic, hydrophobic compounds (R-X) with the thiol, GSH:

Image ch39e1.jpg

Circumstantial evidence has linked the increase in specific GST isozymes or bulk GST activity in cells with resistance to alkylating agents, doxorubicin, and other drugs.164,165,171–174 However, direct evidence that GSTs are responsible for altering drug sensitivities is limited. Another catalytic activity, selenium-independent glutathione peroxidase activity, has been attributed to some isozymes of GST:

Image ch39e2.jpg

This and other GST-mediated reactions are of interest because of their potential to detoxify oxidative damage to membranes and DNA.

Studies using cell-free preparations of GSTs have identified a limited number of antineoplastic drug substrates of these enzymes. These drugs and other substrates possibly associated with drug mediated-oxidative damage are listed in Table 39.5. Whether GST levels in tumor cells are sufficient to detoxify antineoplastic drugs to a clinically significant extent is a matter of considerable debate. Several cancer drugs, particularly reactive electrophilic alkylating agents, can form conjugates with glutathione—both spontaneously and in enzyme-catalyzed reactions.175–182 However, despite these catalytic activities, the role of GSTs in drug resistance remains uncertain due to inconsistent results from different laboratories.173,182–192 Indeed, some investigators have found an association between cellular resistance to some anticancer drugs and expression of a particular isozyme of GST, whereas others have found no such association.

Table 39.5. Some Important Substrates of GSTs Related to Drug Detoxification and Repair of Drug-Mediated Damage.

Table 39.5

Some Important Substrates of GSTs Related to Drug Detoxification and Repair of Drug-Mediated Damage.

The importance of drug/xenobiotic-conjugate transporters for cellular export and detoxification of certain compounds has been increasingly appreciated. Conjugation frequently renders the parent drug more hydrophilc and less able to diffuse the plasma membrane—trapping the drug within the cell. While conjugation with glutathionyl or glucuronosyl groups may render some drugs less toxic, these drug conjugates themselves may retain significant toxicity. For example, the glutathione conjugate formed with cisplatin is itself toxic and an inhibitor of protein synthesis.193 Moreover, drug conjugates may inhibit their conjugating enzyme(s).194 Thus, the relative resistance of cells expressing drug metabolizing enzymes may depend on cellular levels of drug conjugate transporters, including the glutathione conjugate transporters,163,195 such as the MRP family proteins.117,118 Indeed, recent results using model cell lines have demonstrated that combined expression of specific isozymes of GST with MRP1 was necessary to achieve full protection from the toxicities of the cancer drug, chlorambucil,41 or the carcinogen, 4-nitroquinoline 1-oxide.196 In these studies, the expression of either GST or MRP1 alone provided little, if any, protection from toxicity—a finding that illustrates the synergistic interaction of phase II and phase III detoxification processes in the emergence of resistance to some drugs and other xenobiotics.

Emergence of Refractory Tumors Associated with Multiple Resistance Mechanisms

The backbone of many treatment protocols designed to circumvent the proliferation of resistant tumor cells is the administration of multiple drugs with different structural properties and mechanisms of action. The approach supposes that if enough carefully selected drugs are delivered at optimal doses and intervals, individual clones of cells resistant to one class of drug will be effectively killed by another drug in the regimen. The rapid appearance of refractory tumors despite an initially favorable cytoreductive response suggests that the emergence of tumor cell clones with multiple resistance is a common clinical occurrence. We have seen how a single genetic change, such as increased P-glycoprotein or altered topoisomerase II, can mediate cross-resistance to several, but not all, useful antineoplastic drugs. Although these mechanisms provide a molecular explanation for broad-spectrum resistance, it is clear that many refractory tumor clones must simultaneously develop multiple resistance mechanisms. These mechanisms may arise from multiple independent genetic changes in single cell clones or, as suggested by Cadman, from cell-to-cell transfer of genetic information.197

Resistance to Anticancer Genotoxic Treatments Related to Suppression of Apoptotic Pathways

Chemotherapeutic drugs initiate cytotoxicity through their interactions with a variety of molecular targets. For example, epipodophyllotoxins attack topoisomerases II, alkylating agents form adducts with the nucleophilic centers of DNA and proteins, and methotrexate inhibits dihydrofolate reductase, resulting in reduced pyrimidine and purine synthesis. Despite these varied primary targets, most, if not all, cancer drugs effect cell death, at least partially, via downstream events—events that converge upon pathways mediating programmed cell death or apoptosis.

Apoptosis refers to an orderly cellular death program with predictable molecular and morphologic changes, including nuclear pyknosis and fragmentation, internucleosomal endonucleolytic DNA fragmentation, formation of cytoplasmic apoptotic bodies, and plasma membrane changes, such as transposition of phosphatidylserine to the extracellular surface.198 The process is conveniently conceptualized in three phases. First, initiation of apoptosis (e.g., secondary to chemotherapy-mediated DNA damage) is characterized by its reversibility. Second, commitment represents the irreversible decision to complete the death program. The commitment phase may involve mitochondrial changes including the permeability phase transition and the release of cytochrome c and apoptosis inducing factor (AIF)–changes that are hallmarks of apoptosis. Finally, the degradation or execution phase includes downstream events, including DNA fragmentation and morphologic changes. Prior to commitment, apoptosis can be modulated by regulatory elements, such as p53 and the Bcl-2 family proteins.20,198–200 Clearly, such regulation of the apoptotic response can have profound effects on the outcome of chemotherapy and is an area of active investigation germane to drug resistance and sensitivity.

Although apoptosis may be either p53-dependent or independent, frequently the cellular response to DNA damage is regulated by p53.200 As shown in a simplified diagram (Fig. 39.3), cancer therapy–induced DNA damage is sensed by p53 by incompletely understood mechanisms. Depending on the particular cell type and damage, p53 may then initiate one of two possible pathways: apoptosis or a process of cell cycle arrest and repair. In cells where the apoptotic pathway dominates, changes which cause dysfunction or deletion of p53 are likely to result in reduced apoptosis in response to DNA damage, leading to relative resistance and cell survival with damage. Indeed, p53 has been shown to be required for radiation- and etoposide-induced apoptosis in thymocytes, whereas lymphoma cell lines expressing mutant p53 were relatively resistant to DNA damaging agents.201–203 In cells where the p53-dependent cell cycle arrest and repair response dominates, deletion or mutation of p53 might be expected to result in decreased cell cycle arrest and repair leading to accumulated DNA damage and hence sensitivity to the chemotherapeutic agent.200

Figure 39.3. Alternative cellular responses to cancer therapeutic stress.

Figure 39.3

Alternative cellular responses to cancer therapeutic stress.

The mitogen-activated protein kinase (MAPK)–signaling cascades are involved in the regulation of cellular response to exogenous factors, including geno- and cytotoxic cancer treatments.204 The extracellular stimulus regulated kinase (ERK1/2) pathway is implicated in the proliferative response to growth factors. In cells treated with potentially cytotoxic stressors, such as radiation or anticancer drugs, the p38 and stress-activated/c-Jun N-terminal protein kinase (SAPK/JNK) pathways are implicated in mediating cell cycle arrest or apoptosis. Modulation of these interacting pathways can have a profound effect on whether a cancer cell responds to cytotoxin challenge by activation of apoptosis or by cell cycle arrest, repair, and hence relative resistance to treatment.204,205

The Bcl-2 family proteins comprise several important regulators of apoptosis. Although their mechanism(s) of action is incompletely known, the balance of expressed antiapoptotic family members (Bcl-2, Bcl-XL, Bcl-w, A1, and Mcl-1) and proapoptotic family members (Bax, Bak, Bad, Bik, and Bid) can infuence the relative sensitivity of cells to toxic stressors.20,199 Indeed, increased Bcl-2 and its antiapoptotic homologues are associated with increased resistance of lymphoid cells to the cytotoxic effects of corticosteroids, radiation, and DNA damage from chemotherapeutic drugs.199,206–210 It has been proposed that increased levels of antiapoptotic proteins Bcl-2 or Bcl-XL may result in reduced sensitivity to DNA-damaging cancer drugs—a resistance phenotype characterized by cell survival, with increased tolerance of DNA damage and genomic stability. This genomic instability may further lead to mutations activating additional resistance mechanisms and conferring more aggressive tumor behavior.20 Thus, the expression of mutant and wild type p53, Bcl-2 family members, MAPK family members, and other proteins associated with the control of apoptosis may contribute significantly to the clinical sensitivity of tumor cells. These proteins are the targets of investigational agents that may become important in future strategies to overcome clinical drug resistance.

Resistance Factors Unique to Tumor Cells In Vivo: Host-Tumor-Drug Interactions

The failure of chemotherapy to eradicate a tumor in vivo despite exquisite sensitivity to drug in vitro may be due to anatomic or pharmacologic sanctuaries. For example, the failure to deliver adequate amounts of many drugs across blood-brain and -testicular barriers probably accounts for the relatively high frequency of acute lymphoblastic leukemia relapse at these sites.211 In large solid tumors, chemotherapeutic failures are frequently attributed to decreased drug delivery to a tumor that has overgrown its vascular supply. Additionally, development of acidosis and hypoxia in poorly perfused areas of large tumors may interfere with the cytoxicity of some drugs. Altered prodrug activation by liver or other normal tissues may profoundly influence the efficacy of drugs such as cyclophosphamide.

A report by Teicher and colleagues212 suggests that tumor-host interactions may influence drug pharmacokinetics and tumor resistance in unexpected ways. In this study, tumor cells selected for cyclophosphamide and cisplatin resistance in vivo were normally sensitive to drugs in vitro. When the tumor cells were reimplanted into nude mice, in vivo drug resistance was restored. These results suggest that resistant tumors may harbor cellular resistance factors which are operative only in conjunction with host factors and therefore mediate resistance by altered drug pharmacokinetics in vivo only. If this novel host-dependent mechanism of tumor resistance proves common, these results would provide one explanation for the failure of conventional in vitro testing to predict clinical responsiveness in all cases.

Approaches to Overcoming Resistance to Specific Groups of Drugs

Approaches to overcome chemotherapeutic failures include efforts to prevent the emergence of drug resistance (Table 39.6). An appreciation of factors which induce resistance mechanisms may lead to the choice of more efficacious treatment regimens. For example, drugs which may have only sporadic activity against a specific tumor yet are likely to select for cross-resistance to more active agents would be avoided. It is hoped that aggressive combination chemotherapy with non–cross-reacting drugs will eliminate tumor rapidly enough to prevent the selection of tumor cell clones with multiple resistance. Failures of the preventive approach require the incorporation of specific measures aimed at reversing or circumventing drug resistance.

Table 39.6. Approaches to Overcome or Circumvent Drug Resistance.

Table 39.6

Approaches to Overcome or Circumvent Drug Resistance.

Drugs Associated with P-Glycoprotein–and MRP1-Mediated Resistance

Prior to the original descriptions of P-glycoprotein, Tsuruo and co-workers noted that treatment with verapamil of leukemia cells made drug resistant by selection in vincristine or doxorubicin could partially restore antineoplastic drug sensitivity.213 Furthermore, this verapamil-enhanced antineoplastic cytotoxicity, which was specific for drug-resistant but not-sensitive parental cells, was associated with increased accumulation of vincristine and doxorubicin. These results suggested that in the drug-resistant cells, vincristine and doxorubicin share a common transport system which is sensitive to modulation by verapamil. This transport system has now been identified as the P-glycoprotein drug efflux pump. Subsequently, numerous agents have been studied that can partially reverse the drug accumulation defects in classically multidrug-resistant cells, including several calcium channel blockers, calmodulin inhibitors such as phenothiazines, cyclosporin A, and cyclosporin derivatives, and other drugs.214–220 Although the mechanism(s) by which these agents reverse MDR is incompletely understood, it is believed that direct interactions between these agents and P-glycoprotein interfere with antineoplastic drug efflux activity. Since a considerable clinical experience in the use of MDR-reversing agents has existed for the treatment of other disorders, these agents have been included in several clinical trials designed to enhance the antitumor activity of conventional cancer drugs in refractory human neoplasms.

Several clinical trials have used verapamil as a MDRmodifying agent. Some efficacy with these regimens has been reported—especially in the treatment of hematologic malignancies. In one study, verapamil in combination with etoposide resulted in 8 of 11 partial responses in pediatric patients with leukemias refractory to MDR drugs.221 However, the levels of P-glycoprotein in these tumors was not assessed. Thus, the relationship between mdr gene expression and the efficacy of the reversing agent could not be determined. To address this issue, Dalton and collegues examined 8 patients with myelomas and lymphomas refractory to regimens containing vincristine and doxorubicin.82 Patient tumors were analyzed for the presence of P-glycoprotein RNA and protein, as well as for their responses to treatment regimens consisting of verapamil administered with vincristine, doxorubicin, and dexamethasone. Three patient tumors responded to the verapamil-containing regimens (2 transient PRs and I transient CR), and all of these responding tumors were P-glycoprotein positive. A study involving patients with lymphomas demonstrated that P-glycoprotein expression was rare in tumors from newly diagnosed patients but common in refractory tumors from previously treated patients.222 Following treatment with verapamil in combination with doxorubicin- and vincristine-containing regimens, a 72% response rate (28% CR) was observed in refractory patients. Verapamil-containing regimens also showed some efficacy in the treatment with vincristine and doxorubicin of patients with refractory multiple myelomas (5 of 22 patients showed PRs). In this study, a relationship between the administration of verapamil and the reversal of MDR was suggested by the finding that 4 of 10 patient tumors that tested positive for mdr1 expression responded whereas none of 5 patient tumors that tested negative for mdr1 expression responded.223 In contrast, the inclusion of verapamil in treatment regimens for colorectal or refractory ovarian cancer has not been effective in enhancing clinical responses to chemotherapy.224,225 A major factor limiting the usefulness of verapamil as an MDR-reversing agent is dose-limiting cardiac toxicity. It has, therefore, been difficult to achieve clinical levels of verapamil that are predicted, by in vitro testing, to be necessary for optimal MDR reversal—even when the less cardiotoxic D-isomer of verapamil is used.226

While there have been several encouraging studies showing the efficacy of P-glycoprotein/mdr1 inhibitors in murine models of MDR,227–234 most promising clinical trials have been confined to those treating refractory or relapsed hematologic malignancies.222,235,236 Studies involving the use of MDR-reversing agents in the treatment of solid human tumors have been generally disappointing.234

The usefulness of some earlier MDR-reversing agents, such as verapamil, are limited by their relatively low potency (high Ki toward inhibition of P-glycroprotein) and toxicity.237 Indeed, verapamil has significant cardiotoxicity. Cyclosporin A is an immunosuppressant and has significant effects on the metabolism of some drugs. Although cyclosporin A showed considerable promise in the treatment of refractory tumors, such as multiple myeloma,239 it also alters cancer drug pharmacokinetics by slowing renal and nonrenal drug clearance.237 Thus, it is difficult to ascertain whether the role of cyclosporin A in treatment efficacy involves inhibition of tumor P-glycoprotein or pharmacokinetic side effects. Quinidine is a relatively weak inhibitor of P-glycoprotein, but its diasteriomer, quinine, showed some efficacy in acute leukemia.239 Later generation MDR1-reversing agents were designed to be less toxic. For example, when compared with l-verapamil, the r-verapamil isomer has reduced cardiac effects but is an equally potent inhibitor of P-glycoprotein. This drug has shown some effect in the treatment of patients with refractory lymphoma.234 More recently, inhibitors, such as PSC 833, a cyclosporin D analogue, offer high inhibitory potency (low Ki toward P-glycoprotein) without immunosuppression.219

In addition to their actions on P-glycoprotein–positive tumor cells, MDR-reversing agents can have profound effects on the pharmacokinetics and pharmacodynamics of cytotoxic drugs associated with MDR.220,240 As noted above, marked increases in the area under the curve levels, decreased renal and nonrenal clearances, and increased volumes of distribution of etoposide have been observed in patients concomitantly treated with cyclosporin A. The reason for these effects is unknown, but it is suggested they are due to the action of cyclosporins on normal tissues—such as renal, biliary, and endothelial cells—possibly via cyclosporin interactions with the P-glycoprotein resident within these normal tissues. Toxicities of MDR-associated drugs, such as myelosuppression, may be enhanced when administered with reversing agents. These toxicities necessitate appropriate reduction in the dosage of cytotoxic drugs when they are used in combination with cyclosporins. Because P-glycoprotein is found at high levels in CNS endothelium and contributes to the blood-brain barrier,241–243 concomitant administration of MDR-associated chemotherapeutic drugs and P-glycoprotein inhibitors may also enhance neurotoxicities. These pharmacologic issues must be carefully considered in future clinical trials.

Collectively, these trials suggest that the use of MDR-reversing agents may be of some benefit to selected patients with P-glycoprotein–positive refractory tumors. Additional clinical trials are needed before such reversing drugs can be recommended in standard regimens, to clearly establish a correlation between improved antitumor response, using MDR-reversing agents and the presence of P-glycoprotein in those tumors. Moreover, the pharmacodynamic influence of agents, such as cyclosporins, on cytotoxic drugs must be carefully defined in order to achieve appropriate cytotoxic drug dosing. It is necessary to continue the search for reversing agents with improved efficacy and decreased toxicities as well as to determine optimal dosages and schedules.

Alternative strategies for reversing P-glycoprotein–mediated MDR include the use of monoclonal antibodies directed against extracellular epitopes of P-glycoprotein,244 anti–P-glycoprotein antibody-toxin conjugates that target P-glycoprotein expressing MDR tumor cells,245,246 or anti–P-glycoprotein antibodies engineered to recruit activated T-lymphocytes for the cytolysis of P-glycoprotein expressing tumor cells.247 Other approaches to reversing P-glycoprotein–mediated MDR include antisense and ribozyme nucleotides directed against MDR1 mRNA.234 It remains to be determined whether considerable obstacles to the clinical application of inhibitory polynucleotides can be overcome. All the approaches to inhibiting or targeting P-glycoprotein expression may be limited due to the normal expression of this protein pump in normal tissues, including kidney, liver, colon, and endothelial cell of the CNS.

Similar approaches for reversing MRP family–mediated MDR are possible. A number of compounds have been shown to inhibit MRP1-mediated efflux activity, including the organic acids probenicid and sulfinpyrazone (110), the LTD4 antagonist MK571, cyclosporin A, and the cyclosporin derivative, PSC 833.248 Finally, MRP1-mediated transport of some drugs is dependent on intracellular glutathione either as a noncovalent cofactor116,119 or as a moiety covalently linked, nonenzymatically or by GST, to some electrophilic anticancer drugs.41,249 Thus, depletion of tumor cell glutathione or inhibition of GST (see below) offer potential strategies for secondarily reversing MRP1-mediated drug resistance. Some substrates of MRP1 are glucuronide and sulfate derivatives of the parent drug.113,114,250,251 Thus, selective inhibition of tumor cell UDP-glucuronosyl transferases or sulfotransferases could also represent a future avenue for secondary reversal of MRP1-associated drug resistance.

Topoisomerase II Poisons

As discussed above, resistance to topoisomerase II poisons may occur as a consequence of Pglycoprotein overexpression or altered topoisomerase II activities. However, neither of these mechanisms will necessarily result in cross resistance to all the topoisomerase II–directed drugs listed in Table 39.3. For example, resistance to epipodophyllotoxins and anthracyclines on the basis of increased P-glycoprotein is not usually associated with resistance to the acridine derivative, amsacrine. Conversely, resistance to amsacrine and other intercalating drugs due to alterations in topoisomerase II protein is not always associated with resistance to the nonintercalating, epipodophyllotoxin class of topoisomerase II poisons.140 Therefore, these data derived from in vitro studies suggest a rationale for administering an alternative class of topoisomerase II poison in selected cases of clinical resistance to another class of topoisomerase II–directed drug. Additionally, tumor cells resistant to classic topoisomerase II poisons (see Table 39.4) frequently retain sensitivity to the cytotoxicities of the novel class of topoisomerase II–catalytic inhibitors (fostriecin, merbarone, aclarubicin, and bis [2,6-dioxopoperazines]).146,157,158 This class of topoisomerase-directed drug offers an alternative for the treatment of topoisomerase poison–resistant tumors. Finally, structural analogues of parent topoisomerase II poisons may overcome resistance based on altered topoisomerase II.252,253

Resistance to Free Radical–Mediated Drug Cytotoxicity

Several antineoplastic agents form free radical intermediates that are thought to contribute to drug cytotoxicity. Anthracyclines, such as doxorubicin, are among the most important members of this class of compound. While DNA intercalating anthracyclines can damage cells by multiple mechanisms, including inhibition of nucleic acid synthesis, induction of topoisomerase II-mediated DNA strand breaks, and perturbation of cell membranes, these quinone-hydroquinone compounds can also generate toxic free radical species that may cause cell death.254–256 The semiquinone radical so generated may either form a covalently binding free radical derivative or, in the presence of oxygen, may be reoxidized to the quinone species in a reaction producing superoxide anion. Decomposition of hydrogen peroxide formed by dismutation of superoxide anion produces the highly reactive hydroxyl radical, which may directly damage DNA, lipid, and protein. Thus, cellular factors that limit hydrogen peroxide production or repair peroxidative damage to macromolecules could theoretically confer some resistance to anthracyclines.

Several pathways may contribute to protection of tumor cells from anthracycline-mediated free radical damage. First, superoxide anion formation is limited in poorly vascularized, relatively hypoxemic tissues, such as may exist in the centers of large solid tumors. Second, increased intracellular levels of catalase and glutathione peroxidase (GSHPx) can deplete hydrogen peroxide, thus reducing the formation of toxic hydroxyl radicals. Indeed, in comparing parental and MDR MCF7 cells, Sinha and co-workers have reported an association between increased GSHPx activity and reduced doxorubicin-stimulated hydroxyl radical formation.257 Furthermore, lowering GSHPx activity by depleting the enzyme’s cosubstrate, glutathione (GSH), resulted in enhanced doxorubicin-dependent free radical formation and cytotoxicity.258 Additionally, Kramer and colleagues found that GSH depletion with buthionine sulfoximine (BSO) could partially restore the doxorubicin sensitivity of MDR MCF7 cells, presumably by interfering with GSH-dependent reactions, including those catalyzed by GSHPx.259 While these results are consistent with the importance of hydrogen peroxide and hydroxyl radical formation in anthracycline cytotoxicity in MCF-7 cells, other investigators have noted that increased catalase, GSH, and GSHPx levels are not always protective of some cells from doxorubicin-mediated damage.260 Finally, increased repair of peroxidative damage to DNA and unsaturated lipids represents another potentially protective mechanism against doxorubicin-dependent hydroxyl radical toxicity. For example, some isozymes of GST exhibit significant lipid hydroperoxidase activity and may also contain limited DNA hydroperoxidase activity.261 Additionally, the highly toxic 4-hydroxy alkenals formed from the decomposition of lipid hydroperoxides are relatively good substrates for some GSTs.262 Thus, overexpression of particular GST isozymes could conceivably contribute to doxorubicin resistance.

The relative importance of free radical generation in tumor cell kill is unknown, and the protective mechanisms outlined above are speculative. Nevertheless, the GSH-dependent detoxification pathways are of particular interest as they are subject to pharmacologic manipulation. GSHPx and GST activities can be secondarily reduced by depleting tissue GSH with BSO treatment. Furthermore, the activity of GSTs can be inhibited by the administration of competitive substrates, such as ethracrynic acid.263 Such clinical manipulations may enhance tumorcidal activity of doxorubicin but must be viewed cautiously as they may also potentiate drug toxicity toward normal tissues.

Alkylating Agents and Platinum Compounds

Resistance to alkylating agents and platinum compounds can be described by at least three broad mechanistic categories including decreased drug accumulation, increased drug inactivation, and enhanced repair of DNA damage.17,264–266 Additionally, the nature of the tumor cells’ response to alkylating agent damage—whether primarily apoptosis, repair, or survival with damage—will contribute significantly to the outcome of alkylating agent treatment. Preclinical studies have indicated that all these mechanisms may be circumvented, at least partially, by pharmacologic manipulations. Reactions of electrophilic alkylating agents with thiol-containing compounds represent relatively general mechanisms of antineoplastic inactivation or detoxification. For example, GSH forms conjugate with a variety of alkylating agents in both nonenzymatic and in GSTdependent reactions. Table 39.5 lists some of the compounds whose conjugation with GSH is catalyzed by GSTs in vitro.173 Several laboratories have demonstrated an association between increased bulk GST levels or specific GST isozymes with resistance to drugs such as nitrosoureas,267 chlorambucil, and other nitrogen mustards.184,188,268–270 Additionally, increased GSH levels have been correlated with resistance to alkylating agents and cisplatin.271,272 While the electrophilic cisplatin compound can react directly with GSH, it is unknown whether GSTs can catalyze this reaction. This issue is unresolved due to conflicting results which show a correlation between elevated expression of the pi isozyme of GST and resistance to cisplatin in some cells273,274 but not others.187 Perhaps more relevant to the issue of cisplatin resistance is the finding that glutathionyl-platinum complexes, which are themselves toxic, are exported by an ATP-dependent pump probably identical to one of the glutathione conjugate pumps described previously.193 Thus, these drug exporters should be considered in the design of treatments and formulation of strategies to enhance cisplatin efficacy.

The correlations between GSH or GST levels and drug resistance are variable. Indeed, some investigators have been unable to demonstrate a relationship between the overexpression of multiple isozymes of GST and antineoplastic resistance.64,186,187,191 In other studies which have compared paired parental and resistant cell lines, the magnitude of alkylating agent resistance associated with increased GST activity is often modest. As noted above, for some drugs such as chlorambucil, the coexpression of a glutathione conjugate efflux transporter appears to be required for the emergence of GST-mediated resistance in the MCF7 cell model system.41 While the clinical importance of GST and GSH in alkylating resistance is accordingly debated, existing preclinical data has prompted phase I trials using GST inhibitors or the GSH synthesis inhibitor, BSO, in conjuction with alkylating agents.

Aldehyde dehydrogenase is another drug-metabolizing enzyme that has been linked to cyclophosphamide-derivative resistance in murine and human models of drug resistance.275–277 This enzyme converts a metabolite of cyclophosphamide, aldophosphamide, to the inactive compound, carboxyphosphamide, thereby preventing the decomposition of aldophosphamide to its cytotoxic derivative, phosphoramide mustard. Increased expression of aldehyde dehydrogenase has been associated with resistance to cyclophosphamide in vitro. Whether inhibitors of aldehyde dehydrogenase, such as disulfiram and diethylaminobenzaldehyde, can be used therapeutically to enhance the antitumor effect of cyclophosphamide without undue host toxicity remains to be explored.

Cisplatin toxicity is thought to be mediated primarily by the formation of lethal intrastrand DNA cross-links. Several reports have suggested that either increased DNA repair or tolerance of DNA damage is associated with resistance to this compound. In a murine leukemia model, cells selected for cisplatin resistance showed enhanced ability to repair cisplatin-induced intrastrand DNA cross-links.278,279 Aphidicolin can inhibit an enzyme implicated in DNA repair, DNA polymerase alpha. Treatment of ovarian carcinoma cells with aphidicolin potentiated the toxicity of cisplatin in resistant but not sensitive cells.280 These results suggest that the coadministration of DNA polymerase alpha inhibitors with cisplatin may be useful in overcoming cisplatin resistance. Also implicated in platinum sensitivity and resistance are alterations in mismatch repair or of regulators of apoptosis, such as Bcl-2, Bax, p21, or p53.17 Modulation of these pathways by therapeutic agents now in development represents an emerging strategy for overcoming resistance to platinum and other alkylating compounds.


The antimetabolites are a clinically important group of cancer drugs used in the treatment of a variety of solid tumors and hematologic malignancies. The cytotoxicities of the antimetabolites stem from their ability to interfere with key enzymatic steps in nucleic acid metabolism. The discussion which follows concerns three particularly well-studied compounds, the antifolate, methotrexate (MTX), and the pyrimidine analogues, 5-fluorouriacil (5-FU) and cytosine arabinoside (ara-C, 1-ß-D-arabinofuranosylcytosine, cytarabine). Strategies designed to overcome the multiple described mechanisms of cellular resistance to these compounds include dose escalation, pharmacologic manipulation of drug metabolism, and rational design of new antimetabolites.281

The clinically important antifolate, MTX displays significant tumoricidal activity against a variety of human neoplasms, such as acute leukemia, osteogenic sarcoma, choriocarcinoma, breast cancer, head and neck cancers, and others.282 Consideration of MTX metabolism and sites of action (Fig. 39.4) serves as the basis for understanding mechanisms of methotrexate resistance. Following uptake by the folate transport systems, MTX can bind avidly to and inhibit its primary enzyme target, dihydrofolate reductase (DHFR). In the presence of adequate thymidylate synthase activity, inhibition of DHFR results in depletion of the reduced folate pools essential for thymidylate and de novo purine synthesis. The cytotoxicity of MTX is significantly influenced by intracellular polyglutamation. MTX polyglutamates are retained preferentially by cells and bind more effectively to DHFR. Additionally, these polyglutamyl derivatives can inhibit other folate-dependent enzymes, including thymidylate synthase and 5-aminoimadazole-4-carboxamide riboneucleoside (AICAR) transformylase,283 enzymes involved in thymidylate and de novo purine synthesis, respectively. Therefore, resistance to MTX can result from a number of alternative mechanisms, including (1) reduced MTX uptake via a defective folate transport system,6 such as decreased expression of the reduced folate carrier4,284 or of the folate receptors;285,286 (2) increased export via MRP family proteins126,127 or other exporters of polyglutamatable antifolates; (3) reduced polyglutamation leading to decreased drug retention as well as reduced inhibition of thymidylate synthase and AICAR transformylase;287 and (4) either elevated levels of DHFR or reduced affinity of DHFR for MTX.288–291 While all these mechanisms have been described in examples of experimental resistance of cultured cells to MTX, increased DHFR levels secondary to gene amplification has received the greatest study and has been associated with clinical MTX resistance.292–294

Figure 39.4. Methotrexate metabolism and toxicity.

Figure 39.4

Methotrexate metabolism and toxicity. MTx = methotrexate; MTx-(glu)n = plyglutamate methotrexate; DHFR = dehydrofolate reductase; TS = thymidylate synthase; FH2 = dihydrofolate; FH4 = tetrahydrofolate; 5,10 meFH4 = 5,10-methylene tetrahydrofolate.

The use of high-dose MTX (HDMTX) with subsequent rescue of normal tissues by administration of the reduced folate, leucovorin (N5-formyl tetrahydrofolate) has been advocated as an approach which could theoretically circumvent most mechanisms of MTX resistance. At high systemic drug concentrations, cytocidal levels can be achieved by passive diffusion of drug into transport-defective resistant cells. Furthermore, prolonged exposure of cells to high extracellular concentrations of drug can maintain cytotoxic intracellular drug levels in the face of a drug retention defect secondary to decreased polyglutamation. Finally, increased intracellular MTX delivered by HDMTX therapy can saturate DHFR in cells whose resistance is due to amplification of the DHFR gene or due to lowered affinity of DHFR for MTX. Although HDMTX is of proven value in the treatment of ALL and perhaps osteogenic sarcoma, the rationale for the use of this modality in the treatment of other cancers has been recently questioned.295,296 Indeed, some tumors, as well as normal tissues, are rescued from HDMTX toxicity by leucovorin. In these and other cases, the use of HDMTX with leucovorin rescue offers no therapeutic advantage over regimens that use conventional MTX doses. While early studies suggested that HDMTX improved response rates to chemotherapy of osteogenic sarcoma,297 the contribution of HDMTX therapy to the success of recent multi-agent adjuvant protols is unclear. In contrast, HDMTX is indisputably efficacious in the treatment of ALL. The success of HDMTX in this setting is probably due to the penetration of drug across anatomic and pharmacolgic barriers into tumor sanctuaries, such as testes, and, at very high MTX doses, the central nervous system.211

In an effort to improve drug efficacy, other inhibitors of DHFR, such as trimetrexate and piritrexim, have been developed.298–300 These lipid-soluble drugs are taken up by cells independently of the folate-carrier system; consequently their use might obviate transport-mediated antifolate resistance. However, cells which are resistant to MTX on the basis of amplified DHFR will be cross-resistant to trimetrexate. The utility of trimetrexate is further limited by the association of classic MDR with cross-resistance to trimetrexate.301 These results suggest that trimetrexate and drugs of the MDR phenotype share the same P-glycoprotein efflux pump.

Other antifolate compounds capable of inhibiting folate-dependent enzymes besides DHFR have been investigated. One drug, 10-propargyl-5,8-dideazafolate has shown promise as a thymidylate synthase inhibitor.302 Another drug with potential clinical utility, 5,10-dideazatetrahydrofolate is an effective inhibitor of glycinamide ribonucleoside transformylase, the first folate-dependent enzyme in de novo purine synthesis.303 Cells resistant to MTX by virtue of increased DHFR expression would be expected to remain sensitive to these alternative antifolates.

The pyrimidine base, 5-FU and its deoxynucleoside metabolite, 5-fluoro-2’-deoxyuridine (FdUrd) have been used in the treatment of gastrointestinal tumors, breast cancer, head and neck cancer, and some other malignacies. The metabolism of 5-FU is complex and is partially shown in Fig. 39.5.25 The best characterized mechanism of fluoropyrimidine cytotoxicity involves the inhibition of thymidylate synthase by 5-fluoro-2’-deoxyuridine monophosphate (FdUMP). Additionally, the incorporation of the metabolite, 5-fluorouridine triphosphate (FUTP) into RNA has been correlated with cytotoxicity in some systems. While 5-fluro-2’-deoxyuridine triphosphate (FdUTP) can be incorporated into DNA, the relationship between this process and the cytocidal activity of fluoropyrimidines remains undetermined. Resistance to 5-FU may be conferred by alterations in enzymes involved in fluoropyrimidine metabolism, particularly those enzymes associated with the conversion of 5-FU to the thymidylate synthase inhibitor, FdUMP.25 Furthermore, changes in thymidylate synthase level or its affinity for FdUMP have been associated with 5-FU resistance.304–306

Figure 39.5. 5-fluorouracil metabolism and toxicity.

Figure 39.5

5-fluorouracil metabolism and toxicity. 5-FU = 5-fluorouracil; FdUrd = 5-fluoro-2’-deoxyuridine; FdUMP and FdUTP = 5-fluoro-2’-deoxyuridine mono- and triphosphate.

Several strategies to improve fluropyrimidine efficacy and overcome resistance have been advanced. It has been suggested that tumor cell killing may be improved by prolonged or continuous exposure to drug.307,308 Other studies have advocated the coadministration of 5-FU with the reduced folate, leucovorin. The efficacy of this combination stems from leucovorin-dependent increases in intracellular 5,10-methylene tetrahydrofolate (5,10-meTHF), a cofactor that stabilizes the FdUMP-thymidylate synthase inhibitor complex.309,310 Synergy between 5-FU and other agents, which might be exploited clinically, has also been studied. For example, pretreatment of cells with methotrexate enhances the toxicity of 5-FU subsequently administered. Such pretreatment with methotrexate, an inhibitor of de novo purine synthesis (above) has been shown to increase the level of phosphoribosyl pyrophosphate (PRPP). Thus, the expanded pool of PRPP is available for conversion of 5-FU to FUMP and FUTP (see Fig. 39.5). It has been suggested that the increased incorporation of FUTP into RNA that results is responsible for the improved cytotoxicity.311,312 The inhibitor of de novo pyrimidine synthesis, phosphonacetyl-L-aspartate (PALA), has been used with 5-FU in an effort to reduce pyrimidine metabolites that compete for the targets of fluoropyrimidine toxicity.313 Finally, the synergistic interaction between interferon and halogenated pyrimidines has been investigated.314

Ara-C is an important nucleoside antineoplastic agent effective in the treatment of acute leukemias. The metabolism and mechanism of cytotoxicity of ara-C are represented in Fig. 39.6.315 Following its uptake by the nucleoside transport system, ara-C is activated by a series of kinases to ara-CTP, a substrate of DNA polymerase which is incorporated into nascent DNA causing premature chain termination and ultimately cell death. The rate-limiting step in ara-C activation is the S-phase specific reaction catalyzed by deoxycytidine kinase. The cytotoxic compound, ara-CTP or its precursors (ara-CMP and ara-CDP) can be catabolized by phosphatases or they (ara-C and ara-CMP) can be inactivated by deaminases. Several mechanisms of cancer cell resistance to ara-C have been demonstrated, including, but not confined to, the following. Because ara-C activation is cell cycle dependent, quiescent cells or cells that fail to enter the S-phase during the interval of treatment escape the cytotoxicity of ara-C. At suboptimal doses, otherwise drug-sensitive tumor cells located in pharmacologic or anatomic sanctuaries may survive ara-C treatment.316 Decreased nucleoside transport has also been implicated in ara-C resistance.317 Additionally, resistance may be conferred by altered drug metabolism, such as decreased activation by deoxycytidine kinase,315 increased inactivation by cytidine deaminase,15 or altered DNA polymerase affinity for ara-C.318

Figure 39.6. Cytosine arabinoside (ara-C) metabolism and toxicity.

Figure 39.6

Cytosine arabinoside (ara-C) metabolism and toxicity.

Administration of high dose ara-C represents one approach to overcoming resistance to the drug and has been clinically useful in the treatment of some leukemias refractory to conventional doses of ara-C. Resistance based on diminished nucleoside transport and pharmacologic/anatomic sanctuaries can be circumvented with high-dose drug treatment.316 In resistance secondary to increased drug inactivation by cytidine deaminase, coadministration of ara-C with a cytidine deaminase inhibitor, such as tetrahydrouridine, may reverse this mode of drug resistance.319 Alternative pyrimidine analogues such as ara-AC (arabinofuranosyl-5-azacytosine, fazarabine) have shown activity against a broad range of tumor cells in preclinical testing and have been the subject of clinical trials.320,321

Conclusion and Future Directions

Through the kinds of studies done largely in vitro described in this chapter, many of the mechanisms of antineoplastic drug resistance have been identified. While several of these processes operate in vivo, their relative clinical importance must be better clarified in controlled, prospective examinations of patient tumor specimens and correlations with therapeutic responses to chemotherapy. Nevertheless, these mechanisms have suggested potentially useful approaches to overcoming clinical drug resistance. These approaches include the rational choice of conventional agents or design of novel drugs that are less likely to share resistance mechanisms. Additionally, many of the pathways of antineoplastic drug inactivation or transport are targets for pharmacologic manipulations that may reverse or circumvent the resistance of tumors to some drugs. Despite these efforts, many tumors will remain refractory to conventional chemotherapeutic drugs. Their successful treatment may require new modalities including biologic response modifiers, novel immune-based therapeutics, and emerging pharmacologic agents capable of modulating signal transduction and apoptotic pathways of tumor cells in response to both conventional and new therapies.


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