Can multidrug resistance mechanisms be modified? (2000)

The phenomenon of clinical drug resistance has been appreciated for many years as an all too frequent reality in the treatment of patients with cancer. Patients may initially present with resistant tumours, develop resistance during treatment or develop resistance at the time of relapse ( Bradshaw & Arceci, 1998). Another observation that was appreciated from the earliest days of treating patients with cytotoxic drugs was that tumours would usually demonstrate resistance to more than one drug, or even to apparently different classes of drugs. This clinical multidrug resistance phenotype continues to pose significant challenges for physicians trying to find the right combinations of bullets to penetrate a tumour's coat of armour. Although some remarkable advances have been made through empirical testing of new agents and their combinations, clinical drug resistance remains the primary reason for treatment failure in patients with cancer. A critical question is, therefore, whether we have learned enough to be able to deliver therapies that are designed to reverse specific multidrug resistance mechanisms? There is currently much evidence demonstrating that both pharmacological and cellular mechanisms contribute to whether a patient with cancer is cured or not. The modification of either the pharmacokinetics of drug delivery and metabolism or the manipulation of a cellular pathway that results in increased drug sensitivity must be considered in attempting to overcome drug resistance. For example, many clinical trials have demonstrated that adjustments in the dose and schedule of drug delivery must be adapted for very young and older age groups to maximize the benefit and reduce the toxicity of chemotherapeutic agents. Furthermore, there is sound evidence that the different dosing schedules – i.e. continuous infusion, dose intensification and timing intensification – can produce increased tumour responses and improved disease-free survival. Although such approaches are likely to continue as part of therapeutic strategies, further advances would appear to be somewhat limited because of treatment-related morbidity and mortality. In addition, if most dose-intensification approaches can be expected to overcome clinical drug resistance of three- to fivefold, then mechanisms that cause higher degrees of resistance will clearly require other strategies. More recent investigations have led to the elucidation of a variety of drug resistance mechanisms that offer potential new targets in which to direct cancer treatment more specifically. Much of this work arose out of the realization that the goal of ascertaining mechanisms responsible for clinical multidrug resistance was impossible without the development of in vitro models. The generation of tumour cell lines that demonstrated a variety of drug-resistant phenotypes provided the experimental tools needed to elucidate the molecular and cellular mechanisms of resistance to chemotherapy. Cellular factors that are now known to play significant roles in resistance to chemotherapy include: (i) transport-mediated resistance – changes in drug uptake or efflux across the tumour cell membrane [e.g. multidrug resistance (MDR)1 P-glycoprotein transporter] or between the cytoplasm and the nucleus (e.g. LRP/VAULT proteins); (ii) metabolic-mediated resistance – changes in activation or inactivation of drugs within the tumour cell (e.g. glutathione-S-transferase); (iii) drug target-mediated resistance – changes in targeted enzymes through altered levels of those targets within tumour cells or through altered affinity of cellular enzymes for the drug (e.g. topoisomerases); (iv) DNA repair-mediated resistance – changes in DNA repair processes (e.g. mismatch repair genes such as Msh2); and (v) apoptosis-mediated resistance – changes in the ability of tumour cells to execute programmed cell death or apoptotic pathways (e.g. bcl2, p53). Tumour cells are thus able to develop a selective survival advantage by altering the cellular pathways that function to overcome the cytotoxic effects of drugs at one or more of these levels. Understanding such mechanisms has provided the hope that therapeutic strategies could be developed to circumvent chemotherapeutic resistance in patients. The clinical relevance of each of these various mechanisms of drug resistance observed in tumour cell lines has not, however, been well established in most circumstances. Requirements for determining that relevance should include showing that: (i) a specific resistance mechanism is expressed in tumour samples from patients; (ii) the expression of that resistance mechanism is correlated with in vivo resistance; and (iii) the resistance mechanism can be altered to reconstitute a state of increased drug sensitivity and ultimately improve disease-free survival for patients. Probably the largest amount of work has been focused on transport-mediated drug resistance mechanisms and, in particular, drug efflux systems. This review will thus focus on how far work has progressed towards the goal of determining whether drug transport mechanisms can be effectively modified to overcome clinical drug resistance and improve patient outcomes. In an attempt to develop in vitro models that would mimic the multidrug resistance observed in patients, tumour cell lines were generated through repeated exposures to sublethal concentrations of single cytotoxic agents such as vincristine or doxorubicin. Subsequent analysis of some of the resistant clones of cells led to the observation that not only were the surviving cells resistant to the cytotoxic agent used for selection but they were also cross-resistant to a wide range of other unrelated natural product chemotherapeutic drugs ( Ling, 1997). In many ways, this phenotype resembled the resistance to multiple chemotherapeutic agents that patients exhibit and was termed classic multidrug resistance (MDR). Subsequent investigations led to the isolation of the mdr1 gene, which encodes a 170 000-Da membrane protein that was demonstrated to be able to convey the multidrug-resistant phenotype when transfected into drug-sensitive tumour cells ( Ling, 1997). This protein was initially believed to function by altering the permeability of the tumour cell membrane to drugs, and it was therefore termed P-glycoprotein or P-gp-170. However, further work showed that P-gp functioned as an ATP-dependent drug efflux pump. Thus, increased expression of P-gp results in the increased efflux of a variety of natural product drugs, including chemotherapeutic agents; this efflux, in turn, results in decreased intracellular drug accumulation and drug resistance ( Ling, 1997). There is significant evidence that increased expression of MDR1 P-gp is associated with decreased responsiveness to chemotherapy and poor outcome in a variety of solid tumours and, in particular, leukaemia ( Broxterman & Schuurhuis, 1997; Chan et al, 1997 ; Nuessler et al, 1997; Leith, 1998; Leith et al, 1999 ). MDR1 P-gp is also expressed in a number of normal tissues such as the intestine, liver, kidney, adrenal gland, early haematopoietic progenitors and the blood–brain barrier. P-gp appears to function in the protection of normal tissues from environmental toxins, in renal tubule transport and in the secretion of steroids, bile salts and bilirubin ( Sikic et al, 1997; Bradshaw & Arceci, 1998). The discovery that some drug-resistant tumour cells did not express P-glycoprotein but still showed decreased intracellular accumulation of a variety of drugs led to the identification of an MDR1-related family of genes called the multidrug resistance-associated proteins or MRPs ( Cole & Deeley, 1998). The transfection of the MRP1 cDNA into drug-sensitive cells is able to convey a multidrug-resistant phenotype that is similar, but not identical, to that observed in MDR1-transfected cells ( Cole & Deeley, 1998). For example, MDR1 expression appears to be able to convey much higher levels of drug resistance (up to several thousandfold in selected cell lines) and different substrate specificities than MRP1 ( Cole & Deeley, 1998). MRP1 appears to function primarily as a transporter of glutathione conjugates. The MRP2 gene, also termed cMOAT, encodes a multispecific organic anion transporter in biliary canaliculi ( Kool et al, 1997; Cole & Deeley, 1998). Other substrates transported by the MRP proteins include cysteinyl leukotrienes, glutathione disulphide, steroid glucuronides, polyglutamatable antifolates and anionic conjugates of bile salts ( Cole & Deeley, 1998). MRP1 is constitutively expressed in most normal tissues, with relatively higher levels observed in the adrenal glands and testes ( Cole & Deeley, 1998). Although some studies have correlated MRP1 expression with poor prognosis for a variety of cancers, particularly acute myeloid leukaemia (AML), there remains significant controversy ( Norris et al, 1996; Leith et al, 1999 ). Both the mdr1 and mrp1 genes are now known to belong to a large (and growing) gene family of related ABC (ATP binding cassette) drug transport systems that play important physiological roles in the transport of endogenous substrates, as well as in detoxification pathways. That most of these genes and their potential role in drug resistance remain unknown complicates any analysis of the role of specific transporters in studies designed to reverse clinical drug resistance. Much effort has been made to find methods to inhibit these drug efflux pumps and thereby sensitize resistant cells to chemotherapy. In the early 1980s, verapamil and trifluoperazine were shown to increase the intracellular accumulation and cytotoxicity of vincristine ( Ford, 1995). Subsequently, other small molecules with the ability to inhibit P-gp-mediated drug efflux were identified, such as cyclosporin A (CsA) and its d-isomer (PSC-833, Valspodar), as well as, more recently, the inhibitors LY335979, S9788, GF120918, XR9576, VX-70 and OC144-093 ( Ford, 1995; Sikic et al, 1997 ; Bradshaw & Arceci, 1998). Some of these reversal agents (e.g. verapamil and CsA) exert their effect as competitive inhibitors of P-gp-mediated efflux. Others, such as PSC-833, bind to P-gp in a non-competitive fashion and displace cytotoxic agents from interacting with the transporter ( Naito et al, 1996 ). A critical feature that should be noted, however, is that none of these agents have been definitively shown to be specific for a single transporter. In fact, some leukaemia cells, which do not express P-glycoprotein, are able to have drug efflux inhibited with reversal agents such as CsA or PSC-833 ( Leith et al, 1997 ; Leith, 1998). Furthermore, CsA has been shown to augment the P450 system, which may also contribute to more rapid drug inactivation and increased drug sensitivity ( Wacher et al, 1998 ). These observations raise substantial questions about conclusions concerning the specificity of inhibitors of drug efflux. Preclinical animal models have demonstrated that the co-administration of these drug resistance reversal agents with chemotherapy is able to produce significant responses in resistant tumours. These model tumour systems could, in part, address the issue of selectivity because tumour cells engineered to express MDR1 by transfection of its cDNA could be compared with MDR1-negative control cells ( Ford, 1995). However, these studies were not usually controlled for pharmacokinetic alterations of the chemotherapeutic drugs that occurred as a result of exposure to reversal agents, an issue of considerable importance in evaluating clinical trial data. These models have also shown that significantly more toxicity may be encountered when MDR1 reversal agents are used with full dosages of chemotherapeutic drugs ( Ford, 1995). Of particular interest are the results from mice in which either the mdr1 or mrp1 genes have been ‘knocked’ out by homologous recombination. These null-mouse mutants show significantly increased sensitivity to chemotherapeutic agents compared with wild-type mice, but not necessarily in all tissues where MDR1 is highly expressed. For example, the brain was shown to accumulate significantly more drug in the MDR null mice than in other organs and, especially, in wild-type animals ( van Asperen et al, 1996, 1999). These preclinical animal models demonstrated that MDR1 reversal agents could be relatively safely given in vivo and produce an increased drug sensitivity of MDR1-positive tumours, thus leading the way to phase I clinical trials. The initial phase I trials carried out with verapamil in patients, however, demonstrated that the level needed to inhibit MDR1 P-glycoprotein drug efflux was too toxic, secondary to hypotension and heart conduction abnormalities ( Sikic et al, 1997 ; Vossebeld & Sonneveld, 1999). However, subsequent studies with the less cardiovascular-toxic isomer R-verapamil showed that sufficient serum levels could be without toxicity from the reversal with some clinical responses observed in tumours ( Sikic et al, 1997 ; Bradshaw & Arceci, 1998; Vossebeld & Sonneveld, 1999). I studies with CsA and its less and PSC-833 established that serum levels could be effectively for MDR1 P-gp-mediated drug efflux ( Sikic et al, 1997 ; Bradshaw & Arceci, 1998; Vossebeld & Sonneveld, 1999). These phase I as well as subsequent phase also led to the important observation that inhibitors such as CsA or PSC-833 can significantly the pharmacokinetic of chemotherapeutic agents such as and ( et al, ; et al, ; et al, ). Thus, in to pharmacological exposures to that observed with dosing of chemotherapeutic drugs, dosing would have to be by ( et al, ; et al, ; et al, ). The altered pharmacological are believed to be to the of drug transporters in the biliary and as well as effects on the P450 ( Sikic et al, 1997 ). This observation has several of For example, if dose are not carried it has been that the increase in the pharmacological the could be by the of drugs given by continuous with dose such the of the MDR1 P-glycoprotein not function much as a specific of drug as a to the of drug exposure ( Sikic et al, 1997 ; Bradshaw & Arceci, 1998; Vossebeld & Sonneveld, 1999). dose higher serum levels and of drug exposure of patients to the chemotherapeutic drugs would be when a reversal This could therefore be as a of dose than a of the ability to reverse specific drug resistance Some reversal agents, such as R-verapamil ( et al, ( et al, 1998 ( et al, 1997 or ( et al, 1998 do not appear to have significant effects on the of some not chemotherapeutic agents. Thus, tumour responses to a specific drug or drugs that are observed in patients with established resistance and are to the a MDR would clearly demonstrate reversal of clinical drug resistance. Although such responses have been they do not definitively the specificity of the Other clinical studies have to for altered pharmacokinetics of chemotherapeutic agents to exposure to MDR inhibitors ( Sikic et al, 1997 ; Bradshaw & Arceci, 1998; et al, 1999 ; Vossebeld & Sonneveld, 1999). Thus, the of of a tumour to a chemotherapeutic drug or the the MDR reversal agent was of the chemotherapeutic drugs to for decreased because of the was also In this one could also that any clinical responses must be to the of the MDR reversal Although demonstrating that established clinical drug resistance in part, be with the of MDR such studies do not that the increased tumour sensitivity was to P-gp For example, it is the of multiple drug efflux transporters or P450 alterations such studies have provided with some of the factors the of MDR reversal agents and the way for more testing of these approaches in phase trials. phase trials have been that have patients to only or the chemotherapy with the of an MDR reversal agent ( Bradshaw & Arceci, 1998; Vossebeld & Sonneveld, 1999). initial trials did not show a significant advantage of an MDR reversal but levels were not to that serum to inhibit drug efflux, been Furthermore, the MDR1 P-glycoprotein of the tumours or has not been In addition, for the of such studies to be dose of those chemotherapeutic drugs is by the must be into the and this has not been Thus, the question of whether or not drug resistance can be in patients must clearly be the to the question as to whether such trials drug-resistant mechanism is modified and whether patient are improved must at this time be In for to whether of the MDR1 P-glycoprotein is the cause of increased tumour more selective approaches to the question must be For example, one potential would be to develop more specific Although several have been made to this with small it is as to such agents other controlled studies cDNA transfection may elucidate Another would be to drugs or transported by P-glycoprotein into MDR1-positive cells in the or of an of drug efflux. The of conjugates that are towards could this In this the drug or would be targeted and tumour if an MDR1 P-glycoprotein transport were the cytotoxic drug or would be inhibited from out of the an were not the cytotoxic agent would be from an MDR1-positive tumour Furthermore, normal cells that did not have the tumour would not bind to the or the cytotoxic that inhibit MDR1 P-glycoprotein drug efflux have been generated and could also as a to efflux more specifically. Thus, the of chemotherapeutic drugs that are substrates for the MDR1 to a to be in more testing the that selective modification of P-gp drug efflux results in increased tumour responsiveness in patients. However, even this cause significant pharmacological changes in drug that would have to be controlled for by Another for a specific resistance pathway such as MDR1 P-glycoprotein drug efflux has been by the expression and, the function of P-gp through the of In vitro studies have shown that an can be at the expression of MDR1 P-glycoprotein and drug resistance through this mechanism ( et al, ). A related that has very the of called which show specificity in their and of are also relatively in that they may be to clinical trials ( et al, 1999 ; et al, 1999 ). These methods offer more specificity than the of MDR but still require significant in delivery and pharmacological when used in patients. In addition, if such agents were given in one would that normal MDR1 expression would the pharmacokinetics of chemotherapeutic agents in at some designed to MDR1 P-glycoprotein efflux, such as targeted or could also be developed for other drug efflux systems such as most of the inhibitors of MDR1 P-glycoprotein also appear to MRP1 and other ABC family In which intracellular has significantly more selectivity for transport systems on such as MRP ( et al, ). such as have also been shown to inhibit drug resistance to MRP than but appears to be too to be effectively used in vivo ( et al, ). The also selectivity for the transporters ( et al, ). Although some of these agents may have potential clinical they are still in In vitro mechanisms of such as the MDR1 P-glycoprotein appear to have in of their expression in a variety of Although factors are in or groups in of they do not that such will be therapeutic Thus, whether or not specific clinical drug resistance mechanisms transport systems are in patients and result in improved remains an even more advances in therapeutic approaches have been able to overcome drug resistance in a This is by dose timing intensification in the ( et al, ; et al, ; et al, 1999 ). However, therapeutic strategies designed to inhibit selective mechanisms of resistance require inhibitors with specificity for in controlled clinical trials. In that for may not be of specific drug resistance such as the transport system, clearly results in the of other mechanisms of resistance ( et al, ; et al, 1996 ). that as many as ABC transporter genes may in the if a of these transporters is in chemotherapeutic drug the of the ability to a cytotoxic agents is Thus, the of specific inhibitors early in the of a treatment may the of a single of other resistance pathways are likely to this targets that would inhibit classes of drug resistance transporters by be more than specific Furthermore, more pathways that for example, may to be more than trying to pathways leading to drug transport also termed programmed cell a of cellular pathways leading to a of and changes in the death of a cell ( 1997; & 1997). Although is for normal development and of it is also a cellular to ( A wide variety of and in most cells either to or to it from ( 1997). The gene is a not for activation of or treatment with chemotherapy in a variety of normal and cell of the gene are the most frequent known observed in cancers, and function within tumour cells appears to be an important in the to cytotoxic agents ( et al, ). into tumour cells by the of and the have been used to the for in cellular sensitivity to a variety of agents ( et al, ). The were resistant to as well as to and compared with the and or tumours have been shown to exhibit increased sensitivity to or compared with tumours ( et al, ). some tumours from clones which were initially to and developed resistance were shown to but not have ( et al, ). Although these have shown the importance of and in tumour cell sensitivity to and chemotherapy in an in vivo model of it is that such a not the that to treatment of resistance in in patients. In fact, other studies have shown that cancer cells engineered to may develop resistance to some cytotoxic agents but may more to other agents, including ( et al, 1999 ). and also of ( proteins have been and the levels and these proteins can of to ( & The role of and related pathways may also be from For example, in cells from patients with leukaemia has been shown to be in many ( et al, 1996 ). the level of with the of endogenous levels of to with this drug sensitivity has been demonstrated in cells ( et al, 1996 ). The of the amount and the of such is critical for tumour cell survival circumstances. work will be to the importance of of in patients with cancer. of methods to programmed cell death in tumour cells will to be important in cancer The gene is currently the focus of a number of gene strategies. of wild-type into tumour cell lines results in apoptotic changes and ( et al, ). of wild-type an into mice of tumour cells the of the tumour cells ( et al, ). the of which in their normal state may contribute to the development of also specific approaches to tumour For example, a that not express the gene, a protein product that the cellular protein has been showed to in and tumour cells ( et al, 1996 ). that have a including normal cells, are to with the This may a way to tumour cells normal Another that is used in preclinical models is to develop small drugs that are able to the of ( et al, 1999 ). work has also been to other genes important in For example, from acute myeloid leukaemia patients that expressed levels of protein were with ( et al, ). of the samples a significant in expression of by increased in the of approaches may agents but their in vivo delivery in schedules must still be may also is a of the family ( et al, ). is expressed on various cells, including and cells, and has been shown to be expressed on leukaemia cell lines ( et al, ). of cells with in vitro effectively and can further sensitize resistant cells to chemotherapy ( et al, 1999 ). mechanisms of may as an important of treatment in many of there remains significant that it the role in this ( & 1999). Other mechanisms for the of cell and therefore also a potential pathway of drug include the death of tumour cells when they to but have significant abnormalities ( et al, 1999 ; et al, 1999 ). and pathways may contribute to how well a tumour cell such and, whether a will be its Much of the of cancer has been on in some years In addition, a significant part of the of cancer treatment has been on therapeutic dose and which has in treatment-related morbidity and mortality. However, some drug resistance remains the primary reason for treatment A goal for clinical and must be to continue to work towards a more of the multiple mechanisms that cause cancer to The development of more and less therapeutic approaches should be a direct of that improved