The human multidrug-resistance protein (MRP) gene family contains at least six members: MRP1, encoding the multidrug-resistance protein; MRP2 or cMOAT, encoding the canalicular multispecific organic anion transporter; and four homologs, called MRP3, MRP4, MRP5, and MRP6. In this report, we characterize MRP3, the closest homolog of MRP1. Cell lines were retrovirally transduced with MRP3 cDNA, and new monoclonal antibodies specific for MRP3 were generated. We show that MRP3 is an organic anion and multidrug transporter, like the GS-X pumps MRP1 and MRP2. In Two members of the large family of ABC transporters are known thus far to confer multidrug resistance in human cancer cells. These are the MDR1 P-glycoprotein (1) and the multidrugresistance protein MRP1 (2). Both membrane proteins transport a wide range of drugs with different cellular targets and confer resistance by decreasing the intracellular concentration of drugs. P-glycoprotein transports these drugs in unmodified form, whereas MRP1 can transport drugs either conjugated to anionic ligands such as glutathione (GSH), glucuronide, or sulfate, or in an unmodified form, possibly together with GSH. Well known substrates for MRP1 are, for example, cysteinyl leukotrienes, glutathione disulfide, S-(2,4-dinitrophenyl-)glutathione, ethacrynic acid S-glutathione, etoposide glucuronide, certain steroid glucuronides, and bile salt derivatives (3-6). Transporters with the characteristics of MRP1 are known as GS-X pumps (7) or multispecific organic anion transporters (8).Another GS-X pump is MRP2, a homolog of MRP1. Unlike MRP1, which is nearly ubiquitously expressed (9), MRP2 is present mainly in the canalicular membrane of hepatocytes (10), but is also present in other apical domains of polarized cells such as the epithelial cells of the proximal tubules of the kidney (11). Studies with mutant rats (TR Ϫ ͞GY or EHBR), which lack the MRP2 protein in the canalicular membrane of hepatocytes, have shown that the substrate specificity of MRP2 is very similar to that of MRP1 (12, 13). MRP2 also contributes to transport of anticancer drugs and some metals. The mutant rats showed a reduced biliary clearance of methotrexate (14), of the topoisomerase I inhibitor CPT-11 and its metabolites (15), and of mercury, cadmium, and arsenite (refs. 16 and 17; R.O.E., unpublished observation). Cells transduced with an MRP2 cDNA construct transport the cytostatic drug vinblastine (18). Moreover, overexpression of the MRP2 gene has been found in several cisplatinresistant cell lines (19,20), and transfection of an MRP2-antisense construct into liver cells was reported to confer an increased sensitivity to cytotoxic drugs (21). All these observations strongly suggest that MRP2 may confer multidrug resistance, but whether it does so in cancer patients remains to be established.Besides MRP1 and MRP2, there are at least four MRP homologs expressed in humans, called MRP3, MRP4, MRP5, and MRP6 (20,22). Not much is known about the substrate specificity of these putative new transporters...

The proteasome inhibitor bortezomib is a novel anticancer drug that has shown promise in the treatment of refractory multiple myeloma. However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the molecular basis of which remains elusive. Toward this end, we here developed high levels (45-to 129-fold) of acquired resistance to bortezomib in human myelomonocytic THP1 cells by exposure to stepwise increasing (2.5-200 nM) concentrations of bortezomib. Study of the molecular mechanism of bortezomib resistance in these cells revealed (1) an Ala49Thr mutation residing in a highly conserved bortezomib-binding pocket in the proteasome ␤5-subunit (PSMB5) protein, (2) a dramatic overexpression (up to 60-fold) of PSMB5 protein but not of other proteasome subunits including PSMB6, PSMB7, and PSMA7, (3) high levels of cross-resistance to ␤5 subunit-targeted cytotoxic peptides 4A6, MG132, MG262, and ALLN, but not to a broad spectrum of chemotherapeutic drugs, (4) no marked changes in chymotrypsin-like proteasome activity, and (5) IntroductionThe ubiquitin proteasome system (UPS) facilitates the degradation of ubiquitin-tagged intracellular proteins, many of which play a regulatory role in cell proliferation, cell survival, and signaling processes. [1][2][3] As such, proteasome inhibitors have been recognized as a new generation of chemotherapeutic agents and antiinflammatory drugs. [4][5][6][7][8][9][10][11][12][13] The boronic dipeptide bortezomib (PS341, Velcade) is the first proteasome inhibitor that has been approved for the treatment of refractory multiple myeloma. 6,14 Bortezomib is a reversible inhibitor that targets primarily the ␤5-subunit (PSMB5) subunit/chymotrypsin-like activity of the 26S proteasome and to a somewhat lesser extent also caspase-like activity harbored by the ␤1 (PSMB6) proteasome subunit. At higher concentrations, bortezomib inhibits trypsin-like proteolytic activity facilitated by ␤2 (PSMB7) proteasome subunits. [15][16][17] Despite promising clinical activity, some patients with multiple myeloma failed to respond to bortezomib therapy. 18 Moreover, the efficacy for bortezomib may differ between tumor types. 6,[19][20][21] Whether these observations are related to common mechanisms of drug resistance frequently seen for anticancer 22 or anti-inflammatory drugs 23 is largely unknown. However, their characterization is of key importance as it may pave the way for the overcoming of drug resistance, thereby enhancing the efficacy of this new class of proteasome-targeted drugs.One mode of primary resistance to bortezomib is conveyed by constitutively high levels of heat shock protein 27. 24 In the context of acquired resistance, studies aimed at delineating the mechanism of acquired resistance to the tripeptidyl aldehyde proteasome inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal) revealed 2 possible molecular mechanisms: (a) enhanced cellular efflux via the multidrug resistance (MDR) transporter P-glycoprotein (Pgp; ABCC1) 25 or multidrug resistance-related pro...

Purpose Resistance to antiangiogenic tyrosine kinase inhibitors such as sunitinib is an important clinical problem, but its underlying mechanisms are largely unknown. We analyzed tumor sunitinib levels in mice and patients and studied sensitivity and resistance mechanisms to sunitinib. Experimental Design Intratumoral and plasma sunitinib concentrations in mice and patients were determined. Sunitinib exposure on tumor cell proliferation was examined. Resistant tumor cells were derived by continuous exposure and studied for alterations in intracellular sunitinib accumulation and activity. Results Intratumoral concentrations of sunitinib in mice and patients were 10.9 ± 0.5 and 9.5 ± 2.4 μmol/L, respectively, whereas plasma concentrations were 10-fold lower, 1.0 ± 0.1 and 0.3 ± 0.1 μmol/L, respectively. Sunitinib inhibited tumor cell growth at clinically relevant concentrations in vitro, with IC50 values of 1.4 to 2.3 μmol/L. Continuous exposure to sunitinib resulted in resistance of 786-O renal and HT-29 colon cancer cells. Fluorescent microscopy revealed intracellular sunitinib distribution to acidic lysosomes, which were significantly higher expressed in resistant cells. A 1.7- to 2.5-fold higher sunitinib concentration in resistant cells was measured because of increased lysosomal sequestration. Despite the higher intracellular sunitinib accumulation, levels of the key signaling p-Akt and p-ERK 1/2 were unaffected and comparable with untreated parental cells, indicating reduced effectiveness of sunitinib. Conclusion We report that sunitinib inhibits tumor cell proliferation at clinically relevant concentrations and found lysosomal sequestration to be a novel mechanism of sunitinib resistance. This finding warrants clinical evaluation whether targeting lysosomal function will overcome sunitinib resistance.

Thymidylate synthase (TS) is a key enzyme in the de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP), for which 5,10-methylene-tetrahydrofolate (CH(2)-THF) is the methyl donor. TS is an important target for chemotherapy; it is inhibited by folate and nucleotide analogs, such as by 5-fluoro-dUMP (FdUMP), the active metabolite of 5-fluorouracil (5FU). FdUMP forms a relatively stable ternary complex with TS and CH(2)THF, which is further stabilized by leucovorin (LV). 5FU treatment can induce TS expression, which might bypass dTMP depletion. An improved efficacy of 5FU might be achieved by increasing and prolonging TS inhibition, a prevention of dissociation of the ternary complex, and prevention of TS induction. In a panel of 17 colon cancer cells, including several variants with acquired resistance to 5FU, sensitivity was related to TS levels, but exclusion of the resistant variants abolished this relation. For antifolates, polyglutamylation was more important than the intrinsic TS level. Cells with low p53 levels were more sensitive to 5FU and the antifolate raltitrexed (RTX) than cells with high, mutated p53. Free TS protein down-regulates its own translation, but its transcription is regulated by E2F, a cell cycle checkpoint regulator. Together, this results in low TS levels in stationary phase cells. Although cells with a low TS might theoretically be more sensitive to 5FU, the low proliferation rate prevents induction of DNA damage and 5FU toxicity. TS levels were not related to polymorphisms of the TS promoter. Treatment with 5FU or RTX rapidly induced TS levels two- to five-fold. In animal models, 5FU treatment resulted in TS inhibition followed by a two- to three-fold TS induction. Both LV and a high dose of 5FU not only enhanced TS inhibition, but also prevented TS induction and increased the antitumor effect. In patients, TS levels as determined by enzyme activity assays, immunohistochemistry and mRNA expression, were related to a response to 5FU. 5FU treatment initially decreased TS levels, but this was followed by an induction, as seen with an increased ratio of TS protein over TS-mRNA. The clear retrospective relation between TS levels and response now forms the basis for a prospective study, in which TS levels are measured before treatment in order to determine the treatment protocol.

Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the b5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZresistant cells revealed upregulation of mutant b5 subunit of the proteasome. These newly identified b5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the b5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant b5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the a7 subunit of the proteasome can overcome this drug resistance modality.

CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays >200-fold resistance to methotrexate (MTX) due to defective transport via the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, derived by a gradual deprivation of folic acid from 2.3 M to 2 nM (LF) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild type K m value for MTX transport but a 31-fold and 9-fold lower K m values for folic acid and leucovorin, respectively, relative to wild type RFC; 2) a 10-fold RFC1 gene amplification along with a >20-fold increased expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulation of MTX transport by anions (i.e. chloride); and 4) a G 3 A mutation at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, resulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the human RFC. Upon transfer of CEM/MTX-LF cells to folate-replete medium (2.3 M folic acid), the more efficient folic acid uptake in CEM/MTX-LF cells resulted in a 7-and 24-fold elevated total folate pool compared with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol/mg of protein, respectively). This markedly elevated intracellular folate pool conferred a novel mechanism of resistance to polyglutamatable (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. trimetrexate and pyrimethamine) by abolishing their polyglutamylation and circumventing target enzyme inhibition.

Pancreatic cancer is a highly deadly disease: almost all patients develop metastases and conventional treatments have little impact on survival. Therapeutically, this tumor is poorly responsive, largely due to drug resistance. Accumulating evidence suggest that this chemoresistance is intimately linked to specific metabolic aberrations of pancreatic cancer cells, notably an increased use of glucose and the amino acid glutamine fueling anabolic processes. Altered metabolism contributes also to modulation of apoptosis, angiogenesis and drug targets, conferring a resistant phenotype. As a modality to overcome chemoresistance, a variety of experimental compounds inhibiting key metabolic pathways emerged as a promising approach to potentiate the standard treatments for pancreatic cancer in preclinical studies. These results warrant confirmation in clinical trials. Thus, this review summarizes the impact of metabolic aberrations from the perspective of drug resistance and discusses possible novel applications of metabolic inhibition for the development of more effective drugs against pancreatic cancer.

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