Masashi Adachi

Mrp4 Confers Resistance to Topotecan and Protects the Brain from Chemotherapy

ABSTRACT The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined. To explore this role, we generated Mrp4-deficient mice. Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF). Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects. We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane of the endothelial cells of the brain capillaries. Microdialysis sampling of ventricular CSF demonstrated that localization of Mrp4 at the choroid epithelium is integral to its function in limiting drug penetration into the CSF. The topotecan resistance of cells overexpressing Mrp4 and the polarized expression of Mrp4 in the choroid plexus and brain capillary endothelial cells indicate that Mrp4 has a dual role in protecting the brain from cytotoxins and suggest that the therapeutic efficacy of central nervous system-directed drugs that are Mrp4 substrates may be improved by developing Mrp4 inhibitors.

Prognostic significance of angiogenesis in human pancreatic cancer.

SummaryTo evaluate whether angiogenic factors are of clinical relevance to actual human pancreatic cancers, we studied the intratumoral microvessel density (IMD), and PD-ECGF, VEGF protein expression in 40 pancreatic cancers using immunohistochemistry. We also investigated PD-ECGF and VEGF gene expression using reverse transcriptase-PCR (RT-PCR). Of the 40 pancreatic cancers studied, 30 carcinomas (75.0%) were evaluated to be PD-ECGF-positive and 10 carcinomas (25.0%) were determined to be PD-ECGF-negative. In contrast, 27 carcinomas (67.5%) were evaluated to be VEGF-positive, whereas 13 carcinomas (32.5%) were VEGF-negative. VEGF gene expression was moderately associated with an increase in the IMD (r2 = 0.181, P = 0.006), but no significant relationship was found between PD-ECGF gene expression and the IMD (r2 = 0.093, P = 0.059). However, tumours with positive expression for both PD-ECGF and VEGF had a higher IMD (P = 0.027). The results of the immunohistochemistry agreed well with the results of the quantitative RT-PCR. The median survival time of the hypervascular group was significantly shorter than that of the hypovascular group (P < 0.0001). In comparing the survival according to PD-ECGF and VEGF gene expression, the median survival time of the patients with positive PD-ECGF expression was significantly shorter than those with negative PD-ECGF expression (P = 0.040). Furthermore, the median survival time of the patients with positive VEGF expression was significantly shorter than those with negative VEGF expression (P = 0.048). However, the Cox multivariate analysis indicated that the IMD and VEGF expression were independent prognostic factors of the various clinicopathologic variables in pancreatic cancer patients (P = 0.0021 and P = 0.0443, respectively).

Functional Involvement of Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) in the Renal Elimination of the Antiviral Drugs Adefovir and Tenofovir

Acyclic nucleotide phosphonates (adefovir, cidofovir, and tenofovir) are eliminated predominantly into the urine, and renal failure is their dose-limiting toxicity, particularly for adefovir and cidofovir. In this study, we examined the involvement of multidrug resistance-associated protein (MRP)4 (ABCC4) in their luminal efflux in the kidney. ATP-dependent uptake of adefovir and tenofovir but not cidofovir was observed only in the membrane vesicles expressing MRP4. The ATP-dependent uptake of adefovir and tenofovir by MRP4 was not saturated at 1 mM. The ATP-dependent uptake of adefovir by membrane vesicles expressing MRP4 was osmotic-sensitive. No ATP-dependent uptake of either agent was observed in the membrane vesicles expressing human MRP2 or breast cancer resistance protein. These nucleotide analogs were given to mice by constant intravenous infusion, and the plasma, urine, and tissue concentrations were determined. The kidney accumulation of adefovir and tenofovir was significantly greater in Mrp4 knockout mice (130 versus 66 and 191 versus 87 pmol/g tissue, respectively); thus, the renal luminal efflux clearance was estimated to be 37 and 46%, respectively, of the control. There was no difference in the fraction of mono- and diphosphorylated forms of adefovir in the kidney between wild-type and Mrp4 knockout mice. In mice, cidofovir was also eliminated via the urine by tubular secretion as well as glomerular filtration. There was no change in the kinetic parameters of cidofovir in Mrp4 knockout mice. Our results suggest that MRP4 is involved in the luminal efflux of both adefovir and tenofovir, but it makes only a limited contribution to the urinary excretion of cidofovir.

Interactions between Hepatic Mrp4 and Sult2a as Revealed by the Constitutive Androstane Receptor and Mrp4 Knockout Mice

The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.

Correlation of Reduction in MRP-1/CD9 and KAI1/CD82 Expression with Recurrences in Breast Cancer Patients

MRP-1/CD9, KAI1/CD82, and ME491/CD63, have been reported to be associated with the metastatic potential of solid tumors. The aim of this study was to determine whether their expression in tumor tissues is a useful indicator for prognosis in breast cancer patients. We studied 109 breast cancer patients who underwent surgery. Quantitative reverse transcription-polymerase chain reaction analysis was performed to evaluate the expression of these genes. The results were confirmed with immunohistochemistry. All of the carcinomas were ME491/CD63 positive. Thirty-six tumors were MRP-1/CD9 negative. The disease-free survival rate and the 5-year survival rate of patients with MRP-1/CD9-negative tumors were both significantly lower than that in patients with MRP-1/ CD9-positive tumors (P = 0.0005 and P = 0.0380, respectively). Sixty-five tumors were KAI1/CD82 negative. The disease-free survival rate of patients with KAI1/CD82-negative tumors was significantly lower than that of patients with KAI1/CD82-positive tumors (P = 0.0065). Cox regression analysis demonstrated that MRP-1/CD9 status (P = 0.0016) and KAI1/CD82 status (P = 0.0234) were useful indicators for the disease-free survival of breast cancer patients. The disease-free survival rate and 5-year survival rate of patients with either MRP-1/CD9-negative or KAI1/CD82-negative tumors were both significantly lower than patients who were positive for both genes (P = 0.0003 and P = 0.0292, respectively). The expression of MRP-1/CD9 and KAI1/CD82 genes are useful indicators of a poor prognosis in breast cancer patients.

Mrp4−/− mice have an impaired cytoprotective response in obstructive cholestasis†

Mrp4 is a member of the multidrug resistance–associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive upregulation in response to cholestatic injury or bile acid feeding. However, the relative importance of Mrp4 in a protective adaptive response to cholestatic injury is not known. To address this issue, common bile duct ligation (CBDL) was performed in wild‐type and Mrp4−/− mice and animals followed for 7 days. Histological analysis and serum aminotransferase levels revealed more severe liver injury in the absence of Mrp4 expression. Western analyses revealed that Mrp4, but not Mrp3, was significantly increased after CBDL in wild‐type mice. Serum bile acid levels were significantly lower in Mrp4−/− mice than in wild‐type CBDL mice, whereas serum bilirubin levels were the same, suggesting that Mrp4 was required to effectively extrude bile acids from the cholestatic liver. Mrp3 and Ostα‐Ostβ were upregulated in Mrp4−/− mice but were unable to compensate for the loss of Mrp4. High‐performance liquid chromatography analysis on liver extracts revealed that taurine tetrahydroxy bile acid/beta‐muricholic acid ratios were increased twofold in Mrp4−/− mice. In conclusion, hepatic Mrp4 plays a unique and essential protective role in the adaptive response to obstructive cholestatic liver injury. (HEPATOLOGY 2006;43:1013–1021.)

Two new genes from the human ATP-binding cassette transporter superfamily, ABCC11 and ABCC12, tandemly duplicated on chromosome 16q12.

Several years ago, we initiated a long-term project of cloning new human ATP-binding cassette (ABC) transporters and linking them to various disease phenotypes. As one of the results of this project, we present two new members of the human ABCC subfamily, ABCC11 and ABCC12. These two new human ABC transporters were fully characterized and mapped to the human chromosome 16q12. With the addition of these two genes, the complete human ABCC subfamily has 12 identified members (ABCC1-12), nine from the multidrug resistance-like subgroup, two from the sulfonylurea receptor subgroup, and the CFTR gene. Phylogenetic analysis determined that ABCC11 and ABCC12 are derived by duplication, and are most closely related to the ABCC5 gene. Genetic variation in some ABCC subfamily members is associated with human inherited diseases, including cystic fibrosis (CFTR/ABCC7), Dubin-Johnson syndrome (ABCC2), pseudoxanthoma elasticum (ABCC6) and familial persistent hyperinsulinemic hypoglycemia of infancy (ABCC8). Since ABCC11 and ABCC12 were mapped to a region harboring gene(s) for paroxysmal kinesigenic choreoathetosis, the two genes represent positional candidates for this disorder.

Multiple Human Isoforms of Drug Transporters Contribute to the Hepatic and Renal Transport of Olmesartan, a Selective Antagonist of the Angiotensin II AT1-Receptor

Olmesartan, a novel angiotensin II AT1-receptor antagonist, is excreted into both bile and urine, with minimal metabolism. Because olmesartan is a hydrophilic anionic compound, some transporters could be involved in its hepatic and renal clearance. In this study, we characterized the role of human drug transporters in the pharmacokinetics of olmesartan and determined the contribution of each transporter to the overall clearance of olmesartan. Olmesartan was significantly taken up into human embryonic kidney 293 cells expressing organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, organic anion transporter (OAT) 1, and OAT3. We also observed its saturable uptake into human hepatocytes and kidney slices. Estimated from the relative activity factor method and application of specific inhibitors, the relative contributions of OATP1B1 and OATP1B3 to the uptake of olmesartan in human hepatocytes were almost the same, whereas OAT3 was predominantly involved in its uptake in kidney slices. The vectorial transport of olmesartan was observed in OATP1B1/multidrug resistance-associated protein (MRP) 2 double transfectants, but not in OATP1B1/multidrug resistance (MDR) 1 and OATP1B1/breast cancer resistance protein (BCRP) transfectants. ATP-dependent transport into membrane vesicles expressing human MRP2 and MRP4 was clearly observed, with Km values of 14.9 and 26.2 μM, respectively, whereas the urinary excretion of olmesartan in Mrp4-knockout mice was not different from that of control mice. We also investigated the transcellular transport of olmesartan medoxomil, a prodrug of olmesartan. Vectorial basal-to-apical transport was observed in OATP1B1/MRP2, OATP1B1/MDR1 double, and OATP1B1/BCRP double transfectants, suggesting the possible involvement of MRP2, MDR1, and BCRP in the limit of intestinal absorption of olmesartan medoxomil. From these results, we suggest that multiple transporters make a significant contribution to the pharmacokinetics of olmesartan and its prodrug.

Transmembrane 4 superfamily as a prognostic factor in pancreatic cancer

Several members of the transmembrane 4 superfamily (TM4SF) have been reported to be related to tumor progression and metastasis. The aims of our study were to clarify the relationship between TM4SF and pancreatic cancer and to determine the prognostic significance of TM4SF in human pancreatic cancer. The mRNA levels for MRP‐1/CD9, KAI1/CD82 and ME491/CD63, which belong to the TM4SF gene family, were evaluated in 40 resectable pancreatic adenocarcinomas using reverse transcriptase‐PCR. MRP‐1/CD9 gene expression was associated with lymph node status, and with pathological status. Moreover, MRP‐1/CD9 expression was inversely associated with histo‐pathological grading. KAI1/CD82 gene expression was inversely associated with tumor status. ME491/CD63 gene expression, however, was conserved in all pancreatic cancers. The overall survival rate for the 22 patients whose tumors had decreased MRP‐1/CD9 gene expression was strikingly lower than that for the 18 patients with MRP‐1/CD9‐positive tumors. The overall survival rate of the 15 patients who were KAI1/CD82‐positive was significantly higher than that of the 25 patients with decreased KAI1/CD82 gene expression. In a multivariate analysis using the Cox proportional hazards model, MRP‐1/CD9 and KAI1/CD82 status was found to be the most significant Int. J. Cancer (Pred. Oncol.) 79:509–516, 1998.© 1998 Wiley‐Liss, Inc.

Role of MRP4 and MRP5 in biology and chemotherapy

Nucleotide efflux (especially cyclic nucleotides) from a variety of mammalian tissues, bacteria, and lower eukaryotes has been studied for several decades. However, the molecular identity of these nucleotide efflux transporters remained elusive, despite extensive knowledge of their kinetic properties and inhibitor profiles. Identification of the subfamily of adenosine triphosphate (ATP) binding cassette transporters, multidrug resistance protein (MRP) subfamily, permitted rapid advances because some recently identified MRP family members transport modified nucleotide analogs (ie, chemotherapeutic agents). We first identified, MRP4, based on its ability to efflux antiretroviral compounds, such as azidothymidine monophosphate (AZT-MP) and 9-(2-phosphonyl methoxyethyl) adenine (PMEA), in drug-resistant and also in transfected cell lines. MRP5, a close structural homologue of MRP4 also transported PMEA. MRP4 and MRP5 confer resistance to cytotoxic thiopurine nucleotides, and we demonstrate MRP4 expression varies among acute lymphoblastic leukemias, suggesting this as a factor in response to chemotherapy with these agents. The ability of MRP4 and MRP5 to transport 3,5-cyclic adenosine monophosphate (cAMP) and 3,5-cyclic guanosine monophosphate (cGMP) suggests they may play a biological role in cellular signaling by these nucleotides. Finally, we propose that MRP4 may also play a role in hepatic bile acid homeostasis because loss of the main bile acid efflux transporter, sister of P-glycoprotein (SPGP) aka bile-salt export pump (BSEP), leads to a strong compensatory upregulation in MRP4 expression. Cumulatively, these studies reveal that the ATP-binding cassette (ABC) transporters MRP4 and MRP5 have a unique role in biology and in chemotherapeutic response.