Hiroshi Arakawa

Impact of system L amino acid transporter 1 (LAT1) on proliferation of human ovarian cancer cells: A possible target for combination therapy with anti-proliferative aminopeptidase inhibitors

Amino acids activate nutrient signaling via the mammalian target of rapamycin (mTOR), we therefore evaluated the relationship between amino acid transporter gene expression and proliferation in human ovarian cancer cell lines. Expression of three cancer-associated amino acid transporter genes, LAT1, ASCT2 and SN2, was measured by qRT-PCR and Western blot. The effects of silencing the LAT1 gene and its inhibitor BCH on cell growth were evaluated by means of cell proliferation and colony formation assays. The system L amino acid transporter LAT1 was up-regulated in human ovarian cancer SKOV3, IGROV1, A2780, and OVCAR3 cells, compared to normal ovarian epithelial IOSE397 cells, whereas ASCT2 and SN2 were not. BCH reduced phosphorylation of p70S6K, a down-stream effector of mTOR, in SKOV3 and IGROV1 cells, and decreased their proliferation by 30% and 28%, respectively. Although proliferation of SKOV3 (S1) or IGROV1 (I10) cells was unaffected by LAT1-knockdown, plating efficiency in colony formation assays was significantly reduced in SKOV3(S1) and IGROV1(I10) cells to 21% and 52% of the respective plasmid transfected control cells, SKOV3(SC) and IGROV(IC), suggesting that LAT1 affects anchorage-independent cell proliferation. Finally, BCH caused 10.5- and 4.3-fold decrease in the IC(50) value of bestatin, an anti-proliferative aminopeptidase inhibitor, in IGROV1 and A2780 cells, respectively, suggesting that the combined therapy is synergistic. Our findings indicate that LAT1 expression is increased in human ovarian cancer cell lines; LAT1 may be a target for combination therapy with anti-proliferative aminopeptidase inhibitors to combat ovarian cancer.

Enhanced expression of organic anion transporting polypeptides (OATPs) in androgen receptor-positive prostate cancer cells: Possible role of OATP1A2 in adaptive cell growth under androgen-depleted conditions

The biological mechanisms underlying castration resistance of prostate cancer are not fully understood. In the present study, we examined the role of organic anion transporting polypeptides (OATPs) as importers of dehydroepiandrosterone sulfate (DHEAS) into cells to support growth under androgen-depleted conditions. Cell growth and mRNA expression of OATP genes were studied in human prostate cancer LNCaP and 22Rv1 cells under androgen-depleted conditions. The stimulatory effect of DHEAS on cell growth was investigated in LNCaP cells in which OATP1A2 had been silenced. Growth of both cell lines was stimulated by DHEAS and the effect was attenuated by STX64, an inhibitor of steroid sulfatase which can covert DHEAS to DHEA. OATP1A2 mRNA expression was increased most prominently among various genes tested in LNCaP cells grown in androgen-depleted medium. Similar results were obtained with 22Rv1 cells. Furthermore, the characteristics of [(3)H]DHEAS uptake by LNCaP cells were consistent with those of OATP-mediated transport. Knockdown of OATP1A2 in LNCaP cells resulted in loss of the DHEAS sensitivity of cell growth. Our results suggest that enhanced OATP1A2 expression is associated with adaptive cell growth of prostate cancer cells under androgen-depleted conditions. Thus, OATP1A2 may be a pharmacological target for prostate cancer treatment.

Uptake transporter organic anion transporting polypeptide 1B3 contributes to the growth of estrogen-dependent breast cancer.

Estrone-3-sulfate is one of the most abundant estrogen precursors in postmenopausal women. We previously showed that estrone-3-sulfate transporters are present in human breast cancer-derived MCF-7 cells (J. Pharmacol. Exp. Ther. 311 (2004) 1032-1037) and that inhibition of estrone-3-sulfate uptake resulted in the suppression of cell growth (Pharm. Res. 22 (2005) 1634-1641); therefore, estrone-3-sulfate transporter should be a novel target for therapy of hormone-dependent breast cancers. The purpose of the present study is to identify the transporter(s) responsible for the uptake of estrone-3-sulfate in breast cancer cells. We obtained two subclones of MCF-7 cells with different estrone-3-sulfate uptake activities and searched for differentially expressed transporter genes by means of DNA microarray analysis. Among several candidate transporters identified, OATP1B3 was further evaluated, since the uptake characteristics of estrone-3-sulfate by MCF-7 cells seemed consistent with the transport properties of OATP1B3. The contribution of OATP1B3 to estrone-3-sulfate uptake by MCF-7 cells was examined by the relative activity factor (RAF) method, and was calculated to amount to 6%. This result suggests that OATP1B3 is one of the transporters contributing to the supply of the estrogen precursor estrone-3-sulfate to estrogen-dependent breast cancer cells.

Active intestinal absorption of fluoroquinolone antibacterial agent ciprofloxacin by organic anion transporting polypeptide, Oatp1a5

Fluoroquinolone antimicrobial drugs are absorbed efficiently after oral administration despite of their hydrophilic nature, implying an involvement of carrier‐mediated transport in their membrane transport process. It has been that several fluoroquinolones are substrates of organic anion transporter polypeptides OATP1A2 expressed in human intestine derived Caco‐2 cells. In the present study, to clarify the involvement of OATP in intestinal absorption of ciprofloxacin, the contribution of Oatp1a5, which is expressed at the apical membranes of rat enterocytes, to intestinal absorption of ciprofloxacin was investigated in rats. The intestinal membrane permeability of ciprofloxacin was measured by in situ and the vascular perfused closed loop methods. The disappeared and absorbed amount of ciprofloxacin from the intestinal lumen were increased markedly in the presence of 7,8‐benzoflavone, a breast cancer resistance protein inhibitor, and ivermectin, a P‐glycoprotein inhibitor, while it was decreased significantly in the presence of these inhibitors in combination with naringin, an Oatp1a5 inhibitor. Furthermore, the Oatp1a5‐mediated uptake of ciprofloxacin was saturable with a Km value of 140 µm, and naringin inhibited the uptake with an IC50 value of 18 µm by Xenopus oocytes expressing Oatp1a5. Naringin reduced the permeation of ciprofloxacin from the mucosal‐to‐serosal side, with an IC50 value of 7.5 µm by the Ussing‐type chamber method. The estimated IC50 values were comparable to that of Oatp1a5. These data suggest that Oatp1a5 is partially responsible for the intestinal absorption of ciprofloxacin. In conclusion, the intestinal absorption of ciprofloxacin could be affected by influx transporters such as Oatp1a5 as well as the efflux transporters such as P‐gp and Bcrp. Copyright

Preliminary Evaluation of Three-Dimensional Primary Human Hepatocyte Culture System for Assay of Drug-Metabolizing Enzyme-Inducing Potential

Drug-induced liver injury (DILI) is a common reason for withdrawal of candidate drugs from clinical trials, or of approved drugs from the market. DILI may be induced not only by intact parental drugs, but also by metabolites or intermediates, and therefore should be evaluated in the enzyme-induced state. Here, we present a protocol for assay of drug-metabolizing enzyme-inducing potential using three-dimensional (3D) primary cultures of human hepatocytes (hepatocyte spheroids). Hepatocyte spheroids could be used up to 21 d after seeding (pre-culture for 7 d and exposure to inducer for up to 14 d), based on preliminary evaluation of basal activities of CYP subtypes and mRNA expression of the corresponding transcription factor and xenobiotic receptors (aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR)). After 2 d exposure of hepatocyte spheroids to omeprazole, phenobarbital and rifampicin (typical inducers of CYP1A2, 2B6 and 3A4, respectively), CYP1A2, 2B6 and 3A4 mRNA expression levels were significantly increased. The mRNA induction of CYP2B6 remained reasonably stable between days 2 and 14 of exposure to inducers, while induction of both CYP1A2 and 3A4 continued to increase up to day 14. These enzyme activities were all significantly increased compared with the control until day 14. Our findings indicate that our 3D hepatocyte spheroids system would be especially suitable for long-term testing of enzyme activity induction by drugs, either to predict or to verify clinical events.

Role of OATP2A1 in PGE2 secretion from human colorectal cancer cells via exocytosis in response to oxidative stress

Chronic inflammation induced by reactive oxygen species is associated with increased risk of developing colorectal cancer (CRC), and prostaglandin E2 (PGE2), which serves as a key mediator of inflammatory responses, plays an important role in CRC initiation and progression. Therefore, in the present study, we aimed to investigate the role of prostaglandin transporter OATP2A1/SLCO2A1 in the changes of PGE2 disposition in CRC cells in response to oxidative stress. H2O2 induced translocation of cytoplasmic OATP2A1 to plasma membranes in LoVo and COLO 320DM cells, but not in Caco-2 cells. The shift of subcellular OATP2A1 was abolished in the presence of anti-oxidant N-acetyl-L-cysteine or an inhibitor of protein kinase C, which evokes exocytosis. Exposure of LoVo cells to H2O2 caused an increase in the amount of extracellular PGE2 without changing the sum of intra- and extracellular PGE2. OATP2A1 knockdown decreased extracellular PGE2 in LoVo cells. In addition, extracellular PGE2 was significantly reduced by exocytosis inhibitor cytochalasin D, suggesting that H2O2-induced PGE2 release occurs in an exocytotic manner. Furthermore, mRNA expression of vascular endothelial growth factor (VEGF) was significantly reduced in LoVo cells by knockdown of OATP2A1. These results suggest that cytoplasmic OATP2A1 likely facilitates PGE2 loading into suitable intracellular compartment(s) for efficient exocytotic PGE2 release from CRC cells exposed to oxidative stress.

Usefulness of kidney slices for functional analysis of apical reabsorptive transporters

Kidney plays a key role in the elimination and reabsorption of drugs and nutrients, however in vitro methods to evaluate renal disposition are limited. In the present study, we investigated usefulness of isolated kidney slice, which had been used for transport only at basolateral membrane of tubular epithelial cells, for evaluation of apical membrane transporters. As transporters that are easy to discriminate between apical and basolateral transports, apical membrane specific and sodium-dependent transporters (SGLTs and OCTNs) and pH-dependent transporters (PEPTs) are selected. Uptake of ergothioneine, carnitine and methyl-α-D-glucopyranoside, which are substrates of apical Octn1, Octn2, and Sglt1/2, respectively, by mice kidney slices showed clear Na+ dependence and reduction by selective inhibitors. In addition, sodium dependence of ergothioneine uptake was negligible in the kidney slice from Octn1-gene deficient mice. Moreover, uptake of PepT1/2 substrate glycyl-sarcosine, was higher than that in the presence of glycyl-leucine, a non-specific Pept inhibitor. The Km and IC50 values for substrates and inhibitors of each transporter were mostly comparable to those obtained in transporter-transfected cells. In conclusion, it was demonstrated that kidney slices are promising tool to study transporters expressed at the apical membranes as well as basolateral membranes of kidney tubular epithelial cells.

Utility of human hepatocyte spheroids without feeder cells for evaluation of hepatotoxicity

We investigated the utility of three-dimensionally cultured hepatocytes (spheroids) without feeder cells (Sph(f-)) for the prediction of drug-induced liver injury (DILI) in humans. Sph(f-) and spheroids cultured on feeder cells (Sph(f+)) were exposed to the hepatotoxic drugs flutamide, diclofenac, isoniazid and chlorpromazine at various concentrations for 14 days, and albumin secretion and cumulative leakages of toxicity marker enzymes, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (γ-GTP), were measured. The cumulative AST, LDH or γ-GTP leakages from Sph(f-) were similar to or greater than those from Sph(f+) for all drugs tested, although ALT leakages showed no consistent difference between Sph(f+) and Sph(f-). In the case of Sph(f-), significant correlations among all the toxicity markers except for γ-GTP were observed. As regards the drug concentrations causing 1.2-fold elevation of enzyme leakage (F1.2), no consistent difference between Sph(f+) and Sph(f-) was found, although several F1.2 values were undetermined, especially in Sph(f+). The IC50 of albumin secretion and F1.2 of AST leakage from Sph(f-) were equal to or lower than those of Sph(f+) for all the tested drugs. These results indicate that feeder cells might contribute to resistance to hepatotoxicity, suggesting DILI could be evaluated more accurately by using Sph(f-). We suggest that long-term exposure of Sph(f-) to drugs might be a versatile method to predict and reproduce clinical chronic toxicity, especially in response to repeated drug administration.

Effect of tyrosine kinase inhibitors on renal handling of creatinine by MATE1

Creatinine is actively secreted across tubular epithelial cells via organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1). We previously showed that the tyrosine kinase inhibitor (TKI) crizotinib inhibits OCT2-mediated transport of creatinine. In the present work, we examined the inhibitory potency of TKIs, including crizotinib, on MATE1-mediated transport of creatinine. Then, we used the kinetic parameters estimated in this and the previous work to predict the potential impact of TKIs on serum creatinine level (SCr) via reversible inhibition of creatinine transport. Crizotinib inhibited [14C]creatinine uptake by MATE1-overexpressing cells, and the inhibitory effect increased with incubation time, being greater in the case of pre-incubation or combined pre-incubation/co-incubation (pre/co-incubation) than in the case of co-incubation alone. The inhibition was non-competitive, with Ki values of 2.34 μM, 0.455 μM and 0.342 μM under co-, pre- or pre/co-incubation conditions, respectively. Similar values were obtained for inhibition of [3H]MPP+ uptake by MATE1-overexpressing cells. Gefitinib, imatinib, pazopanib, sorafenib, and sunitinib also inhibited MATE1-mediated creatinine uptake. Further, all these TKIs except pazopanib inhibited [14C]creatinine uptake by OCT2-overexpressing cells. In rat kidney slices, the ratio of unbound tissue accumulation of TKIs to extracellular concentration ranged from 2.05 to 3.93. Prediction of the influence of TKIs on SCr based on the renal creatinine clearance and plasma maximum unbound concentrations of TKIs suggested that crizotinib and imatinib might increase SCr by more than 10% in the clinical context. Accordingly, it is necessary to be cautious in diagnosing TKI-induced renal failure only on the basis of an increase of SCr.

Organic cation transporter 1 is responsible for hepatocellular uptake of the tyrosine kinase inhibitor pazopanib

Pazopanib is an orally active tyrosine kinase inhibitor that exhibits hepatotoxicity in some patients. Despite the clinical importance of its hepatic distribution, the transporter(s) responsible for hepatic uptake of pazopanib in humans remain undetermined. To characterize its hepatic uptake mechanism, we screened the effects of several transporter inhibitors, including tetrapentylammonium (TPeA) for organic cation transporters (OCTs) and cyclosporin A (CsA) for organic anion-transporting polypeptides (OATPs), on both plasma disappearance and hepatic distribution of pazopanib in mice after its i.v. administration. Among the inhibitors, TPeA largely reduced hepatic distribution and plasma clearance of pazopanib, whereas CsA showed only partial reduction. Pazopanib uptake by isolated mouse hepatocytes was similarly reduced by these inhibitors, suggesting that OCTs play a major role in the overall hepatic uptake of pazopanib in mice. In human embryonic kidney cell line HEK293 cells stably transfected with human OCT1, pazopanib uptake was significantly higher than that in vector-transfected cells. Moreover, pazopanib uptake by OCT1 became saturated and was inhibited by TPeA, but not by CsA, confirming that pazopanib is also a substrate of human OCT1. Importantly, OCT1-mediated uptake of a typical OCT1 substrate metformin was inhibited by pazopanib with an IC50 value of 0.253 µM, indicating that pazopanib has the potential for clinically relevant inhibition of human OCT1. Finally, pazopanib was taken up by cryopreserved human pooled hepatocytes in a time-dependent manner, and this uptake was largely reduced by TPeA but only partially reduced by CsA. Thus, the present findings suggest that OCT1 is responsible for hepatocellular uptake of pazopanib.