Multidrug Resistance Protein 2 Implicates Anticancer Drug-Resistance ...

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Sorafenib and sunitinib are developed as molecular target medicines for advanced renal cell carcinoma, and known to inhibit certain tyrosine kinases.
March 2011

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Biol. Pharm. Bull. 34(3) 433—435 (2011)

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Multidrug Resistance Protein 2 Implicates Anticancer Drug-Resistance to Sorafenib Yoshihiko SHIBAYAMA,a Kou NAKANO,b Hiroshi MAEDA,c Miyuki TAGUCHI,d Ryuji IKEDA,d Mitsuru SUGAWARA,a Ken ISEKI,*,e Yasuo TAKEDA,d and Katsushi YAMADAd a

Education Research Center for Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Hokkaido University; Laboratory of Clinical Pharmaceutics and Therapeutics, Graduate School of Pharmaceutical Sciences, Hokkaido University; Nishi 6, Kita 12, Kita-ku, Sapporo 060–0812, Japan: b Department of Pharmacy, Central Hospital Tenyoukai; 6–7 Izumi-cho, Kagoshima 892–0822, Japan: c Department of Pharmacy, Kagoshima City Hospital; 20–17 Kajiya-cho, Kagoshima 892–8580, Japan: and d Department of Clinical Pharmacy and Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University; 8–35–1 Sakuragaoka, Kagoshima 890–8520, Japan. Received October 2, 2010; accepted December 16, 2010; published online December 20, 2010 e

Sorafenib and sunitinib is a small molecule inhibitor of certain receptor tyrosine kinases, and have improved outcomes for patients with advanced renal cell carcinoma. Inhibitory concentration of 50% cell growth of sorafenib significantly rose to 6.4-fold in a multidrug resistance protein 2 (MRP2) transfected cell line versus control cell line. The concentration of sorafenib was significantly decreased to 74% of control cells after 3 h treatment. In contrast, a tyrosine kinase inhibitor sunitinib did not show alteration of inhibitory concentration of 50% cell growth and accumulation into the cells of MRP2 transfected cells. The present study suggest that sorafenib is a substrate for MRP2, suggesting that MRP2 may implicate drug resistance to sorafenib. Key words

sorafenib; sunitinib; multidrug resistance protein 2; inhibitory concentration

Sorafenib and sunitinib are developed as molecular target medicines for advanced renal cell carcinoma, and known to inhibit certain tyrosine kinases. The biological effects of receptor tyrosine kinase activation are mediated by a complex cascade of intracellular signaling molecules that are potential targets for therapy, including the Raf, mitogen-activated protein extracellular kinase (MEK) and extracellular signal-regulated kinase (ERK) pathways.1) Sorafenib is a multikinase inhibitor targeting the Raf serine/threonine kinases and the vascular endothelial growth factor (VEGF) receptor 1—3, platelet-derived growth factor (PDGF) receptor-b , c-Kit, Flt3 and p38 tyrosine kinases and, which block the VEGF and PDGF-dependent angiogenesis.2) Sunitinib malate targets receptor tyrosine kinases of VEGFR1-3, PDGFR-a , PDGFRb , FMS-like tyrosine kinase 3, c-Kit, RET and colony-stimulating factor receptor type 1, some of which have been implicated in tumor growth angiogenesis and metastasis.3) Large clinical trials have been demonstrated that sorafenib and sunitinib have improved outcomes for patients with advanced renal cell carcinoma.4,5) The family of ATP binding cassette (ABC) transporters, for example, p-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2) has important roles in the detoxification and excretion of xenobiotics.6) MRP2, an MRP isoform, is a clinically important transporter that functions in the terminal excretion of cytotoxic and carcinogenic substances.7) MRP2 is important clinically as it modulates the pharmacokinetics of many drugs, and its expression and activity are also altered by certain drugs and disease state.8) Sorafenib and sunitinib are largely overlapping specificities; however, it is not clear substrate preferences for MRP2. In this study, we demonstrate substrate preferences for MRP2 about sorafenib and sunitinib. MATERIALS AND METHODS Chemicals Sorafenib and sunitinib were purchased from ∗ To whom correspondence should be addressed.

Toronto Research Chemicals Inc. (North York, Canada). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Invitrogen (Carlsbad, CA, U.S.A.). Other reagents and acetonitrile for high performance liquid chromatography analysis were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Cell Culture A pig transformed renal cell line, LLCPK1 and LLC-PK1/MRP2 cell lines were kindly provided by Dr. Furukawa (Department of Molecular Oncology, Kagoshima University). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine and 100 units/ml of penicillin, 100 m g/ml of streptomycin and 2.5 m g/ml of amphotericin B at 37 °C in a 5% CO2 humidified atmosphere. Cell cultures were maintained at exponential growth by replacing the media every 2—3 d. Culture medium was replaced with fresh medium the day before the experiment. Assays Inhibition concentration of 50% cell growth (IC50) were determined with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) Assay. Cells were incubated in culture medium with various concentrations of the drugs in a final volume of 100 m l. After 3 d, 20 m l of MTT (2.5 mg/ml) was added to each well and the plates were incubated for an additional 3 h. The resulting formazan was dissolved in 80 m l of 20% sodium dodecyl sulfate solution.9) The plates were shaken mechanically for 5 min and read immediately at 570 nm using a model 550 Micro Plate Reader (Bio-Rad, Richmond, CA, U.S.A.). Accumulation of sorafenib in the cells was evaluated with HPLC analysis. The HPLC system consisted of an LC-10AS HPLC pump, SPD6A UV detector, and C-RSA recorder (Shimadzu Corp., Kyoto, Japan). Analysis condition for HPLC analysis was modified Afify et al. report.10) The column temperature was maintained at 30 °C in a CTO-6A column oven (Shimadzu Corp., Kyoto, Japan). Samples were injected onto a reversephase column (Puresil 5m C18, 4.6150 mm, Waters). Oper-

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© 2011 Pharmaceutical Society of Japan

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Vol. 34, No. 3 Table 1. Inhibition Concentration of 50% Cell Growth and Resistance Ratio

MTX Sorafenib Sunitinib

LLC-PK1

LLC-PK1/MRP2

Ratio

18119 3658607 2916276

331542394*** 233761786*** 3761480

183 6.39 1.29

Inhibition concentration of 50% cell growth was evaluated with MTT assay. Each value indicates meansstandard error of the mean (nM) from 6 independent experiments. Resistant ratio of LLC-PK1/MRP2 cell line to LLC-PK1 cell line is shown in the ratio column. LLC-PK1: pig kidney cell line, LLC-PK1/MRP2: human MRP2 gene transfected cell line. Statistical analyses for paired samples were performed by twotailed Student’s t test. Asterisk indicates significant difference from vehicle group (∗∗∗ p0.001).

Fig. 1. A Typical Chromatogram of Sorafenib and Internal Standard The retention times of sorafenib, sunitinib and the internal standard were 1.9 min, 3.6 min and 8.9 min, respectively. Samples were injected onto a reverse-phase column (Puresil 5m C18, 4.6150 mm). Detection wavelength: 254 nm. Flow rate: 1 ml/min. Internal standard: tolnaftate.

ation conditions were as follows: Detector, ultraviolet absorption photometer wavelength of 254 nm; Mobile phases, acetnitrile/distilled water with 0.2% acetic acid (7 : 3); flow rate of 1 ml/min; internal standard solution, 0.03% of tolnaftate in methanol. The retention times of sorafenib, sunitinib and the internal standard were 1.9 min, 3.6 min and 8.9 min, respectively (Fig. 1). The cells were incubated at a dose of 4 m M of sorafenib or sunitinib. The collected cells washed twice with phosphate-buffered saline. Cells homogenate was mixed with 3-fold of the internal standard solution were deproteinized by centrifugation at 9000g for 5 min. The supernatants were deproteinized again with equal volume of with mobile phase solution, and then the mixture was centrifuged at 9000g for 5 min. Ten microliters of supernatant was injected into the HPLC analysis system. Protein concentration was determined by the DC protein assay (Bio-Rad Laboratories, Hercules, CA, U.S.A.). RESULTS AND DISCUSSION Cell viability was evaluated by MTT assay (Table 1). LLCPK1/MRP2 was generated by transfection human MRP2 cDNA into parent cells LLC-PK1.11,12) Methotrexate (MTX) was employed as the positive control experiment for drug resistance against MRP2.13) LLC-PK1/MRP2 cells showed drug resistance to MTX (Table 1). LLC-PK1/MRP2 cells also showed drug resistance to sorafenib, but not sunitinib. To evaluate accumulation of sorafenib into LLC-PK1 and LLC-PK1/MRP2 cells, HPLC analysis was performed (Figs. 1, 2). The cells were incubated for 5, 30, 90 and 180 min at a dose of 4 m M of sorafenib. The concentration of sorafenib in the LLC-PK1/MRP2 cells was significantly decreased following 90 and 180 min incubation (90 min: LLC-PK1, 0.650.04, LLC-PK1/MRP2, 0.520.04; 180 min: LLCPK1, 0.690.06, LLC-PK1/MRP2, 0.510.03 m mol/m g, mean aS.E., Fig. 2). The concentration of sunitinib in the LLC-PK1/MRP2 cells showed no significant difference fol-

Fig. 2. Concentration of Sorafenib in the Cells The cells were incubated at a dose of 4 m M of sorafenib for 5, 30, 90 and 180 min. The collected cells washed twice with phosphate-buffered saline. Concentration of sorafenib and sunitinib is demonstrated as per amount of whole cell homogenate protein. Each value indicates meansstandard error of the mean (m mol/m g protein) from 5 times measurement. Overall differences among treatments were evaluated by one-way analysis of variance (ANOVA), with differences between individual treatments and groups evaluated using the Williams test. Asterisk indicates significant difference from vehicle group (∗ p0.05).

Table 2. Concentration of Sorafenib or Sunitinib in the Cells (m mol/m g Protein) after 180 min Incubation

Sorafenib Sunitinib

LLCPK1

LLCPK1/MRP2

0.690.06 0.670.04

0.510.03* 0.620.09

Intracellular concentration of sorafenib or sunitinib after incubation at a dose of 4 m M of sorafenib or sunitinib for 180 min. Concentration of sorafenib and sunitinib is demonstrated as per amount of whole cell homogenate protein. Each value indicates meansstandard error of the mean (m mol/m g protein) from 5 times measurement. Differences between the treatments were evaluated using the two-tailed Student’s t test. Asterisk indicates significant difference from vehicle group (∗ p0.05).

lowing 180 min incubation (Table 2). Accumulation of sorafenib in the cells were reduced to 74%, resistance ratio rose to 6.4-fold in a multidrug resistance protein 2 (MRP2) transfected cell line versus control cell line. Similarly, Tiwari et al. reported that accumulation of paclitaxel was reduced to 80%, resistance ratio rose to 26-fold in an ABCB1 (P-glycoprotein) overexpressed cell line (KB-C2) versus control cell line (KB-3-1).14) It was assumed that reduction of sorafenib accumulation in the cells may elevate resistance ratio. Accumulating evidences suggest that tyrosine kinase inhibitors transport by the ATP-binding cassette (ABC) transporters. Recently, Lagas et al. reported that sorafenib was moderately transported by P-glycoprotein (P-gp, ABCB1) and more efficiently by breast cancer resistance protein

March 2011

(BCRP, ABCG2).15) Shukla et al. reported that sunitinib also blocks function of P-gp and BCRP.16) It is not clear whether tyrosine kinase inhibitors are transport by MRP2. Marchetti et al. reported that no active transport of erlotinib by MRP2 was observed.17) The present study is the first report a tyrosine kinase inhibitor, sorafenib is a substrate for MRP2. It is reported that pharmacokinetics profiles of sorafenib and sunitinib are different. According to the package insert of sorafenib (Nexavar®), sorafenib may show disposition of enterohepatic circulation. On the other hand, the package insert of sunitinib (Sutent®) indicates that absorption of biliary excretion of sunitinib from intestinal tract was low and sunitinib may not show disposition of enterohepatic circulation. It is well known that MRP2 mediates the canalicular excretion of a broad range of organic anions. It was assumed that substrate preference for MRP2 of the tyrosine kinase inhibitors may affect excretion from liver and enterohepatic circulation. It is known that MRP2 confers drug resistance to anti-cancer drugs. The present study suggests that drug-resistance to sorafenib can be occurred by overexpression of MRP2 and drug resistant tumor with MRP2 overexpression may be effective sunitinib treatment. Consequently, it might be effective to use sorafenib for the initial treatment, and then to use sunitinib for the second-line treatment. In clinical chemotherapy, it was reported that sequential treatment with sorafenib and sunitinib would be effective. Eichelberg et al. reported that sequential tyrosine kinase inhibitors therapy with the sorafenib followed by sunitinib has clinical validity in some patients with advanced renal cell carcinoma when progressive disease occurs under the initial tyrosine kinase inhibitors therapy.18) In conclusion, this study suggests that sorafenib is a substrate for MRP2. In contrast, a tyrosine kinase inhibitor sunitinib is not substrate for MRP2, suggesting that differences of substrate preference sorafenib and sunitinib may implicate clinical drug resistance. Acknowledgement The present study was performed as the training program for intensive course program for oncology pharmacy specialist of oncology professionals in Kyushu, which were supported by the Ministry of Education,

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