Oncology 2014;87:364–370 DOI: 10.1159/000366132 Received: June 13, 2014 Accepted after revision: July 24, 2014 Published online: September 6, 2014
© 2014 S. Karger AG, Basel 0030–2414/14/0876–0364$39.50/0 www.karger.com/ocl
Short Communication
Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2 Katrina L. Mealey
Sandamali Dassanayake
Neal S. Burke
Individualized Medicine Program, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Wash., USA
Key Words ABCG2 · Fluoroquinolone · Retina · Phototoxicity · Tyrosine kinase inhibitors Abstract The tyrosine kinase inhibitor (TKI) class of anticancer agents inhibits ABCG2-mediated drug efflux. ABCG2 is an important component of the blood-retinal barrier, where it limits retinal exposure to phototoxic compounds such as fluoroquinolone antibiotics. Patients treated with TKIs would be expected to be at greater risk for retinal phototoxicity. Using an in vitro system, our results indicate that the TKIs gefitinib and imatinib abrogate the ability of ABCG2 to protect cells against ciprofloxacin-induced phototoxicity. We conclude that the concurrent administration of ABCG2 inhibitors with photoreactive fluoroquinolone antibiotics may result in retinal damage. © 2014 S. Karger AG, Basel
Introduction
Katrina L. Mealey Individualized Medicine Program, Department of Veterinary Clinical Sciences College of Veterinary Medicine, Washington State University 100 Grimes Way, Pullman, WA 99164-6610 (USA) E-Mail kmealey @ vetmed.wsu.edu
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The organs most susceptible to phototoxicity are the skin and the eyes, including the retina [1, 2]. Phototoxic damage to the retina can lead to retinal (macular) degeneration and blindness [3]. Normally, the retina is protected from photoreactive compounds by the bloodretina barrier with the ABC (ATP-binding cassette) transporter ABCG2 playing a critical role [4]. ABCG2 is expressed on the luminal membrane of retinal capillary endothelial cells, where it actively effluxes potentially phototoxic endogenous and exogenous substrates including pheophorbide, protoporphyrin IX and fluoroquinolone antibiotics [4, 5]. The importance of ABCG2 in protecting the retina from fluoroquinolone-induced phototoxicity is dramatically
365
Oncology 2014;87:364–370 DOI: 10.1159/000366132
© 2014 S. Karger AG, Basel www.karger.com/ocl
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
illustrated in the feline species. Owing to a species-wide amino acid change, feline ABCG2 is dysfunctional, resulting in extreme susceptibility to fluoroquinolone-induced retinal degeneration and blindness [6]. Fluoroquinolones have been documented to cause ocular toxicity in other species, including humans [7–9]. Whether or not human patients experience fluoroquinolone-induced retinal degeneration under circumstances of ABCG2 dysfunction has not been investigated. Several commonly used anticancer drugs [tyrosine kinase inhibitors (TKIs) such as imatinib and gefitinib] inhibit ABCG2 function [10]. Concurrent administration of these drugs with fluoroquinolones, which are often recommended for chemotherapy-induced neutropenia [11], could result in fluoroquinolone-induced retinal toxicity in human patients. The goal of this study was to determine if pharmacological inhibition of ABCG2 would enhance fluoroquinolone-induced phototoxicity using a cell culture system. Ciprofloxacin was selected as a representative fluoroquinolone with intermediate phototoxic potential [12].
Materials and Methods Reagents and Cell Lines HEK 293 cells transfected with human ABCG2 (R2 cells) and plasmid control (PC cells) were provided by R. Robey (Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Md., USA). Ciprofloxacin and MTT [3-(4,5 dimethylthiazol-2-thiol)-2,5-diphenyl2H-tetrazolium bromide] were obtained from Sigma Aldrich (St. Louis, Mo., USA), Ko143 from R & D Systems, Inc. (Minneapolis, Minn., USA), gefitinib and imatinib from LC Laboratories (Woburn, Ma., USA), and mitoxantrone (MTX) from Hospira Inc. (Lake Forest, Ill., USA). Western Blot Analysis of ABCG2 Expression in Transfected HEK293 Cells Twenty micrograms of total protein from each cell line were separated on 4–15% Tris-HCl gradient polyacrylamide gels and electrotransferred to polyvinylidene difluoride membranes. Membranes were blocked and then incubated with rat anti-mouse ABCG2 monoclonal antibody (6D170; Santa Cruz Biotechnology, Santa Cruz, Calif., USA). Membranes were washed and incubated with goat anti-rat alkaline phosphatase-conjugated secondary antibody (Santa Cruz Biotechnology). Membranes were washed and binding was detected using Immun-Star AP Substrate (Bio-Rad, Hercules, Calif., USA).
Phototoxicity Assays For phototoxicity assays, R2 and PC cells were plated in 6-well plates and then incubated for 24 h to allow cells to adhere. Cells were then treated with the ABCG2 inhibitor Ko143 (10 μM) for 30 min, followed by treatment with ciprofloxacin (0, 20, 50, 100 and 200 μM) for 1 h. The media was replaced with drug-free media, and cells were then exposed to UVA light (National Biologicals Corporation, Beachwood, Ohio, USA) for 60 min. ‘Dark’ controls included identically treated cells not exposed to UV light. After 24-hour incubation,
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Flow Cytometry To confirm function of transfected ABCG2 in R2 cells, MTX efflux was assessed using flow cytometry. MTX, an intrinsically fluorescent compound, is a prototype substrate for ABCG2 routinely used to assess ABCG2 function [13, 14]. Its efflux from cells is directly measured using flow cytometry to determine intracellular mean fluorescence intensity (MFI). Transfected cells (R2 and PC) were plated in 6-well plates and incubated overnight. Cells were then treated with either vehicle (control) or the ABCG2 inhibitor Ko143 (10 μM) for 30 min, followed by treatment with MTX (10 μM) or no treatment (control) for an additional 60 min. Cells treated with neither Ko143 nor MTX were used to generate the control histograms, which measure cell autofluorescence. After incubation, cells were washed with ice-cold phosphate-buffered saline, placed on ice and immediately analyzed. A FACSCalibur flow cytometer with a 635-nm red diode laser, a 670-nm bandpass filter and Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, Calif., USA) was used to detect intracellular MFI. At least 10,000 events were collected. The mean channel number for each histogram represents the MFI for comparison between different cell lines (PC vs. R2) and treatments (MTX alone vs. MTX plus ABCG2 inhibitor Ko143). Experiments were run in duplicate, and each experiment was repeated on separate days.
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Oncology 2014;87:364–370 © 2014 S. Karger AG, Basel www.karger.com/ocl
DOI: 10.1159/000366132
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Color version available online
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
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Fig. 1. a Immunoblot assessment of ABCG2 expression in HEK 293 cells transfected with empty plasmid (PC cells) or ABCG2 cDNA (R2 cells). b Representative histograms from a functional assay for ABCG2 function in transfected cell lines. Efflux assays were performed with MTX at 10 μM concentrations with or without the ABCG2 inhibitor Ko143 (10 μM). Cell count is represented on the y-axis and fluorescence intensity on the xaxis, with the MFI indicated parenthetically.
cytotoxicity was assessed using the MTT cytotoxicity assay in which viable cells metabolize MTT to a colorimetrically measurable product (formazan). Cell survival was expressed as a fraction of the mean optical density of vehicle-treated controls. Means represent results from experiments performed in duplicate and repeated on separate days. For assessing the effects of TKIs on ciprofloxacin-induced phototoxicity, experiments were repeated as described above, substituting either gefitinib (1 μM) or imatinib (3 μM) for Ko143. For all experiments, each well contained the same concentration of vehicle in order to eliminate any effects the vehicle might have on either intracellular drug concentration or cytotoxicity.
Confirmation of ABCG2 Expression and Function ABCG2 expression was confirmed in R2 cells but was absent in PC cells (fig. 1a). Function of transfected ABCG2 in R2 cells was confirmed using flow cytometry to determine MFI after
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Results
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Oncology 2014;87:364–370 © 2014 S. Karger AG, Basel www.karger.com/ocl
DOI: 10.1159/000366132
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
R2 cells Ciprofloxacin
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Fig. 2. Mean ± SD fractional survival of HEK 293 cells transfected with ABCG2 cDNA (R2 cells) or empty plasmid (PC cells) treated with increasing concentrations of ciprofloxacin alone (white bars) or concurrently treated with the ABCG2 inhibitor Ko143 (black bars).
Cell survival as a fraction of control
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Impact of ABCG2 Inhibition on Ciprofloxacin-Induced Phototoxicity In R2 cells, ABCG2 inhibition by 10 μM Ko143 dramatically increases cytotoxicity of ciprofloxacin in a dose-dependent manner (fig. 2) compared to cytotoxicity of ciprofloxacin alone,
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treatment with MTX. Figure 1b shows very low intrinsic fluorescence of R2 and PC cells (3.9 and 5.5, respectively). When cells are treated with the ABCG2 substrate MTX, both R2 and PC cells display greater MFI reflecting correspondence to intracellular MTX concentration. Figure 1b shows that PC cells treated with MTX were highly fluorescent (MFI 1351), reflecting a high concentration of intracellular MTX. PC cells treated with both Ko143 (ABCG2 inhibitor) and MTX had a negligible effect on MFI (1399) because these cells do not express ABCG2. By comparison, R2 cells treated with MTX exhibited 5.4-fold lower MFI (250) than PC cells, reflecting low intracellular concentrations of MTX resulting from efficient ABCG2-mediated efflux. Co-treatment of R2 cells with Ko143 and MTX dramatically increased MFI (1372) because Ko143 inhibited ABCG2-mediated MTX efflux (fig. 1b). The ABCG2 inhibitor essentially produced the ABCG2 null phenotype displayed by PC cells.
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Oncology 2014;87:364–370 © 2014 S. Karger AG, Basel www.karger.com/ocl
DOI: 10.1159/000366132
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
Gefitinib Ciprofloxacin
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indicating that ABCG2 functions to protect cells from ciprofloxacin-induced phototoxicity. Cytotoxicity was not observed in dark controls, even those treated with ciprofloxacin and Ko143 (data not shown), indicating that ciprofloxacin-induced cytotoxicity is dependent on photoreactive intermediates. Ciprofloxacin-induced cytotoxicity in PC cells is greater than in R2 cells because higher intracellular concentrations of ciprofloxacin are achieved owing to the lack of ABCG2-mediated efflux (fig. 2). Ciprofloxacin-induced cytotoxicity is unaffected by the addition of Ko143 because PC cells lack ABCG2 (fig. 2). Ko143 is an ABCG2 inhibitor that is used for in vitro studies but is not intended for therapeutic use in patients [15]. The TKIs gefitinib and imatinib were evaluated for their ability to inhibit ABCG2 in phototoxicity assays. Gefitinib enhanced ciprofloxacin-induced phototoxicity (fig. 3) in R2 cells in a manner similar to Ko143. Imatinib also caused enhancement of ciprofloxacin-induced phototoxicity in R2 cells but only with high concentrations of ciprofloxacin (fig. 3). Thus, gefitinib appears to be a more potent ABCG2 inhibitor than imatinib. Because PC cells lack ABCG2 and are unable to efflux ciprofloxacin, a dose-dependent increase in cytotoxicity was observed when PC underwent treatment with increasing concen-
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Fig. 3. Mean ± SD fractional survival of HEK 293 cells transfected with human ABCG2 cDNA (R2 cells) treated with increasing concentrations of ciprofloxacin with (black bars) or without (white bars) clinically relevant concentrations of the TKIs gefitinib (1 μM) or imatinib (3 μM).
Cell survival as a fraction of control
1.8
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Oncology 2014;87:364–370 DOI: 10.1159/000366132
© 2014 S. Karger AG, Basel www.karger.com/ocl
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
trations of ciprofloxacin (data not shown but was essentially identical to fig. 2). As expected, cytotoxicity was not enhanced with the addition of either gefitinib or imatinib (data not shown). Discussion
Our data indicate that ABCG2 functions to protect cells from ciprofloxacin-induced phototoxicity. Pharmacological inhibition of ABCG2 by TKI anticancer drugs enhanced ciprofloxacin-induced phototoxicity in this cell culture system. The importance of ABCG2 in protecting the retina from fluoroquinolone-induced phototoxicity is dramatically illustrated in the feline species. Because of a species-wide defect, feline ABCG2 is dysfunctional [6], creating extreme susceptibility to fluoroquinolone-induced retinal degeneration and blindness [5]. Our data suggest that TKI-mediated inhibition of ABCG2 may enhance susceptibility to fluoroquinolone-induced retinal damage in other species, including humans. Because the young eye is more optically transmissive than the adult eye, there is even greater risk for phototoxic injury to the retina in pediatric patients. We evaluated clinically relevant concentrations of gefitinib (1 μM) and imatinib (3 μM) in order to provide a more realistic estimate of the potential for drug-drug interactions in cancer patients. Gefitinib decreased cell survival to 50% when cells were concurrently treated with 50 μM ciprofloxacin and exposed to UV light. This concentration of gefitinib represents plasma concentrations achieved with routine doses used in cancer patients [16], suggesting that fluoroquinolone-induced retinal damage might occur in patients receiving gefitinib concurrently with photoreactive fluoroquinolones. In contrast, clinically relevant concentrations of imatinib [17] caused cytotoxicity only when very high concentrations of ciprofloxacin were used. Thus, imatinib appears less likely to be involved in a drug interaction that would cause retinal damage. In human patient populations, the maximum plasma concentrations of ciprofloxacin after a single dose range from 10 to 25 μM [18, 19]. This does not account for potential increases in ciprofloxacin concentration that might occur with repeated doses or in patients with renal compromise, which substantially decreases ciprofloxacin clearance. Survival of cells treated with gefitinib and 20 μM ciprofloxacin was roughly 77% compared to cells treated with gefitinib alone. Thus, patients receiving this drug combination are at risk for a drug-drug interaction leading to retinal toxicity. In addition to TKIs, there are other drugs that inhibit ABCG2. Considering the ongoing interest in ABCG2 inhibition to enhance chemotherapeutic efficacy in human cancer patients [20], it is important to be aware of the potential for fluoroquinolone-induced retinal degeneration that may result from blocking ABCG2 function. The results of our study highlight the potential for unanticipated adverse effects resulting from drug interactions with ABCG2-inhibiting drugs. Fluoroquinolones are often used to treat and/or prevent infections secondary to chemotherapy-induced neutropenia in cancer patients [11, 21]. Concurrent use of an ABCG2 inhibitor with a fluoroquinolone may cause retinal degeneration and blindness in human cancer patients because of enhanced retinal penetration of the fluoroquinolone. This should be added to the list of other potential drug interactions to consider when designing treatment regimens that include TKIs [22, 23, 24].
This paper was supported by a Washington State University College of Veterinary Medicine Intramural grant.
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Acknowledgment
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Oncology 2014;87:364–370 DOI: 10.1159/000366132
© 2014 S. Karger AG, Basel www.karger.com/ocl
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
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Vassileva SG, Mateev G, Parish LC: Antimicrobial photosensitive reactions. Arch Intern Med 1998;158:1993– 2000. Velpandian TR, Bankoti R, Humayun S, Ravi AK, Kumari SS, Biswas NR: Comparative evaluation of possible ocular photochemical toxicity of fluoroquinolones meant for ocular use in experimental models. Indian J Exp Biol 2006;44:387–391. Roberts JE: Ocular phototoxicity. J Photochem Photobiol 2001;64:136–143. Asashima TS, Hori S, Ohtsuki S, Tachikawa M, Watanabe M, Mukai C, Kitagaki S, Miakoshi N, Terasaki T: ATPbinding cassette transporter G2 mediates the efflux of phototoxins on the luminal membrane of retinal capillary endothelial cells. Pharm Res 2006;23:1235–1242. Wiebe V, Hamilton P: Flouoroquinolone-induced retinal degeneration in cats. J Am Vet Med Assoc 2002;221: 1568–1571. Ramirez CJ, Minch JD, Gay JM, Lahmers SM, Guerra DJ, Haldorson GJ, Schneider T, Mealey KL: Molecular genetic basis for fluoroquinolone-induced retinal degeneration in cats. Pharmacogenet Genomics 2011;21:66–75. Etminan M, Forooghian F, Brophy JM, Bird ST, Maberley D: Oral fluoroquinolones and the risk of retinal detachment. JAMA 2012;307:1414–1419. Rampal S, Kaur R, Sethi R, Singh O, Sood N: Olfloxacin-associated retinopathy in rabbits: role of oxidative stress. Hum Exp Toxicol 2008;27:409–415. Shimoda K, Yoshida M, Wagai N, Takayama S, Kato M: Phototoxic lesions induced by quinolone antibacterial agents in auricular skin and retina of albino mice. Toxicol Pathol 1993;21:554–561. Robey RW, To KK, Polgar O, Dohse M, Fetsch P, Dean M, Bates SE: ABCG2: a perspective. Adv Drug Deliv Rev 2009;61:3–13. Flowers CR, Seidenfeld J, Bow EJ, Karten C, Gleason C, Hawley DK, Kuderer NM, Langston AA, Marr KA, Rolston KV, Ramsey SD: Antimicrobial prophylaxis and outpatient management of fever and neutropenia in adults treated for malignancy: American Society of Clinical Oncology clinical practice guideline. J Clin Oncol 2013;31: 794–810. Yabe K, Goto K, Jindo T, Sekiguchi M, Furuhama K: Structure-phototoxicity relationship in Balb/c mice treated with fluoroquinolone derivatives, followed by ultraviolet-A irradiation. Toxicol Lett 2005;157:203–210. Morisaki K, Robey RW, Ozgegy-Laczka C, Honjo Y, Polgar O, Steadman K, Sarkadi B, Bates SE: Single nucleotide polymorphisms modify the transport activity of ABCG2. Cancer Chemother Pharmacol 2005;56:161–172. Robey RW, Honjo Y, van de Laar A, Miyake K, Regis JT, Litman T, Bates SE: A functional assay for detection of the mitoxantrone resistance protein MXR (ABCG2). Biochim Biophys Acta 2001;1512:171–182. Mealey KL, Barhoumi R, Burghardt RC, Safe S, Kochevar DT: Doxycycline induces expression of P-glycoprotein in MCF-7 breast carcinoma cells. Antimicrob Agents Chemother 2002;46:755–761. Stacy AF, Jansson PJ, Richardson DR: Molecular pharmacology of ABCG2 and its role in chemoresistance. Mol Pharmacol 2013;84:655–669. Nakamura Y, Sano K, Soda H, Takatani H, Fukuda M, Nagashima S, Hayashi T, Oka M, Tsukamoto K, Kohno S: Pharmacokinetics of gefitinib predicts antitumor activity for advanced non-small cell lung cancer. J Thorac Oncol 2010;5:1404–1409. Nikolova A, Peng B, Hubert M, Sieberling M, Keller U, Ho YY, Schran H, Capdeveille R: Bioequivalence, safety, and tolerability of imatinib tablets compared with capsules. Cancer Chemother Pharmacol 2004;53:433–438. Kontou P, Chatzika K, Pitsiou G, Stanopoulos I, Argyropoulou-Pataka P, Kioumis I: Pharmacokinetics of ciprofloxacin and its penetration into bronchial secretions of mechanically ventilated patients with chronic obstructive pulmonary disease. Antimicrob Agents Chemother 2011;55:4149–4153. Lubart E, Berkovitch M, Leibovitz A, Britzi M, Soback S, Bukasov Y, Segal R: Pharmacokinetics of ciprofloxacin in hospitalized geriatric patients: comparison between nasogastric tube and oral administration. Ther Drug Monitor 2013;35:653–656. Henrich CJ, Robey RW, Bokesch HR, Bates SE, Shukla S, Ambudkar SV, Dean M, McMahon JB: New inhibitors of ABCG2 identified by high-throughput screening. Mol Cancer Ther 2007;6:3271–3278. Cullen M, Baijal S: Prevention of febrile neutropenia: use of prophylactic antibiotics. Br J Cancer 2009; 101(suppl 1):S11–S14. Bhullar J, Natarajan K, Shukla S, Mathias TJ, Sadowska M, Ambudkar SV, Baer MR: The FLT3 inhibitor quizartinib inhibits ABCG2 at pharmacologically relevant concentrations, with implications for both chemosensitization and adverse drug interactions. PloS One 2013;8:e71266. Bilbao-Meseguer I, Jose BS, Lopez-Gimenez LR, Gil MA, Serrano L, Castaño M, Sautua S, Basagoiti AD, Belaustegui A, Baza B, Baskaran Z, Bustinza A: Drug interactions with sunitinib. J Oncol Pharm Pract 2014, Epub ahead of print.
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© 2014 S. Karger AG, Basel 0030–2414/14/0876–0364$39.50/0 www.karger.com/ocl
Short Communication
Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2 Katrina L. Mealey
Sandamali Dassanayake
Neal S. Burke
Individualized Medicine Program, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Wash., USA
Key Words ABCG2 · Fluoroquinolone · Retina · Phototoxicity · Tyrosine kinase inhibitors Abstract The tyrosine kinase inhibitor (TKI) class of anticancer agents inhibits ABCG2-mediated drug efflux. ABCG2 is an important component of the blood-retinal barrier, where it limits retinal exposure to phototoxic compounds such as fluoroquinolone antibiotics. Patients treated with TKIs would be expected to be at greater risk for retinal phototoxicity. Using an in vitro system, our results indicate that the TKIs gefitinib and imatinib abrogate the ability of ABCG2 to protect cells against ciprofloxacin-induced phototoxicity. We conclude that the concurrent administration of ABCG2 inhibitors with photoreactive fluoroquinolone antibiotics may result in retinal damage. © 2014 S. Karger AG, Basel
Introduction
Katrina L. Mealey Individualized Medicine Program, Department of Veterinary Clinical Sciences College of Veterinary Medicine, Washington State University 100 Grimes Way, Pullman, WA 99164-6610 (USA) E-Mail kmealey @ vetmed.wsu.edu
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The organs most susceptible to phototoxicity are the skin and the eyes, including the retina [1, 2]. Phototoxic damage to the retina can lead to retinal (macular) degeneration and blindness [3]. Normally, the retina is protected from photoreactive compounds by the bloodretina barrier with the ABC (ATP-binding cassette) transporter ABCG2 playing a critical role [4]. ABCG2 is expressed on the luminal membrane of retinal capillary endothelial cells, where it actively effluxes potentially phototoxic endogenous and exogenous substrates including pheophorbide, protoporphyrin IX and fluoroquinolone antibiotics [4, 5]. The importance of ABCG2 in protecting the retina from fluoroquinolone-induced phototoxicity is dramatically
365
Oncology 2014;87:364–370 DOI: 10.1159/000366132
© 2014 S. Karger AG, Basel www.karger.com/ocl
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
illustrated in the feline species. Owing to a species-wide amino acid change, feline ABCG2 is dysfunctional, resulting in extreme susceptibility to fluoroquinolone-induced retinal degeneration and blindness [6]. Fluoroquinolones have been documented to cause ocular toxicity in other species, including humans [7–9]. Whether or not human patients experience fluoroquinolone-induced retinal degeneration under circumstances of ABCG2 dysfunction has not been investigated. Several commonly used anticancer drugs [tyrosine kinase inhibitors (TKIs) such as imatinib and gefitinib] inhibit ABCG2 function [10]. Concurrent administration of these drugs with fluoroquinolones, which are often recommended for chemotherapy-induced neutropenia [11], could result in fluoroquinolone-induced retinal toxicity in human patients. The goal of this study was to determine if pharmacological inhibition of ABCG2 would enhance fluoroquinolone-induced phototoxicity using a cell culture system. Ciprofloxacin was selected as a representative fluoroquinolone with intermediate phototoxic potential [12].
Materials and Methods Reagents and Cell Lines HEK 293 cells transfected with human ABCG2 (R2 cells) and plasmid control (PC cells) were provided by R. Robey (Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Md., USA). Ciprofloxacin and MTT [3-(4,5 dimethylthiazol-2-thiol)-2,5-diphenyl2H-tetrazolium bromide] were obtained from Sigma Aldrich (St. Louis, Mo., USA), Ko143 from R & D Systems, Inc. (Minneapolis, Minn., USA), gefitinib and imatinib from LC Laboratories (Woburn, Ma., USA), and mitoxantrone (MTX) from Hospira Inc. (Lake Forest, Ill., USA). Western Blot Analysis of ABCG2 Expression in Transfected HEK293 Cells Twenty micrograms of total protein from each cell line were separated on 4–15% Tris-HCl gradient polyacrylamide gels and electrotransferred to polyvinylidene difluoride membranes. Membranes were blocked and then incubated with rat anti-mouse ABCG2 monoclonal antibody (6D170; Santa Cruz Biotechnology, Santa Cruz, Calif., USA). Membranes were washed and incubated with goat anti-rat alkaline phosphatase-conjugated secondary antibody (Santa Cruz Biotechnology). Membranes were washed and binding was detected using Immun-Star AP Substrate (Bio-Rad, Hercules, Calif., USA).
Phototoxicity Assays For phototoxicity assays, R2 and PC cells were plated in 6-well plates and then incubated for 24 h to allow cells to adhere. Cells were then treated with the ABCG2 inhibitor Ko143 (10 μM) for 30 min, followed by treatment with ciprofloxacin (0, 20, 50, 100 and 200 μM) for 1 h. The media was replaced with drug-free media, and cells were then exposed to UVA light (National Biologicals Corporation, Beachwood, Ohio, USA) for 60 min. ‘Dark’ controls included identically treated cells not exposed to UV light. After 24-hour incubation,
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Flow Cytometry To confirm function of transfected ABCG2 in R2 cells, MTX efflux was assessed using flow cytometry. MTX, an intrinsically fluorescent compound, is a prototype substrate for ABCG2 routinely used to assess ABCG2 function [13, 14]. Its efflux from cells is directly measured using flow cytometry to determine intracellular mean fluorescence intensity (MFI). Transfected cells (R2 and PC) were plated in 6-well plates and incubated overnight. Cells were then treated with either vehicle (control) or the ABCG2 inhibitor Ko143 (10 μM) for 30 min, followed by treatment with MTX (10 μM) or no treatment (control) for an additional 60 min. Cells treated with neither Ko143 nor MTX were used to generate the control histograms, which measure cell autofluorescence. After incubation, cells were washed with ice-cold phosphate-buffered saline, placed on ice and immediately analyzed. A FACSCalibur flow cytometer with a 635-nm red diode laser, a 670-nm bandpass filter and Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, Calif., USA) was used to detect intracellular MFI. At least 10,000 events were collected. The mean channel number for each histogram represents the MFI for comparison between different cell lines (PC vs. R2) and treatments (MTX alone vs. MTX plus ABCG2 inhibitor Ko143). Experiments were run in duplicate, and each experiment was repeated on separate days.
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Oncology 2014;87:364–370 © 2014 S. Karger AG, Basel www.karger.com/ocl
DOI: 10.1159/000366132
R2
Color version available online
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
PC
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Fig. 1. a Immunoblot assessment of ABCG2 expression in HEK 293 cells transfected with empty plasmid (PC cells) or ABCG2 cDNA (R2 cells). b Representative histograms from a functional assay for ABCG2 function in transfected cell lines. Efflux assays were performed with MTX at 10 μM concentrations with or without the ABCG2 inhibitor Ko143 (10 μM). Cell count is represented on the y-axis and fluorescence intensity on the xaxis, with the MFI indicated parenthetically.
cytotoxicity was assessed using the MTT cytotoxicity assay in which viable cells metabolize MTT to a colorimetrically measurable product (formazan). Cell survival was expressed as a fraction of the mean optical density of vehicle-treated controls. Means represent results from experiments performed in duplicate and repeated on separate days. For assessing the effects of TKIs on ciprofloxacin-induced phototoxicity, experiments were repeated as described above, substituting either gefitinib (1 μM) or imatinib (3 μM) for Ko143. For all experiments, each well contained the same concentration of vehicle in order to eliminate any effects the vehicle might have on either intracellular drug concentration or cytotoxicity.
Confirmation of ABCG2 Expression and Function ABCG2 expression was confirmed in R2 cells but was absent in PC cells (fig. 1a). Function of transfected ABCG2 in R2 cells was confirmed using flow cytometry to determine MFI after
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Results
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Oncology 2014;87:364–370 © 2014 S. Karger AG, Basel www.karger.com/ocl
DOI: 10.1159/000366132
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
R2 cells Ciprofloxacin
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Fig. 2. Mean ± SD fractional survival of HEK 293 cells transfected with ABCG2 cDNA (R2 cells) or empty plasmid (PC cells) treated with increasing concentrations of ciprofloxacin alone (white bars) or concurrently treated with the ABCG2 inhibitor Ko143 (black bars).
Cell survival as a fraction of control
1.4
Ciprofloxacin + Ko143 (10 μM)
1.2 1.0 0.8 0.6 0.4 0.2 0
0
20 50 100 Concentration of ciprofloxacin (μM)
200
Impact of ABCG2 Inhibition on Ciprofloxacin-Induced Phototoxicity In R2 cells, ABCG2 inhibition by 10 μM Ko143 dramatically increases cytotoxicity of ciprofloxacin in a dose-dependent manner (fig. 2) compared to cytotoxicity of ciprofloxacin alone,
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treatment with MTX. Figure 1b shows very low intrinsic fluorescence of R2 and PC cells (3.9 and 5.5, respectively). When cells are treated with the ABCG2 substrate MTX, both R2 and PC cells display greater MFI reflecting correspondence to intracellular MTX concentration. Figure 1b shows that PC cells treated with MTX were highly fluorescent (MFI 1351), reflecting a high concentration of intracellular MTX. PC cells treated with both Ko143 (ABCG2 inhibitor) and MTX had a negligible effect on MFI (1399) because these cells do not express ABCG2. By comparison, R2 cells treated with MTX exhibited 5.4-fold lower MFI (250) than PC cells, reflecting low intracellular concentrations of MTX resulting from efficient ABCG2-mediated efflux. Co-treatment of R2 cells with Ko143 and MTX dramatically increased MFI (1372) because Ko143 inhibited ABCG2-mediated MTX efflux (fig. 1b). The ABCG2 inhibitor essentially produced the ABCG2 null phenotype displayed by PC cells.
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DOI: 10.1159/000366132
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Gefitinib Ciprofloxacin
Cell survival as a fraction of control
1.4
Ciprofloxacin + gefitinib (1 μM)
1.2 1.0 0.8 0.6 0.4 0.2 0
0
20 50 100 Concentration of ciprofloxacin (μM)
200
Imatinib Ciprofloxacin
Ciprofloxacin + imatinib (3 μM)
1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0
0
20 50 100 Concentration of ciprofloxacin (μM)
200
indicating that ABCG2 functions to protect cells from ciprofloxacin-induced phototoxicity. Cytotoxicity was not observed in dark controls, even those treated with ciprofloxacin and Ko143 (data not shown), indicating that ciprofloxacin-induced cytotoxicity is dependent on photoreactive intermediates. Ciprofloxacin-induced cytotoxicity in PC cells is greater than in R2 cells because higher intracellular concentrations of ciprofloxacin are achieved owing to the lack of ABCG2-mediated efflux (fig. 2). Ciprofloxacin-induced cytotoxicity is unaffected by the addition of Ko143 because PC cells lack ABCG2 (fig. 2). Ko143 is an ABCG2 inhibitor that is used for in vitro studies but is not intended for therapeutic use in patients [15]. The TKIs gefitinib and imatinib were evaluated for their ability to inhibit ABCG2 in phototoxicity assays. Gefitinib enhanced ciprofloxacin-induced phototoxicity (fig. 3) in R2 cells in a manner similar to Ko143. Imatinib also caused enhancement of ciprofloxacin-induced phototoxicity in R2 cells but only with high concentrations of ciprofloxacin (fig. 3). Thus, gefitinib appears to be a more potent ABCG2 inhibitor than imatinib. Because PC cells lack ABCG2 and are unable to efflux ciprofloxacin, a dose-dependent increase in cytotoxicity was observed when PC underwent treatment with increasing concen-
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Fig. 3. Mean ± SD fractional survival of HEK 293 cells transfected with human ABCG2 cDNA (R2 cells) treated with increasing concentrations of ciprofloxacin with (black bars) or without (white bars) clinically relevant concentrations of the TKIs gefitinib (1 μM) or imatinib (3 μM).
Cell survival as a fraction of control
1.8
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Oncology 2014;87:364–370 DOI: 10.1159/000366132
© 2014 S. Karger AG, Basel www.karger.com/ocl
Mealey et al.: Tyrosine Kinase Inhibitors Enhance Ciprofloxacin-Induced Phototoxicity by Inhibiting ABCG2
trations of ciprofloxacin (data not shown but was essentially identical to fig. 2). As expected, cytotoxicity was not enhanced with the addition of either gefitinib or imatinib (data not shown). Discussion
Our data indicate that ABCG2 functions to protect cells from ciprofloxacin-induced phototoxicity. Pharmacological inhibition of ABCG2 by TKI anticancer drugs enhanced ciprofloxacin-induced phototoxicity in this cell culture system. The importance of ABCG2 in protecting the retina from fluoroquinolone-induced phototoxicity is dramatically illustrated in the feline species. Because of a species-wide defect, feline ABCG2 is dysfunctional [6], creating extreme susceptibility to fluoroquinolone-induced retinal degeneration and blindness [5]. Our data suggest that TKI-mediated inhibition of ABCG2 may enhance susceptibility to fluoroquinolone-induced retinal damage in other species, including humans. Because the young eye is more optically transmissive than the adult eye, there is even greater risk for phototoxic injury to the retina in pediatric patients. We evaluated clinically relevant concentrations of gefitinib (1 μM) and imatinib (3 μM) in order to provide a more realistic estimate of the potential for drug-drug interactions in cancer patients. Gefitinib decreased cell survival to 50% when cells were concurrently treated with 50 μM ciprofloxacin and exposed to UV light. This concentration of gefitinib represents plasma concentrations achieved with routine doses used in cancer patients [16], suggesting that fluoroquinolone-induced retinal damage might occur in patients receiving gefitinib concurrently with photoreactive fluoroquinolones. In contrast, clinically relevant concentrations of imatinib [17] caused cytotoxicity only when very high concentrations of ciprofloxacin were used. Thus, imatinib appears less likely to be involved in a drug interaction that would cause retinal damage. In human patient populations, the maximum plasma concentrations of ciprofloxacin after a single dose range from 10 to 25 μM [18, 19]. This does not account for potential increases in ciprofloxacin concentration that might occur with repeated doses or in patients with renal compromise, which substantially decreases ciprofloxacin clearance. Survival of cells treated with gefitinib and 20 μM ciprofloxacin was roughly 77% compared to cells treated with gefitinib alone. Thus, patients receiving this drug combination are at risk for a drug-drug interaction leading to retinal toxicity. In addition to TKIs, there are other drugs that inhibit ABCG2. Considering the ongoing interest in ABCG2 inhibition to enhance chemotherapeutic efficacy in human cancer patients [20], it is important to be aware of the potential for fluoroquinolone-induced retinal degeneration that may result from blocking ABCG2 function. The results of our study highlight the potential for unanticipated adverse effects resulting from drug interactions with ABCG2-inhibiting drugs. Fluoroquinolones are often used to treat and/or prevent infections secondary to chemotherapy-induced neutropenia in cancer patients [11, 21]. Concurrent use of an ABCG2 inhibitor with a fluoroquinolone may cause retinal degeneration and blindness in human cancer patients because of enhanced retinal penetration of the fluoroquinolone. This should be added to the list of other potential drug interactions to consider when designing treatment regimens that include TKIs [22, 23, 24].
This paper was supported by a Washington State University College of Veterinary Medicine Intramural grant.
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Acknowledgment
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Oncology 2014;87:364–370 DOI: 10.1159/000366132
© 2014 S. Karger AG, Basel www.karger.com/ocl
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