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P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model
Accepted Manuscript Title: Nilotinib reverses ABCB1/P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model Author: Zhi-yong Zhou Li-li Wan Quan-jun Yang Yong-long Han Dan Li Jin Lu Cheng Guo PII: DOI: Reference:
S0378-4274(16)33086-7 http://dx.doi.org/doi:10.1016/j.toxlet.2016.07.710 TOXLET 9568
To appear in:
Toxicology Letters
Received date: Revised date: Accepted date:
14-4-2016 6-7-2016 30-7-2016
Please cite this article as: Zhou, Zhi-yong, Wan, Li-li, Yang, Quan-jun, Han, Yonglong, Li, Dan, Lu, Jin, Guo, Cheng, Nilotinib reverses ABCB1/P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2016.07.710 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nilotinib reverses ABCB1/P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model Zhi-yong Zhou 1, 2, Li-li Wan 1, Quan-jun Yang 1, Yong-long Han 1, Dan Li 1, Jin Lu 1, Cheng Guo1
Authors’ Affiliations:
1
Department of Pharmacy, Shanghai Jiao
Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China;
2
College of Medical Science, Three Gorges University,
443002 Yichang, Hubei, China.
Corresponding Author: Cheng Guo, Department of Pharmacy, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China. Phone: +86 21 24058789; Fax: +86 21 24058789; E-mail : [email protected].
1
Highlight 1. Nilotinib can reverse P-gp-mediated multidrug resistance of DOX in vitro and in vivo. 2. Co-administration of nilotinib increase the accumulation of DOX and DOXol in heart. 3. Combination with nilotinib will enhance the cardiotoxicity of DOX in xenograft mice.
Abstract The BCR-Abl tyrosine kinase inhibitor (TKI), nilotinib, was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia-positive chronic myelogenous leukemia. Recent studies have shown that nilotinib induces potent sensitization to anticancer agents by blocking the functions of ABCB1/P-glycoprotein (P-gp) in multidrug resistance (MDR). However, changes in P-gp expression or function affect the cardiac disposition and prolong the presence of both doxorubicin (DOX) and doxorubicinol (DOXol) in cardiac tissue, thus, enhancing the risk of cardiotoxicity. In this study, we used a MDR xenograft model to evaluate the antitumor activity, tissue distribution and cardiotoxicity of DOX when co-administered with nilotinib. This information will provide more insight into the pharmacological role of nilotinib in MDR reversal and the risk of DOX cardiotoxicity. Our results showed that nilotinib significantly enhanced DOX cytotoxicity and increased intracellular rhodamine 123 accumulation in MG63/DOX cells in vitro and strongly enhanced DOX inhibition of growth of 2
P-gp-overexpressing MG63/DOX cell xenografts in nude mice. Additionally, nilotinib significantly increased DOX and DOXol accumulation in serum, heart, liver and tumor tissues. Importantly, nilotinib induced a disproportionate increase in DOXol in cardiac tissue. In the co-administration group, CBR1 and AKR1A1 protein levels were significantly increased in cardiac tissue, with more severe necrosis and vacuole formation. These results indicate that nilotinib reverses P-gpmediated MDR by blocking the efflux function and potentiates DOX-induced cardiotoxicity. These findings represent a guide for the design of future clinical trials and studies of pharmacokinetic interactions and may be useful in guiding the use of nilotinib in combination therapy of cancer in clinical practice. Keywords:
P-glycoprotein,
Doxorubicin,
Cardiotoxicity
3
Multidrug
resistance,
Introduction Doxorubicin (DOX) is an anthracycline first extracted from Streptomyces peucetius var. caesius in the 1970s. It is frequently used in chemotherapy against non-Hodgkin’s and Hodgkin’s lymphomas, multiple myeloma, sarcoma, pediatric cancers and several solid tumors including breast, lung, gastric, ovarian, and thyroid cancers(1-3); however, its clinical use is severely limited due to the development of a progressive dose-dependent cardiomyopathy that results in irreversible congestive heart failure(4,5). Currently, total cumulative dose is the only criteria currently used to predict the toxicity(6,7). The application of DOX is further limited by lack of cytotoxicity against cells expressing multidrug resistance (MDR) proteins leading to the emergence of multidrug resistance(8). Finding ways to maintain the efficacy of DOX and reduce toxicity has now become a major focus of DOX research. P-glycoprotein (P-gp), which is the product of the ABCB1 (or MDR1) gene and is a member of a superfamily of ATP-binding cassette (ABC) membrane transport protein, that was first characterized in multidrug resistant Chinese hamster ovary cells by Juliano and Ling(9). P-gp is a broad-spectrum drug efflux pump that has 12 transmembrane regions and two ATP-binding sites that bind either neutral or positively charged hydrophobic drug substrates(10). P-gp overexpression, which is the most frequent cause of MDR, decreases the cytotoxicity of antitumor drugs including anthracyclines, Vinca alkaloids, podophyllotoxins and taxanes(11), thereby mediating resistance to these agents and affecting their pharmacological behavior. In the past three decades, the search for an effective P-gp inhibitor in the hope of circumventing MDR has become an important focus of research. In recent years, several TKIs including lapatinib(12), gefitinib(13), erlotinib(14), cediranib(15), and sunitinib(16) have been reported to inhibit the functions of P-gp, thereby 4
overcoming chemotherapy resistance in MDR cancer cells. Taken together, these reports suggest that TKIs represent promising MDR inhibitors. However, P-gp is not an abnormal protein, P-gp is a robust detoxification protein that functions as a barrier against xenobiotic entry in the intestine and at the blood–brain barrier and is involved in drug excretion in the kidney, liver, and heart(17,18). Chuan reported that both sodium Tan-IIA sulfonate and sodium danshensu inhibit the expression and efflux functionality of placental P-gp, thereby increasing the transplacental movement of digoxin, and enhancing the effectiveness of the drug used to treat congestive fetal heart failure, while minimizing the risk of maternal toxicity(19). After co-administration with a specific P-gp inhibitor zosuquidar trihydrochloride (LY335979), the concentration of paclitaxel in the brain was significantly increased, while PET-CT imaging showed 2.3-fold increased levels of [18F]-gefitinib in the brain of Abcb1a/1b;Abcg2-/- mice compared to wild-type following intravenous administration of [18F]-gefitinib (1 mg/kg)(20). Based on the results of tissue distribution studies, it has been suggested that P-gp plays an important role in the protection of the organism against potentially toxic agents. Nilotinib, a small-molecule BCR-Abl TKI, was developed to overcome resistance or intolerance to imatinib in patients with Philadelphia-positive chronic myelogenous leukemia(21,22). Previously, it was reported that nilotinib significantly reverses P-gp mediated MDR in cancer cells by inhibiting its activity(23-25). In this study, we used a MDR cancer cell xenograft model to investigate the tissue distribution and risk of cardiotoxicity of DOX administered in combination with nilotinib to reverse drug resistance. Materials and Methods 5
Reagents Doxorubicin was obtained from Pfizer Italia Srl (Latina, Italy). Nilotinib was obtained from Novartis (Stein, Switzerland). Rhodamine 123 (Rho 123), MTT, daunorubicin, and verapamil were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Monoclonal antibodies against P-gp (ab3366), BCRP (108312), CBR1 (ab4148) and AKR1A1 (ab56635) were from Abcam (Hong Kong). All the biochemical assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Acetonitrile and methanol (Tedia, CA, USA) were HPLC grade. Distilled, deionized water was produced by a Milli-Q Reagent Water System (Millipore, MA, USA). All other solvents and chemicals were analytical grade or better. Cell lines All cell lines were cultured in vitro as monolayers in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in an atmosphere of 5% CO2 in air. The human osteosarcoma parental cell line MG63 was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The human MDR osteosarcoma cell line MG63/DOX, which was selected in a step-wise manner by exposing drug-sensitive MG63 cells to increasing doses of adriamycin, was kindly provided by Dr. Shen Zan (Department of Oncology, Affiliated Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai, China). All cells were grown in drug-free culture medium for >2 weeks before use in assays. Animals Athymic nude mice (BALB/c-nude), aged 4–5 weeks and weighing 16–18 g, were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (animal experimental license No. SCXK (hu) 2007-0005), and used for 6
the MG63/DOX cell xenografts. All animals received sterilized food and water ad libitum. An acclimation period of approximately 1 week was allowed between animal arrival and tumor inoculation. All experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals (Center of Experimental Animals, Shanghai Jiao Tong University, China), and all protocols involving animals were approved by the Ethics Committee for Animal Experiments. Cytotoxicity tests MTT assays were conducted as previously described to assess the sensitivity of cells to drugs(26). Drug effects were presented as the IC50 drug concentration and were determined by XLFit software using equation 205. The degree of resistance was estimated by dividing the IC50 of MG63/DOX cells by that of MG63 cells; the fold-MDR reversal factor was calculated by dividing the IC50 of the anticancer agent in the absence of nilotinib by that obtained in the presence of nilotinib. Rho123 accumulation The effect of nilotinib on the intracellular accumulation of Rho 123 between MG63 and MG63/DOX cells was measured by flow cytometry as previously described(27). The P-gp inhibitor verapamil was used as a positive control for MG63/DOX cells. Cells in logarithmic growth were treated with or without nilotinib (2.5, 5, and 10 μM) and verapamil (10 μM) at 37°C for 3 h and then incubated with 5 μM Rho 123 for a further 0.5 h at 37°C. The cells were then collected, centrifuged, and washed twice with ice-cold PBS and resuspended in 500 μL PBS for flow cytometric analysis (BD FACSCalibur, Cellquest PRO, USA). A minimum of 10,000 cells were analyzed for each histogram generated. Nude mouse xenograft model The MG63/DOX inoculated nude xenograft model previously established by us was used in this study(26). The xenograft was found to maintain the 7
MDR phenotype in vivo and was extremely resistant to doxorubicin treatment. Briefly, MG63/DOX cells were suspended at 5×108 cells/mL in serum-free DMEM/Matrigel (1:1). Then, 200 μL of these suspensions (1×107 cells) was injected subcutaneously into the right flank of athymic nude mice. When the tumors reached a mean diameter of 0.5 cm, the mice were randomized into four groups and treated according to various regimens: (a) saline (q3d ×5); (b) doxorubicin (5 mg/kg, i.v., q3d ×5); (c) nilotinib (60 mg/kg, p.o., q3d ×5); and (d) doxorubicin (5 mg/kg, i.v., q3d ×5) + nilotinib (60 mg/kg, p.o., q3d ×5 given 1 h before injecting doxorubicin). The body weights of the animals and the two perpendicular diameters (a and b) were recorded every 3 days, and tumor volume (V) was estimated according to the following formula: V (mm3) = 0.5 a × b2, where V is the tumor volume, a is the long diameter, and b is the short diameter. Tumor growth curves were generated according to tumor volume and days post-inoculation. Tumor tissues were excised from the mice and their weights were measured. The ratio of growth inhibition (IR) was calculated according to the following formula(27): IR(%) = 1- mean tumor weight of experimental group/ mean tumor weight of control group × 100%. Blood and tissue collection Blood and tissue samples were collected at 3 h after the last administration. Blood was obtained by orbital bleeding under CO2 anesthesia and collected in tubes; serum was separated within 1 h. The animals were sacrificed by cervical dislocation to collect the following tissues: heart, liver, spleen, kidney, and tumor. The tissues were dissected out immediately, washed with ice-cold saline, and homogenized with a glass tissue homogenizer in saline to prepare 10% (w/v) homogenates, 8
which were stored at -20°C for analysis. LC/MS/MS assay for doxorubicin and doxorubicinol concentration in serum and tissue homogenates The apparatus and chromatographic analysis conditions were as previously described(28). Samples were prepared by adding 30 μL buffer (aqueous solution of 0.1% formic acid) and 120 μL of internal standard solution (40 ng/mL daunorubicin) to 30 μL of mouse plasma and then vortexing for 1 min. After centrifugation at 15,000 ×g for 5 min, 100 μL of the supernatant was transferred to a sample vial and 5 μL was injected into the LC/MS/MS for quantitative analysis. The LC–MS–MS system was an Agilent 1200 liquid chromatography and a 6410B triple quadrupole mass spectrometer with an electrospray ionization source. Data were analyzed by MassHunter software (Agilent Corporation, MA, USA). Chromatography was performed on a Zorbax XDB-C18 column (2.1mm×150mm i.d., 3.5µm, Agilent Corporation, MA, USA) maintained at 35 ºC using gradient elution with 5mM ammonium formate and 0.1% formic acid in purified water as solvent A and acetonitrile as solvent B. The gradient program was then as follows: 0-1 min, 88%→25% A, 1–2.5 min, 25%→88% A, and 2.5–6 min, 88% A. The flow rate was set at 0.4ml/min. Electrospray ionization (ESI) was performed in the positive ion mode and quantified by the selected reaction monitoring transitions as protonated precursor/product ion transition of m/z 544/397 for doxorubicin, 546/399 for doxorubicinol and 528/321 for IS (Daunorubicin), nebulizer and drying gas were high-purity nitrogen and drying gas, gas temperature and gas flow were set at 350 ◦C and 9.0 L/min. Determination of biochemical indicators To determine specific cardiac tissue toxicity, we investigated parameters 9
including creatine kinase (CK) and lactate dehydrogenase (LDH) in the heart tissue homogenate, which are used as specific markers of cardiac damage in the clinic. One of the most important mechanisms in DOX induced-cardiotoxicity is lipid peroxidation. In the present study, lipid peroxidation was expressed as nmoles of malondialdehyde (MDA, a product formed by the peroxidation of membrane lipids) per milligram of protein. The concentration of superoxide dismutase (SOD, catalyzes the conversion of superoxide radicals to hydrogen peroxide, leading to the formation of hydroxyl radicals) was also measured. The assays were carried out using kits according to the manufacturer’s instructions. Histopathological studies The left ventricular portion of heart tissues was fixed in 10% neutral buffered formalin. Sections (3–5 μm thickness) were stained with hematoxylin
and
eosin
for
histological
examination.
The
histo-morphological evaluation of all the heart sections was carried out in a blinded fashion by a pathologist who was unaware of the treatment groups. Western blot analysis To identify the effects of nilotinib on P-gp expression, the MG63/DOX cells were incubated with different concentrations of nilotinib for 72 h. To clarify the possible mechanism of enhanced cardiotoxicity, we determined the expression of CBR1 and AKR1A1 in heart tissue. The protein concentration was determined by BCA assay, before being separated
by
10%
sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes by electrophoretic transfer. After blocking in 5% non-fat milk at room temperature for 2 h, membranes were subsequently probed with primary antibodies at 4°C overnight. The membranes were then washed three times with TBST buffer and incubated with secondary antibodies at 10
room temperature for 2 h. Immunoreactivity was visualized using an electrochemiluminescence
kit
according
to
the
manufacturer’s
instructions. The density of the immunoreactive bands was analyzed using Image J2x (National Institutes of Health, Bethesda, MD, USA). Statistical analysis All experiments were repeated at least three times and differences were determined by using Student’s t-test and one-way ANOVA with post-hoc analysis. Statistical significance was set at P < 0.05. Results Nilotinib reverses P-gp mediated MDR in MG63/DOX cells To examine the effect of nilotinib on the reversal of P-gp-mediated MDR in cancer cells, we first determined the cytotoxicity of nilotinib alone in MG63/DOX cell line with the MTT assay. More than 90% of MG63/DOX cells were viable when treated with nilotinib up to 3.1 μM; therefore, we used 0.75, 1.5 and 3 μM nilotinib to reverse MDR in vitro. The doxorubicin IC50 in MG63 cells was 0.044 ± 2.71E-05 μM; however, the concentration of doxorubicin required to inhibit MG63/DOX cells by 50% was 20.289 ± 6.82E-03 μM (Table 1). Thus, overexpression of P-gp resulted in a significant increase in resistance to doxorubicin (461-fold). We then investigated the cytotoxic effect of DOX in MG63 and MG63/DOX cells in the presence of 0.75, 1.5, or 3 μM nilotinib. The sensitivity to doxorubicin was unchanged when MG63 cells were co-incubated with nilotinib at concentrations up to 3 μM. In contrast, MG63/DOX cells were sensitized by low concentrations of nilotinib. At concentrations of 0.75, 1.5 and 3 μM, nilotinib induced increasingly effective reversal of resistance to doxorubicin (27-fold, 81-fold and 141-fold, respectively). These results suggested that nilotinib potently reverses P-gp-mediated resistance to doxorubicin in vitro. Cell survival was determined by MTT assay as described in the Materials and 11
Methods. Data represent the mean ± SD of at least three independent experiments performed in triplicate. The fold-MDR reversal (values given in parentheses) was calculated by dividing the anticancer drug IC50 for cells in the absence of nilotinib or verapamil by that obtained in their presence.
Nilotinib enhances the accumulation of Rho 123 in MG63/DOX cells To investigate the mechanism by which nilotinib influences the function of P-gp, we determined the effect of nilotinib on rhodamine accumulation in MG63/DOX cells (Fig. 1). In the absence of nilotinib, the level of Rho 123 accumulation was low in MG63/DOX cells and nilotinib restored the level of Rho 123 accumulation in a dose-dependent manner. These results showed that nilotinib increases intracellular accumulation of the P-gp substrate in cells expressing P-gp. The accumulation of Rho 123 was measured by flow cytometry as described in the Materials and Methods. The intracellular accumulation in MG63/DOX was significantly increased in the presence of nilotinib in a dose-dependent manner. Representative data of least three independent experiments are shown.
Nilotinib reverses P-gp-mediated MDR in the nude mouse MDR xenograft model An established MG63/DOX cell xenograft model in nude mice was used to evaluate the efficacy of nilotinib in reversing doxorubicin resistance in vivo. There were no significant difference in tumor size between animals treated with saline, nilotinib, or doxorubicin, indicating the existence of DOX resistance in vivo. However, the combination of nilotinib and doxorubicin produced a significant inhibition of tumor growth compared with animals treated with saline, nilotinib, or doxorubicin alone (P < 0.05; Fig. 2B and C). The ratio of tumor growth inhibition by the combination was 28.4%. Furthermore, at the doses tested, no mortality or apparent 12
decrease in body weight was observed in the combination treatment groups, suggesting that the combination regimen did not increase the incidence of toxic side-effects (Fig. 2A). The experiment was carried out using athymic mice implanted subcutaneously with MG63/DOX cells. A, changes in body weight with time after inoculation (mean body weight for each group of 10 mice; bar, SD). B, changes in tumor volume with time (mean tumor volume for each group in 10 mice after implantation; bars, SEM). C, tumor sizes on the 29th day after implantation. The doses were as follows: saline (0.2 ml, i.v., q3d ×5); nilotinib (60 mg/kg, p.o., q3d ×5); doxorubicin (5 mg/kg, i.v., q3d ×5) and doxorubicin (5 mg/kg, i.v., q3d ×5) plus nilotinib (60 mg/kg, p.o., q3d ×5, given 1 h before doxorubicin administration).
Tissue levels of DOX and DOXol in tumor-bearing mice after different dosing regimens To evaluate the influence of nilotinib on DOX distribution in mice, the concentrations of DOX and its metabolite DOXol were measured in all the major organs by LC-MS-MS before and after the multiple dose combinations. Compared with the administration of DOX alone (5 mg/kg), the distribution of DOX was significantly increased in serum and heart, liver and tumor tissues following treatment with DOX in combination 60 mg/kg nilotinib (Fig. 3A). The distribution of the DOXol, the main metabolite of DOX was also significantly increased in serum, and heart and tumor tissues (Fig. 3B). Interestingly, the increase in DOXol in heart tissue was very significant (P < 0.005). Mice were treated with doxorubicin (5 mg/kg, i.v., q3d ×5) alone, and doxorubicin (5 mg/kg, i.v., q3d ×5) plus nilotinib (60 mg/kg, p.o., q3d ×5, given 1 h before doxorubicin administration). Columns, mean value (n = 6); bars, SD. *, P < 0.05 versus (vs.) the DOX group; **, P < 0.01 vs. the DOX group; ***, P < 0.005 vs. the DOX group.
Effect of nilotinib on DOX-induced cardiotoxicity in biochemical analysis 13
LDH and CK levels were analyzed as measures of cardiotoxicity. As shown in Figure 4, there was no significant difference between animals treated with saline and those treated with nilotinib alone. However, administration of DOX alone resulted in a significant increase in CK and LDH as compared to the saline group. After co-administration of 60 mg/kg nilotinib, cardiotoxicity was still more obvious, although there was no significant difference compared with the DOX alone group. Furthermore, MDA and SOD levels were significantly elevated in the DOX group and the combination group, confirming the important role of free radicals in DOX-induced cardiotoxicity. Nilotinib enhanced doxorubicin-induced cardiotoxicity evaluated in histopathological analyses As shown in Figure 5, normal morphology of the tissue was seen in the saline group under light microscopy, with no significant difference in cardiac histology between animals treated with nilotinib alone and those in the saline group. In contrast, DOX-induced morphological changes in cardiac tissues were seen in the DOX group. A cross-section of cardiac tissue of the rat showed infiltration by numerous inflammatory cells with marked edema. Severe necrosis and vacuoles were observed in the cardiac tissues of mice following combined administration of nilotinib and DOX.
Nilotinib does not significantly alter P-gp levels in vitro The reversal of P-gp-mediated MDR can be achieved either by inhibiting its function or by lowering its expression. Therefore, we determined the effect of nilotinib on the levels of P-gp and BCRP. There were no significant differences in the expression of BCRP between MG63/DOX and the parent MG63 cells. However, P-gp protein was significantly 14
upregulated in MG63/DOX cells compared to MG63 cells. Furthermore, incubation of cells with nilotinib (0.75–10 μmol/L) for 72 h did not significantly alter the expression of P-gp and BCRP in MG63/DOX cells (Fig. 6). These data suggested that the reversal of MDR is induced by direct inhibition of the efflux function of P-gp as opposed to downregulation of protein expression. AKR1 and CBR1 status and the effects of nilotinib The previous results showed that the concentration of DOX in heart tissue was significantly increased after combined administration with nilotinib (P < 0.05). Even more interestingly, the concentration of DOXol was even more significant (P < 0.005). The two-electron reduction of the C-13 carbonyl group is the important in the metabolization of DOX to DOXol. Candidate genes involved in the formation of DOXol are AKR1A1, AKR1C3, CBR1, and CBR3. Therefore, we examined the expression level of AKR1A1 and CBR1 in cardiac tissue. Our results showed that, compared with the saline and nilotinib groups, AKR1A1 and CBR1 protein levels in the DOX and combination groups were significantly increased, with a more significant effect observed in the combination group (Fig. 7). In contrast, there were no significant differences in the P-gp protein levels among the groups(Fig. 7). These results provide evidence that the nilotinib-induced increase in cardiotoxicity is mediated partly through increased formation of DOXol in a mechanism that involves modulation of aldehyde reductase expression. Discussion In this study, we used a MDR xenograft model to evaluate the antitumor activity,
tissue
co-administered
distribution with
and
nilotinib
cardiotoxicity
to gain
more
of
DOX
insight
into
when the
pharmacological role of nilotinib in MDR reversal and the risk of DOX 15
cardiotoxicity. Our results indicate that nilotinib reverses P-gp mediated MDR by blocking the efflux function and potentiates DOX-induced cardiotoxicity. The main mechanism that gives rise to MDR involved the overexpression of ABC efflux transporters; therefore, one strategy for MDR reversal is based on the use of anticancer drugs in combination with ABC transporter inhibitors. Previous research showed that nilotinib significantly
potentiated
the
cytotoxicity
of
conventional
chemotherapeutic agents in P-gp-overexpressing MDR cells by interacting with P-gp and inhibiting its activity(24,29). In the present study, we showed that nilotinib exerted potent MDR reversal activity in P-gp overexpressing MDR cells both in vitro and in vivo. Nilotinib in non-toxic doses significantly potentiated the cytotoxicity of DOX in MG63/DOX cells, but had no significant reversal effect in the parent MG63 cells (Table 1). In vivo data showed that DOX in combination with nilotinib markedly enhanced the antitumor activity of DOX in a P-gp overexpressing tumor xenograft model (Fig. 2B and 2C). In the mechanistic study, nilotinib significantly enhanced intracellular Rho123 accumulation in MDR cells (Fig. 1). However, expression of P-gp and BCRP proteins in the MG63/DOX cells was not affected by a 72-h treatment with 0.75, 1.5, 3, or 10 μmol/L nilotinib (Fig. 6). Taken together, these data suggest that nilotinib reverses MDR by directly inhibiting the function of P-gp rather than affecting its expression. 16
Early reports raised questions of safety in the co-administration of cytotoxic drugs with P-gp inhibitors(30), since P-gp is not only responsible for the extrusion of antineoplastic drugs from MDR tumoral cells, but also responsible for the biodistribution and excretion of drugs from non-tumoral cells expressing P-gp, such as cardiomyocytes(31). Co-administration of P-gp blockers (amiodarone, verapamil, or PSC 833) significantly increased DOX toxicity in cultured rat cardiomyocytes(32). Furthermore, DOX accumulation was significantly increased in the brain and liver and DOX and DOXol retention in the heart were substantially prolonged in mdr1a-knockout mice(33). These studies indicated that co-administration of P-gp inhibitors may increase the risk of DOX-induced cardiotoxicity by prolonging the exposure of cardiac tissue. Our previous pharmacokinetic studies have shown that single co-administration of 40 mg/kg nilotinib with DOX enhanced AUC(0–t) and Cmax of DOX by 1.24-fold and 1.96-fold, and its primary circulating metabolite DOXol increased by 1.46-fold and 1.68-fold, respectively. However, the cardiac tissue concentrations of DOX and DOXol were significantly decreased when combined with 40 mg/kg nilotinib(28). In accordance with previous reports, it can be speculated that this result may be due to stimulation of P-gp function and expression by short-term exposure to DOX(34). In the present study, we investigated the effect of nilotinib on the MDR reversal activity of DOX in the nude mouse xenograft model. Simultaneously, the concentrations of DOX and DOXol in
the
tissues
were
quantified
by
LC-MS-MS
after
multiple
co-administration of DOX with nilotinib. Significantly increased accumulation of DOX in serum, heart, liver and tumor tissues was observed (Fig. 3A); the same tendency was observed for DOXol accumulation. Interestingly, a disproportionately large increase in the 17
accumulation of DOXol in cardiac tissue was observed (Fig. 3B). LDH and CK levels were measured as markers of cardiotoxicity. The results showed that administration of DOX alone and in combination with nilotinib resulted in a significant increases in CK, LDH, MDA, and SOD as compared to the levels detected in the saline and nilotinib groups; while there was no significant difference between the DOX group and the combination group (Fig. 4). However, histopathological analysis showed more severe necrosis and vacuoles in the cardiac tissue in the combination group (Fig. 5). The exact mechanism of DOX cardiotoxicity is somewhat controversial. There are two main theories involve: (i) iron-related free radicals and formation of the metabolite DOXol and (ii) mitochondrial disruption,
although
these
are
somewhat
interrelated(35).
The
two-electron reduction of the C-13 carbonyl group is the main route of DOX
metabolism,
which
is
implicated
in
DOX-induced
cardiotoxicity(36). Candidate genes for the formation of DOXol are AKR1C3, AKR1A1, CBR1, and CBR3(35,37). To further investigate the role of CBR1 and AKR1A1 in DOX-induced cardiotoxicity, we investigated the levels of CBR1 and AKR1A1 proteins in cardiac tissue. Compared with saline and nilotinib groups, AKR1A1 and CBR1 protein levels were significantly higher in the DOX and combination groups, with the most significant effect observed in the combined treatment group (Fig. 7). These observations indicate that the mechanism underlying the nilotinib-induced increase in cardiotoxicity is mediated by increased formation of DOXol induced via the modulation of aldehyde reductase expression. In conclusion, nilotinib reverses P-gp mediated MDR in vitro and in vivo, with the main reversal mechanism involving the inhibition of P-gp function, resulting in elevated intracellular concentrations of DOX. 18
However, co-administration of nilotinib results in a dramatic increase in systemic DOX and its metabolite DOXol with a concomitantly increased risk of cardiotoxicity. Moreover, the disproportionate increase in DOXol in cardiac tissue indicates that secondary alcohol metabolites play an important role in the development of DOX-induced cardiotoxicity. The upregulation in AKR1A1 and CBR1 expression in cardiac tissue provides further evidence in support of this mechanism. Consequently, our results indicate that co-administration of a P-gp inhibitors may enhance the risk of cardiotoxicity induced by DOX and should therefore be considered carefully in further drug development and application. Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Acknowledgment We thank Dr. Shen Zan (Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China) for providing the MG63/DOX cell line. Grant support The present study was supported by grants from Science and Technology Research Program, Young Talent Foundation of Hubei Provincial Department of Education (No. B2015250, Z-Y. Zhou); China National Natural Sciences Foundation No. 81503155(Q-j Yang). Statement The manuscript does not contain clinical studies or patient data.
19
References
1.
Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 2004;56(2):185-229.
2.
Arcamone F, Cassinelli G, Fantini G, Grein A, Orezzi P, Pol C, et al. Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius. Reprinted from Biotechnology and Bioengineering, Vol. XI, Issue 6, Pages 1101-1110 (1969). Biotechnol Bioeng 2000;67(6):704-13.
3.
Cortes-Funes H, Coronado C. Role of anthracyclines in the era of targeted therapy. Cardiovasc Toxicol 2007;7(2):56-60.
4.
Takemura G, Fujiwara H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog Cardiovasc Dis 2007;49(5):330-52.
5.
Zuppinger C, Timolati F, Suter TM. Pathophysiology and diagnosis of cancer drug induced cardiomyopathy. Cardiovasc Toxicol 2007;7(2):61-6.
6.
Swain SM, Whaley FS, Ewer MS. Congestive heart failure in patients treated with doxorubicin: a retrospective analysis of three trials. Cancer 2003;97(11):2869-79.
7.
Carvalho C, Santos RX, Cardoso S, Correia S, Oliveira PJ, Santos MS, et al. Doxorubicin:
the
good,
the
bad
and
the
ugly
effect.
Curr
Med
Chem
2009;16(25):3267-85. 8.
Booser DJ, Hortobagyi GN. Anthracycline antibiotics in cancer therapy. Focus on drug resistance. Drugs 1994;47(2):223-58.
9.
Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 1976;455(1):152-62.
10.
Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002;2(1):48-58.
11.
Krishna R, Mayer LD. Multidrug resistance (MDR) in cancer. Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. Eur J Pharm Sci 2000;11(4):265-83.
12.
Dai CL, Tiwari AK, Wu CP, Su XD, Wang SR, Liu DG, et al. Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2. Cancer Res 2008;68(19):7905-14.
13.
Kitazaki T, Oka M, Nakamura Y, Tsurutani J, Doi S, Yasunaga M, et al. Gefitinib, an EGFR tyrosine kinase inhibitor, directly inhibits the function of P-glycoprotein in multidrug resistant cancer cells. Lung Cancer 2005;49(3):337-43.
14.
Shi Z, Peng XX, Kim IW, Shukla S, Si QS, Robey RW, et al. Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance. Cancer Res 2007;67(22):11012-20.
15.
Tao LY, Liang YJ, Wang F, Chen LM, Yan YY, Dai CL, et al. Cediranib (recentin, AZD2171) reverses ABCB1- and ABCC1-mediated multidrug resistance by inhibition of their transport function. Cancer Chemother Pharmacol 2009;64(5):961-9. 20
16.
Dai CL, Liang YJ, Wang YS, Tiwari AK, Yan YY, Wang F, et al. Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2. Cancer Lett 2009;279(1):74-83.
17.
Huls M, Russel FG, Masereeuw R. The role of ATP binding cassette transporters in tissue defense and organ regeneration. J Pharmacol Exp Ther 2009;328(1):3-9.
18.
Solbach TF, Paulus B, Weyand M, Eschenhagen T, Zolk O, Fromm MF. ATP-binding cassette transporters in human heart failure. Naunyn Schmiedebergs Arch Pharmacol 2008;377(3):231-43.
19.
Wang C, Li H, Zhou K, Luo C, Li Y, Xie L, et al. Sodium tanshinone IIA sulfonate and sodium danshensu open the placental barrier through down-regulation of placental P-glycoprotein in mice: implications in the transplacental digoxin treatment for fetal heart failure. Int J Cardiol 2014;176(3):1331-3.
20.
Vlaming ML, Lappchen T, Jansen HT, Kivits S, van Driel A, van de Steeg E, et al. PET-CT imaging with [(18)F]-gefitinib to measure Abcb1a/1b (P-gp) and Abcg2 (Bcrp1) mediated drug-drug interactions at the murine blood-brain barrier. Nucl Med Biol 2015;42(11):833-41.
21.
Golemovic M, Verstovsek S, Giles F, Cortes J, Manshouri T, Manley PW, et al. AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, has in vitro activity against imatinib-resistant chronic myeloid leukemia. Clin Cancer Res 2005;11(13):4941-7.
22.
Verstovsek S, Golemovic M, Kantarjian H, Manshouri T, Estrov Z, Manley P, et al. AMN107, a novel aminopyrimidine inhibitor of p190 Bcr-Abl activation and of in vitro proliferation of Philadelphia-positive acute lymphoblastic leukemia cells. Cancer 2005;104(6):1230-6.
23.
Hegedus C, Ozvegy-Laczka C, Apati A, Magocsi M, Nemet K, Orfi L, et al. Interaction of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for altered anti-cancer
effects
and
pharmacological
properties.
Br
J
Pharmacol
2009;158(4):1153-64. 24.
Tiwari AK, Sodani K, Wang SR, Kuang YH, Ashby CR, Jr., Chen X, et al. Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp
and
ABCG2/BCRP/MXR
transporters.
Biochem
Pharmacol
2009;78(2):153-61. 25.
Villar VH, Vogler O, Martinez-Serra J, Ramos R, Calabuig-Farinas S, Gutierrez A, et al. Nilotinib counteracts P-glycoprotein-mediated multidrug resistance and synergizes the antitumoral effect of doxorubicin in soft tissue sarcomas. PLoS One 2012;7(5):e37735.
26.
Zhou Z, Wan L, Han Y, Meng X, Yang Q, Li Y, et al. ABCB1-overexpressing MG63/DOX cell xenograft model: maintain the MDR phenotype in vivo. Pharm Biol 2013;51(8):968-73.
27.
Mi YJ, Liang YJ, Huang HB, Zhao HY, Wu CP, Wang F, et al. Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters. Cancer Res 2010;70(20):7981-91.
28.
Zhou ZY, Wan LL, Yang QJ, Han YL, Li Y, Yu Q, et al. Evaluation of the pharmacokinetics and cardiotoxicity of doxorubicin in rat receiving nilotinib. Toxicol Appl Pharmacol 2013;272(1):238-44.
29.
Tiwari AK, Sodani K, Dai CL, Abuznait AH, Singh S, Xiao ZJ, et al. Nilotinib 21
potentiates
anticancer
drug
sensitivity
in
murine
ABCB1-,
ABCG2-,
and
ABCC10-multidrug resistance xenograft models. Cancer Lett 2013;328(2):307-17. 30.
van Waterschoot RA, Lagas JS, Wagenaar E, van der Kruijssen CM, van Herwaarden AE, Song JY, et al. Absence of both cytochrome P450 3A and P-glycoprotein dramatically increases docetaxel oral bioavailability and risk of intestinal toxicity. Cancer Res 2009;69(23):8996-9002.
31.
Solbach TF, Konig J, Fromm MF, Zolk O. ATP-binding cassette transporters in the heart. Trends Cardiovasc Med 2006;16(1):7-15.
32.
Estevez MD, Wolf A, Schramm U. Effect of PSC 833, verapamil and amiodarone on adriamycin toxicity in cultured rat cardiomyocytes. Toxicol In Vitro 2000;14(1):17-23.
33.
van Asperen J, van Tellingen O, Tijssen F, Schinkel AH, Beijnen JH. Increased accumulation of doxorubicin and doxorubicinol in cardiac tissue of mice lacking mdr1a P-glycoprotein. Br J Cancer 1999;79(1):108-13.
34.
Budde T, Haney J, Bien S, Schwebe M, Riad A, Tschope C, et al. Acute exposure to doxorubicin results in increased cardiac P-glycoprotein expression. J Pharm Sci 2011;100(9):3951-8.
35.
Thorn CF, Oshiro C, Marsh S, Hernandez-Boussard T, McLeod H, Klein TE, et al. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics 2011;21(7):440-6.
36.
Mordente A, Meucci E, Silvestrini A, Martorana GE, Giardina B. New developments in anthracycline-induced cardiotoxicity. Curr Med Chem 2009;16(13):1656-72.
37.
Olson LE, Bedja D, Alvey SJ, Cardounel AJ, Gabrielson KL, Reeves RH. Protection from doxorubicin-induced cardiac toxicity in mice with a null allele of carbonyl reductase 1. Cancer Res 2003;63(20):6602-6.
22
Figure 1 Effect of nilotinib on the intracellular accumulation of Rho 123 in MG63/DOX cells.
23
Figure 2. Potentiation of antitumor effects of doxorubicin by nilotinib in a MG63/DOX cell xenograft model in athymic nude mice.
Figure 3 Tissue levels of DOX and DOXol in tumor-bearing mice after different dosing regimens.
24
Figure 4. Effects of nilotinib on creatine kinase (A, cardiac tissue homogenate), lactate dehydrogenase (B, serum), malondialdehyde (C, cardiac tissue homogenate) and superoxide dismutase (D, cardiac tissue homogenate) activity. Mice were treated with saline (0.2 mL, i.v.), DOX (5 mg/kg, i.v.), nilotinib (60 mg/kg, p.o.) or co-administration of nilotinib. All the treatments were q3d ×5. Columns, mean value (n = 6); bars, SD. One-way ANOVA with post-hoc analysis; *, P < 0.05 vs. the saline group; #, P < 0.05 vs. the nilotinib group.
Figure 5. Microphotographs of H&E sections of cardiac tissue from tumor burdened mice after treatment with doxorubicin (5 mg/kg, i.v., q3d ×5) plus 25
nilotinib (60 mg/kg, p.o., q3d ×5, given 1 h before doxorubicin administration). Mice were sacrificed for pathologic examination 24 h after the last administration. Control mice (A) showed normal morphology; nilotinib-treated mice (B) showed no significant pathological morphological changes; DOX treated mice (C) showed infiltration by numerous inflammatory cells, marked edema and muscle fiber necrosis; mice treated with DOX co-administered with nilotinib (D) showed severe necrosis and vacuoles. Original magnification, ×20.
Figure 6. Effect of nilotinib on the expression of P-gp and BCRP in MG63/DOX cells. 1: MG63; 2: MG63/DOX; 3: MG63/DOX + 0.75 μM nilotinib; 4: MG63/DOX + 1.5 μM nilotinib; 5: MG63/DOX + 3 μM nilotinib; 6: MG63/DOX + 10 μM nilotinib. Incubation of cells with nilotinib (0.75–10 μmol/L) for 72 h did not significantly alter the expression of P-gp or BCRP. This suggests that the MDR reversal effect of nilotinib in MG63/DOX cells is independent of the inhibition of P-gp and BCRP expression.
26
Figure 7. Effect of nilotinib on the expression of CBR1, AKR1A1 and P-gp in cardiac tissue. Equal amount of protein were loaded for Western blot analysis as described in the Materials and Methods. Independent experiments were performed at least three times, and results from a representative experiment are shown.
Table 1. Effects of nilotinib on reversing P-gp mediated DOX resistance in osteosarcoma cells IC10 ± SD, μmol/L Nilotinib
MG63
MG63/DOX
2.267 ± 1.81E-03
3.105 ± 3.85E-04
IC50 ± SD, μmol/L (fold reversal) MG63
MG63/DOX
Doxorubicin
0.044 ± 2.71E-05 (1.00)
20.289 ± 6.82E-03 (1.00)
+0.75 μM nilo
0.026 ± 7.48E-06 (1.69)
0.752 ± 3.84E-04 (26.98)
+1.5 μM nilo
0.025 ± 1.22E-05 (1.76)
0.250 ± 1.79E-04 (81.16)
+3 μM nilo
0.052 ± 4.53E-05 (0.84)
0.144 ± 1.43E-04 (140.90)
+10 μM vera
0.014 ± 1.08E-05 (3.14)
0.191 ± 5.27E-05 (106.23)
27
28
S0378-4274(16)33086-7 http://dx.doi.org/doi:10.1016/j.toxlet.2016.07.710 TOXLET 9568
To appear in:
Toxicology Letters
Received date: Revised date: Accepted date:
14-4-2016 6-7-2016 30-7-2016
Please cite this article as: Zhou, Zhi-yong, Wan, Li-li, Yang, Quan-jun, Han, Yonglong, Li, Dan, Lu, Jin, Guo, Cheng, Nilotinib reverses ABCB1/P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2016.07.710 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nilotinib reverses ABCB1/P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model Zhi-yong Zhou 1, 2, Li-li Wan 1, Quan-jun Yang 1, Yong-long Han 1, Dan Li 1, Jin Lu 1, Cheng Guo1
Authors’ Affiliations:
1
Department of Pharmacy, Shanghai Jiao
Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China;
2
College of Medical Science, Three Gorges University,
443002 Yichang, Hubei, China.
Corresponding Author: Cheng Guo, Department of Pharmacy, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, China. Phone: +86 21 24058789; Fax: +86 21 24058789; E-mail : [email protected].
1
Highlight 1. Nilotinib can reverse P-gp-mediated multidrug resistance of DOX in vitro and in vivo. 2. Co-administration of nilotinib increase the accumulation of DOX and DOXol in heart. 3. Combination with nilotinib will enhance the cardiotoxicity of DOX in xenograft mice.
Abstract The BCR-Abl tyrosine kinase inhibitor (TKI), nilotinib, was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia-positive chronic myelogenous leukemia. Recent studies have shown that nilotinib induces potent sensitization to anticancer agents by blocking the functions of ABCB1/P-glycoprotein (P-gp) in multidrug resistance (MDR). However, changes in P-gp expression or function affect the cardiac disposition and prolong the presence of both doxorubicin (DOX) and doxorubicinol (DOXol) in cardiac tissue, thus, enhancing the risk of cardiotoxicity. In this study, we used a MDR xenograft model to evaluate the antitumor activity, tissue distribution and cardiotoxicity of DOX when co-administered with nilotinib. This information will provide more insight into the pharmacological role of nilotinib in MDR reversal and the risk of DOX cardiotoxicity. Our results showed that nilotinib significantly enhanced DOX cytotoxicity and increased intracellular rhodamine 123 accumulation in MG63/DOX cells in vitro and strongly enhanced DOX inhibition of growth of 2
P-gp-overexpressing MG63/DOX cell xenografts in nude mice. Additionally, nilotinib significantly increased DOX and DOXol accumulation in serum, heart, liver and tumor tissues. Importantly, nilotinib induced a disproportionate increase in DOXol in cardiac tissue. In the co-administration group, CBR1 and AKR1A1 protein levels were significantly increased in cardiac tissue, with more severe necrosis and vacuole formation. These results indicate that nilotinib reverses P-gpmediated MDR by blocking the efflux function and potentiates DOX-induced cardiotoxicity. These findings represent a guide for the design of future clinical trials and studies of pharmacokinetic interactions and may be useful in guiding the use of nilotinib in combination therapy of cancer in clinical practice. Keywords:
P-glycoprotein,
Doxorubicin,
Cardiotoxicity
3
Multidrug
resistance,
Introduction Doxorubicin (DOX) is an anthracycline first extracted from Streptomyces peucetius var. caesius in the 1970s. It is frequently used in chemotherapy against non-Hodgkin’s and Hodgkin’s lymphomas, multiple myeloma, sarcoma, pediatric cancers and several solid tumors including breast, lung, gastric, ovarian, and thyroid cancers(1-3); however, its clinical use is severely limited due to the development of a progressive dose-dependent cardiomyopathy that results in irreversible congestive heart failure(4,5). Currently, total cumulative dose is the only criteria currently used to predict the toxicity(6,7). The application of DOX is further limited by lack of cytotoxicity against cells expressing multidrug resistance (MDR) proteins leading to the emergence of multidrug resistance(8). Finding ways to maintain the efficacy of DOX and reduce toxicity has now become a major focus of DOX research. P-glycoprotein (P-gp), which is the product of the ABCB1 (or MDR1) gene and is a member of a superfamily of ATP-binding cassette (ABC) membrane transport protein, that was first characterized in multidrug resistant Chinese hamster ovary cells by Juliano and Ling(9). P-gp is a broad-spectrum drug efflux pump that has 12 transmembrane regions and two ATP-binding sites that bind either neutral or positively charged hydrophobic drug substrates(10). P-gp overexpression, which is the most frequent cause of MDR, decreases the cytotoxicity of antitumor drugs including anthracyclines, Vinca alkaloids, podophyllotoxins and taxanes(11), thereby mediating resistance to these agents and affecting their pharmacological behavior. In the past three decades, the search for an effective P-gp inhibitor in the hope of circumventing MDR has become an important focus of research. In recent years, several TKIs including lapatinib(12), gefitinib(13), erlotinib(14), cediranib(15), and sunitinib(16) have been reported to inhibit the functions of P-gp, thereby 4
overcoming chemotherapy resistance in MDR cancer cells. Taken together, these reports suggest that TKIs represent promising MDR inhibitors. However, P-gp is not an abnormal protein, P-gp is a robust detoxification protein that functions as a barrier against xenobiotic entry in the intestine and at the blood–brain barrier and is involved in drug excretion in the kidney, liver, and heart(17,18). Chuan reported that both sodium Tan-IIA sulfonate and sodium danshensu inhibit the expression and efflux functionality of placental P-gp, thereby increasing the transplacental movement of digoxin, and enhancing the effectiveness of the drug used to treat congestive fetal heart failure, while minimizing the risk of maternal toxicity(19). After co-administration with a specific P-gp inhibitor zosuquidar trihydrochloride (LY335979), the concentration of paclitaxel in the brain was significantly increased, while PET-CT imaging showed 2.3-fold increased levels of [18F]-gefitinib in the brain of Abcb1a/1b;Abcg2-/- mice compared to wild-type following intravenous administration of [18F]-gefitinib (1 mg/kg)(20). Based on the results of tissue distribution studies, it has been suggested that P-gp plays an important role in the protection of the organism against potentially toxic agents. Nilotinib, a small-molecule BCR-Abl TKI, was developed to overcome resistance or intolerance to imatinib in patients with Philadelphia-positive chronic myelogenous leukemia(21,22). Previously, it was reported that nilotinib significantly reverses P-gp mediated MDR in cancer cells by inhibiting its activity(23-25). In this study, we used a MDR cancer cell xenograft model to investigate the tissue distribution and risk of cardiotoxicity of DOX administered in combination with nilotinib to reverse drug resistance. Materials and Methods 5
Reagents Doxorubicin was obtained from Pfizer Italia Srl (Latina, Italy). Nilotinib was obtained from Novartis (Stein, Switzerland). Rhodamine 123 (Rho 123), MTT, daunorubicin, and verapamil were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Monoclonal antibodies against P-gp (ab3366), BCRP (108312), CBR1 (ab4148) and AKR1A1 (ab56635) were from Abcam (Hong Kong). All the biochemical assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Acetonitrile and methanol (Tedia, CA, USA) were HPLC grade. Distilled, deionized water was produced by a Milli-Q Reagent Water System (Millipore, MA, USA). All other solvents and chemicals were analytical grade or better. Cell lines All cell lines were cultured in vitro as monolayers in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in an atmosphere of 5% CO2 in air. The human osteosarcoma parental cell line MG63 was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The human MDR osteosarcoma cell line MG63/DOX, which was selected in a step-wise manner by exposing drug-sensitive MG63 cells to increasing doses of adriamycin, was kindly provided by Dr. Shen Zan (Department of Oncology, Affiliated Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai, China). All cells were grown in drug-free culture medium for >2 weeks before use in assays. Animals Athymic nude mice (BALB/c-nude), aged 4–5 weeks and weighing 16–18 g, were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (animal experimental license No. SCXK (hu) 2007-0005), and used for 6
the MG63/DOX cell xenografts. All animals received sterilized food and water ad libitum. An acclimation period of approximately 1 week was allowed between animal arrival and tumor inoculation. All experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals (Center of Experimental Animals, Shanghai Jiao Tong University, China), and all protocols involving animals were approved by the Ethics Committee for Animal Experiments. Cytotoxicity tests MTT assays were conducted as previously described to assess the sensitivity of cells to drugs(26). Drug effects were presented as the IC50 drug concentration and were determined by XLFit software using equation 205. The degree of resistance was estimated by dividing the IC50 of MG63/DOX cells by that of MG63 cells; the fold-MDR reversal factor was calculated by dividing the IC50 of the anticancer agent in the absence of nilotinib by that obtained in the presence of nilotinib. Rho123 accumulation The effect of nilotinib on the intracellular accumulation of Rho 123 between MG63 and MG63/DOX cells was measured by flow cytometry as previously described(27). The P-gp inhibitor verapamil was used as a positive control for MG63/DOX cells. Cells in logarithmic growth were treated with or without nilotinib (2.5, 5, and 10 μM) and verapamil (10 μM) at 37°C for 3 h and then incubated with 5 μM Rho 123 for a further 0.5 h at 37°C. The cells were then collected, centrifuged, and washed twice with ice-cold PBS and resuspended in 500 μL PBS for flow cytometric analysis (BD FACSCalibur, Cellquest PRO, USA). A minimum of 10,000 cells were analyzed for each histogram generated. Nude mouse xenograft model The MG63/DOX inoculated nude xenograft model previously established by us was used in this study(26). The xenograft was found to maintain the 7
MDR phenotype in vivo and was extremely resistant to doxorubicin treatment. Briefly, MG63/DOX cells were suspended at 5×108 cells/mL in serum-free DMEM/Matrigel (1:1). Then, 200 μL of these suspensions (1×107 cells) was injected subcutaneously into the right flank of athymic nude mice. When the tumors reached a mean diameter of 0.5 cm, the mice were randomized into four groups and treated according to various regimens: (a) saline (q3d ×5); (b) doxorubicin (5 mg/kg, i.v., q3d ×5); (c) nilotinib (60 mg/kg, p.o., q3d ×5); and (d) doxorubicin (5 mg/kg, i.v., q3d ×5) + nilotinib (60 mg/kg, p.o., q3d ×5 given 1 h before injecting doxorubicin). The body weights of the animals and the two perpendicular diameters (a and b) were recorded every 3 days, and tumor volume (V) was estimated according to the following formula: V (mm3) = 0.5 a × b2, where V is the tumor volume, a is the long diameter, and b is the short diameter. Tumor growth curves were generated according to tumor volume and days post-inoculation. Tumor tissues were excised from the mice and their weights were measured. The ratio of growth inhibition (IR) was calculated according to the following formula(27): IR(%) = 1- mean tumor weight of experimental group/ mean tumor weight of control group × 100%. Blood and tissue collection Blood and tissue samples were collected at 3 h after the last administration. Blood was obtained by orbital bleeding under CO2 anesthesia and collected in tubes; serum was separated within 1 h. The animals were sacrificed by cervical dislocation to collect the following tissues: heart, liver, spleen, kidney, and tumor. The tissues were dissected out immediately, washed with ice-cold saline, and homogenized with a glass tissue homogenizer in saline to prepare 10% (w/v) homogenates, 8
which were stored at -20°C for analysis. LC/MS/MS assay for doxorubicin and doxorubicinol concentration in serum and tissue homogenates The apparatus and chromatographic analysis conditions were as previously described(28). Samples were prepared by adding 30 μL buffer (aqueous solution of 0.1% formic acid) and 120 μL of internal standard solution (40 ng/mL daunorubicin) to 30 μL of mouse plasma and then vortexing for 1 min. After centrifugation at 15,000 ×g for 5 min, 100 μL of the supernatant was transferred to a sample vial and 5 μL was injected into the LC/MS/MS for quantitative analysis. The LC–MS–MS system was an Agilent 1200 liquid chromatography and a 6410B triple quadrupole mass spectrometer with an electrospray ionization source. Data were analyzed by MassHunter software (Agilent Corporation, MA, USA). Chromatography was performed on a Zorbax XDB-C18 column (2.1mm×150mm i.d., 3.5µm, Agilent Corporation, MA, USA) maintained at 35 ºC using gradient elution with 5mM ammonium formate and 0.1% formic acid in purified water as solvent A and acetonitrile as solvent B. The gradient program was then as follows: 0-1 min, 88%→25% A, 1–2.5 min, 25%→88% A, and 2.5–6 min, 88% A. The flow rate was set at 0.4ml/min. Electrospray ionization (ESI) was performed in the positive ion mode and quantified by the selected reaction monitoring transitions as protonated precursor/product ion transition of m/z 544/397 for doxorubicin, 546/399 for doxorubicinol and 528/321 for IS (Daunorubicin), nebulizer and drying gas were high-purity nitrogen and drying gas, gas temperature and gas flow were set at 350 ◦C and 9.0 L/min. Determination of biochemical indicators To determine specific cardiac tissue toxicity, we investigated parameters 9
including creatine kinase (CK) and lactate dehydrogenase (LDH) in the heart tissue homogenate, which are used as specific markers of cardiac damage in the clinic. One of the most important mechanisms in DOX induced-cardiotoxicity is lipid peroxidation. In the present study, lipid peroxidation was expressed as nmoles of malondialdehyde (MDA, a product formed by the peroxidation of membrane lipids) per milligram of protein. The concentration of superoxide dismutase (SOD, catalyzes the conversion of superoxide radicals to hydrogen peroxide, leading to the formation of hydroxyl radicals) was also measured. The assays were carried out using kits according to the manufacturer’s instructions. Histopathological studies The left ventricular portion of heart tissues was fixed in 10% neutral buffered formalin. Sections (3–5 μm thickness) were stained with hematoxylin
and
eosin
for
histological
examination.
The
histo-morphological evaluation of all the heart sections was carried out in a blinded fashion by a pathologist who was unaware of the treatment groups. Western blot analysis To identify the effects of nilotinib on P-gp expression, the MG63/DOX cells were incubated with different concentrations of nilotinib for 72 h. To clarify the possible mechanism of enhanced cardiotoxicity, we determined the expression of CBR1 and AKR1A1 in heart tissue. The protein concentration was determined by BCA assay, before being separated
by
10%
sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes by electrophoretic transfer. After blocking in 5% non-fat milk at room temperature for 2 h, membranes were subsequently probed with primary antibodies at 4°C overnight. The membranes were then washed three times with TBST buffer and incubated with secondary antibodies at 10
room temperature for 2 h. Immunoreactivity was visualized using an electrochemiluminescence
kit
according
to
the
manufacturer’s
instructions. The density of the immunoreactive bands was analyzed using Image J2x (National Institutes of Health, Bethesda, MD, USA). Statistical analysis All experiments were repeated at least three times and differences were determined by using Student’s t-test and one-way ANOVA with post-hoc analysis. Statistical significance was set at P < 0.05. Results Nilotinib reverses P-gp mediated MDR in MG63/DOX cells To examine the effect of nilotinib on the reversal of P-gp-mediated MDR in cancer cells, we first determined the cytotoxicity of nilotinib alone in MG63/DOX cell line with the MTT assay. More than 90% of MG63/DOX cells were viable when treated with nilotinib up to 3.1 μM; therefore, we used 0.75, 1.5 and 3 μM nilotinib to reverse MDR in vitro. The doxorubicin IC50 in MG63 cells was 0.044 ± 2.71E-05 μM; however, the concentration of doxorubicin required to inhibit MG63/DOX cells by 50% was 20.289 ± 6.82E-03 μM (Table 1). Thus, overexpression of P-gp resulted in a significant increase in resistance to doxorubicin (461-fold). We then investigated the cytotoxic effect of DOX in MG63 and MG63/DOX cells in the presence of 0.75, 1.5, or 3 μM nilotinib. The sensitivity to doxorubicin was unchanged when MG63 cells were co-incubated with nilotinib at concentrations up to 3 μM. In contrast, MG63/DOX cells were sensitized by low concentrations of nilotinib. At concentrations of 0.75, 1.5 and 3 μM, nilotinib induced increasingly effective reversal of resistance to doxorubicin (27-fold, 81-fold and 141-fold, respectively). These results suggested that nilotinib potently reverses P-gp-mediated resistance to doxorubicin in vitro. Cell survival was determined by MTT assay as described in the Materials and 11
Methods. Data represent the mean ± SD of at least three independent experiments performed in triplicate. The fold-MDR reversal (values given in parentheses) was calculated by dividing the anticancer drug IC50 for cells in the absence of nilotinib or verapamil by that obtained in their presence.
Nilotinib enhances the accumulation of Rho 123 in MG63/DOX cells To investigate the mechanism by which nilotinib influences the function of P-gp, we determined the effect of nilotinib on rhodamine accumulation in MG63/DOX cells (Fig. 1). In the absence of nilotinib, the level of Rho 123 accumulation was low in MG63/DOX cells and nilotinib restored the level of Rho 123 accumulation in a dose-dependent manner. These results showed that nilotinib increases intracellular accumulation of the P-gp substrate in cells expressing P-gp. The accumulation of Rho 123 was measured by flow cytometry as described in the Materials and Methods. The intracellular accumulation in MG63/DOX was significantly increased in the presence of nilotinib in a dose-dependent manner. Representative data of least three independent experiments are shown.
Nilotinib reverses P-gp-mediated MDR in the nude mouse MDR xenograft model An established MG63/DOX cell xenograft model in nude mice was used to evaluate the efficacy of nilotinib in reversing doxorubicin resistance in vivo. There were no significant difference in tumor size between animals treated with saline, nilotinib, or doxorubicin, indicating the existence of DOX resistance in vivo. However, the combination of nilotinib and doxorubicin produced a significant inhibition of tumor growth compared with animals treated with saline, nilotinib, or doxorubicin alone (P < 0.05; Fig. 2B and C). The ratio of tumor growth inhibition by the combination was 28.4%. Furthermore, at the doses tested, no mortality or apparent 12
decrease in body weight was observed in the combination treatment groups, suggesting that the combination regimen did not increase the incidence of toxic side-effects (Fig. 2A). The experiment was carried out using athymic mice implanted subcutaneously with MG63/DOX cells. A, changes in body weight with time after inoculation (mean body weight for each group of 10 mice; bar, SD). B, changes in tumor volume with time (mean tumor volume for each group in 10 mice after implantation; bars, SEM). C, tumor sizes on the 29th day after implantation. The doses were as follows: saline (0.2 ml, i.v., q3d ×5); nilotinib (60 mg/kg, p.o., q3d ×5); doxorubicin (5 mg/kg, i.v., q3d ×5) and doxorubicin (5 mg/kg, i.v., q3d ×5) plus nilotinib (60 mg/kg, p.o., q3d ×5, given 1 h before doxorubicin administration).
Tissue levels of DOX and DOXol in tumor-bearing mice after different dosing regimens To evaluate the influence of nilotinib on DOX distribution in mice, the concentrations of DOX and its metabolite DOXol were measured in all the major organs by LC-MS-MS before and after the multiple dose combinations. Compared with the administration of DOX alone (5 mg/kg), the distribution of DOX was significantly increased in serum and heart, liver and tumor tissues following treatment with DOX in combination 60 mg/kg nilotinib (Fig. 3A). The distribution of the DOXol, the main metabolite of DOX was also significantly increased in serum, and heart and tumor tissues (Fig. 3B). Interestingly, the increase in DOXol in heart tissue was very significant (P < 0.005). Mice were treated with doxorubicin (5 mg/kg, i.v., q3d ×5) alone, and doxorubicin (5 mg/kg, i.v., q3d ×5) plus nilotinib (60 mg/kg, p.o., q3d ×5, given 1 h before doxorubicin administration). Columns, mean value (n = 6); bars, SD. *, P < 0.05 versus (vs.) the DOX group; **, P < 0.01 vs. the DOX group; ***, P < 0.005 vs. the DOX group.
Effect of nilotinib on DOX-induced cardiotoxicity in biochemical analysis 13
LDH and CK levels were analyzed as measures of cardiotoxicity. As shown in Figure 4, there was no significant difference between animals treated with saline and those treated with nilotinib alone. However, administration of DOX alone resulted in a significant increase in CK and LDH as compared to the saline group. After co-administration of 60 mg/kg nilotinib, cardiotoxicity was still more obvious, although there was no significant difference compared with the DOX alone group. Furthermore, MDA and SOD levels were significantly elevated in the DOX group and the combination group, confirming the important role of free radicals in DOX-induced cardiotoxicity. Nilotinib enhanced doxorubicin-induced cardiotoxicity evaluated in histopathological analyses As shown in Figure 5, normal morphology of the tissue was seen in the saline group under light microscopy, with no significant difference in cardiac histology between animals treated with nilotinib alone and those in the saline group. In contrast, DOX-induced morphological changes in cardiac tissues were seen in the DOX group. A cross-section of cardiac tissue of the rat showed infiltration by numerous inflammatory cells with marked edema. Severe necrosis and vacuoles were observed in the cardiac tissues of mice following combined administration of nilotinib and DOX.
Nilotinib does not significantly alter P-gp levels in vitro The reversal of P-gp-mediated MDR can be achieved either by inhibiting its function or by lowering its expression. Therefore, we determined the effect of nilotinib on the levels of P-gp and BCRP. There were no significant differences in the expression of BCRP between MG63/DOX and the parent MG63 cells. However, P-gp protein was significantly 14
upregulated in MG63/DOX cells compared to MG63 cells. Furthermore, incubation of cells with nilotinib (0.75–10 μmol/L) for 72 h did not significantly alter the expression of P-gp and BCRP in MG63/DOX cells (Fig. 6). These data suggested that the reversal of MDR is induced by direct inhibition of the efflux function of P-gp as opposed to downregulation of protein expression. AKR1 and CBR1 status and the effects of nilotinib The previous results showed that the concentration of DOX in heart tissue was significantly increased after combined administration with nilotinib (P < 0.05). Even more interestingly, the concentration of DOXol was even more significant (P < 0.005). The two-electron reduction of the C-13 carbonyl group is the important in the metabolization of DOX to DOXol. Candidate genes involved in the formation of DOXol are AKR1A1, AKR1C3, CBR1, and CBR3. Therefore, we examined the expression level of AKR1A1 and CBR1 in cardiac tissue. Our results showed that, compared with the saline and nilotinib groups, AKR1A1 and CBR1 protein levels in the DOX and combination groups were significantly increased, with a more significant effect observed in the combination group (Fig. 7). In contrast, there were no significant differences in the P-gp protein levels among the groups(Fig. 7). These results provide evidence that the nilotinib-induced increase in cardiotoxicity is mediated partly through increased formation of DOXol in a mechanism that involves modulation of aldehyde reductase expression. Discussion In this study, we used a MDR xenograft model to evaluate the antitumor activity,
tissue
co-administered
distribution with
and
nilotinib
cardiotoxicity
to gain
more
of
DOX
insight
into
when the
pharmacological role of nilotinib in MDR reversal and the risk of DOX 15
cardiotoxicity. Our results indicate that nilotinib reverses P-gp mediated MDR by blocking the efflux function and potentiates DOX-induced cardiotoxicity. The main mechanism that gives rise to MDR involved the overexpression of ABC efflux transporters; therefore, one strategy for MDR reversal is based on the use of anticancer drugs in combination with ABC transporter inhibitors. Previous research showed that nilotinib significantly
potentiated
the
cytotoxicity
of
conventional
chemotherapeutic agents in P-gp-overexpressing MDR cells by interacting with P-gp and inhibiting its activity(24,29). In the present study, we showed that nilotinib exerted potent MDR reversal activity in P-gp overexpressing MDR cells both in vitro and in vivo. Nilotinib in non-toxic doses significantly potentiated the cytotoxicity of DOX in MG63/DOX cells, but had no significant reversal effect in the parent MG63 cells (Table 1). In vivo data showed that DOX in combination with nilotinib markedly enhanced the antitumor activity of DOX in a P-gp overexpressing tumor xenograft model (Fig. 2B and 2C). In the mechanistic study, nilotinib significantly enhanced intracellular Rho123 accumulation in MDR cells (Fig. 1). However, expression of P-gp and BCRP proteins in the MG63/DOX cells was not affected by a 72-h treatment with 0.75, 1.5, 3, or 10 μmol/L nilotinib (Fig. 6). Taken together, these data suggest that nilotinib reverses MDR by directly inhibiting the function of P-gp rather than affecting its expression. 16
Early reports raised questions of safety in the co-administration of cytotoxic drugs with P-gp inhibitors(30), since P-gp is not only responsible for the extrusion of antineoplastic drugs from MDR tumoral cells, but also responsible for the biodistribution and excretion of drugs from non-tumoral cells expressing P-gp, such as cardiomyocytes(31). Co-administration of P-gp blockers (amiodarone, verapamil, or PSC 833) significantly increased DOX toxicity in cultured rat cardiomyocytes(32). Furthermore, DOX accumulation was significantly increased in the brain and liver and DOX and DOXol retention in the heart were substantially prolonged in mdr1a-knockout mice(33). These studies indicated that co-administration of P-gp inhibitors may increase the risk of DOX-induced cardiotoxicity by prolonging the exposure of cardiac tissue. Our previous pharmacokinetic studies have shown that single co-administration of 40 mg/kg nilotinib with DOX enhanced AUC(0–t) and Cmax of DOX by 1.24-fold and 1.96-fold, and its primary circulating metabolite DOXol increased by 1.46-fold and 1.68-fold, respectively. However, the cardiac tissue concentrations of DOX and DOXol were significantly decreased when combined with 40 mg/kg nilotinib(28). In accordance with previous reports, it can be speculated that this result may be due to stimulation of P-gp function and expression by short-term exposure to DOX(34). In the present study, we investigated the effect of nilotinib on the MDR reversal activity of DOX in the nude mouse xenograft model. Simultaneously, the concentrations of DOX and DOXol in
the
tissues
were
quantified
by
LC-MS-MS
after
multiple
co-administration of DOX with nilotinib. Significantly increased accumulation of DOX in serum, heart, liver and tumor tissues was observed (Fig. 3A); the same tendency was observed for DOXol accumulation. Interestingly, a disproportionately large increase in the 17
accumulation of DOXol in cardiac tissue was observed (Fig. 3B). LDH and CK levels were measured as markers of cardiotoxicity. The results showed that administration of DOX alone and in combination with nilotinib resulted in a significant increases in CK, LDH, MDA, and SOD as compared to the levels detected in the saline and nilotinib groups; while there was no significant difference between the DOX group and the combination group (Fig. 4). However, histopathological analysis showed more severe necrosis and vacuoles in the cardiac tissue in the combination group (Fig. 5). The exact mechanism of DOX cardiotoxicity is somewhat controversial. There are two main theories involve: (i) iron-related free radicals and formation of the metabolite DOXol and (ii) mitochondrial disruption,
although
these
are
somewhat
interrelated(35).
The
two-electron reduction of the C-13 carbonyl group is the main route of DOX
metabolism,
which
is
implicated
in
DOX-induced
cardiotoxicity(36). Candidate genes for the formation of DOXol are AKR1C3, AKR1A1, CBR1, and CBR3(35,37). To further investigate the role of CBR1 and AKR1A1 in DOX-induced cardiotoxicity, we investigated the levels of CBR1 and AKR1A1 proteins in cardiac tissue. Compared with saline and nilotinib groups, AKR1A1 and CBR1 protein levels were significantly higher in the DOX and combination groups, with the most significant effect observed in the combined treatment group (Fig. 7). These observations indicate that the mechanism underlying the nilotinib-induced increase in cardiotoxicity is mediated by increased formation of DOXol induced via the modulation of aldehyde reductase expression. In conclusion, nilotinib reverses P-gp mediated MDR in vitro and in vivo, with the main reversal mechanism involving the inhibition of P-gp function, resulting in elevated intracellular concentrations of DOX. 18
However, co-administration of nilotinib results in a dramatic increase in systemic DOX and its metabolite DOXol with a concomitantly increased risk of cardiotoxicity. Moreover, the disproportionate increase in DOXol in cardiac tissue indicates that secondary alcohol metabolites play an important role in the development of DOX-induced cardiotoxicity. The upregulation in AKR1A1 and CBR1 expression in cardiac tissue provides further evidence in support of this mechanism. Consequently, our results indicate that co-administration of a P-gp inhibitors may enhance the risk of cardiotoxicity induced by DOX and should therefore be considered carefully in further drug development and application. Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Acknowledgment We thank Dr. Shen Zan (Department of Oncology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China) for providing the MG63/DOX cell line. Grant support The present study was supported by grants from Science and Technology Research Program, Young Talent Foundation of Hubei Provincial Department of Education (No. B2015250, Z-Y. Zhou); China National Natural Sciences Foundation No. 81503155(Q-j Yang). Statement The manuscript does not contain clinical studies or patient data.
19
References
1.
Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 2004;56(2):185-229.
2.
Arcamone F, Cassinelli G, Fantini G, Grein A, Orezzi P, Pol C, et al. Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius. Reprinted from Biotechnology and Bioengineering, Vol. XI, Issue 6, Pages 1101-1110 (1969). Biotechnol Bioeng 2000;67(6):704-13.
3.
Cortes-Funes H, Coronado C. Role of anthracyclines in the era of targeted therapy. Cardiovasc Toxicol 2007;7(2):56-60.
4.
Takemura G, Fujiwara H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog Cardiovasc Dis 2007;49(5):330-52.
5.
Zuppinger C, Timolati F, Suter TM. Pathophysiology and diagnosis of cancer drug induced cardiomyopathy. Cardiovasc Toxicol 2007;7(2):61-6.
6.
Swain SM, Whaley FS, Ewer MS. Congestive heart failure in patients treated with doxorubicin: a retrospective analysis of three trials. Cancer 2003;97(11):2869-79.
7.
Carvalho C, Santos RX, Cardoso S, Correia S, Oliveira PJ, Santos MS, et al. Doxorubicin:
the
good,
the
bad
and
the
ugly
effect.
Curr
Med
Chem
2009;16(25):3267-85. 8.
Booser DJ, Hortobagyi GN. Anthracycline antibiotics in cancer therapy. Focus on drug resistance. Drugs 1994;47(2):223-58.
9.
Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 1976;455(1):152-62.
10.
Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002;2(1):48-58.
11.
Krishna R, Mayer LD. Multidrug resistance (MDR) in cancer. Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. Eur J Pharm Sci 2000;11(4):265-83.
12.
Dai CL, Tiwari AK, Wu CP, Su XD, Wang SR, Liu DG, et al. Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2. Cancer Res 2008;68(19):7905-14.
13.
Kitazaki T, Oka M, Nakamura Y, Tsurutani J, Doi S, Yasunaga M, et al. Gefitinib, an EGFR tyrosine kinase inhibitor, directly inhibits the function of P-glycoprotein in multidrug resistant cancer cells. Lung Cancer 2005;49(3):337-43.
14.
Shi Z, Peng XX, Kim IW, Shukla S, Si QS, Robey RW, et al. Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance. Cancer Res 2007;67(22):11012-20.
15.
Tao LY, Liang YJ, Wang F, Chen LM, Yan YY, Dai CL, et al. Cediranib (recentin, AZD2171) reverses ABCB1- and ABCC1-mediated multidrug resistance by inhibition of their transport function. Cancer Chemother Pharmacol 2009;64(5):961-9. 20
16.
Dai CL, Liang YJ, Wang YS, Tiwari AK, Yan YY, Wang F, et al. Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2. Cancer Lett 2009;279(1):74-83.
17.
Huls M, Russel FG, Masereeuw R. The role of ATP binding cassette transporters in tissue defense and organ regeneration. J Pharmacol Exp Ther 2009;328(1):3-9.
18.
Solbach TF, Paulus B, Weyand M, Eschenhagen T, Zolk O, Fromm MF. ATP-binding cassette transporters in human heart failure. Naunyn Schmiedebergs Arch Pharmacol 2008;377(3):231-43.
19.
Wang C, Li H, Zhou K, Luo C, Li Y, Xie L, et al. Sodium tanshinone IIA sulfonate and sodium danshensu open the placental barrier through down-regulation of placental P-glycoprotein in mice: implications in the transplacental digoxin treatment for fetal heart failure. Int J Cardiol 2014;176(3):1331-3.
20.
Vlaming ML, Lappchen T, Jansen HT, Kivits S, van Driel A, van de Steeg E, et al. PET-CT imaging with [(18)F]-gefitinib to measure Abcb1a/1b (P-gp) and Abcg2 (Bcrp1) mediated drug-drug interactions at the murine blood-brain barrier. Nucl Med Biol 2015;42(11):833-41.
21.
Golemovic M, Verstovsek S, Giles F, Cortes J, Manshouri T, Manley PW, et al. AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, has in vitro activity against imatinib-resistant chronic myeloid leukemia. Clin Cancer Res 2005;11(13):4941-7.
22.
Verstovsek S, Golemovic M, Kantarjian H, Manshouri T, Estrov Z, Manley P, et al. AMN107, a novel aminopyrimidine inhibitor of p190 Bcr-Abl activation and of in vitro proliferation of Philadelphia-positive acute lymphoblastic leukemia cells. Cancer 2005;104(6):1230-6.
23.
Hegedus C, Ozvegy-Laczka C, Apati A, Magocsi M, Nemet K, Orfi L, et al. Interaction of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for altered anti-cancer
effects
and
pharmacological
properties.
Br
J
Pharmacol
2009;158(4):1153-64. 24.
Tiwari AK, Sodani K, Wang SR, Kuang YH, Ashby CR, Jr., Chen X, et al. Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp
and
ABCG2/BCRP/MXR
transporters.
Biochem
Pharmacol
2009;78(2):153-61. 25.
Villar VH, Vogler O, Martinez-Serra J, Ramos R, Calabuig-Farinas S, Gutierrez A, et al. Nilotinib counteracts P-glycoprotein-mediated multidrug resistance and synergizes the antitumoral effect of doxorubicin in soft tissue sarcomas. PLoS One 2012;7(5):e37735.
26.
Zhou Z, Wan L, Han Y, Meng X, Yang Q, Li Y, et al. ABCB1-overexpressing MG63/DOX cell xenograft model: maintain the MDR phenotype in vivo. Pharm Biol 2013;51(8):968-73.
27.
Mi YJ, Liang YJ, Huang HB, Zhao HY, Wu CP, Wang F, et al. Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters. Cancer Res 2010;70(20):7981-91.
28.
Zhou ZY, Wan LL, Yang QJ, Han YL, Li Y, Yu Q, et al. Evaluation of the pharmacokinetics and cardiotoxicity of doxorubicin in rat receiving nilotinib. Toxicol Appl Pharmacol 2013;272(1):238-44.
29.
Tiwari AK, Sodani K, Dai CL, Abuznait AH, Singh S, Xiao ZJ, et al. Nilotinib 21
potentiates
anticancer
drug
sensitivity
in
murine
ABCB1-,
ABCG2-,
and
ABCC10-multidrug resistance xenograft models. Cancer Lett 2013;328(2):307-17. 30.
van Waterschoot RA, Lagas JS, Wagenaar E, van der Kruijssen CM, van Herwaarden AE, Song JY, et al. Absence of both cytochrome P450 3A and P-glycoprotein dramatically increases docetaxel oral bioavailability and risk of intestinal toxicity. Cancer Res 2009;69(23):8996-9002.
31.
Solbach TF, Konig J, Fromm MF, Zolk O. ATP-binding cassette transporters in the heart. Trends Cardiovasc Med 2006;16(1):7-15.
32.
Estevez MD, Wolf A, Schramm U. Effect of PSC 833, verapamil and amiodarone on adriamycin toxicity in cultured rat cardiomyocytes. Toxicol In Vitro 2000;14(1):17-23.
33.
van Asperen J, van Tellingen O, Tijssen F, Schinkel AH, Beijnen JH. Increased accumulation of doxorubicin and doxorubicinol in cardiac tissue of mice lacking mdr1a P-glycoprotein. Br J Cancer 1999;79(1):108-13.
34.
Budde T, Haney J, Bien S, Schwebe M, Riad A, Tschope C, et al. Acute exposure to doxorubicin results in increased cardiac P-glycoprotein expression. J Pharm Sci 2011;100(9):3951-8.
35.
Thorn CF, Oshiro C, Marsh S, Hernandez-Boussard T, McLeod H, Klein TE, et al. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics 2011;21(7):440-6.
36.
Mordente A, Meucci E, Silvestrini A, Martorana GE, Giardina B. New developments in anthracycline-induced cardiotoxicity. Curr Med Chem 2009;16(13):1656-72.
37.
Olson LE, Bedja D, Alvey SJ, Cardounel AJ, Gabrielson KL, Reeves RH. Protection from doxorubicin-induced cardiac toxicity in mice with a null allele of carbonyl reductase 1. Cancer Res 2003;63(20):6602-6.
22
Figure 1 Effect of nilotinib on the intracellular accumulation of Rho 123 in MG63/DOX cells.
23
Figure 2. Potentiation of antitumor effects of doxorubicin by nilotinib in a MG63/DOX cell xenograft model in athymic nude mice.
Figure 3 Tissue levels of DOX and DOXol in tumor-bearing mice after different dosing regimens.
24
Figure 4. Effects of nilotinib on creatine kinase (A, cardiac tissue homogenate), lactate dehydrogenase (B, serum), malondialdehyde (C, cardiac tissue homogenate) and superoxide dismutase (D, cardiac tissue homogenate) activity. Mice were treated with saline (0.2 mL, i.v.), DOX (5 mg/kg, i.v.), nilotinib (60 mg/kg, p.o.) or co-administration of nilotinib. All the treatments were q3d ×5. Columns, mean value (n = 6); bars, SD. One-way ANOVA with post-hoc analysis; *, P < 0.05 vs. the saline group; #, P < 0.05 vs. the nilotinib group.
Figure 5. Microphotographs of H&E sections of cardiac tissue from tumor burdened mice after treatment with doxorubicin (5 mg/kg, i.v., q3d ×5) plus 25
nilotinib (60 mg/kg, p.o., q3d ×5, given 1 h before doxorubicin administration). Mice were sacrificed for pathologic examination 24 h after the last administration. Control mice (A) showed normal morphology; nilotinib-treated mice (B) showed no significant pathological morphological changes; DOX treated mice (C) showed infiltration by numerous inflammatory cells, marked edema and muscle fiber necrosis; mice treated with DOX co-administered with nilotinib (D) showed severe necrosis and vacuoles. Original magnification, ×20.
Figure 6. Effect of nilotinib on the expression of P-gp and BCRP in MG63/DOX cells. 1: MG63; 2: MG63/DOX; 3: MG63/DOX + 0.75 μM nilotinib; 4: MG63/DOX + 1.5 μM nilotinib; 5: MG63/DOX + 3 μM nilotinib; 6: MG63/DOX + 10 μM nilotinib. Incubation of cells with nilotinib (0.75–10 μmol/L) for 72 h did not significantly alter the expression of P-gp or BCRP. This suggests that the MDR reversal effect of nilotinib in MG63/DOX cells is independent of the inhibition of P-gp and BCRP expression.
26
Figure 7. Effect of nilotinib on the expression of CBR1, AKR1A1 and P-gp in cardiac tissue. Equal amount of protein were loaded for Western blot analysis as described in the Materials and Methods. Independent experiments were performed at least three times, and results from a representative experiment are shown.
Table 1. Effects of nilotinib on reversing P-gp mediated DOX resistance in osteosarcoma cells IC10 ± SD, μmol/L Nilotinib
MG63
MG63/DOX
2.267 ± 1.81E-03
3.105 ± 3.85E-04
IC50 ± SD, μmol/L (fold reversal) MG63
MG63/DOX
Doxorubicin
0.044 ± 2.71E-05 (1.00)
20.289 ± 6.82E-03 (1.00)
+0.75 μM nilo
0.026 ± 7.48E-06 (1.69)
0.752 ± 3.84E-04 (26.98)
+1.5 μM nilo
0.025 ± 1.22E-05 (1.76)
0.250 ± 1.79E-04 (81.16)
+3 μM nilo
0.052 ± 4.53E-05 (0.84)
0.144 ± 1.43E-04 (140.90)
+10 μM vera
0.014 ± 1.08E-05 (3.14)
0.191 ± 5.27E-05 (106.23)
27
28