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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2016 Jan 15;173(3):613–626. doi: 10.1111/bph.13395

NVP‐TAE684 reverses multidrug resistance (MDR) in human osteosarcoma by inhibiting P‐glycoprotein (PGP1) function

Shunan Ye 1,*,✉, Jianming Zhang 2, Jacson Shen 1, Yan Gao 1, Ying Li 2, Edwin Choy 1, Gregory Cote 1, David Harmon 1, Henry Mankin 1, Nathanael S Gray 3, Francis J Hornicek 1, Zhenfeng Duan 1,✉
PMCID: PMC4728419  PMID: 26603906

Abstract

Background and purpose

Increased expression of P‐glycoprotein (PGP1) is one of the major causes of multidrug resistance (MDR) in cancer, including in osteosarcoma, which eventually leads to the failure of cancer chemotherapy. Thus, there is an urgent need to develop effective therapeutic strategies to override the expression and function of PGP1 to counter MDR in cancer patients.

Experimental Approach

In an effort to search for new chemical entities targeting PGP1‐associated MDR in osteosarcoma, we screened a 500+ compound library of known kinase inhibitors with established kinase selectivity profiles. We aimed to discover potential drug synergistic effects among kinase inhibitors and general chemotherapeutics by combining inhibitors with chemotherapy drugs such as doxorubicin and paclitaxel. The human osteosarcoma MDR cell lines U2OSR2 and KHOSR2 were used for the initial screen and secondary mechanistic studies.

Key Results

After screening 500+ kinase inhibitors, we identified NVP‐TAE684 as the most effective MDR reversing agent. NVP‐TAE684 significantly reversed chemoresistance when used in combination with doxorubicin, paclitaxel, docetaxel, vincristine, ET‐743 or mitoxantrone. NVP‐TAE684 itself is not a PGP1 substrate competitive inhibitor, but it can increase the intracellular accumulation of PGP1 substrates in PGP1‐overexpressing cell lines. NVP‐TAE684 was found to inhibit the function of PGP1 by stimulating PGP1 ATPase activity, a phenomenon reported for other PGP1 inhibitors.

Conclusions and Implications

The application of NVP‐TAE684 to restore sensitivity of osteosarcoma MDR cells to the cytotoxic effects of chemotherapeutics will be useful for further study of PGP1‐mediated MDR in human cancer and may ultimately benefit cancer patients.

Abbreviations

ABC

ATP‐binding cassette

ALK

anaplastic lymphoma kinase

CsA

cyclosporin A

MDR

multidrug resistance

MTT

3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide

PGP1

P‐glycoprotein

Rh123

rhodamine 123

Tables of Links

TARGETS
Catalytic receptors a Enzymes b
ALK Akt (PKB)
EGFR ERK1
IGFR ERK2
Transporters c mTOR
BCRP (ABCG2) PI3K
MRP1 (ABCC1) Src
PGP1 (ABCB1; MDR1)
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LIGANDS
ATP NVP‐TAE684
Cisplatin Paclitaxel
Doxorubicin Verapamil
ET‐743 Vincristine
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These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (a,b,cAlexander et al., 2015a, 2015b, 2015c).

Introduction

For most patients in the advanced stage of cancer, such as in osteosarcoma, chemotherapy is the only treatment option to extend life and palliate symptoms. Despite considerable advances in anticancer drug development, 40% of all human cancers develop multidrug resistance (MDR) after an initial period of response to treatment (Gill et al., 2013; Kim and Helman, 2009; Luetke et al., 2014). The inevitable development of MDR during the course of treatment is a fundamental obstacle associated with cancer care. This resistance is not only to the initial drug but also to other drugs even if the patient has not been previously exposed to them. Studies have shown that MDR affects an estimated 90% of patients treated with chemotherapeutic agents (Agarwal and Kaye, 2003; Fojo and Menefee, 2007; Perez et al., 1993).

Although there are several different intrinsic cellular mechanisms associated with the development of MDR, an important molecular basis for MDR is the overexpression of plasma membrane P‐glycoprotein (PGP1) (Amiri‐Kordestani et al., 2012; Fojo and Menefee, 2007; Gill et al., 2013). MDR is the result of overproduction of PGP1 in the cancer cell membrane, a protein that transports many types of chemicals including chemotherapeutic drugs out of the cancer cell (Amiri‐Kordestani et al., 2012; Goda et al., 2009). This reduces the intracellular concentration of the drug to below that required to achieve therapeutic efficacy. In addition, the drug dose cannot be increased to overcome the PGP1‐mediated resistance (requiring as much as 100‐fold increased drug concentration) because of serious side effects (Agarwal et al., 2003; Fojo and Bates, 2003; Luetke et al., 2014). Therefore, there is an urgent need to find inhibitors of PGP1‐mediated MDR.

Several drugs have been introduced to reverse MDR by inhibiting or blocking PGP1 to confer sensitivity to chemotherapeutic drugs in different model systems (Fojo and Bates, 2003; Goda et al., 2009; Perez et al., 1993; Szakacs et al., 2006). However, none of them have been approved for clinical use to reverse MDR because of deleterious toxicities at the concentrations required to inhibit PGP1. For instance, verapamil is a calcium channel blocker with PGP1 inhibitory activity, but the doses required to override MDR are associated with cardiac toxicity (Ferry et al., 1996). Full reversal of MDR by verapamil requires a concentration of approximately 10 μM in most cell culture models (Duan et al., 1999; Szakacs et al., 2006). However, plasma levels of verapamil above 1 μM will result in atrioventricular block (Duan et al., 1999; Krishna and Mayer, 2000). Another example is cyclosporin A (CsA), which is also extensively used in the clinic as an immunosuppressive agent. However, CsA exerts immunosuppressive effects and nephrotoxicity (Karthikeyan and Hoti, 2015; Twentyman and Bleehen, 1991). Modest PGP1 inhibitors, such as PSC‐833, showed no survival benefit from phase III clinical trials because of their limited therapeutic efficacy on MDR (Amiri‐Kordestani et al., 2012; Fojo and Bates, 2003; Kaye, 2008). In addition, many of these earlier PGP1 inhibitors were developed for other clinical uses and lack sufficient potency and/or specificity (Karthikeyan and Hoti, 2015; Kathawala et al., 2015). Non‐target‐related toxicities of those PGP1 inhibitors compromise the development of therapeutic applications. Few pharmacological agents have been found to completely overcome drug resistance in in vitro models with even fewer examples in vivo (Fojo and Bates, 2003; Luetke et al., 2014). No drug has demonstrated sufficient potency or specificity to be clinically useful to reverse MDR at doses free of limiting side effects. Thus, the development of potent, selective and safe MDR inhibitors still remains a big challenge for cancer therapy (Fojo and Bates, 2003; Kathawala et al., 2015; Szakacs et al., 2006).

Protein kinases are a diverse and large multigene family of enzymes that catalyse the transfer of a phosphate group from ATP to target proteins. Protein kinases play critical roles in many aspects of cancer, including maintaining cell growth, differentiation, adhesion, motility and supporting drug resistance (Kim and Helman, 2009; Shukla et al., 2012). Importantly, kinases can be targeted by pharmaceutical agents to decrease tumour growth and reverse drug resistance. Inhibitions of kinases, such as EGFR, ERK1/2, PI3K/Akt/mTOR, Src, IGFR, ATR or JAK, significantly enhance cell death in the presence of low concentrations of chemotherapeutic drug (Ferry et al., 1996; MacKeigan et al., 2005; Shukla et al., 2012). Protein kinase inhibitors have also been found to reverse MDR by inhibiting the function of ATP‐binding cassette (ABC) transporters, including PGP1 and MRP, or by increasing the efficacy of chemotherapeutic drug‐induced apoptosis (MacKeigan et al., 2005; Shukla et al., 2012; Sodani et al., 2012). These studies suggest that inhibition of the expression and activation of certain kinases may enhance the efficacy of chemotherapeutics and/or reverse MDR.

Screening of chemical libraries for biologically active compounds aimed at specific therapeutic targets is a powerful approach for isolating small molecules that regulate cellular function. We used a kinase‐focused inhibitor library comprising more than 500+ commercially available kinase inhibitors, as well as some inhibitor analogues developed in‐house (Weisberg et al., 2013). This diverse library of ATP‐competitive kinase inhibitors included compounds that target both ‘active’ and ‘inactive’ kinase conformations (Weisberg et al., 2013). The human osteosarcoma MDR cell lines U2OSR2 and KHOSR2 with resistance to chemotherapy drugs doxorubicin and paclitaxel were used for the initial high‐throughput screening. After screening 500+ compounds, we identified NVP‐TAE684, also known as TAE684, initially identified as an anaplastic lymphoma kinase (ALK) inhibitor (Galkin et al., 2007), as one of the most effective MDR reversing compounds. We then systematically evaluated the reversal of MDR by NVP‐TAE684 and further studied the underlying mechanisms. NVP‐TAE684 itself is not a PGP1 substrate, but it can increase the intracellular accumulation of PGP1 substrates in PGP1‐overexpressing cell lines by stimulating PGP1 ATPase activity. Our study indicates that NVP‐TAE684 significantly inhibits PGP1‐mediated MDR in human osteosarcoma. NVP‐TAE684 could be a novel and effective therapy in combination with conventional chemotherapeutic drugs, such as doxorubicin, in the treatment of osteosarcoma patients.

Methods

Kinase inhibitors, chemicals and drugs

The kinase inhibitor library contains 500+ commercially available kinase inhibitors as well as a number of novel kinase inhibitors. These diverse pharmacophores are ATP‐competitive kinase inhibitors that target either active or inactive kinase conformations. These compounds are relatively potent and selective toward a relatively narrow array of kinase targets. This library has been used for screening against a number of different cancer cell lines or cells engineered to be associated with a defined oncogene (BRAFV600E, KRAS, c‐Myc, etc.) (Liu et al., 2013; Weisberg et al., 2013). Doxorubicin, paclitaxel and cisplatin were obtained from residual clinical materials provided by the pharmacy at the Massachusetts General Hospital (Boston, MA, USA). ET‐743 (trabectidin, Yondelis) was supplied by PharmaMar (Madrid, Spain). The fluorescent‐labelled paclitaxel (Oregon Green® 488 Taxol) was purchased from Life Technologies (Carlsbad, CA, USA).

Cell lines and cell culture

Dr Efstathios S. Gonos (National Health Research Foundation, Athens, Greece) kindly provided the human osteosarcoma multidrug‐resistant cell lines KHOSR2, U2OSR2 (established by selection with doxorubicin) and their parental drug‐sensitive cell lines KHOS and U2OS (Lourda et al., 2007). These osteosarcoma‐resistant cell lines have been extensively characterized with the stable MDR phenotype in previous studies. While both drug‐resistant cell lines KHOSR2 and U2OSR2 showed overexpression of PGP1 (also known as MDR1 or ABCB1) as compared with their drug‐sensitive parental cell lines (KHOS and U‐2OS), the expression of MRP1 (also known as ABCC1) or BCRP (also known as ABCG2) was undetectable in these cell lines (Duan et al., 2009a; Duan et al., 2009b,Duan et al., 2009c; Kobayashi et al., 2013; Susa et al., 2010b). All cell lines were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS, 100 U·mL−1 penicillin and 100 μg·mL−1 streptomycin (Invitrogen). Cells were incubated at 37°C in 5% CO2–95% air atmosphere and passaged when nearly confluent using 2% trypsin–EDTA solution. Drug‐resistant cell lines were periodically cultured with the drug to confirm their drug‐resistant characteristics.

Screening of compounds

This assay was based on the restoration of doxorubicin‐ or paclitaxel‐mediated cytotoxicity in PGP1‐overexpressing human osteosarcoma MDR cell lines KHOSR2 and U2OSR2. To investigate the potential of each compound to reverse MDR, U2OSR2 and KHOSR2 cells were treated with different small molecule, kinase inhibitors (1 μM) in the presence or absence of paclitaxel (0.1 μM) or doxorubicin (0.5 μM). These concentrations of paclitaxel and doxorubicin are typically sublethal doses for U2OSR2 and KHOSR2 cells and did not affect their growth. The combinatory drug effect was measured via CellTiter‐Glo® Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA) for automated high‐throughput screening, cell proliferation and cytotoxicity assays. Compounds at 1 μM that could reverse drug sensitivity of U2OSR2 and KHOSR2 to paclitaxel (0.1 μM) and doxorubicin (0.5 μM) but showed no inherent cytotoxicity were identified as ‘hits’ and selected for further study.

MTT cytotoxicity assay

The MTT assay was then used for validation of drug sensitivity and cell growth in the osteosarcoma cell lines with various combinations of different chemotherapy drugs. Briefly, 2 × 103 cells per well were plated in 96‐well plates in RPMI 1640 culture medium containing increasing concentrations of NVP‐TAE684 alone, chemotherapy drug alone or a combination of both. After 96 h of culture, 20 μL of MTT (5 mg·mL−1 in PBS, purchased from Sigma‐Aldrich, St. Louis, MO, USA) were added to each well, and the plates were incubated for 4 h. The resulting formazan product was dissolved with acid–isopropanol, and the absorbance was read at a wavelength of 490 nm on a SPECTRAmax Microplate Spectrophotometer (Molecular Devices, Life Technologies). Dose–response curves were fitted using GraphPad PRISM 4 software (GraphPad Software, San Diego, CA, USA). Drugs at the concentrations utilized in the MTT assay were performed in the absence of cells to verify no change in absorbance. Experiments were performed in triplicate.

Duration of MDR reversal of NVP‐TAE684

The experiment was performed as previously described (Dantzig et al., 1996; Susa et al., 2010a). Briefly, KHOSR2 or U2OSR2 cells were incubated for 24 h with or without NVP‐TAE684 or verapamil (as control) before being washed three times with growth medium. The cells were then incubated for 4 days with increasing concentrations of either doxorubicin or paclitaxel. Final results were determined by MTT assay as described above.

Western blotting

The PGP1 monoclonal antibody C219 was purchased from Covance Inc. (Formerly Signet, Dedham, MA, USA). The mouse monoclonal antibody to human actin was purchased from Sigma‐Aldrich. Western blot analysis was performed as previously described (Yang et al., 2015). The levels of expressed proteins were visualized by scanning the membrane on an Odyssey Infrared Imaging System (LI‐COR Biosciences, Lincoln, NE, USA) with both 680 and 800 nm channels. Densitometric analysis of Western blot results was performed with Image J as described in the software's User Guide. The relative expression of PGP1 was normalized with respect to actin expression.

Drug efflux assay

The Vybrant™ multidrug resistance assay kit (Molecular Devices, Life Technologies) was used to measure drug efflux properties of different resistant cell lines. This assay utilizes the fluorogenic dye calcein acetoxymethyl ester (calcein AM) as a substrate for efflux activity of PGP1 or other ABC membrane pump proteins. Calcein AM is endocytosed and hydrolysed by cytoplasmic esterases to fluorescent calcein. Calcein AM is well retained in the cytosol of cells that do not overexpress PGP1. However, MDR cells expressing high levels of PGP1 or other ABC pump proteins rapidly extrude non‐fluorescent calcein AM from the cellular membrane, reducing the accumulation of fluorescent calcein in the cytosol. Drug‐sensitive and ‐resistant cells (5 × 104 cells per well) were cultured in 96‐well plates for 24 h. Triplicate wells were treated with NVP‐TAE684 or verapamil for 1 h and then incubated in calcein AM. After 30 min, the cells were washed and centrifuged twice with 200 μL cold RPMI 1640 culture medium. Images were acquired with a Nikon Eclipse Ti‐U fluorescence microscope (Nikon Corp., New York, NY, USA) equipped with a SPOT RT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Cell fluorescence was also measured at 490 nm (A490) on a SPECTRAmax® Microplate Spectrofluorometer (Molecular Devices, Life Technologies).

Fluorescence microscopy

For visualization of the effects of NVP‐TAE684 on the intracellular retention of calcein AM, Dioc2, rhodamine 123 (Rh123), doxorubicin and labelled paclitaxel, 1 × 104 resistant cells were seeded on Lab‐Tek 8‐well chamber slides on the day before the assay. Cells were then incubated with 0.25 μM calcein AM, 0.5 μM Dioc2, 5 μM Rh123, 50 μM doxorubicin or 1 μM labelled paclitaxel either alone or in the presence of NVP‐TAE684 in RPMI 1640 media for 1 h at 37°C. After incubation, the medium was removed by aspiration, and cells were counterstained with Hoechst 33342 (1 μg·mL−1 in culture medium) for 2 min. Fluorescence images were acquired as described above.

PGP1 ATPase assay

The Pgp‐Glo™ Assay Systems (Promega) was used for luminescent PGP1 ATPase assays. The PGP1 ATPase assay is a functional tool for determining if a drug interacts with PGP1. The ATPase assay provides a rapid, colorimetric and compound‐independent measure of drugs interacting with Ppg. If a drug does not stimulate PGP1 ATPase activity, the assay can still determine if the drug interacts with PGP1 as an inhibitor of the ATPase activity stimulated by a known substrate, such as verapamil. The effect of NVP‐TAE684 on the ATPase activity of PGP1 was measured according to the manufacturer's protocol.

Statistical data analysis

Values shown are averages of triplicate measurements in two or more experiments. Treatment effects were evaluated using Student's two‐sided t‐test (GraphPadPRISM® 4 software). Values are shown as mean ± SD, and P < 0.05 values were considered to be significant.

Results

NVP‐TAE684 reversal of multiple drug resistance in osteosarcoma cells

In order to investigate potential compounds that can reverse MDR in osteosarcoma, a library of known kinase inhibitors were screened with or without the presence of chemotherapeutic drugs. The screening scheme is illustrated in Supporting Information Fig. S1. Cell viability was assessed after 48 h of drug exposure in the KHOSR2 and U2OSR2 and cell lines. Hit compounds were identified by significantly (with P < 0.05) inhibiting cell proliferation in combination with chemotherapeutics. Top hits were chosen and further verified by a dose–response relationship profiling assay as previously described (Duan et al., 2014). To our surprise, NVP‐TAE684 (5‐chloro‐N4‐(2‐(isopropylsulfonyl)phenyl)‐N2‐(2‐methoxy‐4‐(4‐(4‐methyl‐piperazin‐1‐yl)piperidin‐1‐yl)phenyl)pyrimidine‐2,4‐diamine), also known as TAE684, initially identified as an ALK inhibitor (Galkin et al., 2007), was identified as one of the most effective MDR reversing agents in tandem with doxorubicin or paclitaxel (Figure 1A). At a non‐cytotoxic concentration of 1 μM, NVP‐TAE684 was capable of reversing drug resistance in osteosarcoma MDR cells (Figure 1B). There are no prior reports describing activity of NVP‐TAE684 for reversing drug resistance. When combined with 1 μM NVP‐TAE684, 0.1 μM paclitaxel induced significant cell death in KHOSR2, which grew well in either 0.1 μM paclitaxel or 1 μM NVP‐TAE684 alone (Figure 1B).

Figure 1.

Figure 1

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Chemical structure of NVP‐TAE684 and reversal of drug resistance by NVP‐TAE684. (A) Structure of NVP‐TAE684. (B) NVP‐TAE684 in combination with 0.1 μM paclitaxel induces significant cell death in KHOSR2.

Modulation of drug resistance in MDR cell lines

In PGP1‐overexpressing MDR cell lines, such as KHOSR2 and U2OSR2, NVP‐TAE684 demonstrated a significant (with P < 0.05) reversal of chemoresistance when used in combination with paclitaxel, docetaxel, doxorubicin, vincristine, ET‐743 or mitoxantrone respectively (data not shown). On the other hand, NVP‐TAE684 did not alter the cytotoxicity of cisplatin, carboplatin or methotrexate (Figure 1, and data not shown); these drugs are not PGP1 substrates and are known to be unaffected by PGP1. Complete reversal of MDR was typically observed with NVP‐TAE684 doses between 0.5 and 1 μM (Figure 1). NVP‐TAE684 was capable of reversing MDR at concentrations 20‐ and 50‐fold lower than that required for verapamil and CsA respectively. However, in the parental cell lines KHOS and U2OS, which do not express PGP1, NVP‐TAE684 did not modulate the activity of these cytotoxic agents (Table 1). These results indicate that resistance reversal was attributable to the inhibition of PGP1.

Table 1.

Ability of NVP‐TAE684 to reverse drug resistance in osteosarcoma MDR cell lines

U2OSR2 IC50 (μM) KHOSR2 IC50 (μM)
Paclitaxel 0.067 ± 0.035 0.460 ± 0.012
plus NVP‐TAE684 0.1 μM 0.041 ± 0.018 (1.63) 0.063 ± 0.015 (7.830)
plus NVP‐TAE684 0.5 μM 0.020 ± 0.032 (3.30) 0.021 ± 0.012 (20.90)
plus NVP‐TAE684 1 μM 0.008 ± 0.012 (8.38) 0.010 ± 0.008(46.00)
Doxorubicin 3.125 ± 0.004 1.650 ± 0.008
plus NVP‐TAE684 0.1 μM 1.002 ± 0.024 (3.12) 0.625 ± 0.016 (2.64)
plus NVP‐TAE684 0.5 μM 0.391 ± 0.012 (7.99) 0.462 ± 0.012 (3.57)
plus NVP‐TAE684 1 μM 0.211 ± 0.011 (14.80) 0.141 ± 0.016(11.70)
ET‐743 0.034 ± 0.011 0.260 ± 0.008
plus NVP‐TAE68 0.1 μM 0.020 ± 0.009 (1.7) 0.121 ± 0.011 (2.15)
plus NVP‐TAE68 0.5 μM 0.012 ± 0.006 (2.83) 0.022 ± 0.006 (11.82)
plus NVP‐TAE684 1 μM 0.009 ± 0.002 (3.77) 0.007 ± 0.004 (37.14)
Vincristine 0.641 ± 0.016 1.16 ± 0.024
plus NVP‐TAE684 0.1 μM 0.220 ± 0.014 (2.91) 0.430 ± 0.014 (2.70)
plus NVP‐TAE684 0.5 μM 0.099 ± 0.012 (6.47) 0.160 ± 0.009(7.25)
plus NVP‐TAE684 1 μM 0.035 ± 0.014 (18.31) 0.062 ± 0.019 (18.71)
Cisplatin 3.350 ± 0.011 2.560 ± 0.031
plus NVP‐TAE684 0.1 μM 3.000 ± 0.012 (1.12) 2.032 ± 0.022(1.11)
plus NVP‐TAE684 0.5 μM 2.526 ± 0.013 (1.33) 1.250 ± 0.013 (2.04)
plus NVP‐TAE684 1 μM 2.780 ± 0.010 (1.20) 1.620 ± 0.013 (1.58)
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IC50 is the concentration of drug (μM) that produced 50% inhibition of cell growth.

Results were calculated from a minimum of five experiments with triplicate wells and are presented as mean ± SD.

Numbers in bold in the parentheses represent fold reversal of drug resistance.

Non‐specific toxicity and non‐substrate PGP1 inhibition of NVP‐TAE684

The IC50s for NVP‐TAE684 alone in both KHOS/KHOSR2 and U2OS/U2OSR2 cell lines ranged from 25 to 50 μM, whereas only a concentration of 0.5 to 1 μM of NVP‐TAE684 was required for full reversal of resistance in KHOSR2 or U2OSR2 to various cytotoxic drugs (Figures 2, 3A and 3C). Therefore, the combination of NVP‐TAE684 and chemotherapeutics showed increased toxicity to drug‐resistant cell lines. Furthermore, the IC50 of NVP‐TAE684 for PGP1‐overexpressing MDR cell lines KHOSR2 or U2OSR2 was very similar to their parental cell lines KHOS and U2OS, which lack PGP1 expression (Figure 3A and B). These results indicated that NVP‐TAE684 may not be the direct substrate of PGP1 transport system. On the other hand, there is no evidence to indicate that kinase ALK is essential for KHOSR2, U2OSR2 or their parental cell line KHOS or U2OS's proliferation in literature. Our signal pathway analysis also showed that NVP‐TAE684 did not exert its anti‐cellular proliferation function through inhibiting ERK, JNK, AKT and STAT3 pathways, which are the most well‐characterized effector pathways of ALK (Supporting Information Fig. S2).

Figure 2.

Figure 2

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Effect of NVP‐TAE684 at reversing drug resistance in osteosarcoma MDR cell lines. Cells were treated with chemotherapy drug and NVP‐TAE684 in RPMI 1640 complete media at the indicated concentrations. The relative sensitivity of each line to chemotherapy drug was determined by MTT analysis 6 days post‐treatment. (A) Reversal of drug resistance by NVP‐TAE684 in KHOSR2 cells. (B) Reversal of drug resistance by NVP‐TAE684 in U2OSR2 cells.

Figure 3.

Figure 3

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Non‐specific toxicity of NVP‐TAE684 on KHOS versus KHOSR2, U2OS versus U2OSR2, effect of verapamil on NVP‐TAE684 sensitivity in PGP1‐overexpressing osteosarcoma cells and duration of NVP‐TAE684 activity. Cells were treated with NVP‐TAE684 or verapamil in RPMI 1640 complete medium at the indicated concentrations. The relative sensitivity of each line to NVP‐TAE684 was determined by MTT assay. (A) Non‐specific toxicity of NVP‐TAE684 in KHOS versus KHOSR2. (B) Non‐specific toxicity of NVP‐TAE684 in U2OS versus U2OSR2. (C) Effect of verapamil on NVP‐TAE684 sensitivity in KHOSR2 cells. (D) Duration of reversal of paclitaxel resistance in KHOSR2 cells after incubation and washout of verapamil. (E) Duration of reversal of paclitaxel resistance in KHOSR2 cells after incubation and washout of NVP‐TAE684.

Inhibition of PGP1‐mediated efflux and duration of NVP‐TAE684 activity

To assess whether NVP‐TAE684 is a substrate of PGP1, we determined the effect of the PGP1 substrate competitive inhibitor verapamil on NVP‐TAE684 sensitivity in PGP1‐overexpressing KHOSR2 cells. The results showed that verapamil did not influence the cytotoxic effect of NVP‐TAE684 in KHOSR2 cells (Figure 3C). The ability of NVP‐TAE684 to inhibit PGP1‐mediated action was further investigated using the PGP1 substrate paclitaxel. In a washout experiment, KHOSR2 cells were treated with paclitaxel and verapamil. The removal of verapamil significantly reduced cytotoxicity of paclitaxel (Figure 3D, P < 0.01). However, the combination effect of NVP‐TAE684 and paclitaxel was not significantly affected by washing in KHOSR2 cells (Figure 3E). NVP‐TAE684 was effective at inhibiting PGP1‐mediated drug resistance for at least 3 days after washout. These data collectively demonstrated that NVP‐TAE684 itself is not a PGP1 transporting substrate, and PGP1 does not confer resistance to the cytotoxic effects of NVP‐TAE684 in tumour cells. NVP‐TAE684 may exert its inhibition function through regulation of PGP1 expression or allosteric mode, such as binding of outside of substrate binding pockets, or by modulating the ATPase activity of PGP1, thereby interfering with PGP1‐mediated drug efflux activity.

NVP‐TAE684 modulates PGP1‐mediated uptake and efflux of calcein AM, Dioc2, Rh123, doxorubicin and labelled paclitaxel

MDR is usually manifested as a reduced intracellular drug accumulation resulting from increased drug efflux by PGP1. We next examined the effect of NVP‐TAE684 on the uptake and efflux of PGP1 substrate calcein AM in KHOS and KHOSR2 cells. In the parental drug‐sensitive cell line KHOS, which does not overexpress PGP1, NVP‐TAE684 had no evident effect on accumulation of calcein AM as determined by both image analysis (Figure 4A) and microplate spectrofluorometer analysis (Figure 4B). However, NVP‐TAE684 was shown to significantly increase intracellular accumulation of calcein AM in the KHOSR2 cell line in a dose‐dependent manner (Figure 4C and 4D, P < 0.001). NVP‐TAE684 had a significant effect on the accumulation of calcein AM in KHOSR2 cells starting at a concentration as low as 10 nM.

Figure 4.

Figure 4

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Effects of NVP‐TAE684 on calcein AM efflux from treated KHOS and KHOSR2 cells. The calcein AM assay was optimized and performed using the Vybrant Multidrug Resistance kit and KHOSR2 cells. Cells were seeded at 50 000 cells per well (100 μL of culture medium) in a 96‐well plate and incubated for 24 h. KHOSR2 cells in triplicate were treated with NVP‐TAE684 or verapamil for 1 h and then incubated in calcein AM for 30 min. The cell fluorescence images were acquired by a fluorescence microscope (A, C), and quantities of fluorescence were measured in a SPECTRAmax Microplate Spectrofluorometer (B, D). The data were representative of one of five independent experiments. #P < 0.005 versus cells without verapamil treatment; *P < 0.001 versus cells without NVP‐TAE684 treatment.

To confirm that NVP‐TAE684 inhibition of PGP1‐mediated drug efflux was not specific only to calcein AM, we further evaluated NVP‐TAE684 inhibition of efflux with other PGP1 substrates including Dioc 2, Rh123, doxorubicin and fluorescent‐labelled paclitaxel (Oregon Green 488 Taxol). In PGP1‐overexpressing MDR cell line KHOSR2, NVP‐TAE684 showed an increase in intracellular accumulation and a decrease in efflux of these different PGP1 substrates in a dose‐dependent manner (Figure 5). In the control parental drug‐sensitive cell line KHOS, which does not overexpress PGP1, NVP‐TAE684 had no evident effect on accumulation of these PGP1 substrates (data not shown).

Figure 5.

Figure 5

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NVP‐TAE684 increases PGP1 substrate accumulation in KHOSR2. Images of KHOSR2 cells incubated with various fluorescent substrates of PGP1 in the presence of different concentrations of NVP‐TAE684. For visualization of effects of NVP‐TAE684 on the intracellular retention of calcein AM, Dioc2, rhodamine 123, doxorubicin and Oregon Green 488 Taxol (paclitaxel), 10 000 resistant KHOSR2 cells were seeded on to Lab‐Tek 8‐well chamber slides on the day before the assay. Cells were then incubated with 0.25 μM calcein AM, 0.5 μM Dioc2, 5 μM rhodamine 123, 50 μM doxorubicin or 1 μM Oregon Green 488 Taxol in the presence of 0, 0.1 or 1 μM NVP‐TAE684 for 2 h at 37°C. Images were acquired by a Nikon Eclipse Ti‐U fluorescence microscope (Nikon Corp.) equipped with a SPOT RT digital camera (Diagnostic Instruments, Inc.).

NVP‐TAE684 stimulates PGP1 ATPase activity but does not affect PGP1 expression

Decreasing the expression of PGP1 or blocking the PGP1 efflux pump activity can both lead to increased intracellular drug accumulation. PGP1 has an ATP‐binding region that is essential for substrate transport, and the hydrolysis of ATP by PGP1 ATPase is crucial for restoring the transporter to its active state. Thus, monitoring ATPase activity allows for identification of those compounds that interact with PGP1. PGP1 exhibits a drug‐dependent ATP hydrolysis activity, and a variety of PGP1 inhibitors, as well as PGP1 substrates, can either stimulate or inhibit ATPase activity. PGP1 ATPase activity assay indicated that both NVP‐TAE684 and verapamil significantly increased PGP1 ATPase activity. However, the potency of NVP‐TAE684 was significantly greater than that observed from verapamil, with an EC50 = 6.3 μM for NVP‐TAE684 and an EC50 = 21.4 μM for verapamil respectively (Figure 6A and 6B, P < 0.001). The Western blot analysis indicated that NVP‐TAE684 did not directly interfere with the expression of PGP1 (Figure 6C and 6D). Therefore, these data suggest that NVP‐TAE684 may interact specifically with PGP1 ATPase, which leads to inhibition of the PGP1 efflux pump function by directly modulating the transporter ATPase activity. It also implies that simultaneous administration of NVP‐TAE684 with chemotherapy drugs, especially substrates of PGP1, such as doxorubicin or paclitaxel, may be of clinical benefit for patients bearing tumours that have PGP1‐mediated MDR.

Figure 6.

Figure 6

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Stimulation of PGP1 (Pgp) ATPase activity by NVP‐TAE684 as compared with verapamil and effect of NVP‐TAE684 on the expression of PGP1 (Pgp). Dose‐dependence of verapamil (A) and NVP‐TAE684 (B) stimulation of PGP1 ATPase activity. (C) Effect of NVP‐TAE684 on the expression of PGP1 in osteosarcoma MDR cell lines. The KHOSR2 and U2OSR2 were treated with different concentrations of NVP‐TAE684 for 48 h. Equal amounts (20 μg protein) of total cell lysates were used for each sample. PGP1 expression was determined by Western blot as described in the Methods section. (D) Western blots from (C) were analysed by densitometry as described in the Methods section.

Discussion

The identification and development of specific and potent inhibitors of PGP1 for clinical use in combination with conventional chemotherapy appears to be a promising area in the field of novel anticancer therapeutics in reversing drug resistance (Duan et al., 1999; Ferry et al., 1996; Luetke et al., 2014; Yang et al., 2015). In this study, we identified NVP‐TAE684 as one of the most effective MDR reversing agents, after screening 500+ compounds from a pre‐selected, kinase‐directed small molecule library. NVP‐TAE684 is a previously reported ALK inhibitor with a MW of 614.2 (Galkin et al., 2007). NVP‐TAE684 is able to block the growth of anaplastic large‐cell lymphomas‐derived and ALK‐dependent cell lines at two‐digit nanomolar concentrations. Recently, NVP‐TAE684 has also been found to be a potent LRRK2 (leucine‐rich repeat kinase 2) inhibitor (Zhang et al., 2012). However, there is no prior report describing the ability of NVP‐TAE684 to reverse drug resistance in cancer. We demonstrated that NVP‐TAE684 can reverse PGP1‐mediated MDR of doxorubicin, paclitaxel, ET‐743 and several other commonly used chemotherapeutic drugs in PGP1‐overexpressing cell lines. Our study demonstrated that reversal of MDR by NVP‐TAE684 was through selective and potent inhibition of PGP1 function. In contrast to its activity in osteosarcoma MDR cell lines, NVP‐TAE684 had no significant effect on cytotoxic drug activity in non‐PGP1‐expressing parental cell lines, nor did it affect the cytotoxicity of non‐PGP1 substrate drugs of cisplatin, carboplatin and methotrexate. Furthermore, the concentration of NVP‐TAE684 required to fully reverse drug resistance PGP1‐overexpressed cell lines was 5‐ to 20‐fold lower than the concentration at which any toxicity was observed in the same cell lines.

In MDR cancer cell lines that highly express PGP1, the function of PGP1 is usually as an energy‐dependent membrane transporter that rapidly effluxes chemotherapeutic drugs from cells and therefore prevents the drugs from exerting cytotoxic effects. Studies have shown that MDR cells correlated with reduced accumulation of drugs within the cells due to increased efflux or decreased influx by PGP1 (Duan et al., 1999; Lourda et al., 2007). As PGP1 is an ATP‐dependent transport protein, agents that inhibit ATP‐dependent drug transport PGP1 should inhibit the efflux of drugs from resistant cells and increase intracellular accumulation (Duan et al., 1999; Ferry et al., 1996; Szakacs et al., 2006). We showed that NVP‐TAE684 can also increase the intracellular accumulation of PGP1 substrates by decreasing the efflux of these compounds in resistant cells. However, in comparison with the frequently used reversal agents verapamil and CsA, NVP‐TAE684 has stronger effects and a longer duration inhibiting the PGP1 efflux pump at the same concentration. The potency was confirmed by several assays in two osteosarcoma MDR cell lines with different degrees of drug resistance and PGP1 expression. In these assays, full to partial reversal of resistance in several MDR cell lines to several major classes of chemotherapy drugs was achieved in the presence 0.5 to 1 μM NVP‐TAE684. The strong synergistic effect of NVP‐TAE684 with chemotherapeutic drugs suggests that will be an adequate ‘therapeutic widow’ for future clinical applications, as NVP‐TAE684 has previously been shown to have some favorable pharmacokinetic properties in mice, including high bioavailability, decent half‐life and sufficient distribution into tissues (Galkin et al., 2007; Zhang et al., 2012).

Further mechanistic‐based studies indicate that NVP‐TAE684 significantly increased the PGP1 ATPase activity in a dose‐dependent manner. ATPase activity of PGP1 is required for the proper function of PGP1 (Amiri‐Kordestani et al., 2012; Kathawala et al., 2015). The data also showed that the expression level of PGP1 was unaffected by treatment with NVP‐TAE684. Although ATPase activity is closely associated with the function of PGP1, an increase in ATPase activity does not necessarily lead to augmented PGP1 function. Several reports have demonstrated that certain drug sensitizers or MDR reversing agents increase the ATPase activity of PGP1. For example, verapamil, a common PGP1 inhibitor, stimulates the ATPase activity of PGP1 and inhibits PGP1 function (Garrigues et al., 2002). The MDR inhibitor pentamethoxyflavone increases the ATPase activity in a dose‐dependent manner (Choi et al., 2004). Major cannabinoids from marijuana also show PGP1 inhibitory activity and stimulate PGP1 ATPase (Zhu et al., 2006). Another PGP1 inhibitor, tetrandrine, also known as NSC77037 or CBT‐1, has been demonstrated in several studies to reverse MDR in PGP1‐overexpressing cell lines and in MDR xenograft mouse models (Kelly et al., 2012; Pang et al., 2010; Robey et al., 2008; Susa et al., 2010a; Zhu et al., 2005). Tetrandrine also stimulates ATPase activity in a concentration‐dependent manner (Susa et al., 2010a). The EGFR TK inhibitor gefitinib also stimulates the ATPase activity of PGP1 and directly inhibits the function of PGP1 in MDR cancer cells (Kitazaki et al., 2005). BCRP/MXR inhibitor 6‐prenylchrysin, a flavonoid compound, inhibits BCRP function by stimulating the ATPase activity of BCRP (Ahmed‐Belkacem et al., 2005). In addition, several MDR reversal agents show inhibition of PGP1 ATPase activity (Kopecka et al., 2014; Munagala et al., 2014). In general, some PGP1 inhibitors activate the ATPase of PGP1, whereas others may reduce it. However, the molecular mechanisms of increased or decreased ATPase activity by these PGP1 inhibitors are unknown (Li‐Blatter et al., 2012; Scarborough, 2002, 2003). Identification of the key structural features of PGP1 inhibitors is essential for understanding their interaction with PGP1. Like other PGP1 or MXR inhibitors, the mechanism of how NVP‐TAE684 stimulates PGP1 ATPase activity but inhibits the function of PGP1 deserves further investigation. Molecular docking, site‐directed mutational mapping and quantitative structure–activity relationships may be used to identify NVP‐TAE684's binding site on PGP1.

In the last two decades, a significant effort has been devoted to the field of ABC drug transporters (such as ABCB1, ABCC1 or ABCG2) to identify and evaluate a variety of more specific and potent inhibitors that target the function of these transporters as a means of overcoming tumour resistance (Amiri‐Kordestani et al., 2012; MacKeigan et al., 2005; Perez et al., 1993; Shukla et al., 2012). Physiologically, these transporters may also play a critical role in protecting the cells from xenobiotics (Garrigues et al., 2002; MacKeigan et al., 2005; Shukla et al., 2012). Altering the function of any of these transporters may lead to a severe physiological imbalance resulting in high levels of toxicity. Therefore, it is important to evaluate the toxicity of NVP‐TAE684 in the future to determine whether this novel PGP1 (ABCB1) inhibitor may increase general toxic side effects of chemotherapeutic drugs.

Conclusion

In the present study, we demonstrate that NVP‐TAE684 is a potent, selective and non‐cytotoxic MDR inhibitor that can reverse PGP1‐mediated MDR in PGP1‐overexpressing osteosarcoma cell lines. We anticipate that NVP‐TAE684 will be useful for further studies on PGP1‐mediated MDR in human cancer. As reversal of MDR becomes more important in cancer treatment, MDR inhibitors both for clinical and for laboratory use will become more widespread.

Disclosures

The authors have nothing to disclose.

Author contributions

S. Y., J. Z. and Z. D. performed the research. J. Z. and Z. D. designed the research study. J. Z., H. M., N. S. G., F. J. H. and Z. D. contributed essential tools and reagents. S. Y., J. Z., J. S., Y. G., Y. L. and Z. D. analysed the data. Y. G., E. C., G. C., D. H., H. M., N. S. G. and F. J. H. critically reviewed the manuscript. S. Y., J. Z., J. S. and Z. D. wrote the manuscript.

Conflict of interest

The authors have declared that there is no conflict of interest.

Supporting information

Supplementary Figure 1 Strategy for screening a kinase specific compound library in MDR osteosarcoma cell lines. In step A, the blue colored wells denote the anti‐proliferation effect and red colored wells denote enhanced anti‐proliferation effect. In step B, different colors denote different compounds, and the color gradient denotes strength of cell proliferation inhibition. The kinase inhibitor screening was performed at a concentration of 1 μM with or without the presence of doxorubicin (0.5 μM) or paclitaxel (0.1 μM) in a four‐day cellular proliferation assay. The combinatory drug effect was measured via CellTiter‐Glo® Luminescent Cell Viability Assay Kit as described in Methods.

Supplementary Figure 2 Effect of NVP‐TAE684 on the expression of ERK, JNK, AKT and STAT3 pathways in MDR osteosarcoma cells. KHOSR2 or U2OSR2 cells were treated with NVP‐TAE684 in regular RPMI1640 medium for 48 hours. Total cellular proteins were subjected to Western blotting with specific antibodies as described in Methods. The levels of expressed proteins were visualized by scanning the membrane on an Odyssey Infrared Imaging System.

Supporting info item

Click here for additional data file. (291.7KB, pdf)

Acknowledgements

This study was supported, in part, by grants from the Gategno and Wechsler funds. Z.D. is supported, in part, through a grant from Sarcoma Foundation of America (SFA) and a grant from National Cancer Institute (NCI)/National Institutes of Health (NIH), UO1, CA 151452. Support was also provided by the Jennifer Hunter Yates Foundation and the Kenneth Stanton Fund.

Ye, S. , Zhang, J. , Shen, J. , Gao, Y. , Li, Y. , Choy, E. , Cote, G. , Harmon, D. , Mankin, H. , Gray, N. S. , Hornicek, F. J. , and Duan, Z. (2016) NVP‐TAE684 reverses multidrug resistance (MDR) in human osteosarcoma by inhibiting P‐glycoprotein (PGP1) function. British Journal of Pharmacology, 173: 613–626. doi: 10.1111/bph.13395.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure 1 Strategy for screening a kinase specific compound library in MDR osteosarcoma cell lines. In step A, the blue colored wells denote the anti‐proliferation effect and red colored wells denote enhanced anti‐proliferation effect. In step B, different colors denote different compounds, and the color gradient denotes strength of cell proliferation inhibition. The kinase inhibitor screening was performed at a concentration of 1 μM with or without the presence of doxorubicin (0.5 μM) or paclitaxel (0.1 μM) in a four‐day cellular proliferation assay. The combinatory drug effect was measured via CellTiter‐Glo® Luminescent Cell Viability Assay Kit as described in Methods.

Supplementary Figure 2 Effect of NVP‐TAE684 on the expression of ERK, JNK, AKT and STAT3 pathways in MDR osteosarcoma cells. KHOSR2 or U2OSR2 cells were treated with NVP‐TAE684 in regular RPMI1640 medium for 48 hours. Total cellular proteins were subjected to Western blotting with specific antibodies as described in Methods. The levels of expressed proteins were visualized by scanning the membrane on an Odyssey Infrared Imaging System.

Supporting info item

Click here for additional data file. (291.7KB, pdf)

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