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Introduction

Most non-small cell lung cancers (NSCLC) are already at an advanced stage when diagnosed and are therefore past the optimal timing for surgical resection (1). Seeking effective treatments for these types of cases is particularly important. Docetaxel is a semi-synthetic analogue of paclitaxel, which is widely used as a therapeutic agent in advanced NSCLC (2,3). Docetaxel shows survival benefit when it is used as single agent or in combination with other drugs including chemotherapeutics or vascular endothelial growth factor inhibitors (4–7). However, patients with NSCLC generally develop resistance to docetaxel, and the underlying mechanisms of acquired resistance to docetaxel are not fully understood (8).

The expression of the multidrug resistance (MDR) phenotype is a main mechanism involved in resistance to taxanes (9–12). ABCB1 (P-glycoprotein) is an ATP-binding cassette (ABC) drug pump (13), and is currently the most extensively studied MDR-related transporter protein (14,15), mediating the ATP-dependent efflux of a wide range of hydrophobic drugs such as taxanes (16–18). Several therapeutic agents targeting ABCB1 are available (18). Inhibitors of ABCB1, which reverse the ABCB1 efflux pump, have been studied for more than twenty years, and third-generation drugs, such as elacridar (GF120918), have been developed (9,19). They specifically and potently inhibit ABCB1 and generally do not alter the plasma pharmacokinetics of simultaneously administered antitumor agents, and therefore show potential for combined application with anticancer drugs to combat chemotherapeutic resistance (19–21).

Cellular phenotypes in addition to ABC molecules have been shown to be associated with multidrug resistance (22). Epithelial-mesenchymal transition (EMT) phenotype, loss of epithelial characteristics (E-cadherin), and acquisition of mesenchymal properties (vimentin, fibronectin, or N-cadherin), have been shown to play a crucial role in drug resistance of cancer cells against conventional therapeutics including taxane, vincristine, oxaliplatin, as well as epidermal growth factor receptor (EGFR)-targeted agents (23–25). Cancer stem cell (CSC) features also contribute to this drug resistance (26,27). CSCs drive both continued expansion of malignant cells and resistance to chemotherapy (28–30). However, CSCs in lung cancer remain a subject of ongoing research, and specific markers have not yet been identified. Jiang et al (31) reported that ALDH1 is a lung tumor stem cell-associated marker and that ALDH1-positive cells are highly resistant to chemotherapeutic drugs.

In the present study, we investigated the mechanisms of docetaxel resistance in NSCLC, and examined the effects of the ABCB1 inhibitor elacridar in combination with docetaxel in docetaxel-resistant lung cancer cells.

Materials and methods

Cell lines and reagents

Three NSCLC cell lines (H1299, HCC827, and HCC4006) were used in this study, including one wild-type EGFR cell line (H1299) and two EGFR-mutant cell lines HCC827 (exon19del E746-A750) and HCC4006 (exon19del L747-E749). These cell lines were obtained from Adi F. Gazdar, MD (Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX). These cell lines were proven to have individual genetic origins by using the Powerplex 1.2 system (Promega, Madison, WI, USA) at the University of Texas Southwestern Medical Center at Dallas. All cell lines were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum. They were grown in a humidified incubator with 5% CO2 at 37°C. Docetaxel-resistant sublines (H1299-DR, HCC4006-DR, and HCC827-DR) were established by their parental cells which were cultured with stepwise escalation of concentrations of docetaxel from 0.1 to 100 nmol/l for about 9 months.

Docetaxel, gefitinib, afatinib, and AZD9291 were purchased from Selleck Chemicals (Selleck Chemicals, Houston, TX, USA). Elacridar and Tween 80 (polysorbate 80) were purchased from Sigma, Inc. (Sigma-Aldrich, St Louis, MO, USA).

Determination of cell proliferation

Cells were seeded into 96-well plates at a density of 2×103 cells/well with or without drugs for 72 h and the sensitivities to the drugs were determined by using a modified MTS assay with CellTiter 96 Aqueous One Solution Reagent (Promega), as previously described (32). The anti-proliferative effects are shown as IC50, which is the concentration of the drug required to inhibit cell proliferation by 50%.

Western blot analysis

Resistant cells were cultured in 6 cm dishes for 24 h, and then treated with dimethyl sulfoxide (DMSO) as control, 100 nM docetaxel, and 100 nM docetaxel combined with 0.25 µg/ml elacridar for 48 h. The total cell lysates were extracted with lysis buffer, a mixture of RIPA buffer, phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich), and Complete Mini (Roche, Basel, Switzerland). The primary antibodies were as follows: Monoclonal anti-PARP (46D11) (Catalog #9532), anti-E-cadherin (24E10) (#3195), anti-vimentin (D21H3) (#5741), and anti-ABCB1/MDR1 (E1Y7B) (#13342) (Cell Signaling Technology, Beverly, MA, USA). Monoclonal anti-actin antibody (#MAB1501R), used as an equal loading control, was purchased from Merck Millipore (Billerica, MA, USA). The following secondary antibodies were used: Goat anti-rabbit (#sc-2030) or anti-mouse (#sc-2031) immunoglobulin G (IgG)-conjugated horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX). To detect specific signals, the membranes were examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Amersham, UK) and LAS-3000 (Fujifilm, Tokyo, Japan).

mRNA and siRNA expression analysis by qRT-PCR

Total RNA was extracted by using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. mRNA expression analysis by quantitative reverse transcription PCR (qRT-PCR) was conducted on cDNA by using TaqMan probes and the TaqMan Universal PCR Master Mix (Applied Biosystems). PCR amplification was conducted on an ABI StepOne Real-Time PCR Instrument (Applied Biosystems) and gene expression was calculated using the comparative CT method. Three replicates per sample were assayed for each gene. To quantify the relative changes in gene expression, the (−ΔΔCQ) method was used and reactions were normalized to endogenous control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels in mRNA expression analysis.

Transient transfection

Cells were reverse-transfected with 10 nM small interfering RNAs (siRNAs) mixed with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). The validated siRNAs specific for ABCB1 (si-ABCB1#1, 5-CGA UAC AUG GUU UUC CGA U-3; si-ABCB1#2, 5-GGC UUG CUG UAA UUA CCC A-3) and nonspecific scrambled siRNAs (si-Scr) were purchased from Applied Biosystems. Twenty-four hours after reverse-transfection, the transfected-cells could be used for further experiment according to the manufacturer's protocol. The indicated drugs were added, and cell viability was measured by using the MTS assay after an additional 72 h or made the RNA and protein extraction for real-time PCR and western blot detection.

Results

All established cell lines showed resistance not only to docetaxel but also to EGFR-tyrosine kinase inhibitors

In order to understand the mechanisms of resistance to docetaxel in NSCLC, we first established docetaxel-resistant cell lines. Three cell lines (H1299, HCC827, and HCC4006) were exposed to docetaxel using the stepwise escalation method. Then, the resistance of the established cell lines to docetaxel was confirmed by an MTS assay (Table I). The IC50 values against docetaxel of all the established docetaxel-resistant cells were significantly higher compared with their corresponding parental cells. Moreover, the results were confirmed by western blot analysis for expression of the apoptosis marker, cleaved PARP, and showed that docetaxel treatment effectively induced apoptosis in parental cells, but not in resistant cells (Fig. 1).

Figure 1. Docetaxel treatment effectively induced apoptosis in parental cells, but not in docetaxel-resistant cells. Cells were treated with DMSO or 100 nM of docetaxel for 48 h. Then, lysates were subjected to western blot analysis.

Table I. IC50 values against each drug of parental and docetaxel resistant cell lines.

Table I.

IC50 values against each drug of parental and docetaxel resistant cell lines.

Cell Line	EGFR-mutation	Docetaxel (nM)	Gefitinib (µM)	Afatinib (µM)	AZD9291 (µM)
H1299	Wild-type	3.2	>10	4.3	5.8
H1299-DR	Wild-type	272.1	>10	5.7	5.5
HCC827	Exon 19 del (E746-A750)	0.3	0.0005	<0.001	<0.001
HCC827-DR	Exon 19 del (E746-A750)	>1000	5.0	1.6	2.5
HCC4006	Exon 19 del (L747–A750, P ins)	0.02	0.031	0.002	0.025
HCC4006-DR	Exon 19 del (L747–A750, P ins)	>1000	8.3	2.7	3.1

Next, we explored sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) (gefitinib, afatinib, and AZD9291) both in parental and resistant cell lines. As shown in Table I, the docetaxel-resistant cells with the EGFR mutation showed greater resistance to EGFR-TKIs than their parental cells. The wild-type EGFR cell lines, H1299 parental and H1299-DR, were both insensitive to EGFR-TKI treatment. Although we also treated cells with chemotherapeutic agents such as cisplatin and pemetrexed, no obvious differences between the parental and resistant cells were observed (data not shown).

All resistant cells overexpressed ABCB1, and HCC827-DR and HCC4006-DR exhibited a CSC-like marker and EMT features

Overexpression of ABCB1 is known to be the most common mechanism of cellular resistance to cytotoxic agents. Thus, to explore the mechanism of resistance to docetaxel, expression of ABCB1, ALDH1, and EMT-related markers was examined by real-time PCR and Western blotting in both parental and resistant cell lines. As shown in Fig. 2A, docetaxel-resistant cells highly expressed ABCB1. Moreover, HCC827-DR overexpressed ALDH1, showing a CSC-like marker. To further investigate whether the acquisition of docetaxel resistance induced specific molecular changes consistent with EMT, western blot analysis was performed. As shown in Fig. 2B, lower expression of E-cadherin (epithelial marker) at the protein level was observed in HCC4006-DR cells compared to parental HCC4006 cells.

Figure 2. Docetaxel-resistant cells highly expressed ABCB1. (A) The gene expression of ABCB1 and ALDH1 were determined by real-time PCR in both parental and docetaxel-resistant cells. (B) The protein expression of E-cadherin and vimentin were detected by western blot analysis.

Elacridar, a third-generation ABCB1 inhibitor, overcomes docetaxel resistance, but not to EGFR-TKIs resistance

Having identified that ABCB1 is overexpressed in docetaxel-resistant cells, we examined whether suppression of ABCB1 leads to improved docetaxel resistance. We chose elacridar to inhibit the drug pump function of ABCB1 by competitively combining with ABCB1. Elacridar markedly recovered sensitivity to docetaxel, but not to gefitinib (Table II). The IC50 value of H1299-DR was 9.4 nM at 0.25 µg/ml of elacridar, which was higher than that of parental cells (3.2 nM); thus elacridar only partially recovered the sensitivity to docetaxel. These cells may contain a resistance mechanism other than ABCB1. The expression of the apoptosis marker cleaved-PARP increased in the resistant cells only when both docetaxel and elacridar were administered in combination (Fig. 3). These results suggest that elacridar restored the sensitivity to docetaxel.

Figure 3. Elacridar recovered sensitivity to docetaxel. Docetaxel resistant cells were treated with DMSO, elacridar (0.25 µg/ml) only, or elacridar (0.25 µg/ml) combined with 100 nM of docetaxel for 48 h, and lysates were subjected to western blot analysis with the indicated antibodies.

Table II. IC50 values against docetaxel or gefitinib of docetaxel-resistant cell lines, with or without elacridar treatment.

Table II.

IC50 values against docetaxel or gefitinib of docetaxel-resistant cell lines, with or without elacridar treatment.

	Docetaxel (nM)		Gefitinib (µM)
Elacridar (0.25 µg/ml)	−	+	−	+
H1299DR	272.1	9.4	>10	>10
HCC827DR	>1000	21.9	5.0	1.6
HCC4006DR	>1000	0.3	8.3	4.7

siRNA-mediated knockdown of ABCB1 sensitized resistant cells to docetaxel

To further investigate the function of ABCB1 in the docetaxel resistance mechanism, siRNA-mediated suppression of ABCB1 expression was examined in the docetaxel resistant cell lines. The efficacy of ABCB1 knockdown was confirmed by real-time PCR and Western blot analysis (Fig. 4A and B); siRNA-mediated knockdown of ABCB1 partially sensitized resistant cells to docetaxel (Fig. 4C and Table III). In contrast, ABCB1 depletion did not restore gefitinib sensitivity in docetaxel-resistant cells (Table III).

Figure 4. RNAi-mediated knockdown of ABCB1 modulates the drug sensitivity to docetaxel. (A) The siRNA-mediated suppression of ABCB1 expressions were confirmed by real-time PCR. (B) Seventy-two h after treatment by two different siRNAs against ABCB1 (si-ABCB1#1 or si-ABCB1#2) or scrambled control si-RNA (si-Scr), total cell lysates were harvested and examined by western blot analysis. (C) Cell viabilities against docetaxel with or without siRNA mediated ABCB1 suppression were determined by MTS assay.

Table III. IC50 values against docetaxel or gefitinib of docetaxel resistant cell lines, with or without si-RNA mediated knockdown of ABCB1.

Table III.

IC50 values against docetaxel or gefitinib of docetaxel resistant cell lines, with or without si-RNA mediated knockdown of ABCB1.

	Docetaxel (nM)			Gefitinib (µM)
	si-Scr	si-ABCB1#1	si-ABCB1#2	si-Scr	si-ABCB1#1	si-ABCB1#2
H1299-DR	590.0	25.8	12.6	>10	>10	>10
HCC827-DR	679.4	301.4	382.2	1.2	0.67	0.43
HCC4006-DR	995.8	290.2	328.6	5.7	6.0	4.2

Discussion

In the present study, we established docetaxel-resistant NSCLC cell lines and analyzed their resistance mechanisms in order to understand the biological mechanisms of chemo-resistance in NSCLC cells and to identify reversion opportunities. Several mechanisms for resistance to taxanes have already been reported, such as MDR, CSC-like, and EMT-related marker overexpression (9,25,31,33). The main mechanisms involved in resistance to taxanes are expression of the MDR phenotype and the alteration of their cellular target, namely the tubulin/microtubule system (11,34). Several putative mechanisms of resistance, including alteration of the signaling pathways, altered regulation of the cell cycle, and altered control of apoptosis and cell death signals, have also been described (10,12). Our study confirmed that ABCB1, an MDR molecule, was overexpressed in experimentally established cells. Overexpression of ABCB1 was one of the causes of docetaxel resistance, confirmed by using elacridar and siRNA against ABCB1. However, restoration of sensitivity to docetaxel by ABCB1 siRNA was not perfect. This may be due to incomplete suppression of ABCB1 gene expression in our system, and we therefore inferred that overexpression of ABCB1 is not the only mechanism of docetaxel and multidrug resistance.

We tested EMT- and CSC-related markers in the resistant cell lines because we have recently reported that a gefitinib-resistant cell line was also resistant to docetaxel and exhibited both EMT features and CSC properties (32). E-cadherin and vimentin are EMT-related markers (25), and the expression levels of E-cadherin were slightly lower in HCC4006-DR than in parental cells. The functional implications of EMT include enhanced mobility, invasion, and resistance to apoptotic stimuli (35,36). Moreover, tumor cells can acquire CSC features, secondary tumor initiating, and chemo-resistance properties through EMT (37–39). CSCs, which generate tumors with self-renewal and differentiation abilities, are believed to be highly resistant to conventional chemotherapies owing to various crucial features, including high expression of ABC transporter proteins (40) and ALDH activity (41). HCC827-DR cells overexpressed ALDH1, a CSC-like feature. ABC transporters, including ABCB1, have also been reported to be implicated in promoting CSC-like properties (42).

Notably, EGFR-mutant docetaxel-resistant cells showed cross-resistance to EGFR-TKIs. To our knowledge, we found first that docetaxel treatment led to EGFR-TKIs resistance in EGFR-mutant NSCLC cells. However, ABCB1 expression was not related to sensitivity to EGFR-TKI because elacridar or siRNA against ABCB1 had no effect on sensitivity to EGFR-TKI. We were unable to clarify the mechanism of resistance to EGFR-TKI because well-known mechanisms, such as T790M mutation or MET amplification, were not observed in these cell lines. Although HCC4006-DR cells did not show increased expression of other CSC-like markers, we recently reported a similar observation, that EGFR-TKI resistant cells exhibiting both EMT features and CSC properties with overexpression of ABCB1 were resistant to anti-microtuble agents (32). Mizuuchi et al (43) recently reported similar findings that acquired EGFR-TKI resistance cells became more resistant to anti-microtuble agents. They suggested that this ‘collateral’ cross-resistance to EGFR-TKIs and anti-microtubule agents resulted from two distinct mechanisms, both of which were thought to be a cause of or to result from EMT. Further investigation is required to clarify detailed mechanisms causing this cross-resistance.

Based on our results, elacridar showed a strong effect on docetaxel-resistant NSCLC cells. Elacridar, a third-generation inhibitor of ABCB1, is a non-competitive inhibitor that functions by changing the active allosteric site of ABCB1 (19,44). It shows a relatively minor influence on other members of the ABC family and on the pharmacokinetics of the anti-tumor drugs in clinical use (45).

In conclusion, we have demonstrated that docetaxel-resistant NSCLC cells showed multi-resistance to docetaxel as well as EGFR-TKIs. Molecular analyses showed that all of these resistant cell lines highly expressed ABCB1, and ABCB1 played an important role in acquired resistance to docetaxel in lung cancer. Furthermore, HCC827-DR and HCC4006-DR cells exhibited a CSC-like marker and EMT features, respectively. Thus, elacridar could overcome resistance to docetaxel, suggesting that development of ABCB1 inhibitors show great promise for clinical use to overcome multi-drug resistance.

Acknowledgements

We thank Ms. Fumiko Isobe (Department of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan) for her technical assistance.

Glossary

Abbreviations

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References