Dasatinib reverses the multidrug resistance of breast cancer MCF-7 cells to doxorubicin by downregulating P-gp expression via inhibiting the activation of ERK signaling pathway

Abstract

Multidrug resistance (MDR) is one of the major obstacles to the efficiency of cancer chemotherapy, which often results from the overexpression of drug efflux transporters such as P-glycoprotein (P-gp). In the present study, we determined the effect of dasatinib which was approved for imatinib resistant chronic myelogenous leukemia (CML) and (Ph⁺) acute lymphoblastic leukemia (ALL) treatment on P-gp-mediated MDR. Our results showed that dasatinib significantly increased the sensitivity of P-gp-overexpressing MCF-7/Adr cells to doxorubicin in MTT assays; thus lead to an enhanced cytotoxicity of doxorubicin in MCF-7/Adr cells. Additionally, dasatinib increased the intracellular accumulation, inhibited the efflux of doxorubicin in MCF-7/Adr cells, and significantly enhanced doxorubicin-induced apoptosis in MCF-7/Adr cells. Further studies showed that dasatinib altered the expression levels of mRNA, protein levels of P-gp, and the phosphorylation of signal–regulated kinase (ERK) both in time-dependent (before 24 h) and dose-dependent manners at concentrations that produced MDR reversals. In conclusion, dasatinib reverses P-gp-mediated MDR by downregulating P-gp expression, which may be partly attributed to the inhibition of ERK pathway. Dasatinib may play an important role in circumventing MDR when combined with other conventional antineoplastic drugs.

Keywords:

• dasatinib
• doxorubicin
• ERK pathway
• multidrug resistance
• P-glycoprotein

Abbreviations

MDR	=	multidrug resistance
P-gp	=	P-glycoprotein
DOX	=	doxorubicin
	=	ERKextracellular signal-regulated kinase
P-ERK	=	phosphorylated extracellular signal–regulated kinase

Introduction

Multidrug resistance (MDR) causes resistance of the cytotoxic effects of numerous antitumor drugs that are structurally and mechanistically unrelated, thus significantly decreases the efficacy of cancer chemotherapy. The mechanisms of MDR vary but the most common cause is the overexpression of cell membrane-bound ATP-binding cassette (ABC) transporters. These transporters coupled with energy consumption pump out exogenous toxic substances or metabolite from the inside of cells. Therefore, the effluxes of various chemotherapeutic drugs lead to attenuation of their cytotoxic effects on tumor cells. Among the well-known 48 ABC proteins, P-glycoprotein (P-gp) or ABC subfamily B member 1 (ABCB1), multidrug resistance-associated protein 1 (MRP1) or ABC subfamily C member 1 (ABCC1), and breast cancer resistance protein (BCRP) or ABC subfamily G member 2 (ABCG2) all play significant roles in causing MDR in cancer cells.¹

P-gp, a 170–180 kDa membrane glycoprotein, was first discovered in drug-resistant Chinese hamster ovarian cells.² As an ATP dependent drug efflux pump, it hydrolyses ATP to efflux a broad spectrum of hydrophobic agents that includes lipids, bile acids, xenobiotics and peptides. It also preferentially pumps out large hydrophobic, positively charged compounds such as vincristine, doxorubicin, actinomycin D, etoposide, etc.³ Furthermore, P-gp is known to be overexpressed in many multidrug-resistant cancer cells and thus it plays an important role in the development of resistance to anti-cancer agents.⁴

Breast cancer is a common cancer among women worldwide. Studies showed that estrogen receptor (ER) α-positive breast cancer MCF-7 cells in the presence of anti-estrogen drugs (e.g. tamoxifen) can increase epidermal growth factor receptor (EGFR) expression and the downstream signaling kinases (e.g., mitogen-activated protein kinase (MAPK)).⁵ Extracellular signal–related kinase (ERK), a member of the MAPK pathway, plays an important role in cell proliferation, differentiation and cell migration.⁶ Nevertheless, in breast cancer models, EGFR overexpression alone usually does not constitute tumorigenesis. However, the co-expression of EGFR with the non-receptor kinase c-Src significantly causes tumorigenesis, suggesting that EGFR and c-Src may functionally interact and contribute to the progression of breast cancer.⁷

A protein tyrosine kinase (PTK) is an enzyme that modifies other proteins by transferring a phosphate group from ATP to a protein (i.e. phosphorylation) in cells. Tyrosine kinases have been identified as crucial factors in tumor progression. They promote cell division or inhibit apoptotic processes through the constitutive hyperactivation of downstream signaling cascades.⁸ Furthermore, some tyrosine kinase inhibitors (TKIs) have been confirmed to be effective anticancer agents when administered alone or in combination with other conventional chemotherapeutic agents.⁹ The interactions between TKIs and various ABC transporters have been extensively investigated in recent years. TKIs could selectively modulate the expressions or functions of ABC transporters via different mechanisms. One of the successful antitumor applications of TKIs is the development of BCR-Abl TKIs. It has been reported previously that imatinib¹⁰ and nilotinib¹¹ both interact with specific ABC transporters and inhibit their transport activities significantly. Another type of potential antitumor TKIs is epidermal growth factor receptor (EGFR) TKIs, like gefitinib,¹² erlotinib¹³ and lapatinib.¹⁴ Vascular endothelial growth factor receptor (VEGFR) TKIs, such as vandetanib¹⁵ and cediranib,¹⁶ and the dual Abl-Src kinase inhibitors like bosutinib¹⁷ together with multitargeted TKIs like sunitinib¹⁸ have all been identified to significantly attenuate or reverse ABC transporters–mediated MDR in cancer cells. Therefore, it is possible that TKIs could be used as MDR reversal agents.

Dasatinib (BMS-354825), identified as a highly potent ATP-competitive inhibitor of both Src family kinases and Abl kinases,^19,20 was approved for imatinib resistant chronic myelogenous leukemia (CML) and (Ph⁺) acute lymphoblastic leukemia (ALL) treatment.^21,22 Though it has been reported previously that dasatinib is a dual BCRP/P-gp substrate,²³ no definite study has yet explored the role dasatinib plays in MDR. Dasatinib presents a moderate sensitivity to non-TNBC (“triple-negative” breast cancer) cell line MCF-7,²⁴ which may lead to a poor effect of dasatinib's cytotoxicity property on MCF-7 cells. In this study, we conducted a series of specific experiments to investigate the effect of dasatinib on the reversal of P-gp-mediated MDR in doxorubicin (DOX) – resistant MCF-7/Adr cells. We hope to expand the role of dasatinib in the treatment of breast cancer.

Materials and Methods

Reagents and antibodies

Dasatinib was purchased from Dalian Meilun Biotech Co., Ltd. (China). Verapamil hydrochloride was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (China). Both of them were dissolved in dimethyl sulphoxide (DMSO) and further diluted in culture medium with the final DMSO concentration <0.1%. DOX, obtained from Shenzhen Main Luck Pharmaceuticals Inc.. (China), was dissolved in saline and further diluted in culture medium. Mitogen-activated kinase kinase (MEK) inhibitor U0126 (Selleck Chemicals LLC Houston, TX, USA) was prepared in DMSO at 10 mM stock concentration. Monoclonal antibody against P-gp was from Abcam Company (UK). Phosphorylated extracellular signal–regulated kinase (P-ERK) and extracellular signal-regulated kinase (ERK) antibodies were from Cell Signaling Technology, Inc. (Beverly, MA, USA). Cleaved caspase-3 antibody and secondary antibodies were from Proteintech group (Chicago, USA). RPMI Medium 1640 and fetal bovine serum FBS were from GIBCO® (Grand Island, NY, USA). MTT was purchased from USB Corporation (Cleveland, OH, USA). All other chemicals were of analytical grade or purer.

Cell culture

Human breast carcinoma cell line MCF-7 and DOX-selected P-gp-overexpressing derivative MCF-7/Adr cells were purchased from Nanjing KeyGen Biotech Co. Ltd. (China), and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acid solution, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Vector-MDCK cell line was a generous gift from Professor Yuichi Sugiyama, Graduate School of Pharmaceutical Sciences, University of Tokyo (Japan). P-gp-MDCK transfectant cells were kindly provided by Professor Su Zeng, College of Pharmacy, Zhejiang University (China). Vector-MDCK and P-gp-MDCK cells were routinely maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acid solution, 100 U/ml penicillin and 0.1 mg/ml streptomycin. All cells were cultured at 37°C in a humidified atmosphere of 5% CO2. MCF-7/Adr cells were cultured in medium with 2 mg/mL DOX and were grown in DOX-free culture medium for more than 2 weeks before assay.

MTT cytotoxicity assay

Three–4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay, known as MTT assay, was employed to assess the sensitivity of cells to drugs. Cells were collected and resuspended in a final concentration of 4 × 10⁵ cells/ml, 100 μL aliquots were seeded in 96-well multiplates. After 24 h of culture, cells were exposed to a series of concentrations of DOX alone, or in combination with increasing concentrations of dasatinib or with 10 μM verapamil, and were cultured at 37˚C for 72 h. Then, MTT (5 mg/ml, 10 ml/well) was added to each well and cells were further incubated for 4 h at 37˚C, allowing the viable cells to reduce the yellow MTT into dark-blue formazan crystals, which were dissolved in 100 μl/well of SDS–isobutanol-HCl solution (10% SDS, 5% isobutanol, and 12 μM HCl) overnight at 37˚C. Optical density was measured at 570 nm with the microplate reader (Bio-Rad, San Diego, CA, USA). Six wells were used for each drug concentration and the experiment was replicated 3 times. The IC₅₀ was calculated by Prism (Graphpad Software, La Jolla, CA, USA).²⁵ The fold-reversal factor of MDR was calculated by dividing the IC₅₀ of DOX in the absence of dasatinib or verapamil by those obtained in the presence of dasatinib or verapamil. The resistance index was estimated by dividing the IC₅₀ values of the MDR cells by that of the parental sensitive cells.

Doxorubicin accumulation assay

Cellular accumulation of doxorubicin was determined using the procedure originally notted by Ma MT et al.,²⁶ with minor modifications. Briefly, MDR cells were seeded in 6-well plates. After 24 h incubation of the cells with dasatinib or verapamil, DOX was added and incubated for 1 h at 37˚C. Cells were then washed with phosphate buffered saline (PBS), trypsinized and resuspended in medium at a concentration of 1×10⁶ cells/ml. Intracellular fluorescence of DOX was determined by flow cytometry (FACScalibar, BD, USA) with excitation measured at 488 nm and emission measured at 575 nm.

Doxorubicin efflux assay

Doxorubicin efflux was assayed following a modified method previously described.²⁷ MCF-7/Adr cells were treated with 20 μM doxorubicin for 3h at 37°C, cells were then washed twice with ice-cold PBS, maintained at 37°C with DOX-absent culture media with or without 1.0 μM dasatinib for 0, 15, 30, 60 and 120 min respectively. Subsequent cells were then gathered and washed twice with ice-cold PBS. Finally, the cells were resuspended in ice-cold PBS buffer for flow cytometric analysis (FACScalibar, BD, USA), and the fluorescence intensity of DOX was determined.

TUNEL assay

After pre-incubating the MDR cells with dasatinib for 24 h, cells that grew on microscope slides were treated with DOX for additional 48 h based on IC50 value obtained from cell viability assay. Apoptotic cells were detected by measuring DNA fragmentation using the TdT-mediated dUTP nick-end labeling (TUNEL) method (In Situ Cell Death Detection Kit, POD, Roche Applied Science, Indianapolis, IN, USA) following the manufacturer's protocol. Briefly, cells were fixed with 4% paraformaldehyde for 25 min and washed with phosphate buffered saline (PBS) twice (5 min per time). Samples were treated with cyto-permeabilization solution 0.2% Triton X-100 and then washed again with PBS. A mixture of 5 μL TdT (terminal deoxynucleotidil transferase) enzyme and 45 μL fluorescein marked dUTP was added to each microscope slide. Finally, the slides were incubated for 1 h at 37˚C in a container to ensure proper moisture and darkness. Apoptotic cells were detected through a fluorescent microscope. Ten microscopic fields were randomly selected in each group to count the positive cells, and the extent of apoptosis was evaluated by the average number of positive cells (green spots) in the images.

Quantitative real-time PCR

Cellular P-gp mRNA expression was measured by Quantitative real-time PCR as previously described.²⁸ Total cellular RNA was extracted from cells using RNA isoPlus® Reagent Kit (Takara Biotechnology) according to the manufacturer's instructions. cDNA was generated from 1μg of total RNA using PrimeScript® RT Reagent Kit with gDNA Eraser (Takara Biotechnology) and was amplified using SYBR® Premix Ex Taq™ Kit (Takara Biotechnology) with β-actin as the normalization controls by ABI PRISM® 7500 Real-Time PCR System (Applied Biosystems). The fold change for MDR1 relative to the control was calculated using the comparative ΔΔCt method. Specific primers used for PCR amplification were as follows: MDR1 (Forward: 5′-GGAGCCTACTTGGTGGCACATAA-3′, reverse: 5′-TGGCATAGTCAGGAGCAAATGAAC-3′); β-actin (Forward:5′-ATTGAACACGGCATTGTCAC-3′, reverse: 5′-CATCGGAACCGCTCATTG-3′).

Western blot

As previously described,²⁹ cells were homogenized on ice in lysis buffer for Western blot analysis. Proteins from each cell lysate were mixed with an equal volume of 2 × Loading buffer (125 mM Tris–HCl, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 10% β-mercaptoethanol, and 0.05% bromophenol blue). Equal amounts of proteins were loaded and separated in sodium dodecyl sulfate–polyacrylamide gel by electrophoresis. Proteins were transferred onto PVDF membranes by electroblotting. Membranes were blocked with 5% milk or 5% BSA in Tris-buffered saline with 0.1% Tween 20 for 2 h at 37˚C, and then incubated with an appropriate dilution of antibodies overnight at 4˚C. Finally, blots were developed with corresponding horseradish peroxidase-conjugated anti-IgG antibodies and detected by an enhanced chemiluminescence (ECL) method using Bio-Spectrum Gel Imaging System (UVP, USA). Quantification of protein expression was analyzed through Gel-Pro Analyzer 3.0 software.

Statistical analysis

Data are expressed as means ± SD. All experiments were repeated at least 3 times and the differences were determined by using Student's t test. The value of P < 0.05 was considered to be statistically significant.

Results

Characterization of MCF-7 and MCF-7/Adr cells

To validate the specific property that P-gp is overexpressed in MCF-7/Adr cell line, a comparison between MCF-7 and MCF-7/Adr cells about P-gp expression was provided. As shown in Fig. 1A, MCF-7/Adr cells displayed lower cytotoxicity level of DOX than that of the parental MCF-7 cells. MCF-7/Adr revealed significantly higher levels of the mRNA and protein levels of P-gp in contrast with parental MCF-7 cells in Fig. 1B and C. These results confirmed that P-gp is overexpressed in resistant MCF-7/Adr cells.

Figure 1. Characterization of MCF-7 and MCF-7/Adr cells. After treating cells with increasing concentrations of DOX in sensitive MCF-7 (○) and resistant MCF-7/Adr (•) cells for 48 h, cell viability was determined by MTT assay (A), mRNA expression of P-gp was tested by quantitative real-time PCR analysis (the control group is set at 1) (B) and P-gp protein level was detected by Western blot analysis (C). Data shown are means ± SD of triplicate determinations. ***P < 0.001, versus the control group.

Dasatinib potentiates the sensitivities of P-gp-overexpressing MCF-7/Adr cells to DOX

IC₅₀ values of dasatinib determined by MTT assay for MCF-7 and MCF-7/Adr cells were 17.03 ± 1.93 and 40.10 ± 2.07 μM respectively. Based on the data tested, dasatinib was used at a maximum concentration of 1.0 μM – a concentration at which > 90% of the cells were viable to use in the next MDR reversal study. Dasatinib caused a dose-dependent decrease of the IC₅₀ values of DOX compared to the significant decrease by P-gp inhibitor, verapamil in MCF-7/Adr cells. The fold-reversal of MDR was 1.57, 3.35 and 4.32 in the presence of 0.25, 0.5 and 1.0 μM dasatinib in MCF-7/Adr cells, respectively (Table 1). 1.0 μM dasatinib could also significantly decrease the IC₅₀ values of DOX in stably transfected P-gp-MDCK cells in Table 2. Dasatinib barely altered the sensitization to DOX both in the drug-sensitive parental MCF-7 and MDCK cells. Therefore, the results suggested that dasatinib only significantly enhanced the sensitivity of P-gp-overexpressing cells to DOX.

Table 1. Effect of dasatinib on reversing P-gp-mediated MDR in drug selected cell line

	IC50 ± SD (μM; fold-reversal)
Compounds	MCF-7	MCF-7/Adr (P-gp)	Resistance index
Doxorubicin	0.437 ± 0.258 (1.00)	20.60 ± 2.048 (1.00)	47.13
+ 0.25 μM dasatinib	0.451 ± 0.052 (0.97)	13.10 ± 0.772* (1.57)	29.03
+ 0.5 μM dasatinib	0.424 ± 0.061 (1.03)	6.16± 0.593* (3.35)	14.52
+ 1.0 μM dasatinib	0.413 ± 0.053 (1.06)	4.77 ± 0.284* (4.32)	11.56
+ 10 μM verapamil	0.396 ± 0.029 (1.10)	2.94 ± 0.116* (7.01)	7.41

Reversal activity of dasatinib in drug selected cell lines. Cell survival was determined by MTT assays. MCF-7 cells and P-gp-overexpressing MCF-7/Adr cells were cultured in various doses of doxorubicin alone, or in combination with increasing doses of dasatinib or with 10 μM verapamil for 72 h. Data are the mean ± SD of at least 3 independent experiments done in triplicate. *P < 0.05, vs. the values obtained in the absence of dasatinib or verapamil.

Table 2. Effect of dasatinib on reversing P-gp-mediated MDR in transfected cell line

	IC50 ± SD (μM; fold-reversal)
Compounds	Vector-MDCK	P-gp-MDCK	Resistance index
Doxorubicin	8.50 ± 0.981 (1.00)	29.45 ± 1.128 (1.00)	3.46
+ 1.0 μM dasatinib	7.85 ± 0.903 (1.08)	17.37 ± 0.711* (1.70)	2.21
+ 10 μM verapamil	7.93 ± 1.310 (1.07)	14.70 ± 0.890* (2.00)	1.85

Reversal activity of dasatinib in transfected cell lines. Cell survival was determined by MTT assays. Vector-MDCK cells and P-gp-transfected MDCK cells were cultured in various doses of doxorubicin alone, or in combination with 1.0 μM of dasatinib or with 10 μM verapamil for 72 h. Data are the mean ± SD of at least 3 independent experiments done in triplicate. *P < 0.05, versus the values obtained in the absence of dasatinib or verapamil.

Dasatinib significantly increases the accumulation of DOX in P-gp-overexpressing MCF-7/Adr cells

To understand the mechanism by which dasatinib sensitizes the resistant cells to DOX, we examined the effect of dasatinib on the accumulation of DOX in P-gp-overexpressing MCF-7/Adr cells. The intracellular accumulation levels of DOX in sensitive MCF-7 cells were higher than that of resistant MCF-7/Adr cells (Fig. 2A). Intracellular accumulation of DOX in the absence of dasatinib was low. However, dasatinib significantly increased the intracellular accumulation of DOX in a concentration-dependent manner, compared to the positive control group of verapamil. Dasatinib at 0.25, 0.5 and 1.0 μM concentrations increased the intracellular accumulation of DOX by 1.06-, 1.23- and 1.40-fold in MCF-7/Adr cells, respectively. It had little impact on the intracellular accumulation of DOX in the parental sensitive MCF-7 cells. In summary, these results suggested that dasatinib significantly inhibited P-gp-mediated transport in resistant MCF-7/Adr cells.

Figure 2. Effects of dasatinib on the accumulation and efflux of DOX. (A) Cells were pre-incubated with or without dasatinib or verapamil for 24 h at 37°C and then incubated with 20 μM DOX for another 1h at 37°C. The results are presented as fold change in fluorescence intensity relative to untreated control resistant cells. (B) After a preincubation of 20 μM DOX for 3 h, time course of DOX efflux was measured in MCF-7/Adr cells with the presence (•) or absence (○) of 1.0 μM dasatinib. The accumulation of DOX was measured by flow cytometric analysis. Data shown are means ± SD of triplicate determinations. *P < 0.05 and **P < 0.01 vs. the control group.

Dasatinib inhibits the efflux of DOX in P-gp-overexpressing MCF-7/Adr cells

As dasatinib increased the intracellular accumulation of DOX in MCF-7/Adr cells, we determined that the increased accumulation was due to the inhibition of its efflux. The course of DOX efflux within 2 h after accumulation is shown in Fig. 2B. Dasatinib inhibited DOX efflux of P-gp in MCF-7/Adr cells while it made little difference on DOX efflux in sensitive MCF-7 cells. For instance, at 120 min, 36.9% of accumulated DOX was pumped out of MCF-7/Adr cells in the presence of 1.0 μM dasatinib while 55.4% of accumulated DOX in MCF-7/Adr cells was effluxed in the absence of dasatinib (P < 0.05). The results indicated that dasatinib could effectively inhibit DOX efflux of P-gp.

Dasatinib significantly enhances DOX-induced apoptosis in resistant MCF-7/Adr cells

TUNEL assay showed that dasatinib produced a dose-dependent increase in the number of apoptotic MCF-7/Adr cells induced by DOX in Fig. 3A and B. This may have resulted from an enhanced activation of caspase-3 in Fig. 3C and D. Results confirmed that dasatinib could enhance DOX-induced apoptosis in resistant MCF-7/Adr cells.

Figure 3. Effect of dasatinib on DOX-induced cell apoptosis. (A) After pre-incubating the cells with dasatinib for 24 h at 37°C, cells grown on coverslips were treated with the 20 μM DOX for another 48 h, and apoptosis was analyzed by TUNEL assay as described in Materials and Methods. (B) Quantification of TUNEL fluorescent images. After pre-incubating the cells with dasatinib for 24 h, cleaved caspase-3 expression was performed (C) with quantification (D). Each column shows the mean ± SD of 3 independent experiments, performed in triplicate. *P < 0.05 and **P < 0.01 versus the control group.

Dasatinib significantly alters the mRNA and protein levels of P-gp

To confirm that the reversal of P-gp-mediated MDR is attributed to a reduction of P-gp expression, we determined the effects of dasatinib on the expression levels of mRNA and protein levels of P-gp in resistant MCF-7/Adr cells. Results showed that dasatinib did significantly downregulate the expressions of mRNA and protein levels of P-gp both in time-dependent (before 24 h) (Fig. 4A–C) and dose-dependent (Fig. 4D–F) manners in MCF-7/Adr cells. These data suggested that the reversal effect of dasatinib on P-gp-mediated MDR was ascribed to the downregulation of P-gp expression in MCF-7/Adr cells.

Figure 4. Effects of dasatinib on the mRNA and protein expressions of P-gp in resistant MCF-7/Adr cells. (A, B) Cells were treated with 1.0 μM dasatinib at the indicated time of 0, 12, 24, 48, 72 h, and dasatinib downregulated P-gp in a time-dependent manner before 24 h. (D, E) Cells were treated with 10 μM U0126 and dasatinib at the indicated concentrations of 0, 0.25, 0.5, 1.0 μM for 24 h, and dasatinib downregulated P-gp in a dose-dependent manner. (C, F) The protein expression of P-gp was normalized to β-actin. Each column shows the mean ± SD of 3 independent experiments, performed in triplicate. *P < 0.05 and **P < 0.01, vs. the control group.

Dasatinib significantly blocks the phosphorylation of ERK

To further investigate the potential mechanism involved in the downregulation of P-gp, we determined the effects of dasatinib on the levels of total and phosphorylated forms of ERK in MCF-7/Adr cells. Results showed that dasatinib remarkably decreased the phosphorylated form of ERK both in time-dependent (before 24 h) (Fig. 5A, B) and dose-dependent (Fig. 5C, D) manners compared to the positive group of MEK inhibitor U0126, whereas it had no effect on the total form of ERK. In view of U0126's significant downregulation of P-gp in Fig. 4D–F, the result indicated that the reversal effect of dasatinib on P-gp-mediated MDR might be associated with the inhibition of ERK pathway in MCF-7/Adr cells.

Figure 5. Effect of dasatinib on the expression of phospho-ERK. P-ERK expression was assayed with the treatment of 1.0 μM dasatinib at the indicated time of 0, 12, 24, 48, 72 h (A) and at the indicated concentrations of 0, 0.25, 0.5, 1.0 μM for 24 h (C). U0126 acts as the positive control group. (B, D) The quantification of P-ERK expression was normalized to the total content of ERK. Each column shows the mean ± SD of 3 independent experiments, performed in triplicate. *P < 0.05 and **P < 0.01, versus the control group.

Discussion

Breast cancer is now the second leading cause of cancer among females, next only to lung cancer.³⁰ Despite significant advances in the diagnosis and treatment of breast cancer, metastatic breast cancer is still incurable. Therefore, identification of the key molecular alterations that induce proliferation or drug resistance in breast cancer cells are needed to help discover potential therapeutic targets. Researchers have long understood some of the key molecular drivers of breast cancer, using them as prognostic factors and/or predictive markers for specific therapies such as estrogen receptor (ER), progesterone receptor (PgR), and HER2 and so forth. According to Peyton Rous,³¹ Src is the earliest known oncogene identified as the transforming agent in chicken sarcomas. Previous studies reported that Src kinase activity was 4- to 20- fold higher than normal tissues in human mammary carcinomas.^32-34 Anomalous activation or amplification of SRC and SRC-family kinases (SFK) has been identified to play an important role in proliferation, migration, and invasion of breast cancer cell lines.^35,36 In addition, Abl kinases, one of whose activated forms is BCR-Abl, plays a functional role in breast cancer.^37,38 Thus, both Src and Abl kinases represent effective molecular targets for the development and progression of breast cancer.

Dasatinib is a potent dual Src/Abl kinase inhibitor, implying that dasatinib may be closely associated with the evolvement of breast cancer. As previously reported, dasatinib have an effect on TNBC which is generally the basal-type breast cancer.^24,39 Furthermore, according to Richard S. Finn et al.,³⁵ that basal-type and epithelial-tomesenchymal transition (post-EMT) breast cancer cell lines are more sensitive than luminal-type breast cancer cells to growth inhibition by dasatinib. Thus, based on the low sensitivity of luminal breast cancer cell lines to dasatinib, we on the other side aimed to explore the effect of dasatinib on MDR in one kind of luminal breast cancer cell lines MCF-7.

In the present study, MCF-7/Adr cells were verified to be DOX-resistant (Fig. 1A), via mechanisms that include the overexpression of mRNA and protein levels of P-gp (Fig. 1B, C). Studies also showed that reversal concentrations of dasatinib remarkably potentiated the cytotoxicity of DOX in P-gp-overexpressing MCF-7/Adr and P-gp-transfected MDCK cells, but did not sensitize the parental sensitive MCF-7 or MDCK cells to DOX as shown in Tables 1 and 2. Dasatinib significantly increased the accumulation (Fig. 2A) and inhibited the efflux (Fig. 2B) of DOX in resistant MCF-7/Adr cells.

P-gp, apart from its role as an efflux pump, can also regulate programmed cell death induced by antineoplastic agents like DOX,⁴⁰ UV irradiation and ligation of the cell surface death receptors Fas. Generally, these diverse apoptotic stimuli initiate cell death by activation of caspases like caspase-3.^41-43 However, it has been reported that functional P-gp could inhibit the activation of caspase-3, which could be reversed by inhibiting P-gp.⁴² Therefore, dasatinib could increase cell apoptosis in a dose-dependent manner (Fig. 3A, B) with an enhanced activation of caspase-3 (Fig. 3C, D). These findings suggested that dasatinib could enhance DOX-induced apoptosis in MDR cells.

For initial investigation, we suggested that the MDR reversal effect of dasatinib is due to either the inhibition of P-gp efflux or the downregulation of P-gp expression, or both. Our results showed that dasatinib downregulated the mRNA and protein levels of P-gp both in time-dependent (before 24 h) (Fig. 4A-C) and dose-dependent manners (Fig. 4D-F) in resistant MCF-7/Adr cells. On the other hand, it has been reported that for dasatinib, the ATPase assays did not show a measurable effect on the P-gp ATPase activity up to 1 μM concentration.¹⁷ Therefore, it could be suggested that dasatinib reverses P-gp-mediated MDR by downregulating P-gp expression as opposed to the direct inhibition of P-gp efflux.

Previous studies also demonstrated that dasatinib inhibited cell activation and even induced apoptosis by blocking downstream signaling pathways like phosphorylation of ERK.^{44, 45} And the mechanism of anti-MDR may be associated with the inhibition of ERK pathway.^46-48 In this study, we analyzed the possible association between ERK pathway and the MDR reversal effect of dasatinib. Results revealed that reversal concentrations of dasatinib significantly blocked the phosphorylation of ERK both in time-dependent (before 24 h) (Fig. 5A, B) and dose-dependent (Fig. 5C, D) manners in MCF-7/Adr cells. U0126's significant downregulation of P-gp in Fig. 4D–F indicated that the inhibition of ERK is involved in the reversal of P-gp-mediated MDR by dasatinib.

In conclusion, this study provides an evidence that dasatinib could significantly enhance the efficacy of chemotherapeutic drug in P-gp-overexpressing MDR cells. Furthermore, the reversal of MDR by dasatinib was ascribed to the downregulation of both the mRNA and protein levels of P-gp as opposed to the direct inhibition of P-gp efflux. The downregulation of P-gp might partly depend on the inhibition of ERK pathway. Confirmation of MDR reversal by dasatinib may support the potential benefits of combining dasatinib with other conventional antineoplastic drugs in overcoming drug resistance in cancer chemotherapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We wish to express our heartfelt gratitude to Dr. Yuichi Sugiyama (Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan) for providing vector – MDCK cells, likewise, we also wish to express our sincere gratitude to Professor Zeng Su (College of Pharmacy, Zhejiang University, China) for providing stable transfected ABCB1-MDCK cells.

Funding

This article was supported by a grant from the National Natural Science Foundation of China (No. 81273580, 81072694).

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.