Biology lab report

Biomedicine & Pharmacotherapy 72 (2015) 37–43

Original Article

Lapatinib enhances the cytotoxic effects of doxorubicin in MCF-7 tumorspheres by inhibiting the drug efflux function of ABC transporters

So-Young Chun, Yun-Suk Kwon, Kyung-Soo Nam **, Soyoung Kim *

Department of Pharmacology, School of Medicine and Intractable Disease Research Center, Dongguk University, 87 Dongdae-ro,

Gyeongju-si, Gyeongsangbuk-do 780-350, Republic of Korea

A R T I C L E I N F O

Article history:

Received 9 March 2015

Accepted 20 March 2015

Keywords:

Tumorsphere

Chemoresistance

Lapatinib

MCF-7 breast cancer cells

ATP-binding cassette (ABC) drug

transporters

A B S T R A C T

Increasing evidences indicate that cancer stem cells are resistant to chemotherapy due to their cell

quiescence and the expression of ATP-binding cassette (ABC) transporters. In this study, we utilized

tumorsphere cultures to seek better strategies to overcome chemoresistance since tumorsphere cultures

have been used widely for the enrichment of cancer stem cells. We found that tumorspheres generated

from MCF-7 human breast cancer cells exhibited high proportions of quiescent cells and expressed MDR-

1 at elevated levels, leading to resistance to 5-fluorouracil, paclitaxel, and doxorubicin. Because the

expression of EGFR/HER2 was increased in MCF-7 tumorspheres, we assessed the combinational effect of

the dual ErbB1/ErbB2 inhibitor, lapatinib, with doxorubicin in tumorspheres. The results showed that

inhibition of EGFR/HER2 signaling by lapatinib sensitized MCF-7 tumorspheres to doxorubicin by

inhibiting the expression of the ABC transporters, MDR-1 and BCRP, and thus, enhancing the intracellular

accumulation of doxorubicin. These findings suggest that combinations of lapatinib and cytotoxic

anticancer drugs may offer an advantage for treating the drug-resistant cancers.

� 2015 Elsevier Masson SAS. All rights reserved.

Available online at

ScienceDirect www.sciencedirect.com

1. Introduction

The development of resistance to anticancer drugs is the leading cause of treatment failure in cancer. However, the mechanisms associated with the development of chemoresistance are complex and not fully understood. Recent studies have focused on the linkage between chemoresistance and a small population of cancer cells called cancer stem cells. These cells possess the capacity to self-renew and differentiate to regenerate whole tumors, whereas most cancer cells lack this regenerative capacity [1,2]. Increasing evidences indicate that cancer stem cells are

Abbreviations: Lapa, lapatinib; Dox, doxorubicin; ABC, ATP-binding cassette; MRP-

1, multidrug resistance-associated protein-1; MDR-1, multidrug resistance protein-

1; BCRP, breast cancer resistance protein; HER2, human epidermal growth factor

receptor 2; ER, estrogen receptor; PR, progesterone receptor; EGFR, epidermal

growth factor receptor; TKI, tyrosine kinase inhibitor; PI3K, phosphoinositol-3

kinase; MAPK, mitogen-activated protein kinase; AKT, protein kinase B; ERK,

extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehy-

drogenase.

* Corresponding author. Tel.: +82 54 770 2419; fax: +82 54 770 2477. ** Corresponding author. Tel.: +82 54 770 2412; fax: +82 54 770 2477.

E-mail addresses: namks@dongguk.ac.kr (K.-S. Nam),

soyoungkim@dongguk.ac.kr (S. Kim).

http://dx.doi.org/10.1016/j.biopha.2015.03.009

0753-3322/� 2015 Elsevier Masson SAS. All rights reserved.

resistant to chemotherapy due to their properties expressing ATP- binding cassette (ABC) drug transporters and their quiescence [3,4]. Furthermore, the expressions of ABC drug transporters, such as, p-glycoprotein (MDR-1/ABCB1), multidrug resistance-associat- ed proteins (MRPs), and breast cancer resistance proteins (BCRP/ ABCG2), have been shown to protect cancer stem cells from chemotherapeutic agents [3,5]. Besides the expression of ABC drug transporters, cancer stem cells are expected to be inherently refractory to drugs that target rapidly dividing cells because they are generally regarded as quiescent [4,6].

Tumorsphere culture has been widely utilized as a surrogate in vitro model of cancer stem cells since it has been proposed that cells cultured under non-adherent culture conditions form tumor- sphere that is exhibiting stem cell properties [7–10]. However, several studies have shown that sphere formation from various cancer cell lines or tissues does not always predict the enrichment of cancer stem cells. Accordingly, it appears the enrichment of cancer stem cells in tumorsphere culture is either cell-line dependent or depends on experimental variables, such as, cell density and duration of culture [11–13]. Thus, extensive charac- terization of tumorspheres is required before it can be concluded that tumorspheres confer stem cell properties. In our previous study, although we were unable to show that tumorspheres are

S.-Y. Chun et al. / Biomedicine & Pharmacotherapy 72 (2015) 37–4338

clonally derived from stem cells, we did find that tumorsphere culture provides cell quiescence [14]. Furthermore, in another study, the accumulation of cells in G0/G1 phase was reported for tumorsphere cultured hepatoma cells as compared with the same cells in monolayer culture [15]. Since quiescence is an important mechanism of drug resistance in stem cells, we optimized tumor- sphere cultures for the high-throughput screening of potential therapeutic strategies that are effective against quiescent cells.

In this study, we utilized tumorsphere cultures of MCF-7 human breast cancer cells to identify better means of overcoming chemoresistance. We analyzed the cytotoxic effects of paclitaxel, 5-fluorouracil (5-FU), and doxorubicin in a tumorsphere assay to evaluate the contribution of cell quiescence on chemoresistance. Since we found that enhanced EGFR/HER2 expression is charac- teristic of MCF-7 tumorspheres, we investigated whether combi- natorial treatment based on lapatinib (a dual ErbB1/ErbB2 inhibitor) and cytotoxic therapeutics offers a potential means of overcoming the chemoresistance of tumorspheres.

2. Materials and methods

2.1. Monolayer culture

The MCF-7 human breast cancer cell line was purchased from the Korean Cell Line Bank (Seoul, Korea) and routinely maintained in DMEM (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 10 mg/mL insulin (Welgene), and 1% antibiotic-antimycotic solution (Welgene).

2.2. Tumorsphere culture

The protocol used for tumorsphere culture was as previously described [7,13]. Briefly, MCF-7 cells were suspended in serum- free DMEM/F12 (Welgene) supplemented with 1:50 B27 (Gibco BRL, Grand Island, NY, USA), 10 mg/mL insulin (Welgene), 20 ng/mL recombinant human epidermal growth factor (EGF; R&D systems, Minneapolis, MN, USA), 10 ng/mL recombinant human fibroblast growth factor (FGF; R&D systems), and 1% antibiotic-antimycotic solution (Welgene), and cultured in non-adherent plates.

2.3. Cell kinetic assay

To examine cell proliferation rates, MCF-7 cells were plated at different concentrations (3000–20,000 cells/well) into 96 well plates under tumorspheres (see above) or monolayer culture conditions. After 4 days, premixed cell proliferation reagent WST-8 (DojinDo Laboratories, Kumamoto, Japan) was added to each well and cell viabilities were determined by measuring absorbance at 450 nm according to the manufacturer’s instructions.

2.4. Cytotoxicity assay

MCF-7 cells cultured as monolayers or tumorspheres were treated with doxorubicin (Sigma, St. Louis, MO, USA), 5-fluoroura- cil (Sigma), paclitaxel (Sigma), lapatinib (a dual ErbB1/ErbB2 inhibitor; LC Laboratories, Woburn, MA, USA), U0126 (a MEK1/2 inhibitor; LC Laboratories), or LY294002 (a PI3K/AKT inhibitor; LC Laboratories) for 3 days and cell viabilities were measured with WST-8 reagent (DojinDo Laboratories). To examine the effects of lapatinib and doxorubicin in combination, cells were treated with doxorubicin (0.2–1 mM) in the presence of 5 mM lapatinib.

2.5. Cell cycle analysis by flow cytometry

For cell cycle analysis, cells grown as monolayers or tumor- spheres for 4 days were trypsinized and fixed in cold 70% ethanol.

After centrifugation, cells were washed with PBS containing 2% FBS and stained with 20 mg/mL propidium iodide (Sigma) and 200 mg/ mL RNase A (Sigma) for 30 min at room temperature. The DNA contents labeled with propidium iodide were analyzed by FACSCalibur II flow cytometry (Becton Dickinson Biosciences, San Jose, CA, USA).

2.6. Intracellular doxorubicin accumulation

MCF-7 cells were cultured as monolayers or tumorspheres for 4 days, then treated with 0.5 mM doxorubicin for 1 h, trypsinized, washed twice with PBS containing 2% FBS, and resuspended in PBS containing 2% FBS. Intracellular doxorubicin was measured by its fluorescence intensity using FACSCalibur II flow cytometry (Becton Dickinson Biosciences).

2.7. Measurement of intracellular ROS production

Cells were treated with 0.4 mM doxorubicin with or without 5 mM lapatinib for 3 days, trypsinized, and incubated with 20 mM 20,70-dichlorofluorescein diacetate (DCF-DA, Sigma) for 30 min at 37 8C in the dark. After incubation, cells were immediately washed and resuspended in PBS containing 2% FBS. Intracellular ROS production was assessed by FACSCalibur II flow cytometry (Becton Dickinson Biosciences) by measuring the fluorescent intensity of DCF at 530 nm.

2.8. Semi-quantitative reverse transcription polymerase chain

reaction (RT-PCR)

Cells cultured as monolayers or tumorspheres were treated with 0.4 mM doxorubicin or 5 mM lapatinib for 3 days and then harvested for RNA isolation. Total RNA was extracted using the easy-BLUETM

Total RNA Extraction kit (iNtRON Biotechnology Inc., Sungnam, Korea) and cDNA was synthesized with reverse transcriptase (Takara, Shiga, Japan). RT-PCR for Cyclin D1, MDR-1, MRP-1, BCRP, and GAPDH were conducted as previously described [16]. The primer sequences used for the RT-PCR reactions were as follows:

Cyclin D1 (forward) 50-AGCTCCTGTGCTGCGAAGTGGAAAC-30

and Cyclin D1 (reverse) 50-AGTGTTCAATGAAATCGTGCGGGG-30

MDR-1 (forward) 50-GCCTGGCAGCTGGAAGACAAATACACAAA ATT-30 and MDR-1 (reverse) 50-CAGACAGCAGCTGACAGTCCAAGAACAG- GACT-30; MRP-1 (forward) 50-GCGAGTGTCTCCCTCAAA CG-30 and MRP-1 (reverse) 50-TCCTCACGGTGATGCTGTTC-30; BCRP (forward) 50-GCAGATGCCTTCTTCGTTATG-30 and BCRP (reverse) 50-TCTTCGCCAGTACATGTTGC-30

GAPDH (forward) 50-ATCCCATCACCATCTTCCAG-30 and GAPDH (reverse) 50-TTCTAGACGGCAGGTCAGGT-30.

Densitometric analysis was performed using Scion Image Software (Scion Corporation, CA, USA).

2.9. Western blotting

MCF-7 cells cultured as monolayers or in suspension were lysed with RIPA buffer (50 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris–HCl pH 7.5 and 2 mM EDTA) supplemented with phosphatase and protease inhibitor cocktails (GenDEPOT, Barker, TX, USA). Lysates were centrifuged at 13,000 rpm for 20 min to remove debris, and protein concentra- tions were determined using bicinchoninic acid reagent (Sigma). Equal amounts of protein were separated by SDS-PAGE and

S.-Y. Chun et al. / Biomedicine & Pharmacotherapy 72 (2015) 37–43 39

transferred to polyvinylidene fluoride (PVDF) membranes, which were then blocked with 5% non-fat skim milk in TBS–0.1% Tween 20 (TTBS) for 2 h and incubated with a primary antibody (EGFR, p- EGFR, HER2, AKT, p-AKT, ERK 1/2, p-ERK 1/2, or GAPDH; Cell Signaling, Beverly, MA, USA) overnight. HRP-conjugated secondary anti-rabbit antibody or anti-mouse antibody (Thermo Scientific, Rockford, IL, USA) diluted 1:3000 was incubated with blots for 1 h at room temperature. Blots were developed using Luminescent Image Analyzer LAS-4000 (Fujifilm, Tokyo, Japan).

2.10. Statistical analysis

Statistical significance was determined using the Student’s t-test. All experiments were conducted in triplicate, and results are presented as mean � S.D. P values of <0.05 were considered significant.

3. Results

3.1. The majority of cells in MCF-7 tumorsphere were quiescent

MCF-7 breast cancer cells were cultured in suspension on non- adherent plates for 4 days to test ability to grow as tumorspheres. As described in other studies [13], MCF-7 cells successfully form tumorspheres, which exhibited tightly adherent structures (Fig. 1A). To access proliferation, equal numbers of cells (3000–20,000 cells/ well) were plated into 96 well plates, either non-adherent plates, or regular tissue culture plates, and viable cells were measured using WST-8 reagents after 4 days culture (Fig. 1B). The WST-8 absorbance of monolayer cultured cells was at least three times higher than that of cells cultured as tumorspheres, suggesting that cell growth rate in tumorspheres was much slower. Furthermore, this slow cell growth rate paralleled the accumulation of cell population at G0/G1 phase

Fig. 1. Tumorsphere formation increased MCF-7 quiescence. (A) Images of MCF-7 tumo Comparison of cell proliferation rates of cells grown in monolayers (2D) or tumorspheres

4 days. (C) Cell cycle analysis of cells grown in 2D or TS. (D) The mRNA expression of

in tumorspheres. The cell cycle analysis showed that 10% of cell population was increased at G0/G1 phase in tumorspheres as compared with monolayer cultured cells (Fig. 1C). Moreover, the mRNA expression of cyclin D1, which is the first regulatory protein to drive the G1/S phase transition, was also decreased in tumorspheres (Fig. 1D). Taken together, these results suggest that tumorsphere culture conditions increase numbers of cells in the quiescent state.

3.2. Chemoresistance in tumorspheres

Since tumorspheres exhibited cell quiescence, we investigated the effects of paclitaxel, 5-fluorouracil, and doxorubicin on cells in tumorspheres. To compare cytotoxic effects, MCF-7 was cultured as monolayers or tumorspheres, and their cells were exposed to different concentrations of these agents for 3 days, and then cell viabilities were determined with WST-8. Interestingly, tumor- spheres exhibited significant chemoresistance as compared with monolayer cultured cells. As shown in Fig. 2, cells cultured as monolayers were highly sensitive to paclitaxel (Fig. 2A), 5- fluorouracil (Fig. 2B), and to doxorubicin (Fig. 2C) with IC50 values of 20 nM, 50 mM, and 0.6 mM, respectively, whereas over 80% cells in tumorspheres survived at the highest concentrations tested.

3.3. MDR-1 mediated chemoresistance in tumorspheres

To understand the mechanism underlying the chemoresistance of tumorspheres, we accessed the mRNA expressions of the ATP- binding cassette (ABC) drug transporters including multidrug resistance (MDR-1) and multidrug resistance-associated proteins (MRP-1) in monolayer or tumorsphere cultured MCF-7 cells. The mRNA expression of MDR-1 was significantly higher in tumor- spheres, but MRP-1 expression was similar in tumorsphere and

rspheres grown in non-adherent culture plates for 4 days. Scale bar = 100 mm. (B) (TS). Data represent the absorbance of cells assessed by WST-8 after incubation for

cyclin D1 in cells grown as monolayers or tumorspheres.

Fig. 2. MCF-7 tumorspheres were resistant to chemotherapeutics. MCF-7 cells cultured as monolayers (2D) or tumorspheres (TS) were treated with (A) paclitaxel, (B) 5- fluorouracil, or (C) doxorubicin for 3 days and their viabilities were assessed. Results are the mean � S.D. of three independent experiments performed in triplicate.

Fig. 3. Decreased doxorubicin uptake in tumorspheres mediated by MDR-1. (A) mRNA expressions of MDR-1 and MRP-1 in cells grown as monolayers (2D) or tumorspheres (TS). (B) Comparison of intracellular doxorubicin levels in monolayer or tumorsphere cultured cells. Cells were treated with 0.5 mM doxorubicin (Dox) or vehicle for 1 h and doxorubicin fluorescence was analyzed by flow cytometry.

Fig. 4. Increased EGFR and HER2 signaling in tumorspheres. Western blot analysis of the expressions of (A) p-EGFR, EGFR, HER2, (B) p-ERK 1/2, and (C) p-AKT in cells grown as monolayers (2D) or tumorspheres (TS). Chemotherapeutic responses to (D) lapatinib (a dual ErbB1/ErbB2 inhibitor), (E) U0126 (a MEK 1/2 inhibitor), or (F) LY294002 (a PI3K/

AKT inhibitor) in monolayer or tumorsphere cultured cells. After treatment with each inhibitor for 3 days, cell viabilities were determined. Results are the mean � S.D. of three independent experiments performed in triplicate.

S.-Y. Chun et al. / Biomedicine & Pharmacotherapy 72 (2015) 37–4340

S.-Y. Chun et al. / Biomedicine & Pharmacotherapy 72 (2015) 37–43 41

monolayer cultured cells (Fig. 3A), which suggests that increased MDR-1 expression in tumorspheres plays an important role in protecting cells from cytotoxic agents. To confirm the involvement of MDR-1 in chemoresistance of tumorsphere, we further analyzed intracellular drug accumulation using doxorubicin [17]. Cells grown as monolayers or tumorspheres were treated with 0.5 mM doxorubicin for 1 hr and then doxorubicin fluorescence was

Fig. 5. Lapatinib synergistically increased the cytotoxic effect of doxorubicin on MCF-7 effects of doxorubicin plus lapatinib in tumorspheres. Tumorspheres were treated with v

cell viabilities were determined. Data are the mean � S.D. of three independent experim doxorubicin in tumorspheres. Tumorspheres treated with 5 mM lapatinib or vehicle for 3 day Inhibition of doxorubicin-induced MDR-1, BCRP and MRP-1 expression by lapatinib in tumo

analyzed by RT-PCR. **P < 0.01, ***P < 0.001. (D) Enhancement of ROS levels in MCF-7 cells c

doxorubicin for 3 days in 2D or TS cultured cells. Increased ROS production by lapatinib w

analyzed by flow cytometry. A right-shift of fluorescence intensity was observed after treating monolayer cultured cells with doxorubicin, confirming the accumulation of doxorubicin within monolayer cultured cells. However, this right-shift was much smaller in tumorspheres (Fig. 3B). Taken together, these results suggested that the chemoresistance exhibited by tumorspheres was due to the enhanced expression of MDR-1, leading to the

tumorspheres by inhibiting the expressions of ABC transporters. (A) The cytotoxic

arious concentrations of doxorubicin (Dox) and 5 mM lapatinib (Lapa) for 3 days and ents performed in triplicate. (B) Lapatinib increased the intracellular accumulation of

s were incubated with 0.5 mM doxorubicin for 1 h and analyzed by flow cytometry. (C) rspheres. Cells treated with 0.4 mM doxorubicin and/or 5 mM lapatinib for 3 days were ultured as tumorspheres. The generation of ROS was induced by treatment with 0.4 mM

as observed only in tumorspheres.

S.-Y. Chun et al. / Biomedicine & Pharmacotherapy 72 (2015) 37–4342

decreased intracellular doxorubicin accumulation in tumor- spheres.

3.4. The enhanced EGFR/HER2 signaling in tumorspheres

To identify a better approach for overcoming the chemoresis- tance of tumorspheres, we first analyzed which signaling path- ways are characteristics in MCF-7 tumorspheres. The western blot analysis revealed that the phosphorylation of EGFR and the expression of HER2 were increased in tumorspheres (Fig. 4A). Since the MAPK and PI3K/AKT signaling cascades are the major signaling pathways activated by tyrosine kinase, we examined the phosphorylation of AKT and ERK 1/2 and found that the phosphorylation of AKT and ERK 1/2 were higher in tumorspheres than in monolayer cultured cells (Fig. 4B and C). To explore the role of enhanced EGFR/HER2 signaling in tumorspheres, cells were treated with lapatinib, and cell viability was then accessed. Interestingly, treatment with lapatinib did not elicit different responses in monolayer or tumorsphere cultured cells (Fig. 4D). However, tumorspheres treated with U0126 or LY294002, which were used to block the MAPK and PI3K/AKT signaling pathways, respectively, were less sensitive to U0126 and LY29400 than cells cultured in monolayers (Fig. 4E and F). These findings suggest that the EGFR/HER2 signaling pathway plays an important role in mediating cell survival in the quiescent state rather than its downstream MAPK or PI3K/AKT signaling.

3.5. Lapatinib increased the cytotoxic effects of doxorubicin in

tumorspheres by inhibiting ABC transporters

Since the expressions of EGFR and HER2 were found to be enhanced in tumorspheres, we accessed the combination effects of chemotherapy with lapatinib in tumorspheres assay. Cells were treated with different concentrations of doxorubicin (0.2–1 mM) in the presence of 5 mM lapatinib for 3 days and their cell viability was measured. As shown in Fig. 5A, lapatinib increased the cytotoxic effect of doxorubicin in tumorspheres, for example the >90% cell viability observed after treatment with 0.8 mM doxorubicin for 3 days was reduced to <60% when cells were co-treated with 0.8 mM doxorubicin and 5 mM lapatinib (Fig. 5A). Encouraged by this result, we investigated whether lapatinib modulates the accumulation of intracellular doxorubicin in tumorspheres. Tumor- spheres were pretreated with 5 mM lapatinib for 3 days and then incubated with 0.5 mM doxorubicin for 1 h prior to flow cytometry analysis. We found that lapatinib significantly increased the accumulation of intracellular doxorubicin in tumorspheres (Fig. 5B). Furthermore, the expression of MDR-1 mRNA was suppressed by lapatinib, and although the inhibitory effects of lapatinib on the expression of MRP-1 was not clearly observed, the expression of BCRP was significantly decreased by treatment with lapatinib (Fig. 5C). These results suggest that the inhibition of ABC transporters by lapatinib, resulting in the increased accumulation of intracellular doxorubicin, is a potential mechanism underlying the synergistic effects of lapatinib and doxorubicin.

We further explored the effects of lapatinib on the generation of reactive oxygen species (ROS), which play an important role in triggering apoptosis [18]. Flow cytometry analysis using DCF-DA reagent, which can be converted to fluorescent DCF in a reaction with intracellular ROS, showed that doxorubicin caused a right- shift in DCF fluorescence signal versus untreated cells, thus confirming the generation of ROS by doxorubicin in monolayer and tumorspheres cultures. Interestingly, lapatinib enhanced doxorubicin-induced ROS generation in tumorspheres, but had no effect on monolayer cultured cells (Fig. 5D), which suggests increased ROS generation in tumorspheres by lapatinib enhanced the cytotoxic effect of doxorubicin.

4. Discussion and conclusions

Although tumorsphere assay has been proposed to propagate cancer stem cells [7–10], several authors have reported that sphere formation does not always reflect an enrichment of stem cells because sphere formation is dependent on cell type, density, and duration of culture [11–13]. In the present study, MCF-7 tumor- spheres were found to contain higher proportions of quiescent cells and to overexpress MDR-1 as compared with monolayer cultured MCF-7 cells, and these features mediated resistance to chemotherapeutics such as 5-fluorouracil, paclitaxel, and doxoru- bicin. The expression of ABC transporters and quiescence are important features of cancer stem cells [3,4], and thus, MCF-7 tumorspheres were found to have some of the properties of cancer stem cells. However, their self-renewal abilities were not investigated in the present study.

ABC transporters have been linked to development of resistance to anticancer drugs because the overexpression of efflux trans- porters results in decreased intracellular concentrations of chemotherapeutics [19]. Forty-eight ABC transporters have been identified in human [20], but three of these, namely, P-glycopro- tein (MDR-1/ABCB1) [21], breast cancer resistance protein (BCRP/ ABCG2) [22,23], and multidrug resistance-associated protein (MRP/ABCC) [24] family appear to be the main contributors to drug resistance. The efficacies of ABC transporter inhibitors have been studied extensively with a view toward overcoming drug resistance, but the results have generally been disappointing. However, recent studies reported that lapatinib provides promis- ing results in reversing chemotherapy resistance [25–27]. Lapati- nib is a reversible tyrosine kinase inhibitor (TKI) that inhibits EGFR (ErbB1/HER1) and HER2 (ErbB2), the activation of which triggers PI3K/AKT and MAPK signal transduction cascades, leading to cell proliferation, adhesion, and migration [28,29]. Since the over- expression or dysregulation of EGFR family in human tumor promote tumor growth or progression [30], lapatinib was approved for the treatment of HER2 positive breast cancers in combination with other anticancer agents [31]. Recently, it was shown that lapatinib enhances the cytotoxic effect of vincristine by increasing the intracellular accumulations of therapeutics in multidrug resistant cell lines [25]. Furthermore, the synergistic interactions between lapatinib and chemotherapeutics appear to be associated with its inhibition of ABCG2 activity [25–27]. Con- sistent with other studies, our findings also indicate that lapatinib significantly enhances the cytotoxic effects of doxorubicin in MCF- 7 tumorspheres by inhibiting the drug efflux functions of MDR-1 and BCRP, resulting in the increased intracellular accumulation of doxorubicin. Additionally, lapatinib was also found to stimulate ROS generation in MCF-7 tumorspheres. These findings suggest that the synergistic effects of lapatinib and doxorubicin may come from not only the inhibition of ABC transporters but also increased ROS generation to trigger apoptosis signaling pathways.

Taken together, our findings suggest that combinations of lapatinib and cytotoxic anticancer drugs may offer more effective modalities for the treatment of drug-resistant cancers.

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgement

This study was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (No. 1320060).

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