Reversal of ABCG2-mediated multidrug resistance by human cathelicidin and its analogs in cancer cells

Peptides 40 (2013) 13–21

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Reversal of ABCG2-mediated multidrug resistance by human cathelicidin and its analogs in cancer cells Kenneth K.W. To a,∗ , S.X. Ren b , C.C.M. Wong b , Chi Hin Cho b a School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Room 801N, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, Shatin, New Territories, Hong Kong, China b School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Room 521A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, Shatin, New Territories, Hong Kong, China

a r t i c l e

i n f o

Article history: Received 18 November 2012 Received in revised form 17 December 2012 Accepted 18 December 2012 Available online 27 December 2012 Keywords: Multidrug resistance ABCG2 Efflux transporter Cathelicidin LL37 Host defense peptides

a b s t r a c t Multidrug resistance (MDR) of cancer cells to a wide spectrum of anticancer drugs is a major obstacle to successful chemotherapy. It is usually mediated by the overexpression of one of the three major ABC transporters actively pumping cytotoxic drugs out of the cells. There has been great interest in the search for inhibitors toward these transporters with an aim to circumvent resistance. This is usually achieved by screening from natural product library and the subsequent structural modifications. This study reported the reversal of ABCG2-mediated MDR in drug-selected resistant cancer cell lines by a class of host defense antimicrobial peptides, the human cathelicidin LL37 and its fragments. The effective human cathelicidin peptides (LL17-32 and LL13-37) were found to increase the accumulation of mitoxantrone in cancer cell lines with ABCG2 overexpression, thereby circumventing resistance to mitoxantrone. At the effective concentrations of the cathelicidin peptides, cell proliferation of the parental cells without elevated ABCG2 expression was not affected. Result from drug efflux and ATPase assays suggested that both LL17-32 and LL13-37 interact with ABCG2 and inhibit its transport activity in an uncompetitive manner. The peptides were also found to downregulate ABCG2 protein expression in the resistant cells, probably through a lysosomal degradation pathway. Our data suggest that the human cathelicidin may be further developed for sensitizing resistant cancer cells to chemotherapy. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Cathelicidin is a class of host defense cationic peptides endogenously found in the epithelial cells of colon and stomach, which become highly expressed during infection, inflammation and wound healing. They have been identified in various mammals, including LL37 (human), RL-37 (rhesus monkey), mCRAMP (mouse), rCRAMP (rat), CAP18 (rabbit) and CAP11 (guinea pig). Cathelicidin peptides have been reported to exhibit broad spectrum of antimicrobial activities and they are also known to regulate inflammation and wound healing [14,37].

Abbreviations: FTC, fumitremorgin C; MDR, multidrug resistance; PhA, pheophorbide A. ∗ Corresponding author at: School of Pharmacy, Room 801N, Lo Kwee-Seong Integrated Biomedical Sciences Building, The Chinese University of Hong Kong, Area 39, Shatin, New Territories, Hong Kong, China. Tel.: +852 39438017; fax: +852 26035295. E-mail addresses: [email protected], kenneth kw [email protected] (K.K.W. To), xiang [email protected] (S.X. Ren), clover [email protected] (C.C.M. Wong), [email protected] (C.H. Cho). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.12.019

Human cathelicidin LL37 originates from the proprotein hCAP38, which is stored in neutrophil granules and epithelial cells. The active LL37 is generated after cleavage of the proprotein by a serine protease. Although LL37 is regarded as the sole human cathelicidin, other cleavages sites have been reported, which give rise to different mature peptides. A number of shorter peptides derived from LL37, such as LL-23, KS-27 and LL-29, have been detected in human skin [38]. Besides the natural host defense action of cathelicidin, several new pharmacological uses of cathelicidin have been recently reported including anti-HIV-1 [32], antifungal [32] and protective effects in ulcerative colitis [33]. Recently, the emerging role of cathelicidin in cancer chemotherapy has also been described [35,36]. Multidrug resistance (MDR) to cancer chemotherapy is an old but unresolved obstacle to successful cancer treatment. It is often associated with increased expression of ATP-binding cassette (ABC) transporters on cell surface that mediate energy-dependent transport of substrate drugs out of the cells. There have been enormous efforts all over the world in the search for safe and effective inhibitors of the ABC transporters as a novel strategy to overcome MDR [5]. While most of the inhibitors identified are small chemical entities derived from natural products, the use

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Table 1 Amino acid sequences of human cathelicidin LL37 and its fragments used in the study. Positively charged amino acid residues are labeled in bold whereas negatively charged amino acid residues are underlined. Name

Amino acid sequence

Net charge

LL37 LL17-32 LL13-37

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES FKRIVQRIKDFLRNLV IGKEFKRIVQRIKDFLRNLVPRTES

+6 +4 +4

of antisense oligonucleotides, siRNA, antibodies, and biologically active peptides have also been described [13,34]. The aim of this study was to evaluate whether human cathelicidin peptides exhibit any anticancer activity and that they modulate MDR in cancer chemotherapy. Three peptides (LL37, LL17-32, and LL13-37) were chosen for this study because of their different molecular weight and net charges (Table 1) and potent activity against microbes and cancer cells [12].

2.3. Growth inhibition assay Growth inhibitory effect of various anticancer drugs, with or without the concomitant treatment of the human cathelicidin fragments, on the parental and resistant cell lines were evaluated by the sulforhodamine B assay [27]. Cells were seeded into 96-well microtitre plates in 100 ␮L at a plating density of 3000–5000 cells/well and allowed to incubate overnight. The cells were then treated with various anticancer drugs at a range of concentrations in the presence or absence of a fixed concentration of the human cathelicidin peptides (2, 5, or 10 ␮M) and allowed to incubate at 37 ◦ C in 5% CO2 for 72 h. Each drug concentration was tested in quadruplicate and controls were tested in replicates of eight. Fold resistance was calculated by dividing the IC50 of the anticancer drug with or without the tested cathelicidin in the resistant cells by the IC50 of the anticancer drug alone in the parental cells. Each experiment was carried out independently at least three times. To determine whether differences between IC50 values were significant, the Student’s t-test was performed with p < 0.05 being considered significant.

2. Material and methods 2.4. Flow cytometry-based substrate efflux assay 2.1. Chemicals and reagents Mitoxantrone and paclitaxel were purchased from Sigma Chemical (St Louis, MO). Doxorubicin HCl (PH-1898) was obtained from Yick-Vic Chemicals & Pharmaceuticals (Hong Kong). Pheophorbide A (PhA), fumitremorgin C (FTC) and PSC833 were kind gifts obtained from Dr. Susan Bates (National Cancer Institute, NIH, Bethesda, MD). Calcein AM was purchased from Biotium (Hayward, CA). MK-571 was obtained from EMD Biosciences (San Diego, CA). The human cathelicidin peptide fragments (LL37, LL17-32, LL1337) were obtained commercially (GL Biochem, China) according to our specifications. All these peptides were synthesized with purity ≥95%. Their amino acid sequences are listed in Table 1. Leupeptin and pepstatin A were bought from Sigma (St. Louis, MO).

2.2. Cell culture All cell lines are generous gift from Dr. Susan Bates (National Cancer Institute, Bethesda, MD, USA). Pairs of parental and drug resistant sublines with overexpression of the three major multidrug resistance transporters were used in our study, which include human colon cancer SW620/its P-gp overexpressing SW620 Ad300 subline [10], human breast cancer MCF-7/its ABCG2-overexpressing MCF-7 FLV1000 subline [21], and MCF-7/its MRP1-overexpressing MCF-7 VP-16 subline [25]. The resistant sublines were developed from their respective parental cancer cell lines by stepwise selection in increasing concentrations of selecting agents and were maintained in 300 nM doxorubicin, 1000 nM flavopiridol, and 10 ␮M etoposide, respectively. They have been fully characterized and proven to be useful models for studying multidrug resistant transporters-mediated resistance and their reversal. The resistant cells were allowed to grown in drug-free culture medium for more than 2 weeks before assays. The resistance phenotype was stable for at least 3 months in drug-free medium. The human primary embryonic kidney cell line HEK293 and its stably pcDNA3.1- or ABCG2-transfected cell lines were also used to demonstrate the specific effect of cathelicidin on ABCG2. The transfected cells were cultured in culture medium supplemented with 2 mg/mL G418 [23]. The other cell lines were maintained in DMEM (MCF-7 and its resistant sublines) or RPMI-1640 medium (SW620 and its resistant subline) supplemented with 10% fetal bovine serum, 100 units/mL streptomycin sulfate, and 100 units/mL penicillin G sulfate, and incubated at 37 ◦ C in 5% CO2 .

A flow cytometry-based assay was employed to study the possible efflux inhibition of the three major ABC transporters by the tested human cathelicidin peptide fragments as described previously with minor modification [29]. Briefly, cells were trypsinized and incubated for 30 min in phenol red-free complete medium with the desired fluorescent substrate (0.5 ␮g/mL rhodamine 123, 1 ␮M pheophorbide A or 0.2 ␮M calcein AM) in the presence or absence of the tested cathelicidin peptides. Subsequently, the cells were washed twice with ice-cold PBS and incubated in substrate-free medium for 1 h at 37 ◦ C continuing with the tested inhibitor to generate the inhibitor/efflux histogram, or without the inhibitor to generate the efflux histogram. The inhibited efflux was determined as the difference in mean fluorescence intensity (MFI) between the inhibitor/efflux and efflux histograms. To determine significant difference between intracellular fluorescence values, the Student’s t-test was performed with p < 0.05 being considered significant. Cells were finally washed with cold Dulbecco’s PBS and placed on ice in the dark until analysis by flow cytometry. Inhibitors specific for the three MDR transporters (i.e. FTC, PSC833 and MK-571 for ABCG2, Pgp and MRP1, respectively) were used as control for comparison. Cathelicidin peptides with transporter inhibitory effect will shift the inhibitor/efflux histogram to the right, indicating retention of the fluorescent substrate in the cells. Samples were analyzed on a LSRFortessa Cell Analyzer (BD Biosciences, San Jose, CA). Rhodamine and calcein fluorescence were detected with a 488-nm argon laser and a 530-nm bandpass-filter whereas PhA fluorescence was detected with a 488-nm argon laser and a 670-nm bandpass filter. At least 10,000 events were collected for all flow cytometry studies. Cell debris was eliminated by gating on forward versus side scatter and dead cells were excluded based on propidium iodide staining. All assays were performed in three independent experiments. 2.5. Mitoxantrone accumulation The effect of cathelicidin peptides on the accumulation of mitoxantrone (a known ABCG2 substrate) in MCF-7, MCF-7 FLV1000, pcDNA3.1- or ABCG2-transfected HEK293 cells were determined by flow cytometry. The cells were incubated for 1 h at 37 ◦ C with the cathelicidin peptides, at various concentrations or vehicle. Then, 10 ␮M mitoxantrone was added and incubation was continued for another 1 h. The cells were then collected, washed three times with

K.K.W. To et al. / Peptides 40 (2013) 13–21

ice-cold PBS, and analyzed by flow cytometry as above. Mitoxantrone fluorescence was detected with a 635-red diode laser and a 670-nm bandpass filter. 2.6. Analysis of transporter inhibition kinetics The inhibition kinetic of ABCG2-mediated efflux of mitoxantrone (an ABCG2 substrate anticancer drug) by the cathelicidin peptides was followed by the method as described previously with minor modification [15]. Briefly, ABCG2-stably transfected HEK293/ABCG2 cells were incubated with various concentration of mitoxantrone (1, 2, 5, 10, or 20 ␮M) in the presence of 5 or 10 ␮M of cathelicidin peptides for 3 h at 37 ◦ C. The cells were then collected, centrifuged and washed once with cold PBS, and resuspended in the medium without mitoxantrone in the absence or presence of the cathelicidin peptides. Subsequently, cells were incubated for 10 min at 37 ◦ C to allow for efflux, centrifuged and washed 3 times with cold PBS. In the control samples, the incubations were kept at 0 ◦ C. Finally, the intracellular concentration of mitoxantrone was determined by flow cytometric analysis as above. The quantity of mitoxantrone efflux by ABCG2 was calculated by subtracting values obtained at 37 ◦ C from that at 0 ◦ C. The inhibitory effect of the cathelicidin peptides on ABCG2 was then analyzed by Linewaver–Burk plot. 2.7. 5D3 shift assay for assessing interaction between cathelicidin peptides and ABCG2 The binding of the conformational sensitive 5D3 antibody to intact cells (ABCG2-overexpressing MCF-7 FLV1000) in the presence or absence of the tested cathelicidin peptides was measured by flow cytometer as described previously [16,28]. Cells were preincubated with the tested cathelicidin peptides in 0.5% bovine serum albumin/Dulbecco’s PBS for 10 min at 37 ◦ C before labeling with 0.5 ␮g/mL of either phycoerythrin-conjugated anti-ABCG2 antibody 5D3 (eBioscience, San Diego, CA) or phycoerythrinconjugated mouse IgG2b negative control antibody (eBioscience) for another 45 min at 37 ◦ C. The tested compounds were present during the antibody labeling. As positive control for maximum labeling, 5D3 binding was determined in the presence of 1 ␮M Ko143 (a specific ABCG2 inhibitor). 2.8. ABCG2 ATPase assay The vanadate-sensitive ATPase activity of ABCG2 was determined as previously described [2] with minor modifications. Crude membranes isolated from ABCG2-expressing high five insect cells was kindly provided by Dr. Suresh Ambudkar (National Cancer Institute, NIH, USA). Briefly, the tested cathelicidin peptides (0.4–50 ␮M), Ko143 (control specific ABCG2 inhibitor; 1.5–200 nM), or sulfasalazine (control ABGC2 substrate, 1.56–200 ␮M) were allowed to incubate with the crude membrane (100 ␮g/mL protein) in the presence or absence of 1.2 mM sodium orthovanadate in an ATPase assay buffer (50 mM KCl, 5 mM sodium azide, 2 mM EGTA, 10 mM MgCl2 , 1 mM DTT, pH 6.8) for 5 min at 37 ◦ C. The ATP hydrolysis reaction was then started by the addition of 5 mM ATP and it was allowed to proceed at 37 ◦ C for 40 min. After the incubation, SDS solution (0.1 mL of 5% SDS) was used to terminate the reaction. The liberation of inorganic phosphate was quantified by comparing the absorbance to a phosphate standard curve in a colorimetric assay [2]. 2.9. Reverse transcription and quantitative real-time PCR Total RNA was isolated using the Trizol regent (Invitrogen, Carlsbad, CA). RNA (1 ␮g) was reverse transcribed using the

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Table 2 Antiproliferation activity of human cathelicidin peptides. IC50 ± SD (␮M) LL37 SW620 SW620 Ad300 (P-gp verexpressing) MCF7 MCF7 FLV1000 (ABCG2 overexpressing) MCF7 VP (MRP1 overexpressing)

27.8 31.7 36.7 35.7 35.7

LL17-32 ± ± ± ± ±

3.5 3.7 4.1 3.1 2.9

26.3 24.6 36.8 34.8 36.5

± ± ± ± ±

3.9 2.0 1.9 1.6 1.2

LL13-37 24.5 27.3 33.1 31.8 31.1

± ± ± ± ±

2.4 1.7 2.6 2.3 2.0

PrimeScript First Strand cDNA Synthesis Kit (TaKaRa-Bio, Dalian, China). Quantitative real-time PCR was performed to determine the relative expression level of ABCG2, MDR1, or MRP1 transcripts using the KAPA SYBR FAST qPCR Kit (KapaBiosystems, Woburn, MA) in a LightCycler 480 Instrument I (Roche Applied Science, Indianapolis, IN). The human GAPDH RNA was amplified in parallel as the internal control. The specific primers used are as follows: ABCG2 (forward) 5 -TTTCCAAGCGTTCATTCAAAAA-3 , (reverse) 5 -TACGACTGTGACAATGATCTGAGC-3 ; MDR1 (forward) 5 -CCCATCATTGCAATAGCAGG-3 , (reverse) 5 -GTTCAAACTTCTGCTCCTGA-3 ; MRP1 (forward) 5 -CTACCTCCTGTGGCTGAATCTG3 , (reverse) 5 -CATCAGCTTGATCCGATTGTCT-3 ; and GAPDH (forward) 5 -AGCCACATCGCTCAGACAC-3 , (reverse) 5 GTTCAAACTTCTGCTCCTGA-3 . PCRs were performed at 95 ◦ C for 5 min, followed by 50 cycles of 95 ◦ C for 10 s and 60 ◦ C for 10 s. Fluorescence signal was acquired at the end of the elongation step of every PCR cycle (72 ◦ C for 10 s) to monitor the increasing amount of amplified DNA. Ct was calculated by subtracting the Ct of GAPDH from the Ct of the transcript under investigation. Ct was then calculated by subtracting the Ct of the untreated cells (or parental cells) from the Ct of the treated cells (or resistant cells). Fold change of gene expression was calculated by the equation 2−Ct . 2.10. Western blot analysis ABCG2-overexpressing MCF-7 FLV1000 cells were treated with the cathelicidin peptides for 24 h at a range of concentrations with or without pepstain A and leupeptin (10 ␮M each). The cells were harvested for Western blot analysis. Primary antibody incubation was carried out at 4 ◦ C overnight with a mouse monoclonal antiABCG2 antibody (BXP-21, Kamiya Biomedical, Seattle, WA) diluted at 1:500 in 5% non-fat milk in PBS-T. Afterwards, the membranes were incubated with HRP-conjugated donkey anti-mouse secondary antibody at room temperature for 1 h, and developed using the WesternBright Quantum chemiluminescence detection system (Advansta Corporation, Menlo Park, CA). Anti-GAPDH antibody was used as the loading control (Santa Cruz Biotech, Santa Cruz, CA). Digital chemiluminescence images were captured and analyzed by using the FluorChem Q Imaging System (Alpha Innotech Corporation, Santa Clara, CA). 3. Results 3.1. Reversal of ABCG2-mediated multidrug resistance by LL17-32 and LL13-37 Using three pairs of parental and drug-resistant cancer cell lines with defined overexpression of the major MDR transporters (P-gp, ABCG2, or MRP1), human cathelicidin LL37 and its fragments LL17-32 or LL13-37 were tested for their potential reversal effect of multidrug resistance. First, the cytotoxic effect of the cathelicidin peptides alone was examined. Their IC50 s were found to be between 24 and 37 ␮M in the cell lines tested (Table 2).

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Table 3 Potentiation of ABCG2 substrate anticancer drug (mitoxantrone) cytotoxicity by LL17-32 and LL13-37. (A)

IC50 ± SD (␮M) (fold-resistance) MCF-7

Table 4 The human cathelicidin fragments tested did not affect cytotoxicity of P-gp (paclitaxel) and MRP1 (doxorubicin) substrate anticancer drugs. (A)

MCF-7 FLV1000 (ABCG2-overexpressing)

IC50 ± SD (␮M) (fold-resistance) SW620

SW620 Ad300 (P-gp-overexpressing)

Mitoxantrone +FTC (5 ␮M)

0.0097 ± 0.0035 0.0100 ± 0.0021

(1.0) (1.0)

2.116 ± 0.261 0.023 ± 0.010*

(218) (2.4)

Paclitaxel +PSC833 (0.4 ␮M)

0.043 ± 0.009 0.051 ± 0.012

(1.0) (1.2)

40.66 ± 5.26 0.082 ± 0.026*

(946) (1.9)

+LL37 (2 ␮M) +LL37 (5 ␮M) +LL37 (10 ␮M)

0.0152 ± 0.0251 0.0087 ± 0.0022 0.0110 ± 0.0260

(1.6) (0.9) (1.1)

2.509 ± 0.283 2.311 ± 0.195 2.165 ± 0.175

(259) (238) (223)

+LL37 (2 ␮M) +LL37 (5 ␮M) +LL37 (10 ␮M)

0.062 ± 0.019 0.044 ± 0.011 0.059 ± 0.016

(1.4) (1.0) (1.4)

36.27 ± 4.86 44.26 ± 5.84 39.26 ± 2.98

(843) (1029) (913)

+LL17-32 (2 ␮M) +LL17-32 (5 ␮M) +LL17-32 (10 ␮M)

0.0109 ± 0.0312 0.0081 ± 0.0045 0.0042 ± 0.0044

(1.1) (0.8) (0.4)

2.105 ± 0.246 0.821 ± 0.092* 0.415 ± 0.061*

(217) (85) (43)

+LL17-32 (2 ␮M) +LL17-32 (5 ␮M) +LL17-32 (10 ␮M)

0.109 ± 0.025 0.095 ± 0.033 0.059 ± 0.047

(2.5) (2.2) (1.4)

45.26 ± 5.41 38.22 ± 4.29 51.48 ± 2.19

(1053) (889) (1197)

+LL13-37 (2 ␮M) +LL13-37 (5 ␮M) +LL13-37 (10 ␮M)

0.0126 ± 0.0035 0.0082 ± 0.0041 0.0039 ± 0.0042

(1.3) (0.8) (0.4)

2.065 ± 0.316 1.651 ± 0.175* 0.809 ± 0.082*

(213) (170) (83)

+LL13-37 (2 ␮M) +LL13-37 (5 ␮M) +LL13-37 (10 ␮M)

0.087 ± 0.025 0.052 ± 0.041 0.101 ± 0.052

(2.0) (1.2) (2.3)

33.28 ± 6.58 36.82 ± 5.13 41.28 ± 2.71

(774) (856) (960)

(B)

IC50 ± SD (fold-resistance)

(B)

IC50 ± SD (␮M) (fold-resistance)

HEK293 pcDNA3-transfected

HEK293 ABCG2-transfected

Mitoxantrone +FTC (5 ␮M)

0.78 ± 0.33 nM 0.81 ± 0.23 nM

(1.0) (1.0)

45.8 ± 3.9 nM 1.12 ± 0.21 nM**

(59) (1.4)

Doxorubicin +MK571 (40 ␮M)

0.21 ± 0.06 0.22 ± 0.07

(1.0) (1.0)

3.49 ± 0.98 0.18 ± 0.04**

(17) (0.9)

+LL37 (2 ␮M) +LL37 (5 ␮M) +LL37 (10 ␮M)

0.98 ± 0.18 nM 0.75 ± 0.21 nM 0.84 ± 0.28 nM

(1.3) (1.0) (1.1)

48.5 ± 4.2 nM 44.8 ± 3.4 nM 46.9 ± 2.7 nM

(62) (57) (60)

+LL37 (2 ␮M) +LL37 (5 ␮M) +LL37 (10 ␮M)

0.24 ± 0.09 0.31 ± 0.05 0.35 ± 0.07

(1.1) (1.5) (1.7)

3.27 ± 0.95 4.16 ± 0.87 3.28 ± 0.45

(16) (20) (16)

+LL17-32 (2 ␮M) +LL17-32 (5 ␮M) +LL17-32 (10 ␮M)

0.78 ± 0.22 nM 0.99 ± 0.15 nM 1.01 ± 0.29 nM

(1.0) (1.3) (1.3)

44.9 ± 3.8 nM 10.5 ± 2.1 nM** 4.2 ± 0.8 nM**

(58) (13) (5.4)

+LL17-32 (2 ␮M) +LL17-32 (5 ␮M) +LL17-32 (10 ␮M)

0.38 ± 0.15 0.28 ± 0.08 0.39 ± 0.14

(1.8) (1.3) (1.9)

4.26 ± 1.11 3.82 ± 0.85 5.08 ± 0.85

(20) (18) (24)

+LL13-37 (2 ␮M) +LL13-37 (5 ␮M) +LL13-37 (10 ␮M)

0.97 ± 0.18 nM 0.85 ± 0.24 nM 0.87 ± 0.16 nM

(1.2) (1.1) (1.1)

49.2 ± 4.1 nM 16.5 ± 2.8 nM** 8.5 ± 1.5 nM**

(63) (21) (11)

+LL13-37 (2 ␮M) +LL13-37 (5 ␮M) +LL13-37 (10 ␮M)

0.28 ± 0.05 0.45 ± 0.11 0.33 ± 0.07

(1.3) (2.1) (1.6)

3.28 ± 1.26 3.69 ± 1.12 4.02 ± 0.73

(16) (18) (19)

Cisplatin +FTC (5 ␮M) +LL37 (10 ␮M) +LL17-32 (10 ␮M) +LL13-37 (10 ␮M)

2.26 1.86 2.09 2.35 2.16

(1.0) (0.8) (0.9) (1.0) (1.0)

2.68 2.39 1.98 2.09 2.49

* **

± ± ± ± ±

0.31 ␮M 0.29 ␮M 0.18 ␮M 0.31 ␮M 0.33 ␮M

± ± ± ± ±

0.49 ␮M 0.51 ␮M 0.34 ␮M 0.24 ␮M 0.31 ␮M

MCF-7

(1.2) (1.1) (0.9) (0.9) (1.1)

p < 0.05, compared with mitoxantrone alone in resistant cells. p < 0.05, compared with mitoxantrone alone in pcDNA3-transfected cells.

Of note, the cathelicidin peptides have similar cytotoxic effect in both parental and drug-resistant cells with overexpression of the MDR transporters (Table 2). When used at or below 10 ␮M, all cathelicidin peptides alone did not affect cell proliferation significantly (Supplementary Fig. 1). Therefore, the cathelicidin peptides were tested at 2, 5, or 10 ␮M when combined with other anticancer drug to perform the MDR reversal assay. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.peptides. 2012.12.019. As illustrated in Tables 3 and 4, the resistant cell lines are remarkably resistant to the corresponding transporter substrate anticancer drugs (i.e. ABCG2-overexpressing MCF-7 FLV1000: 218-fold resistant to mitoxantrone; P-gp-overexpressing SW620 Ad300: 946-fold resistant to paclitaxel; MRP1-overexpressing MCF-7 VP: 17-fold resistant to doxorubicin). The cathelicidin peptides LL17-32 and LL13-37, when tested at 5 or 10 ␮M, were found to significantly potentiate the cytotoxicity of known ABCG2 substrate anticancer drugs including mitoxantrone (Table 3A), doxorubicin (Supplementary Table 1) and methotrexate (Supplementary Table 2) in MCF-7 FLV1000 (Table 3A). Their effects are specific because resistance reversal was not observed in the P-gpoverexpressing SW620 Ad300 or MRP-1-overexpressing MCF-7 VP cells (Table 4A and B). In all cell lines tested, no MDR reversal effect was observed for the longest cathelicidin peptide LL37. To further demonstrate the specific effect of LL17-32 and LL13-37 on

MCF-7 VP (MRP1-overexpressing)

*

p < 0.05, compared with paclitaxel alone in P-gp overexpressing resistant cells. p < 0.05, compared with doxorubicin alone in MRP1 overexpressing resistant cells. **

ABCG2-mediated MDR, a ABCG2 stably transfected HEK293 cell line was employed. A similar resistance reversal by LL17-32 and LL13-37 (at 5 and 10 ␮M) was observed (Table 3B). Moreover, the two cathelicidin peptides did not affect the cytotoxicity of cisplatin (which is not an ABCG2 substrate) in both ABCG2-transfected or pcDNA3-transfected HEK293 cells (Table 3B). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.peptides. 2012.12.019.

3.2. Inhibition of ABCG2-mediated drug efflux by the effective cathelicidin peptides Since the cytotoxicity data suggest the specific reversal of ABCG2-mediated MDR by LL17-32 and LL13-37, the possible inhibition of ABCG2 transport activity by these cathelicidin peptides was evaluated. As indicated in Fig. 1A, both LL17-32 and LL13-37 (but not LL37) were found to inhibit the efflux of PhA (a fluorescent ABCG2 probe substrate) in a concentration dependent manner. Moreover, mitoxantrone (a fluorescent ABCG2 substrate anticancer drug) was also used to monitor intracellular drug accumulation. LL17-32 and LL13-37 were found to increase mitoxantrone accumulation concentration dependently in the ABCG2-overexpressing resistant MCF-7 FLV1000 (Fig. 1B), which correlate well with the cytotoxicity data (Table 3A). A similar inhibition of PhA efflux and increased mitoxantrone accumulation by the two cathelicidin peptides were also observed in the ABCG2 stably transfected HEK293 cells (Supplementary Fig. 2A and B).

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Fig. 1. Inhibition of ABCG2-mediated PhA efflux (A) and enhancement of mitoxantrone accumulation (B) in ABCG2-overexpressing resistant MCF-7 FLV1000 cells. (A) Cells were incubated with 1 ␮M PhA alone (black), 1 ␮M PhA with the three cathelicidin peptides at the indicated concentrations (10, 20 or 40 ␮M) or 1 ␮M PhA with 10 ␮M FTC (red) at 37 ◦ C for 30 min. PhA fluorescence retention in the cells after a 1-h PhA-free efflux was measured by flow cytometry. Representative histograms from three independent experiments are shown. (B) Cells were incubated for 1 h at 37 ◦ C with the cathelicidin peptides, at various concentrations (10, 20, or 40 ␮M) or vehicle. Then, 10 ␮M mitoxantrone was added and incubation was continued for another 1 h. The cells were then collected, washed three times with ice-cold PBS, and analyzed by flow cytometry. The results are presented as fold change in fluorescence intensity relative to untreated control MCF-7 FLV1000 cells. Columns, means of triplicate determinations; bars, SD. *p < 0.01, versus the untreated resistant MCF7 FLV1000 group. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.peptides. 2012.12.019. 3.3. Inhibition kinetics of ABCG2-mediated transport by LL17-32 and LL13-37 The Lineweaver–Burk analysis was used to further characterize the inhibition mechanism of LL17-32 and LL13-37 on ABCG2-mediated mitoxantrone efflux. The rate of mitoxantrone efflux was determined in the absence or presence of different concentrations of the cathelicidin peptides in ABCG2 stably transfected HEK293 cells. As shown in Fig. 2, the regression lines of the double reciprocal plot obtained at different concentrations (5 or 10 ␮M) of the cathelicidin peptides have the same slope as the one obtained in their absence, indicating a decrease of both Km and Vmax . The data suggest uncompetitive inhibition of ABCG2-mediated mitoxantrone efflux by the two cathelicidin peptides.

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Fig. 2. Effect of LL17-32 and LL13-37 on the transport kinetics of mitoxantrone mediated by ABCG2. The quantity of mitoxantrone efflux by the ABCG2overexpressing MCF-7 FLV1000 cells was measured for 10 min at 37 ◦ C at various mitoxantrone concentrations (1, 2, 5, 10, or 20 ␮M) in the absence or presence of 5 or 10 ␮M of LL17-32 or LL13-37 by flow cytometry. Data is presented in a double reciprocal Linewaver–Burk plot. The bars represent the SD value calculated from three independent experiments.

3.4. Increased 5D3 labeling by the effective cathelicidin peptides suggest their interaction with ABCG2 5D3 is a conformation sensitive monoclonal antibody, recognizing an extracellular epitope of the human ABCG2. 5D3 binding to an extracellular loop of ABCG2 was known to be increased in certain conformations of the transporter protein, upon substrate/inhibitor binding and ATP hydrolysis (i.e. 5D3 shift) [16,28]. The 5D3 shift assay was therefore performed in MCF-7 FLV1000 cells to demonstrate the interaction of cathelicidin peptides with ABCG2. Using the potent ABCG2 inhibitor (Ko143) as the positive control (set as 100% 5D3 labeling for comparison) (Fig. 3A), other known ABCG2 inhibitors (including FTC, tariquidar and erlotinib) tested were also shown to notably increase 5D3 labeling relative to the untreated control (Fig. 3B). On the other hand, quercetin (a known ABCG2 substrate) was found to increase only slightly the 5D3 shift (∼20% that of Ko143) whereas cisplatin (a non-ABCG2 substrate) did not appreciably affect 5D3 labeling. Interestingly, only the two cathelicidin peptides (LL17-32 and LL13-37) capable of reversing ABCG2-mediated MDR were found to increase 5D3 labeling in a concentration dependent manner (Fig. 3B). The more pronounced increases in 5D3 labeling at 40 ␮M (relative to 10 ␮M) of both LL17-32 and LL13-37 (Fig. 3B) are also consistent with the more significant ABCG2 inhibition at this concentration (Fig. 1). The ineffective LL37 was devoid of this 5D3 shift effect.

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Fig. 4. Effect of cathelicidin peptides on the ATPase activity of ABCG2. The vanadate-sensitive ATPase activity of ABCG2 in crude membranes isolated from ABCG2-expressing High Five insect cells was determined at different concentrations of the cathelicidin peptides. ATP hydrolysis was monitored by measuring the amount of inorganic phosphate released using a colorimetric assay. Ko143 (specific ABCG2 inhibitor) and sulfasalazine (known ABCG2 substrate) were also tested as controls for comparison. Mean of three independent experiments is presented (bars, SD). Fig. 3. 5D3 labeling in intact ABCG2-overexpressing resistant MCF-7 FLV1000 cells, suggesting interaction between cathelicidin peptides (LL17-32 and LL13-37) and ABCG2. (A) A typical 5D3 shift exhibited by ABCG2 inhibitor (Ko143). The solid line represents 5D3 binding of the untreated (native) cells and the dotted line for the cells incubated with 1 ␮M Ko-143. The shaded histogram represents the background fluorescent signal upon labeling with a mouse IgG2b (isotype control). (B) Comparison of 5D3 shift produced by the cathelicidin peptides and other known ABCG2 inhibitors/substrates. Known ABCG2 inhibitors: Ko143 (1 ␮M), FTC (5 ␮M), tariquidar (1 ␮M) and erlotinib (10 ␮M); known ABCG2 substrate: quercetin (25 ␮M); reported non-ABCG2 substrate: cisplatin (50 ␮M). The various tested compounds were present during the 45-min antibody labeling. Fluorescence values are shown as the percentage of maximum labeling obtained in MCF-7 FLV1000 cells incubated with 1 ␮M Ko-143 (set as 100%) and labeled with 5D3. Mean and SD of the mean channel numbers from histograms obtained from three independent experiments were plotted. *p < 0.01, versus the 5D3 shift caused by Ko143.

3.5. Inhibition of ABCG2 ATPase by the MDR reversing cathelicidin peptides Drug efflux function of ABCG2 is associated with ATP hydrolysis that is modulated in the presence of its substrates or inhibitors. To understand further the mechanism of ABCG2 inhibition by the cathelicidin peptides, ABCG2-mediated ATP hydrolysis at a range of different concentrations of the peptides was measured under the condition when the activity of other major membrane ATPases was suppressed by sodium orthovanadate. Like the specific ABCG2 inhibitor (Ko143), both LL17-32 and LL13-37 were found to inhibit the ATPase activity of ABCG2 in a dose dependent manner, albeit the % inhibition was much lower than that mediated by Ko143 (Fig. 4). As a control for comparison, the known ABCG2 substrate (sulfasalazine) was shown to slightly stimulate ABCG2 ATPase activity, which is consistent with other published reports [8]. On the other hand, LL37, the cathelicidin peptide lacking in MDR reversing effect, did not appreciably affect ABCG2 ATPase activity.

3.6. Downregulation of ABCG2 expression by the MDR reversing cathelicidin peptides Reversal of ABCG2-mediated drug resistance by the cathelicidin peptides may also be associated with alteration of the transporter expression. Therefore, ABCG2 mRNA and protein levels were examined in MCF-7 FLV1000 cells after a 24-h incubation with the cathelicidin peptides. Interestingly, ABCG2 mRNA expression was not affected by the treatment with all three cathelicidin peptides (Fig. 5A) whereas ABCG2 protein level was decreased by LL17-32 and LL13-37 in a concentration dependent manner (but not LL37) (Fig. 5B, upper panel). It has been reported that wild type ABCG2 is primarily degraded through a lysosomal-mediated pathway whereas misfolded ABCG2 is degraded by an ubiquitin-mediated proteasomal machinery [31]. To determine whether cathelicidin peptides enhanced ABCG2 lysosomal degradation, the effect of leupeptin and pepstatin (lysosome inhibitors) on LL17-32 and LL13-37-mediated ABCG2 protein downregulation was examined. Leupeptin and pepstatin were found to reverse the ABCG2 protein downregulation by the cathelicidin peptides (Fig. 5B, lower panel). 4. Discussion The emergence of MDR is recognized as a major hurdle in cancer chemotherapy. Overexpression of three major ABC transporters (P-gp, ABCG2 and MRP1) is the most commonly observed mechanism contributing to MDR. Inhibitors of these transporters, P-gp in particular, have been developed and extensively studied with an aim to circumvent MDR. Currently, none of these attempts have been proven to be successful in the clinic to sensitize drug-resistant cancer cells to anticancer drugs, partly because of toxicity, lack of potency, and competitive nature of transporter inhibition leading to unwanted drug–drug interactions and dose reduction [11]. The importance of ABCG2 is increasingly recognized in cancer biology,

K.K.W. To et al. / Peptides 40 (2013) 13–21

Fig. 5. Effect of the tested cathelicidin peptides on ABCG2 mRNA and protein expression. (A) Quantitative real-time RT-PCR analysis showing that all cathelicidin peptides tested did not affect ABCG2 mRNA expression. The effect of LL37, LL17-32, and LL13-37 on ABCG2 mRNA levels were evaluated in the ABCG2-overexpressing resistant MCF-7 FLV1000 cells after a 24-h treatment (at 5, 10, or 20 ␮M). mRNA expression levels were expressed relative to that in the untreated MCF-7 FLV1000 cells, after normalization with GAPDH. (B) Western blot analysis showing that ABCG2 protein levels were decreased by LL17-32 and LL13-37 in a dose-dependent manner in MCF-7 FLV1000 cells after a 24-h treatment, which can be rescued by the concomitant treatment with the lysosome inhibitors (leupeptin and pepstatin, 10 ␮M each). LL37 had no effect on ABCG2 protein expression. ABCG2 protein expression was normalized to the GAPDH loading control and to the untreated sample.

because of its role in mediating MDR in AML [19,22], sustaining cancer stem cells [3] and constituting the blood–brain barrier [30]. To date, only a few ABCG2-specific inhibitors have been reported, most of which are very toxic (e.g. FTC and some analogs of XR9576) and are therefore not suitable for clinical application. In the present study, a few human host defense antimicrobial peptides endogenously expressed in our body (cathelicidin LL37 and its fragments LL17-32 and LL13-37) were evaluated for possible MDR circumvention, where the latter two were found to be specifically targeting ABCG2. The human cathelicidin peptides were first screened for possible MDR reversal of concomitantly administered anticancer drugs in cancer cell line models with defined overexpression of the three major MDR transporters. It is noted that only the designated MDR transporter is overexpressed in one resistant cell line, without the concomitant increased expression of other major transporters. Thus the MDR reversal activity (i.e. drug sensitization) of cathelicidin can be analyzed separately under the condition of different ABC transporter overexpression. Interestingly, among the three cathelicidin peptide fragments tested, only LL17-32 and LL13-37 were found to reverse ABCG2-mediated

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MDR in ABCG2-overexpressing cell lines due to either drug selection (MCF-7 FLV1000) or stable transfection (HEK293/ABCG2) (Table 3A and B, Supplmentary Tables 1 and 2). They appear specific because the cytotoxicity of cisplatin (a non-ABCG2 substrate anticancer drug) was not altered by the cathelicidin peptides in the ABCG2-overexpressing cells (Table 3B). Moreover, P-gp and MRP1mediated MDR were not affected by LL17-32 and LL13-37 (Table 4A and B). It is also important to note that the effective LL17-32 and LL13-37 did not affect cell proliferation at concentrations sufficient for MDR circumvention. Their anti-proliferative effect was only appreciable at higher concentrations (>20 ␮M) (Supplementary Fig. 1). While we have not elucidated the mechanism for their anti-proliferative activity, it has been demonstrated that activation of tumor-suppressing bone morphogenetic protein (BMP) signaling is involved, thus leading to induction of p21Waf1/Cip1 and G0-G1 cell cycle arrest [35]. Consistent with the drug sensitization assay, LL17-32 and LL13-37 were found to inhibit efflux of PhA (an ABCG2-specific fluorescent probe [23]) (Fig. 1A) and increased the accumulation of mitoxantrone (an ABCG2 substrate anticancer drug) in the ABCG2overexpressing MCF-7 FLV1000 cells (but not in the parental MCF-7 cells) (Fig. 1B). Further transport kinetic study revealed that LL1732 and LL13-37 are likely uncompetitive inhibitor on the ABCG2 transporter (Fig. 2), which makes them distinct from most other competitive ABCG2 inhibitors previously reported (e.g. FTC [1] and novobiocin [26]). The uncompetitive nature of transporter inhibition suggests that the cathelicidin peptides did not directly compete with the substrate (i.e. mitoxantrone used in the assay) for the same binding site during the transporter inhibition process, but rather bound to the transporter–substrate complex and change the transporter conformation to elicit the inhibitory effect. However, the data do not exclude the possibility that the cathelicidin peptides may interact with another binding site on ABCG2 not overlapping with the one for mitoxantrone. To this end, the existence of multiple substrate binding sites on ABCG2 has been proposed [4,7]. The uncompetitive mode of ABCG2 inhibition by the cathelicidin peptides may be beneficial due to the less chance of pharmacokinetic drug–drug interaction, though a more in-depth investigation in the in vivo setting is still warranted. The interaction between LL17-32 and LL13-37 was further demonstrated by the 5D3 shift assay. The assay is based on the phenomenon that the binding of a conformation-sensitive antibody 5D3 to ABCG2 could be increased in the presence of an ABCG2 substrate/inhibitor interacting with the transporter [16,28]. In general, ABCG2 inhibitors (or inhibitory concentration of interacting drugs) could lead to higher 5D3 shift effects as compared to those caused by transported substrates [28]. Similar to the non-ABCG2 substrate (cisplatin), LL37, incapable of reversing ABCG2-mediated MDR, did not appreciably affect 5D3 labeling in MCF-7 FLV1000 cells (Fig. 3B). On the other hand, the MDR reversing cathelicidin peptides LL17-32 and LL13-37 were found to increase 5D3 labeling in a concentration-dependent manner (Fig. 3B). The relatively greater 5D3 shift achieved at higher and ABCG2-inhibiting concentrations of LL17-32 and LL13-37 suggested that they may be inhibitors (but not transported substrates). To this end, all cathelicidin peptides were found to be equally cytotoxic in the parental (MCF-7) and ABCG2-overexpressing resistant (MCF-7 FLV1000) cells, thus providing another circumstantial evidence that these peptides are not likely transported by ABCG2 (Supplementary Fig. 1). Measuring ATPase activity is another widely adopted biochemical assay for investigating MDR transporter–drug interactions [24]. Although the high basal ATPase activity of ABCG2 may make the interpretation difficult, the evaluation of ABCG2 ATPase modulatory effect over a range of concentrations of the tested compounds can usually provide some information about the mode of interaction (i.e. inhibitor or transported substrate). Like the control

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ABCG2-specific inhibitor Ko143, the MDR-reversing LL17-32 and LL13-37 were found to decrease the ABCG2 ATPase activity (Fig. 4). Importantly, the ineffective LL37 did not affect appreciably the ATPase activity (Fig. 4), which may expand why it is not capable of inhibiting ABCG2-mediated drug efflux (Fig. 1) and reversing ABCG2-mediated MDR (Table 3). Another distinctive feature of LL17-32 and LL13-37 is that they were found to downregulate ABCG2 protein expression, probably via a lysosomal-dependent degradation of ABCG2 (Fig. 5). A similar mode of ABCG2 modulation has only been reported for another potent ABCG2-specific inhibitor, PZ-39 [17], which was proposed to contribute to its high potency of ABCG2 inhibition. Follow up study is currently underway to investigate whether this lysosomaldependent degradation of ABCG2 enhanced by the cathelicidin peptides is specific to the ABCG2-overexpressing resistant cancer cells. ABCG2 inhibitors are typically hydrophobic, possessing planar structures, capable of forming hydrogen bonding and in some cases positively charged [6,18]. In this study, LL17-32 and LL13-37 (but not the full length LL37) were found to reverse ABCG2mediated MDR, with a more potent reversal effect from LL17-32. Since LL17-32 is a short peptide included within the LL13-37 sequence, the former may be essential for the observed MDR reversal effect. In fact, a LL37 core peptide, composed of amino acid residues 17–29 from the full length LL37, has been identified by a NMR spectroscopy-based assay “TOCSY-trim” as the essential membrane-targeting peptide sequence for antibacterial and anticancer activity [12]. Nonetheless, the ABCG2-inhibiting LL13-37 and LL17-32 identified in this study may be further structurally optimized for MDR reversal. To this end, short mammalian-derived cationic peptides (such as defensins and cecropins) have been reported to augment the in vitro cytotoxicity of doxorubicin in MDR cancer cells [9], though the detailed mechanism has not been elucidated. Recently, another synthetic P-gp-inhibiting peptidomimetic compound has also been described [13]. Since the host defense peptides are also known to induce cell death by enhancing membrane permeability [20], it will be interesting to find out if this cell membrane permeabilization effect is related to ABCG2 inhibition. Contributions Participated in research design: To and Cho. Conducted experiments: To, Ren and Wong. Contributed new reagents or analytical tools: To and Cho. Performed data analysis: To. Wrote or contributed to the writing of the manuscript: To and Cho. Acknowledgements We would like to thank Dr. Susan Bates (National Cancer Institute, NIH) for the MDR transporters-overexpressing cancer cell lines and the various specific transporter inhibitors, and Dr. Suresh Ambudkar (National Cancer Institute, NIH) for providing the ABCG2-expressing High-five insect cell crude membrane. This research was supported partially by a Direct Grant (2041448) provided by the Faculty of Medicine (CUHK) to Kenneth To. References [1] Allen JD, van Loevezijn A, Lakhai JM, van der Valk M, van Tellingen O, Reid G, et al. Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol Cancer Res 2002;1:417–25. [2] Ambudkar SV. Drug-stimulated ATPase activity in crude membranes of human MDR1-transfected mammalian cells. Methods Enzymol 1998;292:504–14. [3] An Y, Ongkeko WM. ABCG2: the key to chemoresistance in cancer stem cells? Expert Opin Drug Metab Toxicol 2009;5:1529–42. [4] Clark R, Kerr ID, Callaghan R. Multiple drug binding sites on the R482G isoform of the ABCG2 transporter. Br J Pharmacol 2006;149:506–15.

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