inhibition of autophagy and induction of breast cancer cell death by mefloquine, an antimalarial...

Cancer Letters 326 (2012) 143–154

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Cancer Letters

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Inhibition of autophagy and induction of breast cancer cell death by mefloquine,an antimalarial agent

Natasha Sharma a, Simmy Thomas b,1, Encouse B. Golden c,2, Florence M. Hofman c, Thomas C. Chen d,Nicos A. Petasis e, Axel H. Schönthal b, Stan G. Louie f,⇑a Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, United Statesb Department of Molecular Microbiology & Immunology, University of Southern California, Los Angeles, CA, United Statesc Department of Pathology, University of Southern California, Los Angeles, CA, United Statesd Department of Neurosurgery, University of Southern California, Los Angeles, CA, United Statese Department of Chemistry, University of Southern California, Los Angeles, CA, United Statesf Department of Clinical Pharmacy and Pharmaceutical Economics and Policy, University of Southern California, Los Angeles, CA, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 May 2012Received in revised form 9 July 2012Accepted 26 July 2012

Keywords:AutophagyChloroquineMefloquineLysosomotropic agentCancer

0304-3835/$ - see front matter � 2012 Elsevier Irelanhttp://dx.doi.org/10.1016/j.canlet.2012.07.029

⇑ Corresponding author. Address: University of SouAvenue, PSC 208 B, Los Angeles, CA 90033, United Stat+1 323 442 1358.

E-mail address: [email protected] (S.G. Louie).1 Present address: Neumedicines Inc., Pasadena, CA,2 Present address: NYU Clinical Cancer Center, NY, U

Autophagy has been recognized as a potential target for cancer therapy. The antimalarial drug chloro-quine (CQ) is able to inhibit autophagy and therefore is being considered for cancer therapeutics. How-ever, the relatively low potency of CQ prompted us to investigate whether other lysosomotropic agentsmight be more effective, and thus potentially more useful. We therefore compared the cytotoxic efficacyof CQ, the quinoline analog mefloquine (MQ), and the fluoroquinolones ciprofloxacin and levofloxacin inseveral human breast cancer cell lines. We found that MQ was the most potent compound tested; itinhibited autophagy, triggered endoplasmic reticulum stress, and caused cell death in T47D and MDA-MB-231. Altogether, our study demonstrates superior potency of MQ over CQ and the ability of MQ toproduce anticancer effects in both hormone receptor positive and negative breast cancer cell lines, sug-gesting its usefulness in treating various types of cancer.

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1. Introduction

Autophagy is a genetically regulated and evolutionary con-served process, which promotes cell survival under stressful condi-tions through the breakdown of damaged and unwanted materialor organelles [1]. This lysosome-dependent degradation process,which involves the formation of a double-membrane vesicle calledautophagosome, encapsulates the cargo to be degraded by thelysosomal enzymes [2]. Recent evidence highlights the importantrole played by autophagy in tumor development, cell death andsurvival. By providing the precursors for macromolecular synthe-sis, autophagy aids in the survival and proliferation of existing tu-mor cells under unfavorable conditions, such as nutrient starvationor hypoxia, which are characteristics of the tumor microenviron-ment [3]. In addition, autophagy can also be induced in responseto chemotherapeutic treatment and has been considered as animportant mechanism contributing to drug resistance [1].

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Further studies have shown the interdependence of autophagywith the endoplasmic reticulum (ER) stress response [4]. For exam-ple, the accumulation of ER resident chaperone proteins in re-sponse to defective autophagy suggests a role for autophagy inlowering ER stress by enhancing lysosomal degradation of un-folded proteins [2]. Also, autophagy is stimulated in response toER stress and assists in mitigating ER stress by virtue of clearingthe unfolded and aggregated proteins [1,5]. Thus, the elevatedautophagic process may support cancer cell survival by providingan ‘‘escape route’’ and protection from proteotoxicity. However,inhibition of autophagy blocks this alternative route which may re-sult in greater susceptibility to cell death-inducing mechanisms,such as ER stress [5].

The antimalarial drug chloroquine (CQ) has been recognized asan inhibitor of autophagy and has been used as a pharmacologicaltool to study the role of autophagy in the laboratory [6–8]. In addi-tion, this drug has been used in clinical trials to evaluate its efficacyas an adjuvant to cancer therapeutic regimens, which revealedpromising, albeit low antitumor activity [1,9]. In an effort to iden-tify and characterize more potent inhibitors of autophagy with po-tential anticancer efficacy, we investigated several lysosomotropicagents that currently are in clinical use for other purposes, such aslevofloxacin, ciprofloxacin, and mefloquine (MQ) [10–13].

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144 N. Sharma et al. / Cancer Letters 326 (2012) 143–154

Levofloxacin and ciprofloxacin are synthetic fluoroquinolonesthat are widely used as broad-spectrum antibiotics [14,15]. MQ isan FDA-approved drug for the prophylaxis and treatment of malar-ia [16]. Previous studies have demonstrated its ability to disruptcalcium homeostasis and increase the transcription of various ERstress-associated proteins such as GRP78 (glucose regulated pro-tein 78) and CHOP (CCAAT/enhancer binding protein homologoustranscription factor) in rat neuroblastoma and human neurons atconcentration around 80 lM [16–18]. In addition, MQ is knownto inhibit MDR1/Pgp (multidrug resistance protein 1/permeabilityglycoprotein) [19,20] and because of which, when used in combi-nation with vinblastine or doxorubicin, MQ was shown to reversethe resistance of cancer cells to the latter [20,21].

For the purpose of this study, we analyzed the effect of MQ onvarious breast cancer cell lines. The current treatment for breastcancer involves surgery, chemotherapy with the hormone therapyand/or targeted therapy. The hormone therapy is used for estrogenand progesterone receptor positive breast cancers. The targetedtherapy focuses on specifically attacking the cancer cells withoutharming the normal cells. Currently monoclonal antibodies againstHER 2 (Herceptin) and the tyrosine kinase inhibitor that blocks theeffect of HER2 (Lapatinib) are used as a targeted therapy for breastcancer. But approximately 15% of globally diagnosed patients havetriple negative breast cancer (TNBC) i.e., they are estrogen, proges-terone receptor negative, and does not overexpress epidermalgrowth factor receptor (Her2/Neu) and hence are unresponsive tothe current chemotherapy [22,23]. The development of resistanceto the current therapy and the ineffectiveness to treat TNBC de-mands the discovery of novel therapy. Hence, we studied the effectof MQ on both hormone receptor positive; T47D and TNBC; MDA-MB-231, which are KRas mutated [24]. Several studies have shownthat Ras oncogene upregulates autophagy, which is required for tu-mor cell survival. The induction of autophagy helps to ensure theenergy balance in tumors with Ras mutation [25–28]. Since Rasmutated and triple negative breast cancers have poor prognosisand are difficult to treat, several studies have suggested to exploitthis autophagy addiction for the development of anticancertherapy.

Here, we present results characterizing MQ as a potent inhibitorof autophagy and inducer of cell death in both hormone receptorpositive and negative cell lines, indicating its potential usage forsuch difficult to treat breast cancers. Hence, we propose that thisagent should be evaluated further as a potentially beneficial ad-junct to cancer therapeutic regimens.

2. Materials and methods

2.1. Materials

MQ, CQ, levofloxacin, ciprofloxacin and 3-methyl adenine (3-MA) were pur-chased from Sigma–Aldrich (St. Louis, MO). MQ and ciprofloxacin were dissolvedin DMSO to produce a 100 mM and 50 mM stock solution, respectively. Levofloxacinand CQ were dissolved in water to produce 100 mM stock solutions. 3-MA was dis-solved in water by heating to produce 200 mM stock solution. Paclitaxel (Taxol�)was obtained from the pharmacy as a 10 mM stock solution. ZVAD-fmk was pur-chased from Promega (Madison, WI) as a 20 mM stock solution. All agents wereadded to the cell culture medium in a manner so that the final concentration of sol-vent (DMSO or water) was considerably less than 1%, but never exceeding 1%.

2.2. Cell lines and culturing

Human breast cancer cell lines MDA-MB-231, MDA-MB-468 and T47D were ob-tained from American Tissue Culture Collection (ATCC, Manassas, VA). MCF7 andMCF7/Dox (doxorubicin-resistant variant of MCF7) were kindly provided by Paris-senti [29,30]. All breast cancer cell lines were propagated in DMEM supplementedwith 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml of streptomycin,and incubated in a humidified atmosphere at 37 �C and 5% CO2.

2.3. MTT assay

Cellular viability was assessed using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-di-phenyl-2H-tetrazolium bromide) assays, where 5.0 � 103 cells were seeded perwell and analyzed as described in detail [31]. In individual experiments, each treat-ment condition was set up in triplicate, and each experiment was repeated from 1to 5 times independently.

2.4. Colony formation assay (CFA)

Cells were seeded into six-well plates at a density of 200 cells per well. Afterovernight incubation, the cells were exposed to the drug treatment for 48 h. There-after, the drug was removed by replacing the medium with fresh growth medium;and the cells were kept in culture undisturbed for 12–14 days, during which timethe surviving cells produced colonies. The colonies were visualized by staining for4 h with 1% methylene blue (in 100% methanol) [31] and were counted.

2.5. Western blot analysis

Total cell lysates were prepared and analyzed by Western blot as described ear-lier [32]. The antibodies against CHOP, LC3B, cleaved caspase 7, and PARP were ob-tained from Cell Signaling Technologies (Beverly, MA; Cat Nos. #2895, #2775,#9491, #9532, respectively). The antibodies against actin, GRP78, beclin1 and ubiq-uitin were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA; Cat Nos.sc-130656, sc-13968, sc-11427, sc-8017, respectively). The antibodies were used atthe dilution of 1:1,000 and 1:500, respectively. The secondary antibodies were cou-pled to horseradish peroxidase and detected by chemiluminescence using SuperSignal West Pico and Femto (Thermo Scientific, Rockford, IL). The membranes wereimaged using FujiFilm LAS-4000 [21].

2.6. siRNA transfection

The siRNA control (scrambled) and siRNA against Beclin 1 were purchased fromCell Signaling Tech., (Beverly, MA; Cat Nos. #6568 and #6246) and used at a finalconcentration of 20 nM. The siRNA against CHOP/GADD153 was purchased fromAmbion (Carlsbad, CA; Cat No. s3996) and used at a final concentration of 5 nM.All siRNA transfections were performed using LipofectAMINE 2000 (LA-2000) pur-chased from Invitrogen (Carlsbad, CA). The cells were plated at 30–50% confluencein a 6-well plate. After an overnight incubation, the medium was changed to anti-biotic-free medium. For transfection, 5 ll of LA-2000 was mixed with 245 ll ofOpti-MEM (Invitrogen) at room temperature for 5 min and then was incubated with250 ll of a mixture of siRNA duplex and Opti-MEM for 20 min at room temperature.The cells were incubated with 500 ll of this mixture for 12 h. After incubation, themedium was replaced with fresh DMEM supplemented with 10% fetal bovine serumand 1% penicillin/streptomycin. The following day, the cells were seeded and har-vested for CFA and western blot respectively.

2.7. Proteasome activity measurement

Chymotrypsin-like activity of the 20S proteasome was analyzed using previ-ously described methods [33]. Briefly, cells were washed with buffer 1 (50 mMTris/HCl pH 7.4, supplemented with 0.1 mM EDTA, 2 mM DTT, 5 mM MgCl2,2 mM ATP). Thereafter, cells were suspended in buffer 2 (50 mM Tris/HCl pH 7.4,supplemented with 0.1 mM EDTA, 20 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.03%SDS) and lysed via multiple passaging (10�) through a syringe fitted with a 25-gauge needle. After lysis, the protein concentration was determined using thebicinchoninic acid (BCA) assay (Pierce, Rockford, IL), and 20 lg of protein was takenfor further analysis. The samples were diluted to 200 ll in a 96-well plate. Then,1.6 ll of fluorogenic proteasome substrate SucLLVY-AMC (Sigma–Aldrich; stocksolution: 10 mM) for chymotrypsin-like activity was added to each well (final con-centration: 80 lM) to start the reaction. Proteolytic activity was measured by mon-itoring the release of the fluorescent 7-amido-4-methylcoumarin (AMC) at 37 �Cusing Spectra Max Gemini Spectrofluorometer at the excitation and emission wave-lengths of 360 and 460 nm, respectively. Readings were taken every 15 min for anhour.

2.8. Cell death ELISA

Cells (4 � 103 cells/well) were seeded in a 96-well plate. After complete adher-ence, the cells were treated with drug for 30 h and then analyzed for the presence ofhistone-complexed DNA fragments using cell death detection ELISA kit (RocheDiagnostics, Indianapolis, IN) according to the manufacturer’s instructions. The kitwas used to specifically quantify apoptotic cell death.

2.9. Quantitative reverse-transcriptase polymerase chain reaction

The gene expression for efflux transporters was quantified using quantitativereverse transcriptase-polymerase chain reaction (qRT-PCR). Total RNA was isolatedfrom cells using RNeasy kit (Qiagen, Valencia, CA) and RNA concentration was

N. Sharma et al. / Cancer Letters 326 (2012) 143–154 145

measured using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilming-ton, DE) at 260 and 280 nm. Following RNA quantification, cDNA was synthesizedby using SuperScript TM III First strand Synthesis SuperMix (Invitrogen, Foster City,CA). The mRNA expression for MRP 2, MRP 4 and P-gp was determined by qRT-PCRusing SYBR Green ER (Invitrogen, Foster City, CA). GAPDH was used as the controlhousekeeping primer for each sample.

2.10. Statistical analysis

Data are presented as mean ± SD. Comparisons were made between differenttreatments using two-way ANOVA, and a p-value of less than 0.05 was consideredsignificant.

3. Results

3.1. Differential cytotoxicity of lysosomotropic agents

We compared the cytotoxic potential of four established lyso-somotropic agents, namely two antimalarial drugs, CQ and MQ,and two fluoroquinolones, levofloxacin and ciprofloxacin. Humanbreast cancer cell lines with different genetic backgrounds and rep-resenting different subtypes of this disease [24] were used in ourexperiments: we chose MCF7 (estrogen receptor positive), T47D(estrogen receptor positive, p53 mutation), MDA-MB-231 (estro-gen receptor, progesterone receptor and Her2/Neu receptor nega-tive, p53 mutation, p16 deletion, K-ras mutation), and MDA-MB-468 (estrogen receptor, progesterone receptor and Her2/Neureceptor negative, p53 mutation, PTEN mutation, Rb mutation).The cells were treated with increasing concentrations of each drug,and cell viability was assessed after 48 h using the MTT assay. Asshown in Fig. 1A, levofloxacin and ciprofloxacin displayed very lit-tle cytotoxicity (IC50 > 100 lM); CQ was more potent, althoughcytotoxic effect appeared to depend on the cell type and the IC50varied considerably from 30 lM to >100 lM. In comparison, MQwas the most cytotoxic compound with an IC50 in the range of3–12 lM (Fig. 1A and B).

The above short-term (48 h) cytotoxicity assays were comple-mented by long-term colony formation assays (CFAs), where weassessed the ability of individual cells to survive 48 h of drug treat-ment and spawn a colony of descendants within the following2 weeks. As shown in Fig. 1C, MQ displayed further increased anti-cancer activity in this type of assay. Concentrations as low as 2.5and 5.0 lM reduced colony formation to below 25% in the case ofT47D and MDA-MB-231 cells, respectively, and 10 lM MQ effec-tively abolished any colony formation. CQ also displayed higherpotency in this assay (Fig. 1D), but it was substantially less potentthan MQ and required 40 lM to reduce colony formation to below25%, and 80 lM to completely inhibit colony formation in the twocell lines tested. Altogether, these data demonstrate potent cyto-toxic activity of MQ in the low micromolar range. Because MQwas substantially more potent than the other lysosomotropicagents including CQ—and thus holding potential for clinical useas an adjuvant to anticancer therapy—we decided to characterizeits cellular and molecular effects in greater detail.

3.2. Mefloquine is effective in multidrug-resistant cancer cells

The development of treatment resistance is among the mostpressing problems in cancer therapy. We therefore investigatedthe effects of MQ on MCF7/DOX cells, which represents a highlydoxorubicin-resistant variant of MCF7 and thus serves as a modelfor breast cancer having become unresponsive to therapy [29].We verified that these cells indeed are unresponsive to treatmentwith doxorubicin (Fig. 2A), and that resistance correlated with>100-fold overexpression of P-glycoprotein (Pgp), as determinedby qRT-PCR measurements of its mRNA levels (Fig. 2B) [34,35].Parental MCF7 and MCF7/DOX cells were both exposed to increas-

ing concentrations of MQ and cellular viability was assessed byMTT assay after 48 h. Remarkably, MQ was similarly effective inkilling MCF7 and MCF7/Dox cells (Fig. 2C), with IC50s of 5 lMand 11 lM, respectively, which were well within the IC50 rangeof 3–12 lM, as determined from the other breast cancer cell lines(see Fig. 1A and B). Thus, the development of Pgp-mediated multi-drug resistance, which generated the unresponsiveness of MCF7/DOX cells to doxorubicin, a commonly employed chemotherapeu-tic drug used in breast cancer therapy, did not lead to protection ofcells from MQ-induced cell death.

3.3. Mefloquine induces apoptosis

We next sought to characterize the central molecular processesunderlying MQ’s cytotoxic effects. In order to determine the effectof MQ on cell death, we investigated the activation of an effectorcaspase, i.e., effector caspase 7, and cleavage of a common targetof caspase activity, poly ADP-ribose polymerase (PARP) [36].MDA-MB-231 and T47D cells were treated with increasing concen-trations of MQ and CQ, and cleaved caspase 7 and PARP wereexamined using Western blot analysis. Because the above MTT as-says had indicated very effective cell death after 48 h of drug treat-ment, we chose earlier time points to examine these proteins forour investigation of the molecular processes leading to cell death.As shown in Fig. 3A, exposure of cells to MQ (15 and 20 lM) andCQ (60 and 80 lM) for 16 h resulted in the appearance of a proteo-lytically cleaved (i.e., activated) fragment of caspase 7, and cleav-age of PARP, as indicated by the accumulation of a smallermolecular weight fragment of this protein. The enhanced cleavageof caspase 7 and PARP were also observed when the cells weretreated with 10 lM MQ for longer times up to 36 h (Fig. 3B). To fur-ther verify ongoing apoptosis, we analyzed the amount of histone-complexed DNA fragments (mono- and oligonucleosomes) afterdrug treatment with the use of a cell death ELISA kit. As shownin Fig. 3C, MQ dose-dependently caused very prominent accumula-tion of these DNA fragments, thus indicating the induction of apop-totsis. The importance of caspase-mediated cell death was furtheranalyzed with the use of a pan-caspase inhibitor, ZVAD-fmk. Cas-pase inhibition resulted in increased cell viability, from 12% to40% in T47D cells, following treatment with 10 lM of MQ for48 h (Fig. 3D). A similar effect was observed in MDA-MB-231 cells(data not shown). However, the observed rescue of cells fromapoptosis in the presence of ZVAD-fmk was incomplete and couldnot be improved with higher dosages of this pan-caspase inhibitor,indicating that other mechanisms of cell death might participate aswell.

3.4. Mefloquine inhibits autophagy

Based on the ability of MQ to accumulate in lysosomes, we sus-pected that it might be a potential inhibitor of autophagy. Wetherefore investigate whether MQ was able to impinge on autoph-agy by analyzing two components of the autophagic process,microtubule-associated protein light chain type 3 (LC3) andsequestosome 1 (p62/A170/SQSTM1) protein. Conversion of thecytosolic, unconjugated form of LC 3 (LC3-I) to the phosphatidyl-ethanolamine-conjugated, membrane-bound form LC3-II is awidely accepted marker for the formation of autophagosomes[37]. In addition, p62 is an indicator as to whether the autophagicprocess is completed (i.e., degradation of p62) or whether autoph-agy is blocked at the stage of autophagosome formation (i.e., accu-mulation of p62) [38].

MDA-MB-231 and T47D cells were treated with MQ in a con-centration- and time-dependent fashion, and LC3-I/-II and p62 lev-els were analyzed by Western blot. As shown in Fig. 4A, treatmentwith MQ resulted in an overall increase of LC3 expression and

Fig. 1. Cytotoxic and antiproliferative effects of MQ Different breast cancer cell lines were treated with increasing concentrations of various lysosomotropic drugs and cellviability was analyzed. (A) Cells were treated with the indicated concentrations of MQ, chloroquine, ciprofloxacin and levofloxacin for 48 h, and cell viability was determinedby MTT assay. (B) Cells were treated with increasing concentrations of MQ for 48 h, and cell viability was determined by MTT assay. Shown is percent cell survival (mean ± SD,n P 3), where the value from untreated control cells was set to 100%. (C and D) Cells were treated with MQ and CQ respectively, for 48 h and cell survival was determined bycolony formation assay after an additional 12 days in culture without drug. Shown is percent colony formation (mean ± SD, n P 2), where the number of colonies derivedfrom untreated cells was set to 100%.


prominent conversion of LC3-I to LC3-II. While the drug effectswere strongest at 20 lM when cells were treated for 16 h, they alsobecame prominent at a lower concentration (10 lM) when treat-ment was prolonged. Similarly, there was obvious accumulationof p62, and these effects could be detected at concentrations aslow as 5 lM MQ. In comparison, CQ increased the conversion ofLC3-I/II and p62 accumulation only at higher concentration(Fig. 4B).

The induction of autophagy by MQ was also verified at the cel-lular level using cells stably transfected with a plasmid encodingLC3 fused to green fluorescent protein (GFP) [39]. In this case,treatment with MQ resulted in the appearance of the typical greenpunctate staining, which is indicative of autophagosome formation

(Fig. 4C). Combined with our data on p62, these results reveal thatMQ arrests autophagy at the stage of autophagosome formation,and thus that MQ is able to block autophagy similar to chloroquine,but at much lower concentrations.

To further investigate inhibitory effects on autophagy in our cellsystem, we used siRNA to knock down the expression of Beclin1, anessential autophagy protein that is necessary for nucleation ofautophagosomes [40]. The efficiency of siRNA-mediated knockdown was verified by Western blot, which indicated an approxi-mate 85% reduction in Beclin1 protein, whereas a non-specific(scrambled) control siRNA had no apparent effect (Fig. 5A). Our re-sults demonstrated no significant effect on LC3-I/II conversion fol-lowing Beclin1 knockdown (Fig. 5A), in line with this protein’s role

Fig. 2. Cytotoxic effects of MQ on multidrug-resistant cells Parental MCF7 anddoxorubicin resistant variant MCF7/DOX were used. (A) Cells were treated withincreasing concentrations of doxorubicin for 48 h. Thereafter, cell survival wasdetermined by MTT assay. (B) mRNA expression of three different transmembraneefflux pumps was determined by quantitative RT-PCR. Shown are the relativeexpression levels of multidrug resistance-associated proteins 2 and 4 (MRP2/ABCC2and MRP4/ABCC4), as well as of P-glycoprotein (Pgp/ABCB1). (C) Cells were treatedwith increasing concentrations of MQ for 48 h, and cell survival was determined byMTT assay. Shown is percent cell survival (mean ± SD, n P 3), where the value fromuntreated control cells was set to 100%.


during the early stages of autophagy, we suspected that Beclin1knockdown would potentiate MQ’s effects. Thus, we treated thesiRNA-transfected cells with MQ, and long-term cellular survivalwas analyzed by CFA. As shown in Fig. 5B, in the absence of MQtreatment, siBeclin1 alone reduced cell survival by about 20%.However, when siBeclin1-transfected cells were treated with MQ,cell survival was significantly (p < 0.05) further decreased. To-gether, these results suggest that combined inhibition of autoph-agy by different approaches, targeting early and late stages, maylead to overall greater tumor cell toxicity. In order to further con-firm that autophagy inhibition results in reduced cell viability ofMDA-MB-231 and T47D cells, we analyzed the effect of 3-methyl-adenine (3-MA), a phosphatidylinositol-3-kinase (PI3K) type-3inhibitor. PI3K type 3 forms essential complexes with Beclin1 to

initiate the nucleation of autophagosome [41]. As shown inFig. 5C, the inhibition of autophagy by 3-MA resulted in a dose-dependent reduction of cell viability, where T47D cells were moresensitive to 3-MA than MDA-MB-231 cells.

3.5. Mefloquine causes accumulation of polyubiquitinated proteins

Autophagy, as well as the ubiquitin–proteasome system (UPS),has critical roles in the elimination of proteins that are markedfor clearance via polyubiquitination, and p62 functions as a recep-tor to deliver ubiquitinated proteins to the autophagosome [38,42].Since we found increased levels of p62 protein following MQ treat-ment (Fig. 4A), indicating that autophagy was inhibited, we pre-dicted that MQ should also lead to the accumulation ofubiquitinated proteins. As shown in Fig. 6A, this appeared to bethe case, i.e., MQ treatment of cells resulted in the accumulationof ubiquitin-conjugated proteins. In order to exclude the possibilitythat the effect on ubiquitinated protein accumulation might be dueto the potential inhibition of the UPS, we studied the effect ofincreasing doses of MQ on the 20S proteasome, where we mea-sured chymotrypsin-like activity (Fig. 6B). Upto 15 lM there wasno effect on the proteasomal activity when MDA-MB-231 cellswere treated for 16 h but there was reduction in the activity atthe toxic dose of 20 lM. We also studied the effect of 10 lM ofMQ on chymotrypsin like activity over time (Fig. 6C). While therewas no inhibition of proteasome activity in MDA-MB-231 cells,there was minor reduction of chymotrypsin-like activity in T47Dcells after 24 h (Fig. 6C). To further investigate this effect, cellswere treated with 10 lM MQ for up to 36 h, and the accumulationof ubiquitinated proteins was analyzed by Western blot. As shownin Fig. 6D, there was no obvious accumulation of ubiquitinatedproteins in either cell line tested, indicating the ability of UPS toeffectively clear the proteins. Altogether, these data indicate thatMQ may only exert a weak, if any, inhibitory effect onto UPS, andotherwise are consistent with inhibition of autophagy as a majormode of action for MQ.

3.6. Mefloquine triggers ER stress

Based on the known interrelation of autophagy and endoplas-mic reticulum (ER) stress, we reasoned that inhibition of autoph-agy by MQ might result in ER stress due to the increasedaccumulation of misfolded and aggregated protein. Thus, we deter-mined the effect of MQ on the expression levels of GRP78 andCHOP, which are markers for ER stress. As shown in Fig. 7A,20 lM MQ triggered ER stress, as evident from the pronounced in-crease in the levels of both proteins. However, lower concentra-tions of MQ (i.e., 5 and 10 lM) did not seem to trigger ER stress.As well, it is noteworthy that increased levels of CHOP, which rep-resents the major executor of the pro-apoptotic function of the ERstress response, were only transient (Fig. 7A, right panel). Becauseelevated levels of CHOP are necessary for ER stress to initiate apop-tosis [43], this result indicates that MQ-induced ER stress may notparticipate in mediating MQ’s cytotoxic effects.

To investigate this aspect further, we knocked down CHOP viatransfection with specific siRNA. Since CHOP is an inducible gene,knock down was analyzed after treating the cells with 1 lM ofthapsigargin (Tg), a model inducer of ER stress and potent stimulusfor CHOP expression [5]. As shown in Fig. 7B, siRNA-mediatedknock down was strikingly effective and prevented CHOP induc-tion by Tg, as compared to control cells transfected with scrambledsiRNA. These cells were treated with increasing concentrations ofMQ and survival was analyzed by long-term CFA. If ER stress/CHOPwere involved in mediating the cytotoxic potency of MQ, we wouldexpect that ablation of CHOP expression would lead to increasedcell survival after drug treatment, as has been demonstrated under

Fig. 3. Induction of apoptosis by MQ Apoptotic cell death was analyzed in MDA-MB-231 and T47D cells after drug treatment. The expression of markers of apoptosis wasexamined by Western blot following treatment of cells (A) with increasing concentrations of MQ and CQ for 16 h, and (B) with 10 lM MQ for different time points. Antibodieswere specific for cleaved (i.e., activated) caspase 7 and for PARP (a target of the caspase cascade). To verify equal loading, the blots were probed with an antibody to actin. (C)Cells were treated with increasing concentrations of MQ for 30 h and apoptosis was determined by cell death ELISA. Shown is the relative increase in apoptotic cell deathfollowing drug treatment. (D) T47D cells were pretreated for 1 h with pan-caspase inhibitor ZVAD-fmk, followed by MQ addition, and cell death was analyzed after 48 h byMTT assay. Shown is percent cell survival (mean ± SD, n P 2), where the value from untreated control cells was set to 100%.


several other conditions [30,44]. However, as shown in Fig. 7C, theknock down of CHOP did not affect the outcome of MQ treatment,i.e., MQ exert similar cytotoxic potency whether or not CHOP wasknocked down. Thus, these results exclude ER stress from playing acentral role during MQ-induced cell death.

3.7. Mefloquine chemosensitizes towards paclitaxel

There are indications that inhibition of autophagy may increasethe chemosensitivity of certain tumor cells [1]. We therefore inves-tigated whether MQ would be able to achieve this outcome in com-bination with paclitaxel (Taxol�), which was selected as arepresentative agent from among several chemotherapeutic drugs

currently in clinical use for the treatment of malignant breast can-cer. Moreover, recent studies have shown its ability to induceautophagy as a protective mechanism [45]. Thus, we combined25 nM of paclitaxel with increasing concentration of MQ and ana-lyzed tumor cell viability by MTT assay. As shown in Fig. 8, theaddition of non-cytotoxic dose (5 lM) of MQ to paclitaxel resultedin further reduced survival of triple-negative MDA-MB-231 andT47D cells, thus indicating chemosensitizing properties of MQ.

4. Discussion

One of the major challenges facing cancer therapy is tumorresistance towards cytotoxic chemotherapy. Among the various

Fig. 4. Inhibition of autophagy by MQ. Various markers of autophagy were analyzed in drug-treated MDA-MB-231 and T47D cells. (A) Cells were exposed to increasingconcentrations of MQ for 16 h (top panel) or to 10 lM MQ for different times (bottom panel) or (B) with increasing concentrations of CQ for 16 h, and cell lysates wereanalyzed by Western blot for the conversion of LC3-I to LC3-II (a marker for the formation of autophagosomes) and for the expression level of p62 (accumulation of whichindicates inhibition of autophagy). Actin was used as a loading control. (C) GFP-LC3-transfected cells were treated with 8 lM MQ or vehicle only (untreated) for 4 h andvisually inspected via fluorescent microscopy. Representative photographs are shown, presenting the characteristic punctate staining (examples marked by arrows), which isindicative of autophagosome formation, in MQ-treated cells only.


cellular processes contributing to this effect is autophagy, whichgenerally is induced in response to metabolic and therapeuticstress, and has been associated with disease relapse [6,46]. Thishas prompted the evaluation of specific autophagy inhibitors, suchas the antimalarial medications chloroquine (CQ) and hydroxy-chloroquine (HCQ), as potential anticancer therapeutic agents[1,9,47]. Several studies using CQ and HCQ as an adjunct to existingchemotherapeutic agents are under preclinical and clinical evalua-tion [9,21,47]. However, presumably due to their low potency, theuse of these compounds has resulted in poor anticancer outcomes.In addition, their low efficacy necessitates higher dosages, which inturn pose the problem of increasingly severe side effects [9]. Takentogether, these limitations highlight the pressing need for the dis-covery of novel autophagy inhibitors exhibiting superior potency.

Lysosomotropic agents, such as CQ and HCQ, inhibit autophagyby virtue of being weak bases that accumulate in lysosomes in

protonated form and increase pH, thereby blunting the activity oflysosomal enzymes and preventing complete degradation of auto-phagolysosomal contents. Based on this mechanism of action, wereasoned other lysosomotropic agents can potentially be moreeffective and possibly better suited for cancer therapeutic pur-poses. And indeed, among the several agents we evaluated, we dis-covered that MQ displayed substantially greater activity than thepreviously established and widely used autophagy inhibitor CQ.In comparison to CQ, which generally requires concentrations of30 to above 100 lM to be effective (Fig. 1) [47], our data show thatMQ displays anticancer activity in breast cancer cell lines at con-centrations as low as 2.5 lM (Fig. 1C), and more generally in therange of 2.5 to 15 lM. Importantly, MQ displayed this pronouncedcytotoxic activity in estrogen receptor positive, triple-negative, anddrug-resistant breast cancer cells, indicating that this agent shouldbe evaluated further for the inclusion in therapies aimed at these

Fig. 5. Altered chemosensitivity to MQ after Beclin1 knockdown. The effect of Beclin1 knockdown on cytotoxic and antiproliferative effects of MQ were analyzed by colonyformation assay. MDA-MB-231 cells were transfected with siRNA directed at Beclin1 (siBeclin) or scrambled siRNA (siControl) or remained untransfected (untreated). (A)After transfection, cell lysates were analyzed by Western blot to confirm downregulation of Beclin1 expression and to study the effect of siBeclin1 on LC3 I/II conversion(marker for autophagosome). Actin served as the loading control. (B) In parallel, transfected cells were treated with increasing concentrations of MQ for 48 h. Thereafter, thedrug was removed and the cells were incubated in the absence of drug for an additional 12 days, after which time the number of colonies formed were counted. Shown is thepercent number of colonies (mean ± SD, n P 2), where the number of colonies derived from untreated cells was set to 100%. The result presents increased sensitivity to MQfollowing Beclin1 knockdown. (C) MDA-MB-231 and T47D cells were treated with increasing concentration of 3-MA for 48 h and cell death was analyzed by MTT assay.Shown is percent cell survival (mean ± SD, n P 2), where the value from untreated control cells was set to 100%.


types of difficult-to-treat cancer. It is interesting to note that MQand CQ were considerably more potent in colony formation assays(CFAs) as compared to MTT assays (Fig. 1). In both cases drug expo-sure was the same (48 h), although MTT assays determined cellularviability at the end of drug treatment (i.e., after 48 h), whereasCFAs measured how many of the drug-exposed cells were able tospawn a colony within 2 weeks after the termination of drug treat-ment. Our results further indicate the higher potency of MQ ascompared to CQ. The increased potency of MQ over time couldbe a consequence of its long intracellular half-life and its abilityto accumulate preferentially in lysosomes [11,48]. As CFAs are rea-sonably good predictors of in vivo activity of anticancer agents, ourresults suggest the exciting possibility that MQ might become amore effective addition to cancer therapeutic regimens in thefuture.

To further study the type of cell death induced by MQ, we ana-lyzed its effect on various markers of apoptosis. As shown inFig. 3A, B and C, MQ induced apoptotic cell death. However, thecombination of MQ with a pan-caspase inhibitor (ZVAD-fmk) onlypartially protected cells against MQ (Fig. 3D), indicating the exis-tence of caspase-independent cell death, as has been previouslyobserved with CQ [49].

Based on the known lysosomotropic nature of MQ, we expectedthat this agent would function as an inhibitor of autophagy. Thisassumption was confirmed by our verification of several estab-lished autophagy markers in response to MQ treatment, such asthe appearance of prominent punctate staining of LC3-GFP-trans-fected cells, induction of LC3 with conversion to its phosphatidyl-ethanolamine-conjugated form (LC3-II), and increased levels of

p62/SQSTM1 (Fig. 4). The latter has been shown to represent read-out for blockage of the autophagic process in particular [38,50].Our observation of increased levels of p62 in response to MQ treat-ment is particularly noteworthy, because the opposite, i.e., thereduction of p62 levels, is being viewed as a tumor cell-protectivemechanism that supports cell survival under hypoxia; as well, ithas been related to the development of resistance to chemothera-peutic agents, such as cisplatin and 5-fluorouracil [37,51,52].

Beclin 1 protein assists in the nucleation of autophagosomes,and thus its knockdown results in inhibition of autophagy [53].Although siRNA-mediated ablation of Beclin 1 reduced overall sur-vival of the MDA-MB-231 cell line (Fig. 5), it was less potent thanexposure of cells to moderate concentrations (5–10 lM) of MQ. Itis unclear why inhibition of autophagy by siBeclin was less cyto-toxic than inhibition of autophagy by MQ, although several possi-bilities exist to explain this discrepancy. For example, knockdownof Beclin 1 expression may have been incomplete, as indicated bythe residual signal in our Western blot shown in Fig. 5. Or else, itmay point to the involvement of Beclin 1-independent autophagy[54,55], as supported by our observation that knockdown of Be-clin1 had no effect on LC3-I/II conversion or formation of auto-phagosomes (Fig. 5A). In addition, inhibition of PI3K type 3 by5 mM 3-MA, a concentration that suffices for autophagy inhibition,diminished MDA-MB-231 cell viability by approximately 20%,which correlated with the effects of siBeclin in these cells. Thus,our results suggests the possibility that even complete ablationof nucleation of autophagosomes (by Beclin1 knockdown andPI3K type 3 inhibition) would only partially block the entireautophagic process, whereas inhibition by MQ would be more

Fig. 6. Accumulation of ubiquitinated proteins and proteasome activity. MDA-MB-231 and T47D cells were treated with the indicated concentrations of MQ for 16 h, or withMQ 10 lM for the indicated times, and the effect of drug treatment on ubiquitinated proteins and proteasome activity was determined. (A) Cell lysates were analyzed byWestern blot with an antibody specific to ubiquitin (i.e., recognizing ubiquitinated proteins). Equal loading was verified by using an antibody against actin. (B and C) Thechymotrypsin-like proteasome activity of the 20S proteasome was determined by assessing the release of a fluorescent substrate 60 min after the addition of fluorescentprobe (mean ± SD, n P 2). (D) Cell lysates were analyzed as in (A).


comprehensive. Alternatively, MQ may exert additional, non-autophagy related effects that may result in cytotoxic outcomes.However, because the combination of siBeclin with MQ treatmentresulted in overall only limited further reductions in colony sur-vival, we surmise that other, non-autophagic cytotoxic effects ofMQ, if indeed present, probably do not play a major role in theobserved anticancer effects.

Previous studies have shown induction of ER stress markerssuch as CHOP and GRP 78 by MQ [16,17]. Combined with theknown interdependence of ER stress and autophagy [56,57], thisled us to postulate a key role for ER stress in MQ-induced cytotox-icity. However, this does not appear to be the case for the followingreasons. Although we did confirm the induction of ER stress mark-ers GRP78 and CHOP, this effect required very high concentrationsof MQ (20 lM). Moreover, the elevation in CHOP levels was only

transient (Fig. 7A). This latter observation is noteworthy, becauseit had been demonstrated by others that high CHOP levels haveto be maintained in order for ER stress to become cytotoxic [43].Thus, declining levels of CHOP expression, as observed in ourexperimental conditions, are not consistent with ER stress-inducedcell death. As well, several reports [30] have demonstrated previ-ously that CHOP knockdown effectively protects cells from under-going death in those cases where ER stress represents a keymediator of drug toxicity. This is not the case in our experimentalsystem, i.e., despite effective CHOP knowdown there was nodetectable protection of cells from MQ toxicity. Thus, combinedour results exclude ER stress as a major component of MQ-inducedcytotoxicity.

MQ displayed striking ability to kill MCF/DOX cells (Fig. 2),which is an MCF7 variant that has been selected for high resistance

Fig. 7. Induction of ER stress by MQ. The expression of ER stress markers in MDA-MB-231 cells in response to MQ treatment was analyzed by Western blot. (A) Cells weretreated with increasing concentrations of MQ for 16 h (left panel) or with 20 lM MQ for various time points (right panel). Cell lysates were analyzed by Western blot usingspecific antibodies to GRP78 (a pro-survival ER stress marker) and CHOP (a pro-apoptotic ER stress marker). To verify equal loading, the blots were also probed for actin. (B)MDA-MB-231 cells were transfected with 5 nM siRNA directed against CHOP (siCHOP) or with scrambled siRNA (siControl). Knockdown of CHOP expression was confirmedby Western blot after treatment of cells with 1 lM thapsigargin (to trigger elevated CHOP levels). (C) In parallel, transfected cells were treated with MQ for 48 h and cellsurvival was analyzed by colony formation assay after keeping cells in culture for an additional 12 days without drug. Shown is the percent number of colonies (mean ± SD,n P 2), where the number of colonies in the control untreated cells was set to 100%.

Fig. 8. Increased chemosensitivity by MQ treatment. MDA-MB-231 and T47D cells were simultaneously treated with 25 nM paclitaxel and increasing concentrations of MQfor 48 h and cell survival was assessed by MTT assay. Shown is percent cell survival (mean ± SD, n P 2), where survival of untreated cells was set to 100%.


to doxorubicin, paclitaxel, and other chemotherapeutics commonlyused during breast cancer therapeutic regimens [29,30]. Multi-drug-resistance of these cells is due to the overexpression of drugefflux membrane transporters, such as P-glycoprotein (Pgp) (Fig. 2and [34]), which is noteworthy because MQ is able to inhibit suchmembrane pumps [20,21]. The presence of this feature indicatesthe possibility that MQ may exert anticancer activity via a highlydesirable multi-thronged approach that includes drug efflux pumpinhibition, stimulation of p62 expression, and autophagy blockade.Thus, MQ’s ability to lead to tumor cell chemosensitization, as alsoshown in combination with paclitaxel (Fig. 8), may be based onadditional features of this agent, in addition to its ability to blockautophagy.

In summary, our study introduces MQ as a pharmacologicalinhibitor of autophagy that appears substantially more potent than

CQ. Its efficacy in highly drug-resistant cells, in combination withits chemosensitizing potential, makes this agent a promising can-didate for further evaluation as part of cancer therapeutic regi-mens. As MQ already is in clinical use for other indications,repositioning MQ as an anticancer agent, in particular as an adju-vant for difficult-to-treat subtypes of breast cancer, should bepursued.

Acknowledgements

We are grateful to Dr. Amadeo M. Parissenti (Northeastern On-tario Regional Cancer Centre, Sudbury, Ontario, Canada) for provid-ing MCF7 and MCF7/DOX cell lines. We thank Hee-Yeon Cho, DolphEllefson and Jared Russell for helpful discussion.


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