cytotoxic and antitumor activity of lactaptin in...

Research ArticleCytotoxic and Antitumor Activity of Lactaptin in Combinationwith Autophagy Inducers and Inhibitors

Anastasia V Bagamanshina 1 Olga S Troitskaya 1 Anna A Nushtaeva 1

Anastasia Yu Yunusova1 Marina O Starykovych2 Elena V Kuligina 1

Yuri Ya Kit2 Max Richter3 FabianWohlfromm3 Thilo Kaumlhne3 Inna N Lavrik3

Vladimir A Richter1 and Olga A Koval 14

1 Institute of Chemical Biology and Fundamental Medicine Siberian Branch Russian Academy of Sciences Lavrentiev Ave 8630090 Novosibirsk Russia

2Institute of Cell Biology Dragomanova Str 16 79000 Lrsquoviv Ukraine3Otto von Guericke University Universitatspl 2 39106 Magdeburg Germany4Novosibirsk State University Pirogova Str 1 630090 Novosibirsk Russia

Correspondence should be addressed to Olga A Koval o kovalngsru

Received 3 April 2019 Revised 16 May 2019 Accepted 23 May 2019 Published 17 June 2019

Academic Editor Yujiang Fang

Copyright copy 2019 Anastasia V Bagamanshina et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellularmetabolism Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processesfor cell vitality Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics Earlier we havedemonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancercells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3) In this study we investigated whetherautophagy inhibitorsmdashchloroquine (CQ) Ku55933 (Ku) and 3-methyladenine (3MA)mdashor inducermdashrapamycin (Rap)mdashcanenhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model Western Blot analysisrevealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that thesedata were confirmed by the transmission electron microscopy of autophagosomeautophagolysosome formation RL2 stimulatesreactive oxygen species (ROS) production autophagosomes accumulation upregulation of ATG5 with processing of LC3I to LC3IIand downregulation of p62sequestosome 1 (p62) We have shown that autophagy modulators CQ Ku and Rap synergisticallyincreased cytotoxicity of RL2 and RL2 with CQ induced autophagic cell death In addition CQ Ku and Rap in combination withRL2 decreased activity of lysosomal protease Cathepsin D More importantly combining RL2 with CQ we improved antitumoreffect inmice Detected synergistic cytotoxic effects of both types of autophagy regulators inhibitors and inducerswith RL2 againstcancer cells allow us to believe that these combinations can be a basis for the new anticancer approach Finally we suppose that CQand Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death

1 Introduction

Autophagy is a cellular process which is essential for allmulticellular organismsWhen autophagy is initiated cellularorganelles and proteins are engulfed by autophagosomesdigested in autophagolysosomes and recycled to restorehomeostasis and cellular metabolism There is no doubt

that targeting autophagy is a very promising strategy forthe treatment of various diseases including cancer [1ndash7] Incancer biology autophagy usually promotes tumor progres-sion as being one of the fundamental mechanisms whichallows tumors to survive in nutrient-deprived or hypoxicconditions [8 9]Moreover anticancer drugs can also activateautophagy in cancer cells which results in the decrease of

HindawiBioMed Research InternationalVolume 2019 Article ID 4087160 16 pageshttpsdoiorg10115520194087160

2 BioMed Research International

phagophore Isolation membranewith cargo

Autophagosome

Lysosome

Degradation

Autophagolysosome

3-Methyladenine

PI3K-III complex ULK complex

mTorc1

Rapamycin

Ku55933

Chloroquine

Figure 1 Key points of autophagy modulation by various drugs

efficiency of chemotherapeutics [7 10 11] For convenientanticancer chemotherapeutics such as doxorubicin cisplatinand methotrexate [8] activation of prosurvival autophagyhas already been demonstrated But in some cases autophagyaccelerates cell death and can stimulate tumor suppression[12] Therefore correct regulation of autophagy is an impor-tant antineoplastic strategy [9]

Earlier we showed that recombinant analog of lactaptinRL2 suppresses tumor growth and metastasis in mice withno signs of toxic effects [13] Besides apoptosis RL2 inducedprocessing of microtubule-associated protein 1 light chain3 (LC3) which is referred to as a marker of autophagyWhen RL2 was used in vitro in MDA-MB-231 cells withautophagy inhibitor chloroquine this combination wasmorecytotoxic than RL2 or CQ alone [14]Therefore we supposedthat treatment of lactaptin analog with various autophagyinducers or inhibitors has the potential for improving ofcytotoxic and anticancer effect of RL2

In this study we used a set of various autophagy in-hibitors and inducers which switch over diverse steps inautophagy pathway (see Figure 1) 3-Methyladenine (3MA)is a widely used inhibitor of autophagy which suppressesphosphoinositide-3-kinases (PI3Ks) activity [15 16] leadingto suppression of autophagosome formation [17] Chloro-quine prevents fusion of autophagosomes with lysosomes[16 18] while Ku55933 (Ku) an ATM kinase inhibitor [19]acts like 3MA by blocking class III PI3K [20] Spermidineinduces macroautophagy by inhibiting the acetyltransferaseEP300 [21] Rapamycin activates autophagy inhibiting mTORsignaling pathway [22]

Here we tried to reveal which autophagy inhibitor orinducer enhances cytotoxic activity of lactaptin analog RL2in vitro and in vivo with the highest degree and to dis-cover activated death pathways by these combinations ofcompounds

2 Experimental Section

21 Materials

211 Cell Lines and Mice MCF-7 human breast adenocarci-noma cells andMDA-MB-231 human breast adenocarcinomacells were obtained from the Russian cell culture collection(RussianBranch of the ETCS St Petersburg Russia)TheRLSmurine lymphosarcoma cells were generously provided byDr V I Kaledin (Institute of Cytology and Genetics SB RASNovosibirsk Russia) Cells were maintained as previouslypublished [23]

Female CBA mice (8-9 weeks old) were obtained fromSPF vivarium of the Institute of Cytology and Genetics SBRAS Novosibirsk Russia

212 Antibodies and Chemicals The following antibodiesand chemicals were obtained from commercial sourcesanti-LC3B and anti-ATG5 (Abcam USA) anti-p62SQSTM(Sigma-Aldrich USA) polyclonal rabbit-anti-mouse andmouse-anti-rabbit HRP-conjugated secondary antibodies(Biosan Russia) trypsin (Gibco USA) inhibitor of trypsinfrom soybean (Paneco USA) FITC Annexin V Apoptosisdetection kit (BD Pharmingen USA) and Ku55933 (Ku)(Tocris Bioscience UK) Chloroquine 3-methyladeninerapamycin and spermidine were purchased from Sigma-Aldrich USA

22 Methods

221 MTT Assay Cells were seeded in 96-well plates at 2 times103 cells per well in a volume of 100 120583L and cultivated understandard conditions in RPMI medium with 10 FBS 03mgmL L-glutamine 100 120583gmL streptomycin and 100UmLpenicillin After 24 hours the cells were added to 100 120583L of

BioMed Research International 3

test compounds dissolved in RPMI medium After 48 hoursthe medium was replaced with 200 120583L of serum-free RPMImediumwithMTT (025mgmL) and cell were incubated for4 hours at 37∘C Finally the mediumwas removed fromwellsand formazan crystals were dissolved in DMSO The opticaldensity of the solution was measured at 120582 = 570 nm using amultichannel spectrophotometer The effect of combinationtreatment was calculated with CompuSyn soft version 10The MTT assay data of monotreatment and combinationtreatment were used for the calculation of CI by the softwareSynergy (CIlt1) or additivity (CI=1) effects were revealedaccording to the Chou-Talalay range

222 Reactive Oxygen Species (ROS) Measurement ForROS measurement two fluorescent probes were used di-hydroethidium (DHE O

2

--specific probe) and 2101584071015840-dichlo-rofluorescin diacetate (DCFDA detecting mainly H

2O2)

ROS content was measured in control (untreated) or RL2-treated (for 6 12 and 24 h) cells after they were stainedby fluorescent probes (10 120583M) for 30 min at 37∘C usingFACScan flow cytometer (BD Biosciences Mountain ViewCA) All experiments were repeated three times with threeparallel repeats TheAnalysis of Variance (ANOVA) was usedas a statistical test for the comparison of the experimentalgroups Two-way ANOVAwith Bonferroni posttests in orderto compare replicated means by rows using GraphPad Prismv60 software was applied

For ROS visualization MDA-MD-231 cells were grownon glass slides treated with RL2 or PBS (control) and finallyincubated with 10 120583M DCFDA for 30 min at 37∘C Afterincubation cells were immediately analyzed by fluorescencemicroscope Zeiss Axio Imager A1 (Carl Zeiss Germany) atwavelength ExEm= 495529 nm

223 Western Blot Analysis Cells were lysed in lysis buffer(20 mM Tris 150 mM NaCl 1 mM EDTA 1 NP40 pH 75)Aliquots of cell lysates were analyzed by 15 SDS-PAGE ina buffer for 50 mM Tris-HCl 100 mM 120573-mercaptoethanol1 SDS 10 glycerol bromophenol blue (75 ngmL) andxylene cyanol (75 ngmL pH68) and transferred to aTrance-blot nitrocellulose membrane (Bio-Rad Laboratories USA)by a wet blotting procedure (150V 1 h 10∘C) in NuPAGEbuffer (Novex Life Technologies USA) The nitrocellulosemembrane was processed by buffer iBind Solution Kit (NovexLife Technologies USA) then the membrane was incubatedin iBind Western apparatus (Life Technologies USA) withprimary antibodies and HRP-conjugated secondary anti-bodies for 18h at +4∘C Visualization of bound antibodieswas achieved using a chemiluminescent substrate for HRP(Invitrogen USA) on a C-Digit device (LI-COR USA) witha software package Image Studio Ver 40 C-DiGit

224 Apoptosis Detection MDA-MB-231 and MCF-7 cellswere seeded in 24-well plates and cultivated under standardconditions When cells grew up to 80monolayer they weretreated with tested compounds for various time intervalsBefore trypsinization detached cells were collected Adhesivecells were rinsed with PBS and detached from the plate with

trypsin Trypsin inhibitor from soybean was used to inhibittrypsin-initiated proteolysis Detached and trypsinized cellswere combined and then they were centrifuged for 5 min(08times103 rpm)To detect cell death FITCAnnexin VApopto-sis detection kit and FACSCanto II (BD Biosciences San JoseCA) flow cytometer were used

225 Transmission Electron Microscopy MDA-MB-231 cellswere seeded in 6-well plates at 105 cells per well and culturedunder standard conditions After 24 hours drugs were addedto the cells After 35 hours of incubation at + 37∘C 100120583L of FBS was added to the cells and the incubation wascontinued After 4 8 and 24 hours cells were detachedwith 05 trypsin diluted by L-15 medium (1 mL) with 10FBS and centrifuged for 5 min at 3000 rpm Precipitatedcells were fixed with 4 paraformaldehyde Cell pellets wereextra fixed with 1 osmium tetroxide solution for 2 h andthen they were sequentially dehydrated in ethanol solutionsrising concentration from 30 to 96 The precipitates werewashed with absolute acetone solution 5 times for 15 minand then they were heated in a mixture of resin (Araldite502SPI-PON 812 DDSA in a ratio of 11537) and acetone(11) for 24 h Air-dried cell pellets were placed in the conicalmolds and poured with resin mixed with catalyst (DMP-30)Obtained blockswere cut on ultramicrotome EMUC7 (LeicaGermany) the sections were contrasted with uranyl acetateand plumbum citrate solutions Contrast ultrathin sectionswere examined in a transmission electron microscope JEM1400 (JEOL Japan) equipped with a Veleta digital camera(Olympus Soft Imaging Solution Germany)

226 External ATP Assay Cells were seeded in 24-wellsplates in IMDMmediumwithout a phenol indicator with 10FBS 2 mM L-glutamine and antimycotic antibiotic solutionThe next day cells were treated with drugs and after theend of incubation the medium was collected ExtracellularATPwasmeasured in the conditionedmedia using ENLITENATP Assay System Bioluminescence Detection kit (PromegaUSA) based on luciferin-luciferase conversion according tothe following reaction

ATP+D-Luciferin+O2997888rarrOxyluciferin+AMP+PPi+

CO2+LightFor the measurement of the light intensity (560 nm)

a luminometer CLARIOstar (BMG Labtech Germany) wasused

227 Cathepsin D Activity Cells (2x105) were plated in six-well plates treated with investigated compounds for 24 h and48 h Cathepsin D activity in the cell lysates was assayed usingaCathepsin D activity assay kit (Abcam England ab65302)The kit is a fluorescent-based assay that utilizes the pre-ferred Cathepsin D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2) labeled with methyl coumaryl amide (MCA) Asample preparation was made according to the manufac-turerrsquos instructions The resulting mixtures were analyzedusing a fluorimeter (Cary Eclipse Varian Australia) atExEm=328400 nm using 5 nm excitation and emissionslits


228 Protein Immunoprecipitation and Mass SpectrometryAnalysis The analysis of RL2-binding partners was per-formed by 120581-Casein immunoprecipitation The 120581-Caseinantibody (Anti-CSN3 antibody ab111406 Abcam) was cova-lently coupled to sepharose beads using Pierce Co-Immu-noprecipitation Kit according to manufacturerrsquos instructions(Thermo Fisher Scientific Inc USA) and incubated withtotal cell lysates of RL2-treated MDA-MB-231 cells Controlsamples were incubated with total cell lysates of RL2-treatedpancreatic cancer cell line SUIT-020 The precipitates wereanalyzed by nano-LC-tandem mass spectrometry and West-ern Blot

Sample preparation for mass spectrometry was per-formed via on-bead digestion [24] In brief beads wererehydrated in 50 mM NH

4HCO3 pH 80 and subsequently

incubated with 1 mM DTT at 56∘C for 45 min Afterwardsreduced cysteine residues were szlig-methylthiolated via theaddition of 5 mM methyl methanethiosulfonate at roomtemperature for 30 min Proteins were digested by adding05 120583g trypsin (Trypsin Gold Promega) and incubated at37∘C overnight The generated tryptic peptides were elutedfrom the beads with two washing steps using 50 120583l of 25mM NH

4HCO3for each wash The washes corresponding

to one sample were pooled together and dried in a vac-uum centrifuge The peptides were dissolved in 5 120583l 01trifluoroacetic acid (TFA) and purified on ZIP-TIP C18-nanocolumns (Millipore Billerica USA) The peptides wereeluted in 7 120583l of 70 (vv) ACN and subsequently dried in avacuum centrifuge LC-MSMS was performed on a hybriddual pressure linear ion TrapOrbitrap mass spectrometer(LTQ Orbitrap Velos Pro Thermo Scientific San Jose CA)equipped with an EASY-nLCUltraHPLC (Thermo ScientificSan Jose CA) The peptide samples were dissolved in 10 120583lof 2 acetonitrile (ACN)01 trifluoroacetic acid (TFA) andfractionated on a 75 120583m ID 25 cm PepMap C18-columnpacked with 2 120583m resin (Dionex Germany) Separationwas achieved by applying a gradient from 2 ACN to 35ACN in 01 formic acid (FA) over a 150 min gradient ata flow rate of 300 nlmin The LTQ Orbitrap Velos Pro MSexclusively used CID-fragmentation when acquiring MSMSspectra consisting of an Orbitrap full MS scan followed byup to 15 LTQ MSMS experiments (TOP15) on the mostabundant ions detected in the full MS scan The essential MSsettings were as follows full MS (FTMS resolution 60000mz range 400ndash2000)MSMS (Linear Trap minimum signalthreshold 500 isolation width 2 Da dynamic exclusiontime setting 30 s singly charged ions were excluded fromselection) Normalized collision energy was set to 35 andthe activation time was set to 10 ms Raw data processingand protein identification of the high resolution Orbitrapdata sets were performed with de novo sequencing algorithmsof PEAKS Studio 80 (Bioinformatics Solutions) The falsediscovery rate was set to lt1

229 Mouse Experiment All procedures involving animalswere performed in compliance with the protocols and rec-ommendations for proper use and care of laboratory animals(ECC Directive 86609EEC) The protocol was approved

by the Committee on the Ethics of Animal Experiments ofthe Administration of the Siberian Branch of the RussianAcademy of Science ( 40 from April 4 2018) Female 8-9-week-old CBAmice were intramuscularly inoculated in rightleg with RLS cells Tumor volumes were monitored every 2days On the 8th day after the tumor transplantation RL2(12 mgkg) was injected intravenously via the tail vein andsuch injections were repeated every 2-3 days with a total of4 injections CQ (50 mgkg) was injected intraperitoneallydaily with a total of 10 injections When tumors reached anaverage volume of 50mm3 animals were divided into groupsControl mice received iv saline (02 mL 4 injections each 2-3 days) After the first injection tumor volume and mouseweight were measured twice a week

2210 Statistical Analysis The Mann-Whitney U-test wasused to define statistically significant differences for in vitroexperiments Studentrsquos t-test was used in in vivo experimentsfor comparing between two groups Results are reported asthemeanplusmn standard deviation (SD) P value less than or equalto 005 was considered as significant

3 Results

31 RL2 Activates Concentration-Dependent ROS Productionin Treated Cells Earlier we demonstrated that lactaptinanalog RL2 induces apoptotic markers such as caspase-3-7 activation and mitochondrial membrane depolarization incancer cells [14 25]

Besides classical apoptotic pathways excessive reactiveoxygen species (ROS) can lead to cell damage and apoptosisUsing the ROS-sensitive fluorescent probes DCFDA (detect-ing mainly H

2O2) and DHE (O

2-specific) to monitor cellular

oxidative stress we found that RL2 increased the productionof ROS in MDA-MB-231 and MCF-7 cells in concentration-dependent manner but not in a time-dependent manner (seeFigures 2(a)ndash2(c))The increase of ROS production was well-detected after 3 h and it stayed high with no statistically validchanges during 12 h of incubation

32 RL2 Stimulates Changes of Autophagy-Related ProteinsRL2 effects on autophagy-related proteins were analyzed Weobserved a reduction of p62 levels in MDA-MB-231 cellsafter 8 h of incubation (see Figure 2(d)) Autophagy-relatedLC3-II form was detected at 3 h and 8 h of incubationthough its levels decreased by 24 h The latter can be dueto the proteolytic degradation of LC3II We observed thatBeclin 1 started to decrease from 8 h of incubation with RL2ATG5 level was slightly higher in treated cells than in controlcells To summarize RL2 activates autophagy-like molecularchanges inMDA-MB-231 cells whichweremost prominentlyobserved at 8 h after treatment with RL2

33 CQ Increases Cytotoxicity of RL2-Treated Cells andChanges Autophagy and Apoptosis Features MDA-MB-231and MCF-7 cells were incubated with CQ alone and incombinationwith RL2 for 48 hours and then cytotoxic effectswere measured by MTT assay (see Figures 3(a)ndash3(c))


control + RL2 (01 mgmL)

+ RL2 (02 mgmL) + RL2 (03 mgmL)

(a)

DCF

DA

fluo

resc

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lativ

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MDA-MB-231

0

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0 100 200 300 400RL2 ugmL

0255075

100125150175200225

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MCF-7

(b)

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175

Time h

DCF

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fluo

resc

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lativ

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0255075

100125150175

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Time h

MCF-7

MDA-MB-231

DCF

DA

fluo

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lativ

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(c)

MDA-MB-231

MCF-7

62

62

P62

P62

Beclin1

Tubulin

Tubulin

ATG5

ATG5

LC3-ILC3-II

LC3-I

LC3-II

60

53

53

50

50

1816

18

16

0h 3h 8h 24h

0h 5h 12h 24h kDa

kDa

(d)

Figure 2 Analysis of ROS production and changes in autophagy-relatedproteins (a) Representative fluorescencemicroscopy images of ROSproduction (green) in RL2-treatedMDA-MB-231 cells (b) (c) Dependence of ROS production on RL2 concentration and time of incubationFor a kinetic study of ROS production cells were treated with RL2 (300 120583gmL) (d) The representative Western Blot of MCF-7 and MDA-MB-231 cells treated with RL2 (300 120583gmL) is showing a level of autophagy-related proteins

We varied CQ concentration from 05 120583M to 100 120583Mwhile RL2 concentration was constant The dose-dependentdecrease of viability of MDA-MB-231 and MCF-7 cellstreated with CQ was observed CQ enhanced the cytotoxic

effect of RL2 in MDA-MB-231 and MCF-7 cells comparedwith CQ alone (see Figures 3(b) and 3(c)) These differenceswere more substantial if CQ concentration was used in thenoncytotoxic range (05-20 120583M interval) The combinatorial


(a)

(b)

(c)

(d)

(e)

(f)

CI=065-095 (synergy)CI=1 (additivity)

CI=1 (additivity)

P=005Ku+01 mgml RL2Ku

CI=047-064 (synergy)P=005Ku+01 mgml RL2Ku

CI=077-08 (synergy)P=005CQ+01 mgml RL2CQ

CI=097 (synergy)P=005CQ+01 mgml RL2CQ

MDA-MB-231 MDA-MB-231

MCF-7MCF-7

MDA-MB-231 CQMCF-7 CQ

MDA-MB-231 KuMCF-7 Ku

0102030405060708090

100110120

Viab

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()

0102030405060708090

100110120

Viab

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()

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

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ility

()

0102030405060708090

100110120

Viab

ility

()

Figure 3 The influence of CQ and Ku with RL2 on viability of MDA-MB-231 cells and MCF-7 cells Detection of viability was produced byMTT assay (a)ndash(c) Cytotoxic effect of CQ (0-100 120583M) alone and in combination with RL2 (01 mgmL) on MDA-MB-231 and MCF-7 cellsCombinatorial index (CI) is 077-1 (d)ndash(f) Cytotoxic effect of Ku (0-40120583M) alone and in combinationwith RL2 (01mgmL) onMDA-MB-231and MCF-7 cells Combinatorial index (CI) is 047-1 CI was calculated with CompuSyn version 10


RL2

CQ RL2 + CQ

control

101

96

275

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142

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486

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control

PE-A

RL2

RL2 + CQCQ

MCF-7

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por

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()

Q1Q2Q3Q4

Q1Q2Q3Q4

(b)

Figure 4 RL2 in combination with CQ increases percentage of dead cells MCF-7 and MDA-MB-231 were treated with RL2 (015 mgmL)CQ (20 120583M) or both compounds After 24 h of incubation cells were collected stained with FITC Annexin V Apoptosis detection kit (BD)and analyzed with flow cytometry (a)The typical cell distributions in Q1-Q4 quadrants (b)The histograms show typical cell distributions ofthree independent experiments

index (CI) for MDA-MB-231 cells was 097 and for MCF-7cells was 077 this indicates a synergistic effect of CQ andRL2

Next we analyzedwhether a combination of CQwith RL2activates apoptosis in treated cells MDA-MB-231 andMCF-7cells were treated with RL2 CQ alone and the combinationAfter 24 h of incubation cells were stained with AnnexinVPI and analyzed by flow cytometry We showed thatAnnexin V+PI+ (Q2 late apoptotic) populations in MDA-MB-231 cells and Annexin V+PI- (Q4 early apoptotic)populations in MCF-7 cells were significantly higher aftercombinatorial treatment in comparing (see Figure 4) As we

observed the live cells population was the lowest in combi-natorial treatment and this is in good correlation with MTTdata

Next we examined autophagy markers in MDA-MB-231cells treated with RL2 orand CQ by immunoblot assayThis combinatorial treatment led to the accumulation of p62protein with a maximum amount occurring at 3 hours inthe treatment of MDA-MB-231 cells (see Figure 5(a)) Theincrease of p62 could indicate the autophagy suppressionThus CQ in combination with RL2 effectively decreased theviability of MDA-MB-231 and MCF-7 cells via inhibitingautophagy and amplifying cell death


0 248324832483 Time (h)RL2CQ

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(b)

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3 2453 2453

(e)

Figure 5 Changes in the cellular proteins after their incubation with indicated compounds Representative Western Blots showing changesin autophagy-related proteins Whole cell lysates were prepared for analysis using tubulin as a loading control (a) (c) (e) Western Blots withMDA-MB-231 cell lysates Cells were treated with RL2 (03 mgmL) CQ (10 120583M) 3MA (10 mM) and Rap (10 120583M) for various time points(0ndash24h) (b) (d) Western Blots with MCF-7 cell lysates Cells were treated with RL2 (03 mgmL) Ku (30 120583M) and Rap (10 120583M) for theindicated time points (0ndash24h)

34 Combination of RL2 with Ku55933 Increases CytotoxicEffect Ku55933 (Ku) an ATM specific inhibitor was alsodemonstrated to inhibit autophagy [20] Ku decreased viabil-ity of MCF-7 cell more effectively compared to MDA-MB-231 with inhibitory concentration 50 (IC50) values of 261120583M and 572 120583M respectively (see Figures 3(d)ndash3(f)) Strongsynergistic effects with RL2 were observed when Ku wasused up to IC50 concentrations (see Figures 3(e) and 3(f))The highest increase of cytotoxicity of such combination wasobserved with the lowest concentrations of Ku in the MCF-7cells The combinatorial index for 1-20 120583M Ku with RL2 (01mgml) was 065 which indicates synergistic effects Despitethe fact that the cytotoxicity of RL2 with Ku was lower forMDA-MB-231 cells than for MCF-7 cells a high synergisticeffect (combinatorial index CI=047-064) was observed forthese compounds in MDA-MB-231 cells

Because MCF-7 cells were more sensitive to Ku thanMDA-MB-231 cells and also more sensitive to the combina-tion of Ku with RL2 the autophagy-related proteins p62 andLC3 were analyzed in treated MCF-7 cells (see Figure 5(b))We revealed that RL2 (03 mgml) in combination with Ku(30 120583M) led to the accumulation of p62 when compared tocontrol nontreated cells which indicates autophagy suppres-sion Ku alone slightly induced LC3II formation Comparedto RL2-treated cells LC3I to LC3II processing was less strongin samples treated with the combination of Ku and RL2(see Figure 5(b)) We concluded that this finding confirmedautophagy-suppressive action of Ku

35 3-Methyladenine (3MA) Does Not Increase Cytotoxicity ofRL2-Treated Cells The cytotoxic effect of RL2 (01 mgml)


in combination with 3MA (0-40 mM) was investigated onMCF-7 and MDA-MB-231 cells We observed no increaseof cytotoxic effect in the combination of 3MA and RL2in both MCF-7 and MDA-MB-231 cells when compared tomonotreatment (see Figures 6(a)ndash6(c)) Increase of p62 levelsappeared when cells were treated with RL2 and 3MA (10mM)(see Figure 5(c)) LC3 analysis revealed that combinatorialtreatment of 3MA and RL2 led to less LC3II than RL2 aloneindicating a decrease of autophagy level

36 Cytotoxicity of RL2 in Combination with AutophagyInducers Rapamycin (Rap) and Spermidine To examinewhether rapamycin increases or decreases viability of RL2-treated cells we incubated MDA-MB-231 cells and MCF7cells with a combination of drugs and performed MTTanalysis Galluzzi et al state that rapamycin concentrationshigher than 10 120583M are likely to promote acute cell deathmainly through off-target mechanisms [26] Keeping thisrestriction in mind we limited Rap concentration to 1-10120583M We revealed that both cell lines were low sensitiveto rapamycin (see Figures 6(d)ndash6(f)) Under combinatorialtreatment synergistic effects were observed for the full rangeof Rap concentrations used (see Figures 6(e) and 6(f))Performing Western Blot analysis we observed that thecombination of RL2 with Rap decreased p62 expression (seeFigure 5(d))

The disappearance of LC3II accompanied by the decreaseof LC3I could be a consequence of proteasomal degradationof LC3 during autophagy which was initiated by both com-pounds Under combinatorial treatment a decrease in ATG5levels was also observed (see Figures 5(d) and 5(e)) Takentogether a high level of autophagy induced by RL2 and Rapled to cell death

Besides Rap another autophagy inducer spermidine wasanalyzed in combination with RL2We revealed that RL2 didnot increase cytotoxicity of spermidine

37 Autophagy Modulators Change Cathepsin D Activityin RL2-Treated Cells Earlier we have demonstrated RL2decreased cellular Cathepsin D (CatD) activity in MDA-MB-231 cells when used in combination with CQ [14] RL2 slightlydecreased CatD activity whereas CQ decreased up to 2-foldTesting here other autophagy modulators we showed thatCatD activity in cells treated with combination of RL2 and Kuor 3MA was not changed compared to control cells or RL2-treated cells (see Figure 7)

Next we investigated if autophagy inducers Rap andspermidine modulate CatD activity We observed that Rapand spermidine have opposite effects on CatD activity a validdecrease of its activity in Rap-treated cells was detected whilein spermidine-treated cells its activity was increased Inter-estingly the addition of RL2 normalized (decreased) CatDactivity in spermidine-treated cells to control cellrsquos activityThe addition of RL2 to Rap-treated cells slightly decreasedCatD activity compared with mono-Rap treatment Takentogether the combinatorial treatment with RL2 and Rap orwith spermidine showed a decrease of Cathepsin D activityImportantly we can show the interaction between CatD and

RL2 by bothmass spectrometry andWesternBlot (see Figures7(b) and 7(d)) suggesting that RL2might triggerCatD activityvia direct interactions with CatD

38 Autophagy Modulators Decrease ATP Release from RL2-Treated Cells Cell death associated with autophagy can alsodisplay signals of immunogenic death [1] It was statedthat autophagy is required for chemotherapeutics to induceATP release We investigated if RL2 CQ Rap or theircombinations activate ATP release from treated cells Weobserved that ATP release from RL2-incubated cells wasabout 10 times higher than that from nontreated cells (seeFigure 8) Chloroquine and rapamycin did not change ATPrelease compared to nontreated cells When we incubatedcells with a combination of these compounds with RL2the amount of external ATP was slightly higher than thatfor samples incubated only with CQ or Rap but it wassubstantially lower than that for RL2 alone This indicates thatCQ and Rap prevent massive ATP release from dying cellsincubated with RL2

39 Estimation ofAutophagosomeAutophagolysosomeForma-tion inTreatedCells For direct visualization of autophagy weanalyzed cellular ultrastructure by the electron microscopy(see Figures 9(a)ndash9(f)) After 4 h of incubation onlycells treated with RL2 alone showed an elevated amountof autophagosomesautophagolysosomes (see Figure 9(g))Cells treated with RL2 and CQ for 8-24 h contained atwofold increase of autophagosomes compared to controlcells or RL2-treated cells CQ alone also led to an increaseof autophagosomesautophagolysosomes after 8-24 h of incu-bation These data conform with the fact that CQ preventsproductive autophagy with complete catabolic processes thatlead to the accumulation of autophagosome into the cells

310 RL2 in Combination with CQ Suppresses RLS AllograftGrowth To test whether RL2 in combination with CQdelays tumor growth in vivo more efficiently than RL2 andCQ alone a mouse allograft model of cyclophosphamide-resistant lymphosarcoma RLS was used A suspension ofRLS cells was intramuscularly injected into the micersquos rightleg to induce tumor development Animals received RL2 (12mgkg) iv andor CQ (50 mgkg) ip while control tumor-bearing mice received NaCl ip We showed that RL2 incombination with CQ significantly delayed tumor growthcompared to the control group (plt0025) There were nosignificant differences between the CQ-treated group andthe RL2+CQ-treated group (pgt01) (see Figure 10(a)) Thetumor growth inhibition rate was calculated as 865 (groupRL2 and CQ) 249 (group RL2) and 784 (group CQ)Another supervised characteristic was the lifespan of thetumor-bearing mice (see Figure 10(b)) By day 23 after thetumor transplantation all animals which received RL2 andCQ were alive The percentage of survival for other groupswas lower 571 in CQ-receiving group 50 in RL2-treatedgroup and 222 in control group By this date the compari-son of tumor sizes between the groups was nonnegligible due


0102030405060708090

100110120

Viab

ility

()

P=005CI=09-097 (synergy)

MDA-MB-231MDA-MB-231

MCF-7MCF-7

RapRap + 01 mgml RL2

3MA3MA+01 mgml RL2

10 20 300 403MA (mM)

0102030405060708090

100110120

Viab

ility

()

102030405060708090

100110120

Viab

ility

()

10 20 300 403MA (mM)

10 200 40303MA (mM)

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast



3MA3MA+01 mgml RL2

lowast

(a)

(b)

(d)

(e)

(c) (f)

P=005lowast

MDA-MB-231 3MAMCF-7 3MA

MDA-MB-231 RapMCF-7 Rap

Figure 6 Combination of 3MA and Rap with RL2 influence on viability of MDA-MB-231 cells and MCF-7 cells Detection of viability wasproduced by MTT assay (a)ndash(c) Cytotoxic effect of 3MA (0ndash40 mM) alone and in combination with RL2 (01 mgmL) on MDA-MB-231 andMCF-7 cells (d)ndash(f) Cytotoxic effect of Rap (0-10 120583M) alone and in combination with RL2 (01 mgmL) on MDA-MB-231 and MCF-7 cellsCombinatorial index (CI) is 017-097 CI was calculated with CompuSyn version 10


MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)

-Casein immunoprecipitation withMDA-MB-231 cell lysateCovalent binding

of -Casein antibody

to protein A sepharose beads

mass spectrometry analysis

control -Casein immunoprecipitation with

SUIT cell lysate

protein A sepharose beads


(b)

n1 n2 n3 n1 n2 n3Cathepsin D P07339 9 6 11 - - 11

protein ID

-Casein immunoprecipitationMDA-MB-231 cell lysates SUIT-020 cell lysates

unique peptides unique peptides

(c)

larr Cathepsin D (le)

lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -

larr Procathepsin D (le)

larr Cathepsin D (se)

larr Procathepsin D (se)

(d)

Figure 7 Cathepsin D effects (a) Changes in CatD activity MDA-MB-231 and MCF-7 cells were treated with RL2 (02 mgmL) CQ (5120583M)Ku (30 120583M) 3MA (15 mM) spermidine (6 120583M) and Rap (15 120583M) for 24 h and after that CatD activity in lysates was measured usingfluorescent substrate Starvation (starv) was initiated by serum deprivation The data are presented as percentage from control (nontreatedcells) and shown as the mean plusmn SD of three independent experiments (b) Workflow for RL2-based mass spectrometry analysis 120581-Caseinantibody protein A sepharose beads were loaded with 120581-Casein antibody and incubated with RL2-treated MDA-MB-231 cell lysates or RL2-treated SUIT-020 cell lysates (control) Precipitated proteins were identified by nano-LC-tandem mass spectrometry (c) Mass spectrometryidentified unique peptides for each protein in the course of identificationThe absolute values of unique peptides for Cathepsin D identifiedby mass spectrometry analysis are shown hereThe abbreviations n1 n2 and n3 indicate absolute values for every experimental replicate (d)RL2-pull-down precipitates were analyzed by Western Blot and probed for indicated proteins (bead con control IP without antibody)


0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU

Figure 8 Analysis of drug-related ATP release MDA-MB-231 cellswere treatedwith RL2 (02mgmL) CQ (5120583M) and Rap (15 120583M) for20 or 44 h after that medium was collected and tested with ATP-specific luminescent substrate RLU relative luminescence unitsThe data are representative of three independent repeats and areshown as the mean plusmn SD

to the death of a major part of the animals in the controlgroup

4 Discussion

Nowadays the fundamental value of autophagy in livingorganisms is obvious Autophagy enables cells to adapt tostressful conditions including cancer cell survival underhypoxic and low-nutrient conditions Its role in cancerprogression and chemoresistancemakes autophagy an appro-priate target for anticancer therapy [27 28] Meanwhileseveral conventional chemotherapeutics induce cancer celldeath through autophagy-related mechanisms [29] Here weinvestigated whether analog of proapoptotic protein lac-taptin (RL2) activates autophagy-related pathways in cancercells and whether autophagy modulators improve cytotoxicactivity of RL2 Autophagic cell death (type II programmedcell death) is primarily independent of the activation ofcaspases and morphologically different from apoptosis [17]In comparison with apoptosis autophagic cell death is char-acterized by the massive accumulation of autophagosomeslimited chromatin condensation and lysosomal protein LC3maturation

In the current study we have demonstrated that RL2induced a short-term autophagy at the initial stage of incu-bation in vitro Western Blot analysis revealed changes ofthe major autophagy-related proteins in RL2-treated cellsElectron microscopy confirmed autophagosome formationwhich was induced by RL2 Moreover we have detected thatRL2 stimulated dose-dependent ROS production Excessivecellular levels of ROS can cause damage to molecules andorganelles such as mitochondria [30] For example H

2O2

causes initial mitochondrial membrane potential (MMP)

hyperpolarization leading to the decrease ofMMPmitochon-drial translocation of Bax and Bad and cytochrome c release[31] In conjunction with our previous data on the disruptionof the innermitochondrial membrane potential following theRL2 treatment we suggested that ROS can be engaged in RL2cytotoxicity

Chemicals which activate or deactivate autophagy path-ways can govern therapeutic-dependent death of cancer cellsWe investigated cytotoxic effects of RL2 in combination withdifferent autophagy inhibitors and inducers on MDA-MB-231 and MCF-7 cells We have used well-described specificautophagy inhibitors (CQ 3MA) and inducer (Rap) as wellas nonspecific autophagy modulators like inhibitor Ku andinducer spermidine We have shown that only CQ Ku andRap synergized prodeath activity of RL2 It was not clear whyautophagy activator Rap became so effective in combinationwith RL2 to be an autophagy inhibitor When we used CQwith RL2 the increase of cytotoxicity was accomplished byeffective suppression of RL2-induced autophagy according toWestern Blot analysis However electron microscopy showedhigher amounts of autophagosomes in CQ-treated MDA-MB-231 cells compared with nontreated cells CQ-dependentaccumulation of autophagosomes is well described and origi-nates from the prevention of the degradation process [32 33]Massive accumulation of autophagosomes is one of crucialcharacteristics of autophagic cell death [34 35] An analysisof the phosphatidylserine externalization in cells treated withCQ and RL2 by flow cytometry showed that the decrease ofliving cells was accomplished by the substantial increase ofsecondary apoptotic (Annexin V+PI+) cells but not earlyapoptotic (Annexin V+PI-) cells compared to RL2-treatedcells (see Figure 4) These data indicate that the combinationof CQ and RL2 amplified cell death and that this is diversefrom apoptosis and is more likely autophagic cell deathIn comparison with CQ 3MA blocks the early stage ofautophagy It can inhibit the formation of Beclin 1-PI3KC3complexes as well as conversion of soluble LC3-I to lipidbound LC3-II and the formation of autophagosomes Wesuppose that the suppression of autophagosome formation by3MA excludes the possibility of autophagic cell death whichapparently takes place in CQRL2-treated cells

Autophagy inducer rapamycin is a macrolide immuno-suppressant which is licensed for use against solid organand bone marrow rejection [36] Rap inhibits immune cellgrowth and proliferation by blocking signal transduction[37] Usually the amount of LC3II correlates with the amountof autophagosomes and we expected that Rap could intensifyRL2-dependent autophagy When we used Rap with RL2we observed that Rap efficiently induced LC3I to LC3II(lipidation) processing at an early stage of incubation (3-5h) but when subjected to longer incubation both LC3I andLC3II disappeared (see Figures 5(d) and 5(e))This is becauseunder Rap-initiated autophagy LC3II is also degraded whichcorrelates with other investigators [38]

We found that p62 was sensitive to combination of RL2with all autophagy inhibitors used CQ Ku and 3MA butonly CQ and Ku synergized cytotoxic effect with RL2 Henceautophagy inhibition is not sufficient for the increase of cyto-toxicity The amounts of LC3II and p62 are reliable markers


ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)

Figure 9 RL2 and autophagy modulators affect autophagosomeautophagolysosome formation in MDA-MB-231 cells (a)ndash(f) Electronmicroscopy images of cross-sections of cells treated with RL2 (03 mgmL) CQ (20120583M) and Rap (100 nM) (a) phagophore formation inRL2-treated cells (b) (c) Fragment of RL2-treated cell with multiple autolysosomes (d) Autophagosomes in Rap-treated cells (8h) (e) Deadcell after Rap+RL2 treatment (24 h) (f) Autophagosomes in a dead cell after CQ+RL2 treatment (24 h) Asterisks indicate phagophore (Ph)multivesicular body (MVB) or autolysosome (AL) (g) Percentage of autophagosomesautophagolysosomes in MDA-MB-231 cells treatedwith RL2 and CQ

of autophagy but only the morphological assessment withelectron microscopy can confirm autophagosome involve-ment The increased number of autophagic vesicles whichwere observed in the cells incubated with combinations ofRapRL2 or CQRL2 may be a result of autophagy inductionfor Rap and its blockade at downstream steps for CQ [18]In both cases the accumulation of autophagosomes led to asignificant increase in cell death

The activity of lysosomal proteases produces autophagicprotein degradation Cathepsin D is a lysosomal aspartyl

protease involved in the degradation of unfolded damagedand unused proteins Alteration of CatD enzymatic activityleads to changes in modulation of diverse enzymes andgrowth factors which are crucial in various proteinopathiesand cancer [39ndash41] It is known that lysosomal cathepsinsare involved in apoptotic and nonapoptotic cell death [42]The release of CatD increases the release of cytochromeC and subsequently activates caspase-3-7 thus inducingthe depolarization of the mitochondrial membrane linkingdefective autophagy to caspase-dependent apoptosis [41]


controlCQRL2RL2 + CQ

13 14 15 16 17 18 19 2012days after tumor transplantation

0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ

5 10 15 20 250days after tumor transplantation

0102030405060708090

100

Perc

ent S

urvi

val

(b)

Figure 10 RL2 in combination with CQ delays tumor development Female CBA mice aged 8-9 weeks were used for intramuscular RLS celltransplantation The mice were randomly divided into 4 groups (6-9 mice in group) RL2 (12 mgkg) dissolved in saline was administerediv via the tail vein every 2-3 days starting on day 8 after the tumor transplantation CQ (50 mgkg) dissolved in saline was administered ipdaily RL2 in combination with CQ was administered as described above respectively (a) The growth rate of RLS tumors (b) Lifespan ofcontrol and experimental mice Error bars represent mean plusmn standard deviationThe statistical difference in median tumor volume betweenthe control and the experimental groups was lowastPlt005 Studentrsquos t-test was used for statistical analysis Results are reported as the mean plusmnstandard deviation (SD)

Alternatively cytoprotective activity of CatD against exces-sive aggregated proteins was demonstrated as well and thisactivity was independent of autophagy [43]We consider thatwhen entering into the cells RL2 can stimulate proteases inparticular CatD responsible for the degradation of excessiveand unused proteins However we revealed a decrease ofCatD activity in the cells incubated with RL2 This findingindicates that RL2 is more likely to act as CatD suppressorand that this can be realized through their direct physicalinteraction Among autophagy modulators used in this studyspermidine and 3MA prevent a RL2-dependent decrease ofCatD activity while CQ and Rap intensify CatD deactivationWe suppose that the decrease of CatD enzymatic activity isessential for high cytotoxic activity of the combination ofRL2 with CQ or Rap In our body there is an endogenouspool of spermidine with no toxicity under physiological con-centration An exogenous supply of spermidine can inducemacroautophagywith increased autophagic flux by inhibitingthe acetyltransferase EP300 [21] By this pathway spermidinereduces global protein acetylation of cellular proteins thatcan lead to CatD activation [44] According to our dataspermidine acts as CatD activator and it was the least potentfor increase of cytotoxic activity of RL2

Chemotherapy-induced release of ATP from dying cellsleads to the increase of extracellular ATP acting as a ldquofindmerdquo signal for the recruitment of phagocytes Autophagycontributes to this phagocytic targeting of apoptotic cancercells Moreover dying autophagic cells can release ATP [45]In our experiments a treatment of MDA-MB-231 cells withRL2 resulted in a fivefold increase of extracellular ATP thiscould be indirect evidence of autophagy activation When wecoincubated the cells with RL2 and autophagy modulators

we observed that CQ and Rap prevent massive ATP releasefromdying cellsThis is in line with other studies where it wasdemonstrated that the suppression of autophagy inhibitedthe release of adenosine triphosphate from chemotherapy-treated tumor cells [1] It was also demonstrated that inhi-bition of autophagy with Rap failed to trigger ATP releaseTherefore in spite of the high cytotoxicity of the combinationof RL2 and CQ it is more expected that cells after suchcombinatorial treatment will fail to prime T cells in vivo andto activate antitumor immunity

We have also tried to translate our in vitro results toan animal model Antitumor potential of CQ with variousefficiencies has been demonstrated in several works [46ndash48] This compound has a low toxicity itself and can beinjected intraperitoneally intomice whichmakesCQ therapyof a mice cancer model a very suitable investigation ofcombinatorial treatment We showed that the treatment ofthe tumor-bearing mice with RL2 and CQ improved theantitumor effect of RL2 Taking into account the high speedof tumor progression and the mortality rate of the mice ofa nontreated group we did not observe statistically validdifferences between CQ-treated and CQ-with-RL2-treatedanimals However the viability rate of animals receiving RL2and CQ was significantly higher than that in animals treatedwith CQ alone

5 Conclusions

This study provides new insights into the mechanismsinvolved in recombinant lactaptin RL2 cytotoxic activityBased on our results we suggest that the combination of RL2


with chloroquine is a promising antitumor approach againstbreast cancers

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon request

Ethical Approval

All animal experiments were carried out in compliance withthe protocols and recommendations for the proper use andcare of laboratory animals (EEC Directive 86609EEC) Theprotocol was approved by the Committee on the Ethics ofAnimal Experiments of the Administration of SB RAS ( 40April 4 2018)

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Olga A Koval Inna N Lavrik Yuri Ya Kit and Vladimir ARichter designed the study Anastasia V Bagamanshina OlgaS Troitskaya Anna A Nushtaeva Marina O StarykovychElena V Kuligina Max Richter and Fabian Wohlfrommperformed the experiments Thilo Kahne performed massspectrometry analysis Anastasia Yu Yunusova analyzed cellultrastructure Olga A Koval Inna N Lavrik and MaxRichter analyzed the dataThe draftmanuscript was preparedby Olga A Koval with input from Inna N Lavrik Yuri Ya Kitand Vladimir A Richter All authors read and approved thefinal version of the manuscript

Acknowledgments

We acknowledge Volkswagen Foundation (VW 90315) Rus-sian State funded budget project of ICBFM SB RAS 0000-017-117020210023-1 for supporting our work FEBSYouth Travel Fund (YTF) provided the participation inAdvanced Lecture Course on Oncometabolism 18-24 June2017 Figueira da Foz Portugal with presenting pilot resultsof this investigation

References

[1] M Michaud I Martins A Q Sukkurwala et al ldquoAutophagy-dependent anticancer immune responses induced bychemotherapeutic agents in micerdquo Science vol 334 no6062 pp 1573ndash1577 2011

[2] F M Menzies A Fleming A Caricasole et al ldquoAutophagy andneurodegeneration pathogenic mechanisms and therapeuticopportunitiesrdquo Neuron vol 93 no 5 pp 1015ndash1034 2017

[3] G R YDeMeyerMO J Grootaert C FMichiels A Kurdi DM Schrijvers andWMartinet ldquoAutophagy in vascular diseaserdquoCirculation Research vol 116 pp 468ndash479 2015

[4] M Condello E Pellegrini M Caraglia and S Meschini ldquoTar-geting autophagy to overcome human diseasesrdquo InternationalJournal of Molecular Sciences vol 20 no 3 p 725 2019

[5] L Wilde K Tanson J Curry and U Martinez-OutschoornldquoAutophagy in cancer a complex relationshiprdquo BiochemicalJournal vol 475 no 11 pp 1939ndash1954 2018

[6] A L Swampillai P Salomoni and S C Short ldquoThe role ofautophagy in clinical practicerdquo Clinical Oncology vol 24 no6 pp 387ndash395 2012

[7] H-Q Zhang N Fang X-M Liu et al ldquoAntitumor activity ofchloroquine in combination with cisplatin in human gastriccancer xenograftsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 9 pp 3907ndash3912 2015

[8] X Xiao WWang Y Li et al ldquoHSP90AA1-mediated autophagypromotes drug resistance in osteosarcomardquo Journal of Experi-mental amp Clinical Cancer Research vol 37 no 1 p 201 2018

[9] A Duffy J Le E Sausville and A Emadi ldquoAutophagy mod-ulation a target for cancer treatment developmentrdquo CancerChemotherapy and Pharmacology vol 75 no 3 pp 439ndash4472015

[10] N Eritja B-J Chen R Rodrıguez-Barrueco et al ldquoAutophagyorchestrates adaptive responses to targeted therapy in endome-trial cancerrdquo Autophagy vol 13 no 3 pp 608ndash624 2017

[11] X Sui N Kong X Wang et al ldquoJNK confers 5-fluorouracilresistance in p53-deficient and mutant p53-expressing coloncancer cells by inducing survival autophagyrdquo Scientific Reportsvol 15 no 4 p 4694 2014

[12] R A Gonzalez-Polo P Boya A L Pauleau et al ldquoThe apop-tosisautophagy paradox autophagic vacuolization beforeapoptotic deathrdquo Journal of Cell Science vol 118 no 14 pp3091ndash3102 2005

[13] D V Semenov A S Fomin E V Kuligina et al ldquoRecombinantanalogs of a novel milk pro-apoptotic peptide lactaptin andtheir effect on cultured human cellsrdquo The Protein Journal vol29 no 3 pp 174ndash180 2010

[14] O A Koval A V Tkachenko A S Fomin et al ldquoLactaptininduces p53-independent cell death associated with features ofapoptosis and autophagy and delays growth of breast cancercells in mouse xenograftsrdquo PLoS ONE vol 9 no 4 Article IDe93921 2014

[15] V Vinod C J Padmakrishnan B Vijayan and S Gopala ldquoHowcan i halt thee the puzzles involved in autophagic inhibitionrdquoPharmacological Research vol 82 pp 1ndash8 2014

[16] B Pasquier ldquoAutophagy inhibitorsrdquo Cellular and Molecular LifeSciences vol 73 no 5 pp 985ndash1001 2016

[17] D J Klionsky H Abeliovich P Agostinis et al et al ldquoGuide-lines for the use and interpretation of assays for monitoringautophagy in higher eukaryotesrdquo Autophagy vol 4 no 2 pp151ndash175 2008

[18] N Mizushima T Yoshimori and B Levine ldquoMethods inmammalian autophagy researchrdquo Cell vol 140 no 3 pp 313ndash326 2010

[19] A Nadkarni M Shrivastav A C Mladek et al ldquoATM inhibitorKU-55933 increases the TMZ responsiveness of only inherentlyTMZ sensitive GBM cellsrdquo Journal of Neuro-Oncology vol 110no 3 pp 349ndash357 2012

[20] T Farkas M Daugaard and M Jaattela ldquoIdentification ofsmall molecule inhibitors of phosphatidylinositol 3-kinase andautophagyrdquoThe Journal of Biological Chemistry vol 286 no 45pp 38904ndash38912 2011


[21] F Pietrocola S Lachkar D P Enot et al ldquoSpermidine inducesautophagy by inhibiting the acetyltransferase EP300rdquo CellDeath amp Differentiation vol 22 no 3 pp 509ndash516 2015

[22] K A Tekirdag G Korkmaz D G Ozturk R Agami andD Gozuacik ldquoMIR181A regulates starvation-and rapamycinin-duced autophagy through targeting of ATG5rdquo Autophagy vol9 no 3 pp 374ndash385 2013

[23] O Koval G Kochneva A Tkachenko et al ldquoRecombinantvaccinia viruses coding transgenes of apoptosis-inducing pro-teins enhance apoptosis but not immunogenicity of infectedtumor cellsrdquo BioMed Research International vol 2017 ArticleID 3620510 14 pages 2017

[24] O Sokolova T Kahne K Bryan and M Naumann ldquoInterac-tome analysis of transforming growth factor-120573-activatedkinase1 in Helicobacter pylori -infected cells revealed novel regulatorstripartite motif 28 and CDC37rdquo Oncotarget vol 9 no 18 pp14366ndash14381 2018

[25] O A Koval A S Fomin V I Kaledin et al ldquoA novel pro-apoptotic effector lactaptin inhibits tumor growth in micemodelsrdquo Biochimie vol 94 no 12 pp 2467ndash2474 2012

[26] L Galluzzi E Morselli O Kepp et al ldquoEvaluation ofrapamycin-induced cell deathrdquo Methods in Molecular Biologyvol 821 pp 125ndash169 2012

[27] C Yun and S Lee ldquoThe roles of autophagy in cancerrdquo Interna-tional Journal ofMolecularSciences vol 19 no 11 p e3466 2018

[28] S Datta D Choudhury A Das et al ldquoAutophagy inhibitionwith chloroquine reverts paclitaxel resistance and attenuatesmetastatic potential in human nonsmall lung adenocarcinomaA549 cells via ROSmediatedmodulation of120573-catenin pathwayrdquoApoptosis vol 24 no 5-6 pp 434-434 2019

[29] C D Graham N Kaza B J Klocke et al ldquoTamoxifen inducescytotoxic autophagy in glioblastomardquo Journal ofNeuropathologyamp Experimental Neurology vol 75 no 10 pp 946ndash954 2016

[30] M Redza-Dutordoir and D A Averill-Bates ldquoActivation ofapoptosis signalling pathways by reactive oxygen speciesrdquoBiochimica et Biophysica Acta (BBA) - Molecular Cell Researchvol 1863 no 12 pp 2977ndash2992 2016

[31] H Stridh M Kimland D P Jones S Orrenius and M BHampton ldquoCytochrome c release and caspase activation inhydrogen peroxide- and tributyltin-induced apoptosisrdquo FEBSLetters vol 429 no 3 pp 351ndash355 1998

[32] S Avniel-Polak G Leibowitz V Doviner D J Gross and SGrozinsky-Glasberg ldquoCombining chloroquine with RAD001inhibits tumor growth in a NEN mouse modelrdquo Endocrine-Related Cancer vol 25 no 6 pp 677ndash686 2018

[33] Y Geng L Kohli B J Klocke and K A Roth ldquoChloroquine-induced autophagic vacuole accumulation and cell death inglioma cells is p53 independentrdquo Neuro-Oncology vol 12 no5 pp 473ndash481 2010

[34] A L Edinger and C BThompson ldquoDeath by design apoptosisnecrosis and autophagyrdquoCurrentOpinion in Cell Biology vol 16no 6 pp 663ndash669 2004

[35] W Bursch ldquoThe autophagosomal-lysosomal compartment inprogrammed cell deathrdquoCell DeathampDifferentiation vol 8 no6 pp 569ndash581 2001

[36] C Cutler and J H Antin ldquoSirolimus for GVHD prophylaxis inallogeneic stem cell transplantationrdquo Bone Marrow Transplan-tation vol 34 no 6 pp 471ndash476 2004

[37] A T Waickman and J D Powell ldquomTOR metabolism and theregulation of T-cell differentiation and functionrdquo Immunologi-cal Reviews vol 249 no 1 pp 43ndash58 2012

[38] N Mizushima and T Yoshimori ldquoHow to interpret LC3immunoblottingrdquo Autophagy vol 3 no 6 pp 542ndash545 2007

[39] S Chen H Dong S Yang andH Guo ldquoCathepsins in digestivecancersrdquoOncotarget vol 8 no 25 pp 41690ndash41700 2017

[40] C ZhangM Zhang and S Song ldquoCathepsinD enhances breastcancer invasion and metastasis through promoting hepsinubiquitin-proteasomedegradationrdquoCancer Letters vol 438 pp105ndash115 2018

[41] A Aufschnaiter V Kohler and S Buttner ldquoTaking out thegarbage Cathepsin D and calcineurin in neurodegenerationrdquoNeural Regeneration Research vol 12 no 11 pp 1776ndash1779 2017

[42] U Repnik V Stoka V Turk and B Turk ldquoLysosomes andlysosomal cathepsins in cell deathrdquo Biochimica et BiophysicaActamdashProteins and Proteomics vol 1824 no 1 pp 22ndash33 2012

[43] A Aufschnaiter L Habernig V Kohler et al ldquoThe coordinatedaction of calcineurin and cathepsin D protects against 120572-synuclein toxicityrdquo Frontiers in Molecular Neuroscience vol 10p 207 2017

[44] E Morselli G Marino M V Bennetzen et al ldquoSpermidine andresveratrol induce autophagy by distinct pathways convergingon the acetylproteomerdquoThe Journal of Cell Biology vol 192 no4 pp 615ndash629 2011

[45] G Ayna D V Krysko A Kaczmarek G Petrovski P Vanden-abeele and L Fesus ldquoATP release from dying autophagic cellsand their phagocytosis are crucial for inflammasome activationin macrophagesrdquo PLoS ONE vol 7 no 6 Article ID e400692012

[46] N Seiler M Chaabi S Roussi F Gosse A Lobstein andF Raul ldquoSynergism between apple procyanidins and lyso-somotropic drugs potential in chemopreventionrdquo AnticancerReseach vol 26 no 5A pp 3381ndash3385 2006

[47] YWang R-Q Peng D-D Li et al ldquoChloroquine enhances thecytotoxicity of topotecan by inhibiting autophagy in lung cancercellsrdquo Chinese Journal of Cancer vol 30 no 10 pp 690ndash7002011

[48] X Liang J C Tang Y L Liang R A Jin and X J Cai ldquoSup-pression of autophagy by chloroquine sensitizes 5-fluorouracil-mediated cell death in gallbladder carcinoma cellsrdquo Cell ampBioscience vol 4 no 1 p 10 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


phagophore Isolation membranewith cargo

Autophagosome

Lysosome

Degradation

Autophagolysosome

3-Methyladenine

PI3K-III complex ULK complex

mTorc1

Rapamycin

Ku55933

Chloroquine

Figure 1 Key points of autophagy modulation by various drugs

efficiency of chemotherapeutics [7 10 11] For convenientanticancer chemotherapeutics such as doxorubicin cisplatinand methotrexate [8] activation of prosurvival autophagyhas already been demonstrated But in some cases autophagyaccelerates cell death and can stimulate tumor suppression[12] Therefore correct regulation of autophagy is an impor-tant antineoplastic strategy [9]

Earlier we showed that recombinant analog of lactaptinRL2 suppresses tumor growth and metastasis in mice withno signs of toxic effects [13] Besides apoptosis RL2 inducedprocessing of microtubule-associated protein 1 light chain3 (LC3) which is referred to as a marker of autophagyWhen RL2 was used in vitro in MDA-MB-231 cells withautophagy inhibitor chloroquine this combination wasmorecytotoxic than RL2 or CQ alone [14]Therefore we supposedthat treatment of lactaptin analog with various autophagyinducers or inhibitors has the potential for improving ofcytotoxic and anticancer effect of RL2

In this study we used a set of various autophagy in-hibitors and inducers which switch over diverse steps inautophagy pathway (see Figure 1) 3-Methyladenine (3MA)is a widely used inhibitor of autophagy which suppressesphosphoinositide-3-kinases (PI3Ks) activity [15 16] leadingto suppression of autophagosome formation [17] Chloro-quine prevents fusion of autophagosomes with lysosomes[16 18] while Ku55933 (Ku) an ATM kinase inhibitor [19]acts like 3MA by blocking class III PI3K [20] Spermidineinduces macroautophagy by inhibiting the acetyltransferaseEP300 [21] Rapamycin activates autophagy inhibiting mTORsignaling pathway [22]

Here we tried to reveal which autophagy inhibitor orinducer enhances cytotoxic activity of lactaptin analog RL2in vitro and in vivo with the highest degree and to dis-cover activated death pathways by these combinations ofcompounds

2 Experimental Section

21 Materials

211 Cell Lines and Mice MCF-7 human breast adenocarci-noma cells andMDA-MB-231 human breast adenocarcinomacells were obtained from the Russian cell culture collection(RussianBranch of the ETCS St Petersburg Russia)TheRLSmurine lymphosarcoma cells were generously provided byDr V I Kaledin (Institute of Cytology and Genetics SB RASNovosibirsk Russia) Cells were maintained as previouslypublished [23]

Female CBA mice (8-9 weeks old) were obtained fromSPF vivarium of the Institute of Cytology and Genetics SBRAS Novosibirsk Russia

212 Antibodies and Chemicals The following antibodiesand chemicals were obtained from commercial sourcesanti-LC3B and anti-ATG5 (Abcam USA) anti-p62SQSTM(Sigma-Aldrich USA) polyclonal rabbit-anti-mouse andmouse-anti-rabbit HRP-conjugated secondary antibodies(Biosan Russia) trypsin (Gibco USA) inhibitor of trypsinfrom soybean (Paneco USA) FITC Annexin V Apoptosisdetection kit (BD Pharmingen USA) and Ku55933 (Ku)(Tocris Bioscience UK) Chloroquine 3-methyladeninerapamycin and spermidine were purchased from Sigma-Aldrich USA

22 Methods

221 MTT Assay Cells were seeded in 96-well plates at 2 times103 cells per well in a volume of 100 120583L and cultivated understandard conditions in RPMI medium with 10 FBS 03mgmL L-glutamine 100 120583gmL streptomycin and 100UmLpenicillin After 24 hours the cells were added to 100 120583L of




2


2O2)






















3 Results



2O2) and DHE (O







+ RL2 (02 mgmL) + RL2 (03 mgmL)

(a)

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

MDA-MB-231

0

25

50

75

100

125

0 100 200 300 400RL2 ugmL

0255075

100125150175200225

0 100 200 300 400RL2 ugmL

MCF-7

(b)

0

25

50

75

100

125

150

175

Time h

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

0255075

100125150175

0 1263

0 1263

Time h

MCF-7

MDA-MB-231

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

(c)

MDA-MB-231

MCF-7

62

62

P62

P62

Beclin1

Tubulin

Tubulin

ATG5

ATG5

LC3-ILC3-II

LC3-I

LC3-II

60

53

53

50

50

1816

18

16

0h 3h 8h 24h

0h 5h 12h 24h kDa

kDa

(d)





(a)

(b)

(c)

(d)

(e)

(f)


CI=1 (additivity)






MCF-7MCF-7



0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()



RL2

CQ RL2 + CQ

control

101

96

275

201

142

122

486

211

control

PE-A

RL2

RL2 + CQCQ

MCF-7

16

138

FITC-A

MDA-MB-231

142

242

52

307

138

439

102

103

104

105

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

(a)


20

40

60

80

100


MCF-7 MDA-MB-231

Cell

por

tion

()

0

20

40

60

80

100

Cell

por

tion

()

Q1Q2Q3Q4

Q1Q2Q3Q4

(b)







0 248324832483 Time (h)RL2CQ

MDA-MB-231

62

53

1816

kDa

- + + - - - + ++ +- - - - + + + + + +

P62

Tubulin

LC3-ILC3-II

(a)

Time (h)RL2Ku

MCF-7

62

53

1816

P62

Tubulin

LC3-ILC3-II

kDa

- + + - - - + ++ +- - - - + + + + + +0 24125 24125 24125

(b)

MDA-MB-231

53

Time (h)RL23MA

621816

P62

Tubulin

LC3-ILC3-II

kDa0 248324832483

- + + - - - + ++ +- - - - + + + + + +

(c)

MCF-7

53

50

1816

Time (h)RapRL2

Tubulin

ATG5

LC3-ILC3-II

kDa0 245

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(d)MDA-MB-231

62

53

50

1816

RL2Rap

0 245 Time (h)

P62

Tubulin

LC3-ILC3-II

ATG5

kDa

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(e)

















0102030405060708090

100110120

Viab

ility

()



MCF-7MCF-7


3MA3MA+01 mgml RL2

10 20 300 403MA (mM)

0102030405060708090

100110120

Viab

ility

()

102030405060708090

100110120

Viab

ility

()

10 20 300 403MA (mM)

10 200 40303MA (mM)

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast



3MA3MA+01 mgml RL2

lowast

(a)

(b)

(d)

(e)

(c) (f)

P=005lowast





MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)


of -Casein antibody




SUIT cell lysate



(b)


protein ID



(c)


lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -




(d)



0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















2


2O2)






















3 Results



2O2) and DHE (O







+ RL2 (02 mgmL) + RL2 (03 mgmL)

(a)

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

MDA-MB-231

0

25

50

75

100

125

0 100 200 300 400RL2 ugmL

0255075

100125150175200225

0 100 200 300 400RL2 ugmL

MCF-7

(b)

0

25

50

75

100

125

150

175

Time h

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

0255075

100125150175

0 1263

0 1263

Time h

MCF-7

MDA-MB-231

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

(c)

MDA-MB-231

MCF-7

62

62

P62

P62

Beclin1

Tubulin

Tubulin

ATG5

ATG5

LC3-ILC3-II

LC3-I

LC3-II

60

53

53

50

50

1816

18

16

0h 3h 8h 24h

0h 5h 12h 24h kDa

kDa

(d)





(a)

(b)

(c)

(d)

(e)

(f)


CI=1 (additivity)






MCF-7MCF-7



0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()



RL2

CQ RL2 + CQ

control

101

96

275

201

142

122

486

211

control

PE-A

RL2

RL2 + CQCQ

MCF-7

16

138

FITC-A

MDA-MB-231

142

242

52

307

138

439

102

103

104

105

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

(a)


20

40

60

80

100


MCF-7 MDA-MB-231

Cell

por

tion

()

0

20

40

60

80

100

Cell

por

tion

()

Q1Q2Q3Q4

Q1Q2Q3Q4

(b)







0 248324832483 Time (h)RL2CQ

MDA-MB-231

62

53

1816

kDa

- + + - - - + ++ +- - - - + + + + + +

P62

Tubulin

LC3-ILC3-II

(a)

Time (h)RL2Ku

MCF-7

62

53

1816

P62

Tubulin

LC3-ILC3-II

kDa

- + + - - - + ++ +- - - - + + + + + +0 24125 24125 24125

(b)

MDA-MB-231

53

Time (h)RL23MA

621816

P62

Tubulin

LC3-ILC3-II

kDa0 248324832483

- + + - - - + ++ +- - - - + + + + + +

(c)

MCF-7

53

50

1816

Time (h)RapRL2

Tubulin

ATG5

LC3-ILC3-II

kDa0 245

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(d)MDA-MB-231

62

53

50

1816

RL2Rap

0 245 Time (h)

P62

Tubulin

LC3-ILC3-II

ATG5

kDa

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(e)

















0102030405060708090

100110120

Viab

ility

()



MCF-7MCF-7


3MA3MA+01 mgml RL2

10 20 300 403MA (mM)

0102030405060708090

100110120

Viab

ility

()

102030405060708090

100110120

Viab

ility

()

10 20 300 403MA (mM)

10 200 40303MA (mM)

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast



3MA3MA+01 mgml RL2

lowast

(a)

(b)

(d)

(e)

(c) (f)

P=005lowast





MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)


of -Casein antibody




SUIT cell lysate



(b)


protein ID



(c)


lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -




(d)



0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





























3 Results



2O2) and DHE (O







+ RL2 (02 mgmL) + RL2 (03 mgmL)

(a)

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

MDA-MB-231

0

25

50

75

100

125

0 100 200 300 400RL2 ugmL

0255075

100125150175200225

0 100 200 300 400RL2 ugmL

MCF-7

(b)

0

25

50

75

100

125

150

175

Time h

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

0255075

100125150175

0 1263

0 1263

Time h

MCF-7

MDA-MB-231

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

(c)

MDA-MB-231

MCF-7

62

62

P62

P62

Beclin1

Tubulin

Tubulin

ATG5

ATG5

LC3-ILC3-II

LC3-I

LC3-II

60

53

53

50

50

1816

18

16

0h 3h 8h 24h

0h 5h 12h 24h kDa

kDa

(d)





(a)

(b)

(c)

(d)

(e)

(f)


CI=1 (additivity)






MCF-7MCF-7



0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()



RL2

CQ RL2 + CQ

control

101

96

275

201

142

122

486

211

control

PE-A

RL2

RL2 + CQCQ

MCF-7

16

138

FITC-A

MDA-MB-231

142

242

52

307

138

439

102

103

104

105

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

(a)


20

40

60

80

100


MCF-7 MDA-MB-231

Cell

por

tion

()

0

20

40

60

80

100

Cell

por

tion

()

Q1Q2Q3Q4

Q1Q2Q3Q4

(b)







0 248324832483 Time (h)RL2CQ

MDA-MB-231

62

53

1816

kDa

- + + - - - + ++ +- - - - + + + + + +

P62

Tubulin

LC3-ILC3-II

(a)

Time (h)RL2Ku

MCF-7

62

53

1816

P62

Tubulin

LC3-ILC3-II

kDa

- + + - - - + ++ +- - - - + + + + + +0 24125 24125 24125

(b)

MDA-MB-231

53

Time (h)RL23MA

621816

P62

Tubulin

LC3-ILC3-II

kDa0 248324832483

- + + - - - + ++ +- - - - + + + + + +

(c)

MCF-7

53

50

1816

Time (h)RapRL2

Tubulin

ATG5

LC3-ILC3-II

kDa0 245

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(d)MDA-MB-231

62

53

50

1816

RL2Rap

0 245 Time (h)

P62

Tubulin

LC3-ILC3-II

ATG5

kDa

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(e)

















0102030405060708090

100110120

Viab

ility

()



MCF-7MCF-7


3MA3MA+01 mgml RL2

10 20 300 403MA (mM)

0102030405060708090

100110120

Viab

ility

()

102030405060708090

100110120

Viab

ility

()

10 20 300 403MA (mM)

10 200 40303MA (mM)

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast



3MA3MA+01 mgml RL2

lowast

(a)

(b)

(d)

(e)

(c) (f)

P=005lowast





MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)


of -Casein antibody




SUIT cell lysate



(b)


protein ID



(c)


lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -




(d)



0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















+ RL2 (02 mgmL) + RL2 (03 mgmL)

(a)

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

MDA-MB-231

0

25

50

75

100

125

0 100 200 300 400RL2 ugmL

0255075

100125150175200225

0 100 200 300 400RL2 ugmL

MCF-7

(b)

0

25

50

75

100

125

150

175

Time h

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

0255075

100125150175

0 1263

0 1263

Time h

MCF-7

MDA-MB-231

DCF

DA

fluo

resc

ence

re

lativ

e uni

ts

(c)

MDA-MB-231

MCF-7

62

62

P62

P62

Beclin1

Tubulin

Tubulin

ATG5

ATG5

LC3-ILC3-II

LC3-I

LC3-II

60

53

53

50

50

1816

18

16

0h 3h 8h 24h

0h 5h 12h 24h kDa

kDa

(d)





(a)

(b)

(c)

(d)

(e)

(f)


CI=1 (additivity)






MCF-7MCF-7



0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()



RL2

CQ RL2 + CQ

control

101

96

275

201

142

122

486

211

control

PE-A

RL2

RL2 + CQCQ

MCF-7

16

138

FITC-A

MDA-MB-231

142

242

52

307

138

439

102

103

104

105

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

(a)


20

40

60

80

100


MCF-7 MDA-MB-231

Cell

por

tion

()

0

20

40

60

80

100

Cell

por

tion

()

Q1Q2Q3Q4

Q1Q2Q3Q4

(b)







0 248324832483 Time (h)RL2CQ

MDA-MB-231

62

53

1816

kDa

- + + - - - + ++ +- - - - + + + + + +

P62

Tubulin

LC3-ILC3-II

(a)

Time (h)RL2Ku

MCF-7

62

53

1816

P62

Tubulin

LC3-ILC3-II

kDa

- + + - - - + ++ +- - - - + + + + + +0 24125 24125 24125

(b)

MDA-MB-231

53

Time (h)RL23MA

621816

P62

Tubulin

LC3-ILC3-II

kDa0 248324832483

- + + - - - + ++ +- - - - + + + + + +

(c)

MCF-7

53

50

1816

Time (h)RapRL2

Tubulin

ATG5

LC3-ILC3-II

kDa0 245

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(d)MDA-MB-231

62

53

50

1816

RL2Rap

0 245 Time (h)

P62

Tubulin

LC3-ILC3-II

ATG5

kDa

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(e)

















0102030405060708090

100110120

Viab

ility

()



MCF-7MCF-7


3MA3MA+01 mgml RL2

10 20 300 403MA (mM)

0102030405060708090

100110120

Viab

ility

()

102030405060708090

100110120

Viab

ility

()

10 20 300 403MA (mM)

10 200 40303MA (mM)

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast



3MA3MA+01 mgml RL2

lowast

(a)

(b)

(d)

(e)

(c) (f)

P=005lowast





MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)


of -Casein antibody




SUIT cell lysate



(b)


protein ID



(c)


lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -




(d)



0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a)

(b)

(c)

(d)

(e)

(f)


CI=1 (additivity)






MCF-7MCF-7



0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0 40 60 8020 100CQ (M)

0 20 30 4010Ku 55933 (M)

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()

0102030405060708090

100110120

Viab

ility

()



RL2

CQ RL2 + CQ

control

101

96

275

201

142

122

486

211

control

PE-A

RL2

RL2 + CQCQ

MCF-7

16

138

FITC-A

MDA-MB-231

142

242

52

307

138

439

102

103

104

105

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

(a)


20

40

60

80

100


MCF-7 MDA-MB-231

Cell

por

tion

()

0

20

40

60

80

100

Cell

por

tion

()

Q1Q2Q3Q4

Q1Q2Q3Q4

(b)







0 248324832483 Time (h)RL2CQ

MDA-MB-231

62

53

1816

kDa

- + + - - - + ++ +- - - - + + + + + +

P62

Tubulin

LC3-ILC3-II

(a)

Time (h)RL2Ku

MCF-7

62

53

1816

P62

Tubulin

LC3-ILC3-II

kDa

- + + - - - + ++ +- - - - + + + + + +0 24125 24125 24125

(b)

MDA-MB-231

53

Time (h)RL23MA

621816

P62

Tubulin

LC3-ILC3-II

kDa0 248324832483

- + + - - - + ++ +- - - - + + + + + +

(c)

MCF-7

53

50

1816

Time (h)RapRL2

Tubulin

ATG5

LC3-ILC3-II

kDa0 245

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(d)MDA-MB-231

62

53

50

1816

RL2Rap

0 245 Time (h)

P62

Tubulin

LC3-ILC3-II

ATG5

kDa

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(e)

















0102030405060708090

100110120

Viab

ility

()



MCF-7MCF-7


3MA3MA+01 mgml RL2

10 20 300 403MA (mM)

0102030405060708090

100110120

Viab

ility

()

102030405060708090

100110120

Viab

ility

()

10 20 300 403MA (mM)

10 200 40303MA (mM)

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast



3MA3MA+01 mgml RL2

lowast

(a)

(b)

(d)

(e)

(c) (f)

P=005lowast





MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)


of -Casein antibody




SUIT cell lysate



(b)


protein ID



(c)


lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -




(d)



0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















RL2

CQ RL2 + CQ

control

101

96

275

201

142

122

486

211

control

PE-A

RL2

RL2 + CQCQ

MCF-7

16

138

FITC-A

MDA-MB-231

142

242

52

307

138

439

102

103

104

105

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

PE-A

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

FITC-A10

210

310

410

5

(a)


20

40

60

80

100


MCF-7 MDA-MB-231

Cell

por

tion

()

0

20

40

60

80

100

Cell

por

tion

()

Q1Q2Q3Q4

Q1Q2Q3Q4

(b)







0 248324832483 Time (h)RL2CQ

MDA-MB-231

62

53

1816

kDa

- + + - - - + ++ +- - - - + + + + + +

P62

Tubulin

LC3-ILC3-II

(a)

Time (h)RL2Ku

MCF-7

62

53

1816

P62

Tubulin

LC3-ILC3-II

kDa

- + + - - - + ++ +- - - - + + + + + +0 24125 24125 24125

(b)

MDA-MB-231

53

Time (h)RL23MA

621816

P62

Tubulin

LC3-ILC3-II

kDa0 248324832483

- + + - - - + ++ +- - - - + + + + + +

(c)

MCF-7

53

50

1816

Time (h)RapRL2

Tubulin

ATG5

LC3-ILC3-II

kDa0 245

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(d)MDA-MB-231

62

53

50

1816

RL2Rap

0 245 Time (h)

P62

Tubulin

LC3-ILC3-II

ATG5

kDa

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(e)

















0102030405060708090

100110120

Viab

ility

()



MCF-7MCF-7


3MA3MA+01 mgml RL2

10 20 300 403MA (mM)

0102030405060708090

100110120

Viab

ility

()

102030405060708090

100110120

Viab

ility

()

10 20 300 403MA (mM)

10 200 40303MA (mM)

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast



3MA3MA+01 mgml RL2

lowast

(a)

(b)

(d)

(e)

(c) (f)

P=005lowast





MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)


of -Casein antibody




SUIT cell lysate



(b)


protein ID



(c)


lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -




(d)



0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0 248324832483 Time (h)RL2CQ

MDA-MB-231

62

53

1816

kDa

- + + - - - + ++ +- - - - + + + + + +

P62

Tubulin

LC3-ILC3-II

(a)

Time (h)RL2Ku

MCF-7

62

53

1816

P62

Tubulin

LC3-ILC3-II

kDa

- + + - - - + ++ +- - - - + + + + + +0 24125 24125 24125

(b)

MDA-MB-231

53

Time (h)RL23MA

621816

P62

Tubulin

LC3-ILC3-II

kDa0 248324832483

- + + - - - + ++ +- - - - + + + + + +

(c)

MCF-7

53

50

1816

Time (h)RapRL2

Tubulin

ATG5

LC3-ILC3-II

kDa0 245

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(d)MDA-MB-231

62

53

50

1816

RL2Rap

0 245 Time (h)

P62

Tubulin

LC3-ILC3-II

ATG5

kDa

- + + - - - + ++ +- - - - + + + + + +

3 2453 2453

(e)

















0102030405060708090

100110120

Viab

ility

()



MCF-7MCF-7


3MA3MA+01 mgml RL2

10 20 300 403MA (mM)

0102030405060708090

100110120

Viab

ility

()

102030405060708090

100110120

Viab

ility

()

10 20 300 403MA (mM)

10 200 40303MA (mM)

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast



3MA3MA+01 mgml RL2

lowast

(a)

(b)

(d)

(e)

(c) (f)

P=005lowast





MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)


of -Casein antibody




SUIT cell lysate



(b)


protein ID



(c)


lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -




(d)



0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































0102030405060708090

100110120

Viab

ility

()



MCF-7MCF-7


3MA3MA+01 mgml RL2

10 20 300 403MA (mM)

0102030405060708090

100110120

Viab

ility

()

102030405060708090

100110120

Viab

ility

()

10 20 300 403MA (mM)

10 200 40303MA (mM)

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

0102030405060708090

100110

Viab

ility

()

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

2 4 6 8 100Rap (M)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast



3MA3MA+01 mgml RL2

lowast

(a)

(b)

(d)

(e)

(c) (f)

P=005lowast





MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)


of -Casein antibody




SUIT cell lysate



(b)


protein ID



(c)


lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -




(d)



0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















MDA-MB-231MCF-7

RL2

Con

tr RL2

Con

tr

CQ

RL2+

CQ Ku Ku

Ku +

RL2

Ku+R

L2

3MA

+ R

L2

SP +

RL2

Rap

+ RL

2

starv

RapSP

SP+R

L2

Rap+

RL2

RapSP

3MA

0

40

80

120

160

200

0

40

80

120

160

200

(a)


of -Casein antibody




SUIT cell lysate



(b)


protein ID



(c)


lysa

te

RL2

pull-

dow

n

Bead

cont

rol

larr RL215 -

50 -

37 -

50 -

37 -




(d)



0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

20000

ATP-

free w

ater

L-15

med

ium

cont

rol

RL2

20 h

RL2

44 h

RL2

+ CQ

20

h

RL2+

Rap

20 h

RL2+

Rap

44 h

Rap

20 h

Rap

44 h

RL2

+ CQ

44

h

CQ 2

0 h

CQ 4

4 h

40000

60000

80000

100000

120000

Extr

acel

lula

r ATP

RLU



4 Discussion



2O2







ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















ControlRL2CQRL2+CQ

Auto

phag

osom

es

MVB

MVB

AL

AL

AL

MVB

MVB

Ph

Ph

AL

(a) (b) (c)

(d) (e) (f)

0

20

40

60

80

100

auto

phag

olys

osom

es (

)

8h 24h4h

(g)








0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















0200400600800

10001200140016001800

tum

or v

olum

e (ＧＧ

3)

(a)

ControlCQRL2RL2+CQ


0102030405060708090

100

Perc

ent S

urvi

val

(b)






5 Conclusions




Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Data Availability


Ethical Approval






Acknowledgments


References






















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















cytotoxic and antitumor activity of lactaptin in...

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