ResearchArticle Angelica gigas Nakai Has Synergetic ...downloads.hindawi.com/journals/bmri/2018/6716547.pdf · ResearchArticle Angelica gigas Nakai Has Synergetic Effects on Doxorubicin-Induced

Research ArticleAngelica gigas Nakai Has Synergetic Effects onDoxorubicin-Induced Apoptosis

Yong-Joon Jeon 1 Jong-Il Shin 1 Sol Lee1 Yoon Gyeong Lee1 Ji Beom Kim1

Hak Cheol Kwon2 Sung Hun Kim2 Inki Kim3 Kyungho Lee 14 and Ye SunHan 5

1Department of Biological Sciences Konkuk University Neungdong-ro 120 Gwangjin-gu Seoul 05029 Republic of Korea2Korea Institute of Science and Technology Gangneung Gangwondo 25451 Republic of Korea3Department of Convergence Medicine Asan Institute for Life Sciences Asan Medical Center Seoul 05505 Republic of Korea4Korea Hemp Institute Konkuk University Konkuk University Neungdong-ro 120 Gwangjin-gu Seoul 05029 Republic of Korea5Department of Advanced Technology Fusion Konkuk University Neungdong-ro 120 Gwangjin-gu Seoul 05029 Republic of Korea

Correspondence should be addressed to Kyungho Lee kyunghokonkukackr and Ye Sun Han yshankonkukackr

Received 7 March 2018 Revised 11 July 2018 Accepted 24 July 2018 Published 1 August 2018

Academic Editor Claudio Tabolacci

Copyright copy 2018 Yong-Joon Jeon et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Natural products are valuable sources for drug discovery because they have a wide variety of useful chemical components andbiological properties A quick reevaluation of the potential therapeutic properties of established natural productswasmade possibleby the recent development of the methodology and improvement in the accuracy of an automated high-throughput screeningsystem In this study we screened natural product libraries to detect compounds with anticancer effects using HeLa cells Ofthe 420 plant extracts screened the extract of Angelica gigas Nakai (AGN) was the most effective in reducing cell viability ofHeLa cells Markers of apoptosis such as exposure of phosphatidylserine and cleavage of caspase-7 and PARP were increasedby treatment with the AGN extract Treatment of the AGN extract increased expression of PKR as well as ATF4 and CHOP theunfolded protein response genes In addition cotreatment of doxorubicin and theAGNextract significantly increased doxorubicin-induced apoptosis in HeLa cells Decursin and decursinol angelate which were known to have anticancer effects were the maincomponents of the AGN extract These results suggest that the extract of AGN containing decursin and decursinol angelateincreases doxorubicin susceptibility

1 Introduction

Doxorubicin (adriamycin) belonging to the anthracyclinegroup was initially derived from Streptomyces peucetius inthe 1960s [1] Owing to its wide range of anticancer effectsagainst various types of cancers including solid tumors andhematological malignancies doxorubicin has occupied animportant place in chemotherapy [2 3] The application ofdoxorubicin however hasmany side effects including cardiactoxicity therefore the dose of doxorubicin has been limited[4] In addition multidrug resistance or chemoresistanceprompted by chemotherapy reduced doxorubicin susceptibil-ity further limiting its use [5] Despite these disadvantagesdoxorubicin is still an attractive chemotherapeutic drug Touse doxorubicin more efficiently various therapies have beenproposed including the use of combination therapy as a

treatment strategy Consequently chemotherapy regimensusing doxorubicin such as FAC (fluorouracil doxorubicinand cyclophosphamide) TAC (docetaxel doxorubicin andcyclophosphamide) and R-CHOP (rituximab cyclophos-phamide doxorubicin vincristine and prednisone) havebeen administered to cancer patients [6 7]

Ever since natural products have been recognized askey components of traditional medicines many drugs andtherapies using natural products have been developed [89] Consequently 24 natural products were developed intoapproved novel drugs between 1970 and 2006 [10] Howeverit is very difficult to select the biologically useful natural prod-ucts from among the wide diversity of natural productsAlthough high-throughput screening (HTS) is an efficientmethod for selecting various natural products it is knownto have drawbacks during natural product screening [11] In

HindawiBioMed Research InternationalVolume 2018 Article ID 6716547 11 pageshttpsdoiorg10115520186716547

2 BioMed Research International

this study we screened natural product libraries via HTS andapplied the MTT assay to select the extract of Angelica gigasNakai (AGN) which exhibited anticancer properties

The genus Angelica L belonging to the Umbelliferae fam-ily is distributed in Asia Europe and North America andcomprises more than 60 species [12 13] In China Japanand Korea Angelica L has been used as a traditional herbalmedicine for curing colds pain and anemia and has beenknown as the ldquofemale ginsengrdquo due to its beneficial effects onfemale health [12 14 15] Angelica L contains a varietyof bioactive metabolites such as pyranocoumarins essen-tial oils and polyacetylenes which exhibit many beneficialeffects including anticancerous anti-inflammatory antifun-gal antioxidant and neuroprotective properties [13 14]Pyranocoumarins which are known to be associated withthe anticancer effect of Angelica L are more abundant inthe roots of the Angelica gigas Nakai (local name dang-gui inKorea) growing inKorea than in the species growing inChinaand Japan [16] Pyranocoumarins are the major componentsof the alcoholic extract of AGN [17] Decursin decursinolangelate and decursinol are representative pyranocoumarinsin the AGN extract Decursin the most abundant pyra-nocoumarin in the AGN extract has been reported toshow anticancer effects in various cancer cells [13 14] Inaddition the alleviation of neurotoxicity and nephrotoxicityvia its antioxidant properties is the other beneficial effect ofdecursin [18 19] As neurotoxicity and nephrotoxicity aresome of the side effects of doxorubicin [20] the combinationof doxorubicin and the AGN extract could offer a strategy forincreasing the effectiveness of doxorubicin

Integrated stress response (ISR) is a cellular stressresponse induced by various stress stimuli leading to thephosphorylation of eukaryotic translation initiation factor2 alpha (eIF2120572) The phosphorylation of eIF2120572 is mediatedby four kinases General Control Nondepressible protein 2(GCN2) Protein Kinase R (PKR) PKR-like ER localizedeIF2120572 Kinase (PERK) and Heme-Regulated Inhibitor kinase(HRI) [21 22] Although each kinase recognizes differentstimuli ISR is initiated by very diverse stress stimuli viathe four kinases and prompts a cellular response for deter-mining cell fate The upregulation of eIF2120572 phosphorylationattenuates global protein translation to reduce cellular stress[21] However the rates of translation of mRNAs includingthe second 51015840-uORF such as activating transcription factor4 (ATF4) are further increased by eIF2120572 phosphorylation[23] ATF4 activates the expression of genes that regulatecellular homeostasis in order to protect cells or increasesthe expression of downstream transcription factors such asCEBP-homologous protein (CHOP) [24] In conditions ofsevere stress ATF4 and CHOP induce cell death by activatingdownstream death factors or by producing ROS via increasedprotein synthesis [24 25] Therefore ISR is an importantmechanism for determining cell fate by inducing a cellularresponse by various cellular stimuli via the eIF2120572-ATF4pathway

In this study the AGN extract effectively induced apop-tosis in HeLa cells As the cell death induced by doxorubicinwas related to eIF2120572 phosphorylation we investigated thesynergetic effect between doxorubicin and the AGN extract

The administration of the AGN extract enhanced ATF4and CHOP expression in doxorubicin-treated HeLa cellsresulting in an increase in doxorubicin-induced apoptosis

2 Materials and Methods

Doxorubicin was purchased from Sigma-Aldrich (St LouisMO) The PKR inhibitor C16 was purchased from Cal-biochem (La Jolla CA) The anti-phospho eIF2120572 anti-eIF2120572 and anti-CHOP antibodies were purchased from CellSignaling Technology (Beverly MA)The anti-GAPDH anti-actin anti-caspase-3 anti-PKR anti-Bcl-2 and anti-PARPantibodies were purchased from Santa Cruz Biotechnology(Santa Cruz CA) The annexin V probe was purchasedfrom Bioacts (Korea) Hoechst 33342 was purchased fromInvitrogen (Carlsbad CA)

21 Cell Culture The HeLa cell line was obtained from theKorean Cell Line Bank (KCLB) The HeLa cells were cul-tured in Dulbeccorsquos modified Eaglersquos media (Welgene DaeguKorea) supplemented with 10 heat-activated fetal bovineserum (Biowest Nuaille France) and 1 penicillin or strep-tomycin mixtures (GIBCO ThermoFisher MA USA) TheHeLa cells were incubated in a humidified atmosphere at aCO2concentration of 5 and a temperature of 37∘C

22 Cell Viability Assay The cells were seeded in 48-wellplates and incubated for 16 h The cells were treated withdifferent concentrations of doxorubicin for 24 h Cell survivalwas measured using the MTT [3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide] assay (Sigma-AldrichST Louis MO) In brief PBS containing 5mgml MTT wasdiluted with the media at a concentration of 05mgml andincubated in a humidified chamber containing CO

2for 2 h

Themedium was aspirated from each well and 200120583l DMSOwas added to dissolve the Formazan crystals The absorbanceof each well was measured using a UVM 340 plate reader at awavelength of 570nm

23 Immunoblot Analyses The cells were harvested usingRIPA lysis buffer [containing 150mMNaCl 1 Triton X-1001 sodium deoxycholate 01 SDS 50mM Tris-HCl and2mM EDTA] along with 1 phosphatase inhibitor andprotease inhibitor cocktail (Roche Diagnostics Germany)The protein concentration was quantified using the PierceBCAprotein assay kit (Thermo Scientific Australia) Proteinsboiled in 1x sample buffer [containing 500mM Tris-HCl(pH 68) 10 SDS 20 glycerol 005 bromophenol blueand 1 120573-mercaptoethanol] for 5 minutes at 100∘C wereseparated on SDS-polyacrylamide gels The proteins wereelectrotransferred to Immobilon-P membranes (MilliporeTemecula CA) and blotted with the indicated antibodiesat 4∘C overnight in Tris-Buffered saline containing 008Tween 20 (TBST) and 1 nonfat milk The membraneswere then incubated with horseradish peroxidase-conjugatedantibodies at room temperature for 2 h and the band signalwas detected using a LAS-3000 Luminescent Image Analyzer(Fujifilm Japan) To determine the equal loading of sam-ples the blots were stripped in stripping buffer [containing

BioMed Research International 3

Table 1 EC50 values of the natural products

Natural product extract 478 622 1114 1197EC 50 value 4677 1036 2912 4326

100mM 120573-mercaptoethanol 2 SDS and 625mMTris-HCl(pH 68)] at 50∘C for 20 minutes followed by washing twicewith TBST buffer for 15minutes each time and reprobedwithan antibody specific for 120573-actin or GAPDH

24 Measurement of Apoptosis The cells were cultured in aconfocal dish and treated with doxorubicin andor the AGNextract for 24 h The cells were washed with PBS and bindingbuffer (20mM Hepes at pH 74 150mM NaCl and 25mMCaCl2) The staining solution was prepared by diluting the

annexin V probe and Hoechst 33342 with the binding bufferat concentration ratios of 1200 and 15000 respectively Thecells were stained with the staining solution for 20 minutesThe stained cells were observed using a confocal microscope(Zeiss LSM 800 Carl Zeiss)

25 Preparation of Crude Extract The crude extract samplesused in this study were provided by Natural Product Libraryof Korea Institute of Science and Technology GangneungInstitute Gangneung KoreaThe natural product library wasmade from Korean native plants The preparation of A gigasextract is as follows The roots of A gigas were purchasedin a local oriental medicine market in Bonghwa Korea in2015The plant materials were authenticated by Professor DSJang at College of Korean Medicine at Kyung Hee UniversityThe specimenwas deposited inKISTNatural Product Library(Deposit number BS0622A1) The dried materials (100 g)were cut and extracted twice with 1 L of ethanol by refluxat 60∘C for 2 hours Thereafter the extract was filtered andconcentrated using a rotary evaporator under vacuum at35∘C

26 Chemical Composition of the AGN Extract To investi-gate the chemical constituents of the AGN extract LCMSanalyses were performed on an Agilent 1200 HPLC systemequipped with UV and ESI-MS detection using a Phe-nomenex LunaC18 column (150times 46mm 5120583m)Themobilephase used was a linear gradient of 10-100 acetonitrile inwater (containing 005 formic acid) for over 30 minutesat a flow rate of 07mlmin The HPLC chromatogram wasmonitored at a UV wavelength of 254 nm Mass analysiswas performed using the positive-ion mode After analysesthe peaks in the HPLC chromatogram were identified bycomparing the obtained UV spectra and mass spectra withthose of compounds previously reported from A gigas

27 Quantitative Real-TimePCR Quantitative real-time PCRwas accomplished with HiPi Real-Time PCR 2timesMaster MixSYBR Green (ELPiS Biotechnology Korea) with 40 cyclesThe cycle threshold (cT) was observed in extension step andused for calculation of relative gene expression Analysis ofmelting curve was carried out in order to convict specificamplification

28 Statistical Analyses The values in the figures are ex-pressed as the mean plusmn SD The figures in this study representthe results of experiments performed more than three timesStatistical analyses of the data obtained from the control andthe treated groups were performed by using Studentrsquos t-testValues of P lt 005 indicate statistical significance

3 Results

31 Screening of the Most Effective Anticancer Candidate fromthe KIST Natural Product Library Initially natural productextract libraries were selected to obtain components withanti-inflammatory effects Since recent studies have shownthat cancer and inflammation are closely related [26] in thisstudywe investigated the anticancer effects of natural productextracts from the KIST Natural Product Library To selectextracts with anticancer properties from among approxi-mately 420 natural products HeLa cells were treated with 50120583gml of eachnatural product extract for 24 hours and naturalproduct extracts that markedly reduced cell viability to below50 were identified Through this process four extractsBE0478A1 BE0622A1 BE1114A1 and BE1197A1 wereselected In order to compare the anticancer effects of the fourextractswith EC50 valueHeLa cellswere treatedwith variousconcentrations of each extract for 24 hours and EC50 valueof the four natural product extracts was calculated (Table 1and Figure 1(a)) The results showed that the BE0622A1extract was the most efficient in reducing HeLa cell viabilitydepending on the concentration (Figure 1(a)) and the EC

50

value was the lowest compared to that of the other extracts(Table 1) Apoptosis has been recognized as an importantmechanism for cancer therapy and many anticancer drugsare known to induce apoptosis in cancer cells [27] Theactivation of caspase-7 (cCas-7) and the cleavage of PARPare representative markers of apoptosis Thus we comparedthe apoptosis induced by the extracts by observing cCas-7 activation and PARP cleavage (Figure 1(b)) Similarly tothe cell viability cCas-7 activation and PARP cleavage wereenhanced more after treatment with the BE0622A1 extractthan with extracts BE1114A1 and BE1197A1

The apoptosis mediated by the extract BE0622A1 wasalso confirmed by annexin V staining (Figure 1(c)) Amongthe natural products evaluated BE0622A1 proved to be themost effective anticancer extract in HeLa cells The extractBE0622A1 was prepared from root of Angelica gigas Nakai(AGN)

32 Activation of ISR by Treatment with the Angelica gigasNakai Extract ISR has been known to cope with diversestresses resulting in cell death or adaptation [21] Fourkinases sensing various stress stimuli phosphorylate eIF2120572to initiate ISR As the AGN extract is a crude mixture wespeculated that the AGN extract can offer numerous kinds of

4 BioMed Research International

0

039

078

156

312

5

625

125 25 50 100

(gml)0

20

40

60

80

100

120

Cel

l via

bilit

y (

of C

ontr

ol)

6221114

1197

(a)

0 25 375

GAPDH

25 375 25 375

PARP

cCas-7

622

(g

ml)

111

4(

gm

l)

111

97(

gm

l)

(b)

0 10622 (gml)

(c)

Figure 1 Angelica gigas Nakai (BE0622A1) one of the 420 natural products showed the most effective anticancer effect (a) The HeLa cellswere treated with various concentrations of the indicated natural products for 24 h and the MTT assay was subsequently performed (b)The HeLa cells were treated with various concentrations of BE0622A1 BE1114A1 and BE1197A1 for 24 h and cell lysates were subjectedto immunoblot analyses using specific antibodies for cleaved form of caspase-7 PARP and GAPDH (c) The HeLa cells were treated withvarious concentrations of BE0622A1 for 24 h and apoptosis was analyzed by annexin V staining with FITC conjugation

stimuli to HeLa cells Accordingly we investigated whetherthe AGN extract could increase phosphorylation of eIF2120572The EC

50value of the AGN extract was approximately 10

120583gml (Figure 1(a)) Based on these results the level of eIF2120572phosphorylation wasmeasured after treatment with the AGNextract for 16 h (Figure 2(a))The level of eIF2120572 phosphoryla-tion was not affected by concentration of the AGN extractHowever the expression of ATF4 and CHOP downstreamfactors of eIF2120572 was enhanced by treatment with the AGNextract Phosphorylation of eIF2120572 was increased in a time-dependent manner (Figure 2(b)) The phosphorylation ofeIF2120572was increased after 2 h treatmentwith theAGN extractAt the same time the expression of PKR one of the eIF2120572kinases was also increased The transcription of ATF4 andCHOP was increased in a time-dependent manner andexpression of ATF4 and CHOP was also increased sequen-tially after 4 h and 8 h respectively (Figures 2(b)ndash2(d))We therefore concluded that the AGN extract-mediatedapoptosis was associatedwith ISR via the eIF2120572-ATF4-CHOPpathway Treatment of the AGN extract also increased thesplicing of XBP1 mRNA suggesting activation of the IRE1120572pathway (data not shown)

33 The AGN Extract Showed a Synergetic Effect on theDoxorubicin-Induced Apoptosis Previous studies have dem-onstrated that phosphorylation of eIF2120572 improved doxorub-icin-mediated cell death in cancer cells [28 29] In additioncombination therapy has been widely used as a method forovercoming the limitations of chemotherapy in the treatmentof cancer [30] Therefore we hypothesized that doxorubicinand theAGNextract could exhibit a synergetic effect based oneIF2120572 phosphorylation To investigate whether AGN affectsdoxorubicin-induced cell death doxorubicin was coadmin-istered with the AGN extract for 24 h (Figure 3(a)) Althoughcotreatment with 05 120583gml AGN extract and various concen-tration of doxorubicin hardly affected doxorubicin-inducedcell viability cotreatment with 1 120583gml AGN extract and 1120583M doxorubicin significantly reduced the cell viability Wethen examined the effect of the AGN extract on doxorubicin-induced apoptosis through cCas-7 and PARP Cotreatmentwith 1120583M doxorubicin with 1 120583gml or 2 120583gml AGN extractmarkedly increased the activation of caspase-7 and thecleaved form of PARP (Figure 3(b)) which means that thedoxorubicin-mediated apoptosis was greatly enhanced bythe administration of the AGN extract Furthermore the

BioMed Research International 5

0 5 2010

CHOP

GAPDH

ATF4

p-eIF2

eIF2

AGN (gml)

(a)

0h 2h 4h 8h 16h 24h

CHOP

GAPDH

ATF4

PKR

Fold increase 1 33 29 62 95 71

AGN (10gml)

p-eIF2

eIF2

(b)

0 2h 4h 8h 16h 24hAGN (10gml)

005

115

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335

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5

ATF4

(Rel

ativ

e exp

ress

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lowastlowastlowast

(c)

0 2h 4h 8h 16h 24hAGN (10gml)

0

10

20

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60

70

CHO

P(R

elat

ive e

xpre

ssio

n)lowastlowastlowast

(d)

Figure 2 The AGN extract activated the integrated stress response (ISR) in HeLa cells The HeLa cells were treated with the indicatedconcentrations of the AGN extract for 16 h (a) or with 10 120583gml of the AGN extract for the indicated time periods (b) Cell lysates weresubjected to immunoblot analyses using the indicated antibodiesThe indicated fold increase in CHOP expression is the ratio of the CHOP toGAPDH (cd)ThemRNA levels of ATF4 and CHOPwere determined by real-time quantitative PCR Statistical significance of the differenceas calculated by Studentrsquos t-test is with lowastlowastlowastplt0001

administration of the AGN extract along with doxorubicinconsiderably increased the cleaved form of caspase-8 in con-trast to the administration of doxorubicin alone (Figure 3(b))The upregulation of the cleaved form of caspase-8 indicatesthat the receptor-mediatedextrinsic apoptotic pathway isactivated [31] The fluorescence intensity of the apoptoticmarker as indicated by annexin V staining in the Hoechst-stained cells was also stronger when the cells were cotreatedwith doxorubicin and theAGNextract thanwith doxorubicinalone (Figure 3(c)) These results suggest that doxorubicin-induced apoptosis was enhanced via the extrinsic apoptoticpathway by the administration of the AGN extract

34 Upregulation of Apoptosis via the ATF4-CHOP PathwayThe abovementioned results demonstrate that the AGNextract has a synergetic effect on doxorubicin-induced apop-tosis We further speculated whether ISR is correlated to theAGN extract-mediated synergy To confirm the correlationHeLa cells were cotreated with doxorubicin and the AGN

extract (Figure 4) Figures 4(a)ndash4(c) represent the time-dependent changes in transcriptional and translational levelexpression of ATF4 and CHOP at the indicated concen-trations and Figure 4(d) depicts the changes at variousconcentrations of doxorubicin and the AGN extract over24 hours Although the treatment with doxorubicin alonehardly enhanced the expression of ATF4 and CHOP cotreat-ment with the AGN extract and doxorubicin increased theexpression of ATF4 and CHOP However in comparison tothe administration of the AGN extract alone the expressionlevel of ATF4 and CHOP was low by cotreatment (Figures4(a) and 4(d)) The expression of death receptor 5 (DR5)which is a downstream factor of CHOP and related tocaspase-8 activation was not changed in correspondencewith the expression of CHOP (Figure 4) The expression ofDR5 therefore was not related to the ATF4-CHOP pathwayNevertheless these results suggest that the synergetic effect ofthe AGN extract is related to the upregulation of ATF4 andCHOP expression

6 BioMed Research International

0 05 1 2Dox (M)

lowast

0

20

40

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80

100

120C

ell v

iabi

lity

( o

f Con

trol

)

DMSOAGN-05

AGN-1

(a)

0 1 0 1 0 1

c-Cas-7

GAPDH

Caspase-8

PARP

0 1 2AGN (gml)Dox (gml)

(b)

Control Dox AGN Dox + AGN

(c)

Figure 3 Coadministration of the AGN extract with doxorubicin enhanced doxorubicin-induced apoptosis in HeLa cells (a) The cells werecotreatedwith the indicated concentrations of doxorubicin and theAGNextract for 24 h and cell viability wasmeasured using theMTT assayThe statistical significance of the differences as calculated by Studentrsquos t-test was determined with lowastp lt 001 (b)The cells were cotreated withthe indicated concentrations of doxorubicin and the AGN extract for 24 h and subjected to immunoblot analyses using specific antibodiesas indicated (c) The cells were cotreated with 1 120583M doxorubicin and 1 120583gml AGN extract for 16 h and apoptosis was analyzed by costainingwith annexin V-FITC and Hoechst probes

35 Chemical Composition of the AGN Extract Sownd-hararajan et al reported that there are differences in majorcomponents depending on the plant part yield and extrac-tionmethod in essential oil of various species ofAngelica [32]Therefore to clarify the major components and biologicallyactive components of the AGN extract the AGN extract wasdivided into eight fractions When HeLa cells were treatedwith various concentrations of each fraction for 24 hoursfraction 3 reduced HeLa cell viability most effectively (Sup-plementary Materials Figure S1a) Also fraction 3 not onlyactivated caspase 7 but also increased expression of CHOPin HeLa cells (Supplementary Materials Figure S1b) Wethen checked the HPLC-MS chromatogram and the 1H NMRspectrum of each fraction to find major components of eachfraction Figure 5(b) revealed that fraction 3 of the AGNextract contained three coumarins 7-demethylsuberosin(mz 231) decursin (mz 329) and decursinol angelate (mz329) Among these substances decursin and decursinolangelate were the main constituents (Figure 5(b)) Thesecompounds are known as the principal constituents of Agigas which have significant anticancer effects in various

cancer models [8 9] Therefore the results proposed thatthese compounds play an important role in induction ofapoptosis in HeLa cells

4 Discussion

Although doxorubicin is an efficient anticancer drug variousside effects such as drug resistance and cytotoxicity have lim-ited the use of doxorubicinMany attempts have beenmade toovercome the limitations of doxorubicin usage and enhanceits efficiency and combination therapy has frequently beenused as a strategy for the efficient use of doxorubicinIn the present study the combination of doxorubicin andthe AGN extract markedly enhanced doxorubicin-inducedapoptosis in HeLa cells (Figure 3) This event was associatedwith the AGN extract-mediated expression of ATF4 andCHOP (Figures 2 and 4) Particularly unlike in HeLa cellscotreatment of doxorubicin and the AGN extract did notsignificantly increase doxorubicin-induced apoptosis in wildtypeWI-38 cells (Supplementary Materials Figure S2)Theseresults demonstrate that administration of the AGN extract

BioMed Research International 7

- ++-

--++

++-

-++

16h8h

ATF4

GAPDH

DR5

CHOP

AGN (1gml)Dox (1 M)

(a)

8h 16h

- ++-

--++

++-

-++

lowastlowastlowast lowastlowast

0

05

1

15

2

25

3

ATF4

(Rel

ativ

e exp

ress

ion)

AGN (1gml)Dox (1 M)

(b)

8h 16h

- ++-

--++

++-

-++

lowastlowast

0102030405060708090

100

CHO

P(R

elat

ive e

xpre

ssio

n)

AGN (1gml)Dox (1 M)

(c)

ATF4

GAPDH

DR5

CHOP

0 1 0 1 0 10 1 2AGN (gml)

Dox (M)

(d)

Figure 4 Administration of the AGN extract activated the ATF4-CHOP pathway in doxorubicin-treated HeLa cells (a) The cells werecotreated with 1 120583M doxorubicin and 1 120583gml AGN extract for the indicated time periods and immunoblot analyses were performed usingthe indicated antibodies (b c) The mRNA level of ATF4 and CHOP were determined by real-time quantitative PCR Statistical significanceof the difference as calculated by Studentrsquos t-test is with lowastlowastplt001 or lowastlowastlowastplt0001 (d)The cells were cotreated with 1 120583M doxorubicin and 1-2120583gml AGN extract for 24 h as indicated and immunoblot analyses were performed using the indicated antibodies

increased the efficiency of doxorubicin in HeLa cells throughthe activation of ISR suggesting that the AGN extract workssynergistically with doxorubicin

The AGN extract was the most effective among thenatural products screened in inducing apoptosis in HeLacells (Figure 1) AGN-mediated apoptosis was associated withthe eIF2120572-ATF4-CHOP pathway (Figure 2) Accordinglywe investigated the activity of eIF2120572 kinases PERK andPKR under conditions that the cells were treated with theAGN extract alone PERK which is known to be activatedby ER stress [21] was not activated by treatment with 10120583gml AGN extract (data not shown) On the other handthe AGN extract increased the expression of PKR whichcorresponded to the time when eIF2120572 phosphorylation isincreased (Figure 2(b)) Treatment with the PKR inhibitorC16 significantly inhibited the activation of cCas-7 andrestored cell viability that had been reduced by treatmentwiththe AGN extract (Supplementary Materials Figures S3a andS3b) These data show that the AGN extract seems to induceapoptosis by activating PKR in HeLa cells Treatment withC16 however neither inhibited nor increased the expression

of ATF4 and CHOP (Supplementary Materials Figure S3c)Previous studies reported that the inhibition of PKR usingC16 decreased caspase-3 activation by inhibiting NF-120581B-induced inflammation and FADD phosphorylation [33 34]p53 is another PKR-mediated apoptotic signal transducer [2935] Therefore it seems that there is no correlation betweenthe activity of PKR and expression of ATF4-CHOP in AGNextract-treated HeLa cells upregulation of the eIF2120572-ATF4-CHOP pathway by treatment with C16 shows the possibilityof correlation to another eIF2120572 kinase

Expression of ATF4 and CHOP was not induced bytreatment of doxorubicin alone in HeLa cells but coad-ministration with the AGN extract markedly increased theexpression of both genes Indeed the expression level ofATF4 and CHOP was higher when the cells were treatedwith the AGN extract alone than with cotreatment (Figure 4)Treatment of breast cancer cells with doxorubicin effectivelyincreases the phosphorylation of eIF2120572 but suppresses theexpression of ATF4 at the transcription level [28 36] Nev-ertheless administration of the AGN extract increased theexpression of ATF4 and CHOP in doxorubicin-treated HeLa

8 BioMed Research International

O OHO

(1)

OOO

O

O

(2)

OOO

O

O

(3)

(a)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Time (min)

0

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232 2694296176 24802245 2414 264921861983 209519281805169115841511143113451263118910721012853811724668581533459125

1583233 26952110296 16051562 18551794150 1952 2349 2397 26461190 229314151070 1315694 992945882804635584475418079

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917 956737 1559 15711230232 269513811016753 1168594 879 1673295 1391704 20021816 1879118 232322182133 2405 26392524560407027

667 957232 1557 2695993918737 784 1570295107 1230 23302159 2411 2490 26431379 2109 22791169 199718151509318 593535044

693

231 857 2695719296 666098 2048 2150 2320 2520 265323901560977 1987534 1863181216491280 151313981221447 1135387

694688232 2694739296091 26162343 2452448 228621941557 2106200418841278 1649 183715131398786 1069 1232914356 623 984210

232 1560 26951572814296 696531 659080 2330 2408 2635253022181889 217019931840960 1350886 1503128211961059360 469181

CXcaliburdataKGIAg_crude 6282018 85806 PM

RT000 - 3000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Time (min)

0

100000

200000

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uAU

15641577

7001356

231 567 752 2694925 966677 850 1132299 1211 1236 1864 21231388 1504 1659 1770 2008 2312 23642254 2439999 2560458370 543179030 095

Crude Ex

F1 (Subfraction)

F2

F3

F4

F5

F6

F7

F8

(2) and (3)(1)

Crude Ex Conc 7mgmlSubfractions Conc 2mgml

(b)

Figure 5 Chemical composition of theAGN extract (a) Chemical structures of the three components (1) 7-demethylsuberosin (2) decursinand (3) decursinol angelate (b) HPLC-MS chromatograms of the AGN extract and the fractions

cells indicating that ATF4 and CHOP play a role in thesynergetic effect of doxorubicin and the AGN extract BothATF4 and CHOP are recognized as key transcription factorsfunctioning downstream of eIF2120572 which regulate the expres-sion of genes associated with cellular homeostasis and celldeath [24 37] Although many studies indicate that ATF4plays a role in cellular protection in association with theexpression of redox enzymes autophagy translation andmultidrug-resistant gene expression [24] ATF4 promotesproapoptotic factors such as Puma and Noxa [38 39]Therefore it is likely that increased expression of ATF4 by

the AGN extract enhanced doxorubicin-induced apoptosis inHeLa cells Indeed an increase in protein synthesis by ATF4expression induced ROS-mediated apoptosis [25] Howevertreatment with N-acetyl-cystein (NAC) which is a precursorof glutathione did not affect the cell viability in HeLa cellscotreatment with doxorubicin and the AGN extract (data notshown)

CHOP is a transcription factor functioning downstreamof ATF4 and is a well-known death factor in ISR CHOP-mediated apoptosis is associated with several apoptotic fac-tors including the anti- and proapoptotic Bcl-2 families

BioMed Research International 9

microRNAs TRB3 DR5 and GADD34 [40 41] DR5 isknown to induce apoptosis by activating caspase-8 [42] Infact cotreatment with doxorubicin and the AGN extractsignificantly increased the activation of caspase-8 (Figure 3)However administration of the AGN extract did not enhancedoxorubicin-mediated DR5 expression (Figure 4) Althoughthe increase in caspase-8 activation by the AGN extractwas not related to the expression of CHOP-DR5 the AGNextract is known to activate caspase-8 by enhancing theexpression of the DR5 ligand TRAIL [43] Accordingly it ispossible that upregulation of caspase-8 activation was causedby the expression of TRAIL in AGN extract-treated cellsInhibition of the anti-apoptotic protein Bcl-2 and activationof the proapoptotic protein BaxBak are known as CHOP-mediated apoptotic mechanisms [44 45] Treatment withthe ANG extract resulted in downregulation of Bcl-2 andupregulation of Bax expression [46] Also decursin anddecursinol angelate which aremajor components of theAGNextract effectively decreasedBcl-2 expression [43]Thereforethere is a possibility that CHOP functions as an apoptoticfactor in AGN extract-treated cells However knockdownof CHOP using specific shRNA did not affect the apoptosisin AGN extract-treated HeLa cells even in cotreated condi-tionswith doxorubicin (SupplementaryMaterials Figure S4)Therefore as mentioned above CHOP plays an importantrole in ISR-mediated apoptosis but does not seem to affectAGN extract-mediated apoptosis

Many studies have shown that decursin and decursinolangelate are major compounds of Angelica gigas Nakai andthey are known to have primary responsibility for the anti-cancer effect ofAngelica gigasNakai [13 14 46]Therefore wealso analyzed the composition of the AGN extract and foundthat decursin and decursinol angelate are major componentsof the AGN extract (Figure 5) Previous studies have shownthat decursin has synergetic effects with doxorubicin [4748] Decursin enhanced caspase-9-mediated apoptosis indoxorubicin-treated multiple myeloma cells via the mTORand STAT3 pathways [47] and other reports showed thatdecursin increased caspase-8-mediated apoptosis by increas-ing TRAIL sensitivity [49] Thus the apoptotic pathwayinduced by decursin might activate different pathwaysdepending on the cellrsquos characteristics and conditions Fur-ther studies to characterize the relationship between decursinand doxorubicin are needed Decursin inhibits the expressionof P-glycoprotein which is an efflux pump that reduces theefficiency of doxorubicin by lowering its cellular concentra-tion [48] Decursin is also known to inhibit cancer cell metas-tasis and angiogenesis [50 51] Therefore it could be used asan efficient component in combination therapies along withseveral other anticancer drugs including doxorubicin

5 Conclusions

Collectively our results showed that theAGNextract inducedexpression of PKR ATF4 and CHOP as well as phospho-rylation of eIF2120572 It significantly increased apoptosis andenhanced doxorubicin susceptibility in HeLa cells We alsoanalyzed the composition of the AGN extract and found thatdecursin and decursinol angelate were the main components

of the extract Consequently the AGN extract comprisingdecursin and decursinol angelate could be an effective mate-rial for coadministration in combination therapies along withdoxorubicin

Data Availability

All other data arising from this study are contained within thearticle and supplementary information files

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Yong-Joon Jeon and Jong-Il Shin contributed equally to thiswork

Acknowledgments

The natural product samples were provided by the KISTNatural Product Library supported by the KIST Institu-tional Program (2Z05320) This study was supported bya grant from the Korea Health Technology RampD Projectprovided to the Korea Health Industry Development Insti-tute (KHIDI) funded by the Ministry of Health amp Wel-fare Republic of Korea (Grant no HI15C1540) along withthe National Research Foundation of Korea (NRF) grantfunded by the Korean government (MSIP) (no NRF-2015R1C1A2A01053623)

Supplementary Materials

Figure S1 fraction 3 of AGN extract markedly inducedapoptosis and increased expression of CHOP in HeLa cells(a) The MTT assay was performed for measurement of cellviability (b) The cells were treated with 20 120583gml fractionof the AGN extract for 16 h Immunoblot analyses wereperformed using specific antibodies as indicated Figure S2AGNextract did not enhance doxorubicin-induced apoptosisin wild type WI-38 cells (a and b) WI-38 cells were treatedwith the indicated concentrations of the AGN extract for24 h (a) The MTT assay was performed for measurementof cell viability (b) Immunoblot analyses were performedusing specific antibodies as indicated (c) The cells werecotreated with the indicated concentrations of doxorubicinand the AGN extract for 24 h and cell viability was measuredusing the MTT assay (d) The cells were cotreated with 1120583M doxorubicin and 1 120583gml AGN extract for the indicatedtime periods and subjected to immunoblot analyses usingspecific antibodies as indicated Figure S3 C16 restoredthe AGN-mediated apoptosis regardless of the eIF2120572-ATF4-CHOP pathway (a and b) The cells were cotreated with theindicated AGN extract and C16 for 24 h (a) The MTT assaywas performed for measurement of cell viability Statisticalsignificance of the difference was calculated by Studentrsquos t-test with lowastplt001 (b) Immunoblot analyses were performedfor measurement of apoptosis using specific antibodies (c)

10 BioMed Research International

The cells were cotreated with 10 120583gml AGN extract and500 nM C16 for 4 h (top) or 24 h (bottom) and immunoblotanalyses were performed using specific antibodies FigureS4KnockdownofCHOPdid not affect the apoptosis inAGNextract-treated HeLa cells HeLa cells were transfected withEGFP- or CHOP-specific shRNA (a) The cells were treatedwith the indicated concentrations of the AGN extract for24 h and an MTT assay was performed to determine cellviability (b)The cells were treated with 10 120583gml AGN extractfor 16 h and subjected to immunoblot analyses using specificantibodies as indicated (c) The cells were cotreated with theindicated concentrations of doxorubicin and theAGN extractfor 24 h and cell viability was measured using the MTTassay (d) The cells were cotreated with 1 120583M doxorubicinand 1120583gml AGN extract for the indicated time periods andsubjected to immunoblot analyses using specific antibodiesas indicated (Supplementary Materials)

References

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[2] J V McGowan R Chung A Maulik I Piotrowska J MWalker and D M Yellon ldquoAnthracycline Chemotherapy andCardiotoxicityrdquo Cardiovascular Drugs and Therapy vol 31 no1 pp 63ndash75 2017

[3] S Rivankar ldquoAn overview of doxorubicin formulations incancer therapyrdquo Journal of Cancer Research and Therapeuticsvol 10 no 4 pp 853ndash858 2014

[4] SM Swain F SWhaley andM S Ewer ldquoCongestive heart fail-ure in patients treatedwith doxorubicin a retrospective analysisof three trialsrdquo Cancer vol 97 no 11 pp 2869ndash2879 2003

[5] L Bao A Haque K Jackson et al ldquoIncreased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistancein the Metastatic 4T1 Breast Cancer Modelrdquo The AmericanJournal of Pathology vol 178 no 2 pp 838ndash852 2011

[6] K R Hess et al ldquoPharmacogenomic Predictor of Sensitivity toPreoperative Chemotherapy With Paclitaxel and FluorouracilDoxorubicin andCyclophosphamide in Breast Cancerrdquo Journalof Clinical Oncology vol 24 no 26 pp 4236ndash4244 2007

[7] W Hiddemann M Kneba M Dreyling et al ldquoFrontline ther-apy with rituximab added to the combination of cyclophos-phamide doxorubicin vincristine and prednisone (CHOP)significantly improves the outcome for patients with advanced-stage follicular lymphoma compared with therapy with CHOPalone results of a prospective randomized study of the GermanLow-Grade Lymphoma Study Grouprdquo Blood vol 106 no 12 pp3725ndash3732 2005

[8] M S Butler ldquoThe role of natural product chemistry in drug dis-coveryrdquo Journal ofNatural Products vol 67 no 12 pp 2141ndash21532004

[9] M Kartal ldquoIntellectual property protection in the naturalproduct drug discovery traditional herbal medicine and herbalmedicinal productsrdquo Phytotherapy Research vol 21 no 2 pp113ndash119 2007

[10] A Ganesan ldquoThe impact of natural products upon moderndrug discoveryrdquo Current Opinion in Chemical Biology vol 12no 3 pp 306ndash317 2008

[11] JW Li and J CVederas ldquoDrug discovery and natural productsend of an era or an endless frontierrdquo Science vol 325 no 5937pp 161ndash165 2009

[12] S D Sarker and L Nahar ldquoNatural medicine the genusAngelicardquoCurrent Medicinal Chemistry vol 11 no 11 pp 1479ndash1500 2004

[13] J Zhang L Li C Jiang C Xing S-H Kim and J LuldquoAnti-cancer and other bioactivities of Korean Angelica gigasNakai (AGN) and its major pyranocoumarin compoundsrdquoAnti-Cancer Agents in Medicinal Chemistry vol 12 no 10 pp1239ndash1254 2012

[14] C Reddy S Kim M Hur et al ldquoNatural Korean MedicineDang-Gui Biosynthesis Effective Extraction and Formula-tions of Major Active Pyranocoumarins Their MolecularAction Mechanism in Cancer and Other Biological ActivitiesrdquoMolecules vol 22 no 12 p 2170 2017

[15] J H Park Y J Lee and S J Keon ldquoPharmacognostical studieson the Dang Gui from Koreardquo Korean Journal of Pharmacog-nosy vol 36 no 2 pp 141ndash144 2005

[16] S-KChoAMA El-Aty J-HChoiMRKim and JH ShimldquoOptimized conditions for the extraction of secondary volatilemetabolites in Angelica roots by accelerated solvent extractionrdquoJournal of Pharmaceutical and Biomedical Analysis vol 44 no5 pp 1154ndash1158 2007

[17] M A Yoo Y K Song H Jang D M Kim and S Y ByunldquoProfiling of skin anti-aging related proteins in human dermalfibroblasts by decursin in Angelica gigas NakairdquoKorean Journalof Chemical Engineering vol 28 no 3 pp 880ndash885 2011

[18] J H Kim S-J Jeong H-Y Kwon et al ldquoDecursin preventscisplatin-induced apoptosis via the enhancement of antioxidantenzymes in human renal epithelial cellsrdquo Biological amp Pharma-ceutical Bulletin vol 33 no 8 pp 1279ndash1284 2010

[19] L Li W Li S Jung Y Lee and Y Kim ldquoProtective Effects ofDecursin and Decursinol Angelate against Amyloid 120573-Protein-Induced Oxidative Stress in the PC12 Cell Line The Role ofNrf2 and Antioxidant Enzymesrdquo Bioscience Biotechnology andBiochemistry vol 75 no 3 pp 434ndash442 2014

[20] C Carvalho R X Santos S Cardoso et al ldquoDoxorubicin thegood the bad and the ugly effectrdquoCurrentMedicinal Chemistryvol 16 no 25 pp 3267ndash3285 2009

[21] K Pakos-Zebrucka I Koryga K Mnich M Ljujic A Samaliand A M Gorman ldquoThe integrated stress responserdquo EMBOReports vol 17 no 10 pp 1374ndash1395 2016

[22] N Donnelly et al ldquoThe eIF2alpha kinases their structures andfunctionsrdquo Cellular and Molecular Life Sciences vol 70 no 19pp 3493ndash3511 2013

[23] P D Lu H P Harding and D Ron ldquoTranslation reinitiation atalternative open reading frames regulates gene expression in anintegrated stress responserdquoThe Journal of Cell Biology vol 167no 1 pp 27ndash33 2004

[24] J Han and R J Kaufman ldquoPhysiologicalpathological ramifica-tions of transcription factors in the unfolded protein responserdquoGenes amp Development vol 31 no 14 pp 1417ndash1438 2017

[25] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013

[26] DHanahan andR AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011

[27] R S Y Wong ldquoApoptosis in cancer from pathogenesis to treat-mentrdquo Journal of Experimental amp Clinical Cancer Research vol30 no 1 article 87 2011

BioMed Research International 11

[28] Y J Jeon et al ldquoSalubrinal-Mediated Upregulation of eIF2120572Phosphorylation Increases Doxorubicin Sensitivity in MCF-7ADR Cellsrdquo Molecules and Cells vol 39 no 2 pp 129ndash1352016

[29] R L Bennett A L Carruthers T Hui K R Kerney X Liu andW S May ldquoIncreased Expression of the dsRNA-ActivatedProtein Kinase PKR in Breast Cancer Promotes Sensitivity toDoxorubicinrdquo PLoS ONE vol 7 no 9 Article ID e46040 2012

[30] A C Palmer and P K Sorger ldquoCombination Cancer TherapyCan Confer Benefit via Patient-to-Patient Variability withoutDrug Additivity or Synergyrdquo Cell vol 171 no 7 pp 1678ndash1682e13 2017

[31] S Elmore ldquoApoptosis a review of programmed cell deathrdquoToxicologic Pathology vol 35 no 4 pp 495ndash516 2007

[32] K Sowndhararajan P Deepa M Kim S J Park and S Kim ldquoAReview of the Composition of the Essential Oils and BiologicalActivities of Angelica Speciesrdquo Scientia Pharmaceutica vol 85no 3 p 33 2017

[33] J Couturier M Morel R Pontcharraud et al ldquo Interaction ofDouble-stranded RNA-dependent Protein Kinase (PKR) withthe Death Receptor Signaling Pathway in Amyloid 120573 (A120573)-treated Cells and in APP rdquoThe Journal of Biological Chemistryvol 285 no 2 pp 1272ndash1282 2010

[34] J Couturier M Paccalin M Morel et al ldquoPrevention of the120573-amyloid peptide-induced inflammatory process by inhibitionof double-stranded RNA-dependent protein kinase in primarymurine mixed co-culturesrdquo Journal of Neuroinflammation vol8 no 1 p 72 2011

[35] C-H Yoon E-S Lee D-S Lim and Y-S Bae ldquoPKR ap53 target gene plays a crucial role in the tumor-suppressorfunction of p53rdquoProceedings of theNational Acadamy of Sciencesof the United States of America vol 106 no 19 pp 7852ndash78572009

[36] S J Kim KM Park N Kim and Y I Yeom ldquoDoxorubicin pre-vents endoplasmic reticulum stress-induced apoptosisrdquo Bio-chemical and Biophysical ResearchCommunications vol 339 no2 pp 463ndash468 2006

[37] C Hetz ldquoThe unfolded protein response controlling cell fatedecisions under ER stress and beyondrdquo Nature Reviews Molec-ular Cell Biology vol 13 no 2 pp 89ndash102 2012

[38] G Qing B Li A Vu et al ldquoATF4 Regulates MYC-MediatedNeuroblastomaCell Death upon Glutamine Deprivationrdquo Can-cer Cell vol 22 no 5 pp 631ndash644 2012

[39] J L Armstrong R Flockhart G J Veal P E Lovat and C P FRedfern ldquoRegulation of endoplasmic reticulum stress-inducedcell death byATF4 in neuroectodermal tumor cellsrdquoThe Journalof Biological Chemistry vol 285 no 9 pp 6091ndash6100 2010

[40] Y Li Y Guo J Tang J Jiang and Z Chen ldquoNew insightsinto the roles of CHOP-induced apoptosis in ER stressrdquo ActaBiochimica et Biophysica Sinica vol 46 no 8 pp 629ndash640 2014

[41] Z Xu Y Bu N Chitnis C Koumenis S Y Fuchs and JA Diehl ldquomiR-216b regulation of c-Jun mediates GADD153CHOP-dependent apoptosisrdquoNature Communications vol 7 p11422 2016

[42] A R Safa andK E Pollok ldquoTargeting the anti-apoptotic proteinc-FLIP for cancer therapyrdquo Cancers vol 3 no 2 pp 1639ndash16712011

[43] N-H Yim J H LeeW-K ChoM C Yang D H Kwak and JY Ma ldquoDecursin and decursinol angelate from Angelica gigasNakai induce apoptosis via induction of TRAIL expression oncervical cancer cellsrdquo European Journal of Integrative Medicinevol 3 no 4 pp e293ndashe301 2011

[44] K D McCullough J L Martindale L O Klotz T Y Awand N J Holbrook ldquoGadd153 sensitizes cells to endoplasmicreticulum stress by down-regulating Bc12 and perturbing thecellular redox staterdquoMolecular and Cellular Biology vol 21 no4 pp 1249ndash1259 2001

[45] T Gotoh K Terada S Oyadomari and M Mori ldquohsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-inducedapoptosis by inhibiting translocation of Bax to mitochondriardquoCell Death amp Differentiation vol 11 no 4 pp 390ndash402 2004

[46] Y Jiang J Piao H-J Cho W-S Kang and H-Y KimldquoImprovement in antiproliferative activity of Angelica gigasNakai by solid dispersion formation via hot-melt extrusionand induction of cell cycle arrest and apoptosis in HeLa cellsrdquoBioscience Biotechnology and Biochemistry vol 79 no 10 pp1635ndash1643 2015

[47] J Jang S-J Jeong H-Y Kwon et al ldquoDecursin and Doxoru-bicin Are in Synergy for the Induction of Apoptosis via STAT3andor mTOR Pathways in Human Multiple Myeloma CellsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 506324 13 pages 2013

[48] H S Choi S-G Cho M K Kim et al ldquoDecursin in Angelicagigas Nakai (AGN) Enhances Doxorubicin Chemosensitivityin NCIADR-RES Ovarian Cancer Cells via Inhibition of P-glycoprotein Expressionrdquo Phytotherapy Research vol 30 no 12pp 2020ndash2026 2016

[49] J Kim M Yun E-O Kim et al ldquoDecursin enhances TRAIL-induced apoptosis throughoxidative stressmediated- endoplas-mic reticulum stress signalling in non-small cell lung cancersrdquoBritish Journal of Pharmacology vol 173 no 6 pp 1033ndash10442016

[50] S H SonM-J KimW-Y Chung et al ldquoDecursin and decursi-nol inhibit VEGF-induced angiogenesis by blocking the acti-vation of extracellular signal-regulated kinase and c-Jun N-terminal kinaserdquoCancer Letters vol 280 no 1 pp 86ndash92 2009

[51] S H Son K-K Park S K Park et al ldquoDecursin and decursinolfromAngelica gigas inhibit the lungmetastasis of murine coloncarcinomardquo Phytotherapy Research vol 25 no 7 pp 959ndash9642011

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