Research ArticleBaicalein Inhibits Orthotopic Human Non-Small Cell LungCancer Xenografts via SrcId1 Pathway

Zhengxiao Zhao 1 Baojun Liu 12 Jing Sun 12 Linwei Lu12 Lumei Liu 12

Jian Qiu12 Qiuping Li12 Chen Yan12 Shan Jiang12 Nabijan Mohammadtursun12

WenjuanMa3 Mihui Li12 Jingcheng Dong 1 andWeiyi Gong 1

1e Department of Integrative Medicine Huashan Hospital Fudan University 12 Middle Urumqi Road Shanghai 200040 China2e Institutes of Integrative Medicine of Fudan University 12 Middle Urumqi Road Shanghai 200040 China3Department of dermatology Huashan Hospital Fudan University Shanghai 200040 China

Correspondence should be addressed to Jingcheng Dong jcdong2004126com andWeiyi Gong onlygwy119163com

Received 29 October 2018 Revised 12 February 2019 Accepted 24 February 2019 Published 4 March 2019

Academic Editor Mohammed S Ali-Shtayeh

Copyright copy 2019 ZhengxiaoZhao et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Non-small cell lung cancer (NSCLC) is one of the most lethal cancers worldwide Inhibitor of differentiation 1 (Id1) is themember mostly linked to tumorigenesis in Id family and a potential molecular target in cancer therapy In the current studywe established an orthotopic lung cancer model by injecting athymic nude mice with A549 cells and evaluated the antitumoreffect of baicalein and expression of Id1-related proteins in vivo and in vitro Micro-CT images showed that tumor volume inbaicalein group was significantly reduced Western blot analysis revealed that baicalein suppressed the expression of Id1 proteinepithelial-to-mesenchymal transition (EMT) related molecules (N-Cadherin vimentin) and angiogenesis related protein (VEGF-A) accompanied by upregulation of epithelial markers (such as E-cadherin) In addition phosphorylation of upstream molecularSrc was significantly restrained after baicalein treatment This study firstly demonstrates that baicalein inhibits tumor growth inorthotopic human NSCLC xenografts via targeting SrcId1 pathway

1 Introduction

Lung cancer is currently the leading cause of cancer-relatedmortality worldwide with more than 13 million deathseach year [1] Although many new systemic therapies fortreating lung cancer have been developed in recent yearsthe prognosis has little improvement in the past decadesIn particular lung cancer is notorious for its propensity todevelop metastasis The five-year survival rates for patientswith metastatic lung cancer vary globally but are consistentlyless than 15 indicating that new approaches are urgentlyneeded for managing lung cancer [2 3]

Recently the role of inhibitor of differentiation 1 (Id1)in lung cancer pathogenesis has been gaining interest Id1belongs to the helix-loop-helix (HLH) family of transcrip-tional regulatory proteins which comprises four membersId1-4 and functions as dominant negative regulators of basicHLH transcriptional factors by preventing them binding toDNA [4] Among all the Id family members Id1 is the most

linked to carcinogenesis and associated with decreased celldifferentiation and induced cell proliferation Overexpressionof Id1 protein has been found inmany types of human cancerwhich correlated with tumor progression and unfavorableprognosis [5] Recently Id1 has been shown to be expressed ina variety of tissue microarray samples in non-small cell lungcancer (NSCLC) [6] Studies have shown that Src signalingpathways are necessary for expression of Id1 [7] Induction ofId1 has been reported to facilitate the growth and metastasisof NSCLC while knockdown of Id1 significantly suppressesthe proliferation migration and invasion of NSCLC cells[8 9] Genetic loss of Id1 in the host tissue also increasedsurvival and impaired liver colonization of Id1-- lung cancermice [10] These studies suggest that therapeutic targeting ofId1 is an attractive strategy for combating lung cancer

Scutellaria baicalensis Georgi (Lamiaceae family) one ofthe most popular and multipurpose herbs used in China hasa potential role in themanagement of cancer [11 12] Baicalein(Figure 1) is one of the major bioactive flavones derived

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 9806062 7 pageshttpsdoiorg10115520199806062

2 Evidence-Based Complementary and Alternative Medicine

OH O

O

HO

HO

Figure 1 Chemical structure of baicalein

from the root of Scutellaria It is cytotoxic to various tumorcells and suppresses tumor growth in vivo without systemictoxicity Several studies showed that baicalein inhibited lungcancer growth arrested cell cycle and decreased metastasisformation [13ndash16] Although baicalein has numerous pur-ported properties the link to Id1 activity in lung cancer is stillunknown

Our published data demonstrated that flavonoid com-ponents in Scutellaria baicalensis inhibit nicotine-inducedproliferation migration of NSCLC cells and lung cancer-associated inflammation in vitro [14] In the current studyour purpose was to determine whether baicalein was activeagainst Id1 in orthotopic lung cancer model Here wereported for the first time baicalein reduced tumor growthof orthotopic human NSCLC xenografts in treated athymicmice In parallel baicalein reduced Id1 protein expressionreversed the process of epithelial-to-mesenchymal transition(EMT) and suppressed expression of vascular endothelialgrowth factor-A (VEGF-A)which is essential to angiogenesisFurthermore the above effects of baicalein might be imple-mented by inhibition of Src phosphorylation These findingssuggest that identification of baicalein as modulators of Id1function may be a useful strategy in the treatment of cancer

2 Material and Methods

21 Chemicals Baicalein (purity gt 95 HPLC) was pur-chased from Meilun Bio (Dalian China) and preparedwith 05 CMC-Na solution Matrigel was purchased fromBD Biocoat (New Jersey USA) Sodium pentobarbital wasfrom Merck Drugs amp Biotechnology (New Jersey USA) Id1rabbit monoclonal antibody was from BioChek (San Fran-cisco USA) Antibodies including anti-E-Cadherin anti-N-cadherin anti-vimentin anti-120573-actin anti-Src and anti-p-Src(Tyr416) were purchased from Cell Signaling Technology(Massachusetts USA) and VEGF-A from Abcam (Cam-bridge UK) Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit-IgG were from Pierce Biotechnology(Rockford IL)

22 Animals Six-week-old Balbc male thymic nude mice(weighed 18-20 g) were obtained from BampK Laboratory Ani-mal Co Ltd (Shanghai China) The animals were housed ina temperature-controlled room (22∘C) with a 12 h light-darkcycle under pathogen-free conditions and had free access tofood and water All studies were performed in accordancewith the recommendations of the Guide for the Care andUse of Laboratory Animals of Fudan University of ChineseMedicine and all procedures were performed under the

supervision of the Animal Experimental Ethical Committeeof Fudan University (Approval Number 2015-01-HSYY-DJC-01)

23 Orthotopic Lung TumorModel and Treatments To estab-lish orthotopic xenografts subconfluent A549 cells wereharvested by a brief treatment with trypsinEDTA washedwith cold PBS by centrifugation and then resuspended inPBSmatrigel (11) and kept on ice before used The micewere anesthetized with sodium pentobarbital (ip 10mgkg)A 5mm incision was made on dorsal side over left lungabout 08-1 cm above the lower rib line Fat and muscleswere separated to visualize lung movement Tumor cells (1 times106 cells in 50120583L PBSMatrigel) were injected into left lungparenchyma directly at the depth of 3mm The wound wasclosed with suture which was removed five days later Bodyweights were recorded twice a week

Mice were randomly assigned to three equal groups (n = 8per group 4 per cage) normal control and baicalein groupMice in control group and baicalein group accepted thesurgery to establish orthotopic tumor model Normal groupaccepted a sham surgery Thirty days later baicalein groupwas intragastrically administered with baicalein (dissolvedin 05 CMC-Na 40mgkg∙d) for 28 days Control groupwas intragastrically administered with equal CMC-Na Inaddition 09 physiological saline at the same volume wasused as a normal control At the end of the experimentanimalswere sacrificed by cervical dislocation and their lungswere harvested and weighted

24 Micro-CT Scanning Micro-CT was performed fourweeks later after the start of treatment as described Brieflyserial lung imaging was scanned on an in vivo micro-CTsystem (SkyScan1076 BrukermicroCT Kontich Belgium)Data were acquired at 38120583m isotropic resolution 49 kV200120583A 360∘ rotation around the vertical axis rotation stepof 05∘ and camera exposure time of 250 millisecondsDuring in vivo imaging the animals were anesthetized with2 isoflurane in medical air and kept at a constant 37∘Ctemperature by regulated warm airflow Acquired projectionimages were reconstructed with NRecon v169 software(BrukermicroCT) with a beam hardening correction of 40and ring artifact correction of 10 resulting in the acquisitionof 518 cross sections per lung

25 Immunohistochemistry Lungs were excised from eachmouse fixed in 4 formalin and embedded in paraffinfor immunohistochemical staining Briefly sections 3 umthin cut from blocks were stained with hematoxylin andeosin stain (HE) Immunohistochemical staining was carriedout manually using rabbit monoclonal IgGs specific Id1antibody (1100 BioCheck) Sections were cut and submergedin EDTA buffer and heated in a microwave oven (100∘C)for antigen retrieval Endogenous peroxidase activity wasblocked by 15min of treatment with 03 hydrogen peroxideat 37∘C After rinsing the sections were further blocked by30min of treatment with 5 goat serum at 37∘C and thenincubated with the primary antibody at 4∘C overnight fol-lowed by biotin labeled secondary antibody developed with

Evidence-Based Complementary and Alternative Medicine 3

Normal

Control

Baicalein

(a)

HampE

(b)

Normal

Control

Baicalein

microCT

lowastlowast40

30

20

10

0

Tum

or v

olum

e (m

m3)

CMC-Na

Baicalein

(c)

08

06

04

02

00

Wei

ght o

f lun

g (g

)

Normal

Control

Baicale

in

(d)

35

30

25

20

150 20 40 60 80

Day

Mic

e wei

ght (

g)

NormalControl lowastBaicalein

lowastlowastlowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

(e)

Figure 2 Baicalein inhibited tumor growth of A549 orthotopic NSCLC xenografts (a) Gross images of normal lungs and tumor bearinglungs Nodules can be seen in control group (white arrows) (b) Hematoxylin-eosin (HampE) staining (200X 400X magnifications) of normallungs and tumor bearing lungs Black arrows indicated the tumor cells (c) Representative micro-CT images of mice in each group showedthe lung anatomy and tumor load (yellow arrows) after treatment (d) Weight of lungs (e) Changes of mice weight in each group over timeData was shown as mean plusmn SEM lowastPlt005 lowastlowastPlt001

331015840-diaminobenzidine For a negative control a nonspecificantibody was used instead of the primary antigen Stainingwas visualized and quantified by light microscope

26Western Blot Tissues or cells were lysed at 4∘C accordingto instructions of protein extraction kit (Beyotime Biotech-nology Haimen China) Protein concentrations were deter-mined by BCA protein assay kit (Beyotime) The proteinextract (20 120583g) was mixed with SDS-PAGE Sample LoadingBuffer and heated at 100∘C for 15 minutes Equal amountsof protein were separated by 12 SDS-polyacrylamide gelelectrophoresis and transferred to polyvinylidene difluoride(PVDF) membranes The membranes were then blockedat room temperature for 1 h with 5 (wv) nonfat milkin TBST buffer and incubated with primary antibodies in1 BSA overnight at 4∘C with continuous shaking Afterthree washes in TBST membranes were incubated with sec-ondary antibodies conjugated with horseradish peroxidasefor one hand visualized by enhanced chemiluminescence

using Supersignal West Femto Chemiluminescent Substrate(Pierce Biotechnology Inc Rockford IL USA) Band inten-sities were analyzed by NIH ImageJ software and normalizedto 120573-actin The western blot data were replicated three times

27 Statistical Analysis Mean values and standard errors ofthe mean were calculated for each point from the poolednormalized data The one-way analysis of variance (ANOVA)test (SPSS software package SPSS Inc Chicago IL USA)was used for evaluating the differences between groupsStatistical significance (Plt005) was established with post hoccomparison by the Dunnettrsquos test unless otherwise statedDatawere graphed byPrism60 (GraphPad Software La JollaCA)

3 Results

31 Baicalein Inhibited the Growth of Orthotopic HumanNSCLC Xenogras To assess the anti-tumor effect of

4 Evidence-Based Complementary and Alternative Medicine

1 1 12 2 2

Normal Control Baicalein

Id1

-actin

lowastlowastlowastlowast lowastlowastlowastlowast

Normal

Baicale

in

Control

0

20

40

60

Relat

ive e

xpre

ssio

n le

vel o

f Id1

(a)

Normal Control Baicaleinlowastlowast lowastlowast

Normal

Baicale

in

Control

0

20

40

60

80

o

f id1

pos

itive

cells

(b)

Figure 3 Baicalein inhibited Id1 expression of A549 orthotopic NSCLC xenografts (a)Western blot analysis was performed to determine theId1 protein Two representative mice were presented (b) Immunohistochemical analysis for Id1 of normal lung or xenograft tissues The totalnumber of Id1 positive cells (brown-stained nuclei regardless of staining intensity were counted as positive) in three random microscopicfields was counted by Image-Pro Plus 60 Data was shown as mean plusmn SEM (lowastlowastPlt 001 lowast lowast lowastlowastPlt 00001 compared with the control group)

baicalein we built an orthotopic lung cancer model in Balbcnude mice by A549 cells implantation Except for normalgroup mice were injected with 1x106 A549 cells into the leftlungs Thirty days following inoculation mice in baicaleingroupwere intragastrically administeredwith baicalein (05CMC-Na solution 40mgkg) Control group was intra-gastrically administered with CMC-Na (05 02mL permouse) and normal group was with 09 physiologicalsaline After 28 days of treatment mice were scanned withmicro-CT and sacrificed Lungs of mice were harvested graynodules could be seen in control group (Figure 2(a) whitearrows) In control and baicalein groups HE staining oflungs showed heterogeneous cells with larger eosinophilicnucleoli and vacuoles in the cytoplasm Visible glandularcavities could be seen among these cells (Figure 2(b) blackarrows) which indicated the success of orthotopic lungcancer model Micro-CT scanning exhibited much smallernodules in left lung of baicalein group comparing to con-trol group (Figure 2(c) yellow arrows) and the differenceswere significant (Plt001) We also measured the weightof lungs in each group Results showed that the lungs incontrol group were heavier than the other two groupsbut significant difference was not observed (Figure 2(d))Mice weight in normal group increased gradually over timeand was higher than that of control and baicalein-treatedgroups after 20th day of inoculation the difference between

normal group and control group was significant (Plt001)(Figure 2(e))

32 Baicalein Inhibited Id1 Expression To exploremechanismof the anti-tumor effect of baicalein we detected Id1 expres-sion in each group as Id1 is an essential tumor promoterwhich promotes the proliferation of tumor cells and facilitatestumor growth [17] When compared with lungs of normalmice lungs of tumor bearing mice expressed higher Id1protein (Plt00001) and baicalein significantly inhibited theId1 expression level (Plt00001) (Figure 3(a)) The immuno-histochemistry also showed much more Id1 positive cells incontrol group (Plt001) baicalein significantly reduced thenumber of Id1 positive cells (Plt001) (Figure 3(b))

33 Baicalein Abrogated Epithelial-Mesenchymal Transition(EMT) and Angiogenesis through SrcId1 Signaling PathwayTissue microarrays containing 532 NSCLC patientsrsquo samplesshow that Id1 is significantly correlated with EMT-relatedproteins [10]The loss of Id1 reduces the levels of EMT-relatedproteins [18] which indicate that EMT-related proteins maybe the downstream of Id1 in NSCLC Here we obtained theinhibiting effect of baicalein to Id1 protein thus we examinedthe expression of EMT-associated proteins (vimentin E-cadherin and N-cadherin) Results showed baicalein abro-gated the increase of N-cadherin (Plt001) and vimentin

Evidence-Based Complementary and Alternative Medicine 5

-actin

1 1 12 2 2

Normal Control Baicalein

VEGF-A

Vimentin

N-Cadherin

E-Cadherin

000510152025 lowastlowastlowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n of

Vim

entin

Normal

Control

Baicale

in

Normal

Control

Baicale

in

Relat

ive e

xpre

ssio

n of

E-C

adhe

rin

Relat

ive e

xpre

ssio

n of

N-C

adhe

rin

Normal

Control

Baicale

in

Normal

Control

Baicale

in00

05

10

15lowastlowast lowastlowast

0

1

2

3

4 lowastlowast lowastlowast

Relat

ive e

xpre

ssio

n of

VEG

F-A

000510152025 lowastlowast lowastlowast

(a)

-actin

p-Src(Tyr416)

Src

1 1 12 2 2Normal Control Baicalein

Normal

Control

Baicale

in

Normal

Control

Baicale

in

Relat

ive e

xpre

ssio

n le

vel o

f Src

00

05

10

15

Relat

ive e

xpre

ssio

n le

vel o

f p-S

rc (T

yr41

6)

00

05

10

15

20

25 lowastlowastlowastlowastlowastlowastlowastlowast

(b)

Figure 4 Baicalein suppressed the EMTprocedure VEGF-A andphosphorylation of SrcWestern blot was performed to analyze the (a) EMTrelated markers VEGF-A and (b) Src p-Src (Tyr416) Two representative mice were presented Data was shown as mean plusmn SEM lowastPlt005lowastlowastPlt001 lowast lowast lowastlowast Plt00001

(Plt001) and the decrease of E-cadherin in tumor bearinglung tissue (Plt001) (Figure 4(a)) A study showed that Id1has been implicated in VEGF-A regulation during tumorangiogenesis [19] and peritoneal expression of VEGF-A isregulated by TGF-1205731 through the ID1 pathway in womenwith endometriosis [20] In our research we observed thatVEGF-A was overexpressed in control group and baicaleinsignificantly inhibited the expression of VEGF-A (Plt001)(Figure 4(a)) To assess how baicalein inhibits the expressionof Id1 protein we analyzed the effect of baicalein to Srcand its phosphorylation as studies show that Src regulatesthe expression of Id1 in human lung adenocarcinoma andpancreatic adenocarcinoma [7 21] Our results showed thatbaicalein significantly reversed high phosphorylation of Srcin tumor bearing mice indicating that baicalein inhibits Id1in an Src dependent manner (Plt00001) (Figure 4(b))

34 Regulation of Baicalein on SrcId1 Signaling PathwayWas Verified In Vitro To verify the effect of baicalein toSrcId1 signaling pathway A549 cells were treated withbaicalein (10120583M) in different time points and expressions ofp-Src (Tyr416) Id1 E-cadherin N-cadherin vimentin and

VEGF-Awere determined by using western blot As shown inFigure 5 baicalein inhibited Id1 expression time dependentlyWhen A549 cells were treated with baicalein for 24 h or 36 hthe phosphorylation of Src and expressions of N-cadherinand vimentin were significantly suppressed (Plt001) Whentreated for 36 h VEGF-A was also reduced significantlyAs for E-Cadherin baicalein significantly upregulated itsexpression (Plt00001)

4 Discussion

We established an orthotopic lung cancer model through avisible transthoracic injection with A549 cells and demon-strated that baicalein was effective in this model We iden-tified Id1 as the pivotal respondent to baicalein in itsanti-tumor effect The downstream molecules of Id1 (N-cadherin vimentin and VEGF-A) were further suppressedand epithelial marker (E-cadherin) was increased We alsodemonstrated that baicalein might function through inhibit-ing the phosphorylation of Src as it was the upstreamof Id1 In light of our results we propose baicalein as apromising adjuvant therapy phytochemical and Id1 and its

6 Evidence-Based Complementary and Alternative Medicine

Id1

Relat

ive e

xpre

ssio

n le

vel

Src00

05

10

15

5

10

15

0 h12 h

24 h36 h

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

-actin

VEGF-A

Vimentin

N-Cadherin

E-Cadherin

p-Src(Tyr416)

Src

0 hBaicalein 12 h 24 h 36 h

p-Src(T

yr416

)

E-Cad

herin

N-Cad

herin

Vimentin

VEGF-A Id 1

Figure 5 Baicalein regulated SrcId1 signaling pathway A549 cells were treated with baicalein (10120583M) for 0 h 12 h 24 h or 36 h and westernblot was performed to determine expressions of p-Src (Tyr416) Id1 E-cadherin N-cadherin vimentin and VEGF-A Data was shown asmean plusmn SEM lowastPlt005 lowastlowastPlt001

mediators as candidates for targeted anti-tumor therapeuticstrategies

Nowadays lung cancer is the most lethal cancer type inwhich NSCLC accounts for approximately 85 of all lungcancers [22 23] Except for chemotherapy drugs a varietyof other medicines for instance EGFR inhibitors ALKinhibitors angiogenesis inhibitors and immune checkpointinhibitors are available in the market [24] However despitethe advances in therapies the five-year survival of NSCLCremains lowThis highlights the necessity for alternative treat-ments for unresectable NSCLC bearing patients Baicaleinis a main component of Scutellaria baicalensis which hasbeen used to treat diseases for thousands of years in ChinaProperties of its effectiveness and low toxicity have gainedadmiration of patients The anticancer potential of baicaleinwas observed in lung cancer bladder cancer breast cancerovarian cancer etc [25] Nevertheless most of these studiesobtained the results through experiments in vitro or subcu-taneous xenografts in vivo model Our study firstly demon-strates its anticancer potential by using orthotopic lungcancer model which mimics the clinical situation preferably(Figure 2) This is of significant importance for preclinicaldrug research and provides more powerful evidence forbaicalein of being a promising anticancer therapeutic drug

Researchers have shown that baicalein may functionthrough apoptosis induction autophagy triggering cell cyclearrest or inhibition of 12-lipoxygenase (LOX) [12] Ourstudy identified Id1 as a critical respondent to baicaleinfor the first time Id1 is a member of HLH family whichfunctions as a differentiation inhibitor and it is overexpressedin many cancers and facilitates the growth and metastasisof cancer [5] In our study tumor burden increased the

expression of Id1 and baicalein significantly inhibited itsexpression time dependently (Figures 3 and 5) and thusproliferation and metastasis of tumor cells were suppressedWe also showed that mesenchymal markers (vimentin andN-cadherin) and angiogenesis marker (VEGFA) increasedand epithelial markers (E-cadherin) significantly decreasedin lungs of tumor bearing mice and baicalein regressedthese effects in vivo and in vitro This indicated that EMTprocedure and angiogenesis of lung cancer were abrogated bybaicalein Studies have shown that Src is the upstream of Id1We observed that baicalein could inhibit the p-Src (Tyr416)expression which indicated that its effect to Id1 might bethrough inhibiting Src phosphorylation (Figures 4 and 5)

In conclusion our data demonstrated the antitumoreffect of baicalein in orthotopic NSCLC model for the firsttime and clarified that SrcId1 pathway was involved inthe baicalein-induced inhibition of tumor growth providingfurther insight into the therapeutic strategies

Data Availability

All data generated or analyzed during this study are includedin this article

Conflicts of Interest

The authors declare no conflicts of interest in relation to thework described

Acknowledgments

This study was funded by grants from National Natu-ral Science Foundation of China [Grant nos 81403148

Evidence-Based Complementary and Alternative Medicine 7

81673916 8150150396 and 81703829] Shanghai MunicipalCommission of Health and Family Planning [Grant noZYKC201602001] and Development Project of ShanghaiPeak Disciplines Integrative Medicine [Grant no 20150407]

References

[1] L A Torre F Bray R L Siegel J Ferlay and J Lortet-Tieulent ldquoGlobal cancer statistics 2012rdquo CA A Cancer Journalfor Clinicians vol 65 no 2 pp 87ndash108 2015

[2] C Gridelli A Rossi D P Carbone et al ldquoNon-small-cell lungcancerrdquo Nature Reviews Disease Primers vol 1 article 150092015

[3] C Liam S Andarini P Lee J C Ho N Q Chau and JTscheikuna ldquoLung cancer staging now and in the futurerdquoRespirology vol 1 pp 526ndash534 2015

[4] R Benezra R L Davis D Lockshon D L Turner and HWeintraub ldquoThe protein Id a negative regulator of helix-loop-helix DNA binding proteinsrdquoCell vol 61 no 1 pp 49ndash59 1990

[5] J Perk A Iavarone and R Benezra ldquoId family of helix-loop-helix proteins in cancerrdquo Nature Reviews Cancer vol 5 no 8pp 603ndash614 2005

[6] S I Rothschild A Kappeler D Ratschiller et al ldquoThe stem cellgene ldquoinhibitor of differentiation 1rdquo (ID1) is frequently expressedin non-small cell lung cancerrdquo Lung Cancer vol 71 no 3 pp306ndash311 2011

[7] J G Trevino S Pillai S Kunigal et al ldquoNicotine inducesinhibitor of differentiation-1 in a Src-dependent pathway pro-motingmetastasis and chemoresistance in pancreatic adenocar-cinomardquo Neoplasia vol 14 no 12 pp 1102ndash1114 2012

[8] R Bhattacharya J Kowalski A R Larson M Brock and RM Alani ldquoId1 promotes tumor cell migration in nonsmall celllung cancersrdquo Journal of Oncology vol 2010 Article ID 8561058 pages 2010

[9] J Li Y Li B Wang Y Ma and P Chen ldquoId-1 promotes migra-tion and invasion of non-small cell lung cancer cells throughactivating NF-120581B signaling pathwayrdquo Journal of BiomedicalScience vol 24 article 95 2017

[10] E Castanon A Soltermann I Lopez et al ldquoThe inhibitor ofdifferentiation-1 (Id1) enables lung cancer liver colonizationthrough activation of an EMT program in tumor cells andestablishment of the pre-metastatic nicherdquo Cancer Letters vol402 pp 43ndash51 2017

[11] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009

[12] W-Y Gong Z-X Zhao B-J Liu L-W Lu and J-C DongldquoExploring the chemopreventive properties and perspectives ofbaicalin and its aglycone baicalein in solid tumorsrdquo EuropeanJournal of Medicinal Chemistry vol 126 pp 844ndash852 2017

[13] C Naveenkumar S Asokkumar S Raghunandhakumar et alldquoPotent antitumor and antineoplastic efficacy of baicalein onbenzo(a)pyrene-induced experimental pulmonary tumorigen-esisrdquo Fundamental amp Clinical Pharmacology vol 26 no 2 pp259ndash270 2012

[14] W-Y I Gong J-F Wu B-J Liu et al ldquoFlavonoid componentsin Scutellaria baicalensis inhibit nicotine-induced proliferationmetastasis and lung cancer-associated inflammation in vitrordquoInternational Journal of Oncology vol 44 no 5 pp 1561ndash15702014

[15] J Gao W A Morgan A Sanchez-Medina and O CorcoranldquoThe ethanol extract of Scutellaria baicalensis and the activecompounds induce cell cycle arrest and apoptosis includingupregulation of p53 and Bax in human lung cancer cellsrdquoToxicology and Applied Pharmacology vol 254 no 3 pp 221ndash228 2011

[16] G Su H Chen and X Sun ldquoBaicalein suppresses non smallcell lung cancer cell proliferation invasion and Notch signalingpathwayrdquo Cancer Biomarkers vol 22 no 1 Article ID 170673pp 13ndash18 2018

[17] C Roschger and C Cabrele ldquoThe Id-protein family in develop-mental and cancer-associated pathwaysrdquo Cell Communicationand Signaling vol 15 article 26 2017

[18] E Cubillo A Diaz-Lopez E P Cuevas et al ldquoE47 and Id1interplay in epithelial-mesenchymal transitionrdquo PLoS One vol8 article e59948 2013

[19] M-T Ling T C M Lau C Zhou et al ldquoOverexpression ofId-1 in prostate cancer cells promotes angiogenesis throughthe activation of vascular endothelial growth factor (VEGF)rdquoCarcinogenesis vol 26 no 10 pp 1668ndash1676 2005

[20] V J Young S F Ahmad J K Brown W C Duncan and AWHorne ldquoPeritoneal VEGF-A expression is regulated by TGF-beta1 through an ID1 pathway in women with endometriosisrdquoScientific Reports vol 5 article 16859 2015

[21] S I Rothschild M P Tschan E A Federzoni et alldquoMicroRNA-29b is involved in the Src-ID1 signaling pathwayand is dysregulated in human lung adenocarcinomardquoOncogenevol 31 no 38 pp 4221ndash4232 2012

[22] World Health Organization ldquoGLOBOCAN 2012 Estimatedcancer incidence mortality and prevalence worldwiderdquo 2012httpglobocaniarcfrPagesfact sheets canceraspx

[23] D S Ettinger W Akerley and G Bepler ldquoNonsmall cell lungcancerrdquo Journal of the National Comprehensive Cancer Networkvol 8 pp 740ndash801 2010

[24] J Minguet K H Smith and P Bramlage ldquoTargeted therapiesfor treatment of non-small cell lung cancer - Recent advancesand future perspectivesrdquo International Journal of Cancer vol138 no 11 pp 2549ndash2561 2016

[25] H Liu Y Dong Y Gao et al ldquoThe fascinating effects ofbaicalein on cancer a reviewrdquo International Journal ofMolecularSciences vol 17 article 168110 2016

Evidence-Based Complementary and Alternative Medicine 3

Normal

Control

Baicalein

(a)

HampE

(b)

Normal

Control

Baicalein

microCT

lowastlowast40

30

20

10

0

Tum

or v

olum

e (m

m3)

CMC-Na

Baicalein

(c)

08

06

04

02

00

Wei

ght o

f lun

g (g

)

Normal

Control

Baicale

in

(d)

35

30

25

20

150 20 40 60 80

Day

Mic

e wei

ght (

g)

NormalControl lowastBaicalein

lowastlowastlowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

(e)

Figure 2 Baicalein inhibited tumor growth of A549 orthotopic NSCLC xenografts (a) Gross images of normal lungs and tumor bearinglungs Nodules can be seen in control group (white arrows) (b) Hematoxylin-eosin (HampE) staining (200X 400X magnifications) of normallungs and tumor bearing lungs Black arrows indicated the tumor cells (c) Representative micro-CT images of mice in each group showedthe lung anatomy and tumor load (yellow arrows) after treatment (d) Weight of lungs (e) Changes of mice weight in each group over timeData was shown as mean plusmn SEM lowastPlt005 lowastlowastPlt001

331015840-diaminobenzidine For a negative control a nonspecificantibody was used instead of the primary antigen Stainingwas visualized and quantified by light microscope

26Western Blot Tissues or cells were lysed at 4∘C accordingto instructions of protein extraction kit (Beyotime Biotech-nology Haimen China) Protein concentrations were deter-mined by BCA protein assay kit (Beyotime) The proteinextract (20 120583g) was mixed with SDS-PAGE Sample LoadingBuffer and heated at 100∘C for 15 minutes Equal amountsof protein were separated by 12 SDS-polyacrylamide gelelectrophoresis and transferred to polyvinylidene difluoride(PVDF) membranes The membranes were then blockedat room temperature for 1 h with 5 (wv) nonfat milkin TBST buffer and incubated with primary antibodies in1 BSA overnight at 4∘C with continuous shaking Afterthree washes in TBST membranes were incubated with sec-ondary antibodies conjugated with horseradish peroxidasefor one hand visualized by enhanced chemiluminescence

using Supersignal West Femto Chemiluminescent Substrate(Pierce Biotechnology Inc Rockford IL USA) Band inten-sities were analyzed by NIH ImageJ software and normalizedto 120573-actin The western blot data were replicated three times

27 Statistical Analysis Mean values and standard errors ofthe mean were calculated for each point from the poolednormalized data The one-way analysis of variance (ANOVA)test (SPSS software package SPSS Inc Chicago IL USA)was used for evaluating the differences between groupsStatistical significance (Plt005) was established with post hoccomparison by the Dunnettrsquos test unless otherwise statedDatawere graphed byPrism60 (GraphPad Software La JollaCA)

3 Results

31 Baicalein Inhibited the Growth of Orthotopic HumanNSCLC Xenogras To assess the anti-tumor effect of

4 Evidence-Based Complementary and Alternative Medicine

1 1 12 2 2

Normal Control Baicalein

Id1

-actin

lowastlowastlowastlowast lowastlowastlowastlowast

Normal

Baicale

in

Control

0

20

40

60

Relat

ive e

xpre

ssio

n le

vel o

f Id1

(a)

Normal Control Baicaleinlowastlowast lowastlowast

Normal

Baicale

in

Control

0

20

40

60

80

o

f id1

pos

itive

cells

(b)

Figure 3 Baicalein inhibited Id1 expression of A549 orthotopic NSCLC xenografts (a)Western blot analysis was performed to determine theId1 protein Two representative mice were presented (b) Immunohistochemical analysis for Id1 of normal lung or xenograft tissues The totalnumber of Id1 positive cells (brown-stained nuclei regardless of staining intensity were counted as positive) in three random microscopicfields was counted by Image-Pro Plus 60 Data was shown as mean plusmn SEM (lowastlowastPlt 001 lowast lowast lowastlowastPlt 00001 compared with the control group)

baicalein we built an orthotopic lung cancer model in Balbcnude mice by A549 cells implantation Except for normalgroup mice were injected with 1x106 A549 cells into the leftlungs Thirty days following inoculation mice in baicaleingroupwere intragastrically administeredwith baicalein (05CMC-Na solution 40mgkg) Control group was intra-gastrically administered with CMC-Na (05 02mL permouse) and normal group was with 09 physiologicalsaline After 28 days of treatment mice were scanned withmicro-CT and sacrificed Lungs of mice were harvested graynodules could be seen in control group (Figure 2(a) whitearrows) In control and baicalein groups HE staining oflungs showed heterogeneous cells with larger eosinophilicnucleoli and vacuoles in the cytoplasm Visible glandularcavities could be seen among these cells (Figure 2(b) blackarrows) which indicated the success of orthotopic lungcancer model Micro-CT scanning exhibited much smallernodules in left lung of baicalein group comparing to con-trol group (Figure 2(c) yellow arrows) and the differenceswere significant (Plt001) We also measured the weightof lungs in each group Results showed that the lungs incontrol group were heavier than the other two groupsbut significant difference was not observed (Figure 2(d))Mice weight in normal group increased gradually over timeand was higher than that of control and baicalein-treatedgroups after 20th day of inoculation the difference between

normal group and control group was significant (Plt001)(Figure 2(e))

32 Baicalein Inhibited Id1 Expression To exploremechanismof the anti-tumor effect of baicalein we detected Id1 expres-sion in each group as Id1 is an essential tumor promoterwhich promotes the proliferation of tumor cells and facilitatestumor growth [17] When compared with lungs of normalmice lungs of tumor bearing mice expressed higher Id1protein (Plt00001) and baicalein significantly inhibited theId1 expression level (Plt00001) (Figure 3(a)) The immuno-histochemistry also showed much more Id1 positive cells incontrol group (Plt001) baicalein significantly reduced thenumber of Id1 positive cells (Plt001) (Figure 3(b))

33 Baicalein Abrogated Epithelial-Mesenchymal Transition(EMT) and Angiogenesis through SrcId1 Signaling PathwayTissue microarrays containing 532 NSCLC patientsrsquo samplesshow that Id1 is significantly correlated with EMT-relatedproteins [10]The loss of Id1 reduces the levels of EMT-relatedproteins [18] which indicate that EMT-related proteins maybe the downstream of Id1 in NSCLC Here we obtained theinhibiting effect of baicalein to Id1 protein thus we examinedthe expression of EMT-associated proteins (vimentin E-cadherin and N-cadherin) Results showed baicalein abro-gated the increase of N-cadherin (Plt001) and vimentin

Evidence-Based Complementary and Alternative Medicine 5

-actin

1 1 12 2 2

Normal Control Baicalein

VEGF-A

Vimentin

N-Cadherin

E-Cadherin

000510152025 lowastlowastlowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n of

Vim

entin

Normal

Control

Baicale

in

Normal

Control

Baicale

in

Relat

ive e

xpre

ssio

n of

E-C

adhe

rin

Relat

ive e

xpre

ssio

n of

N-C

adhe

rin

Normal

Control

Baicale

in

Normal

Control

Baicale

in00

05

10

15lowastlowast lowastlowast

0

1

2

3

4 lowastlowast lowastlowast

Relat

ive e

xpre

ssio

n of

VEG

F-A

000510152025 lowastlowast lowastlowast

(a)

-actin

p-Src(Tyr416)

Src

1 1 12 2 2Normal Control Baicalein

Normal

Control

Baicale

in

Normal

Control

Baicale

in

Relat

ive e

xpre

ssio

n le

vel o

f Src

00

05

10

15

Relat

ive e

xpre

ssio

n le

vel o

f p-S

rc (T

yr41

6)

00

05

10

15

20

25 lowastlowastlowastlowastlowastlowastlowastlowast

(b)

Figure 4 Baicalein suppressed the EMTprocedure VEGF-A andphosphorylation of SrcWestern blot was performed to analyze the (a) EMTrelated markers VEGF-A and (b) Src p-Src (Tyr416) Two representative mice were presented Data was shown as mean plusmn SEM lowastPlt005lowastlowastPlt001 lowast lowast lowastlowast Plt00001

(Plt001) and the decrease of E-cadherin in tumor bearinglung tissue (Plt001) (Figure 4(a)) A study showed that Id1has been implicated in VEGF-A regulation during tumorangiogenesis [19] and peritoneal expression of VEGF-A isregulated by TGF-1205731 through the ID1 pathway in womenwith endometriosis [20] In our research we observed thatVEGF-A was overexpressed in control group and baicaleinsignificantly inhibited the expression of VEGF-A (Plt001)(Figure 4(a)) To assess how baicalein inhibits the expressionof Id1 protein we analyzed the effect of baicalein to Srcand its phosphorylation as studies show that Src regulatesthe expression of Id1 in human lung adenocarcinoma andpancreatic adenocarcinoma [7 21] Our results showed thatbaicalein significantly reversed high phosphorylation of Srcin tumor bearing mice indicating that baicalein inhibits Id1in an Src dependent manner (Plt00001) (Figure 4(b))

34 Regulation of Baicalein on SrcId1 Signaling PathwayWas Verified In Vitro To verify the effect of baicalein toSrcId1 signaling pathway A549 cells were treated withbaicalein (10120583M) in different time points and expressions ofp-Src (Tyr416) Id1 E-cadherin N-cadherin vimentin and

VEGF-Awere determined by using western blot As shown inFigure 5 baicalein inhibited Id1 expression time dependentlyWhen A549 cells were treated with baicalein for 24 h or 36 hthe phosphorylation of Src and expressions of N-cadherinand vimentin were significantly suppressed (Plt001) Whentreated for 36 h VEGF-A was also reduced significantlyAs for E-Cadherin baicalein significantly upregulated itsexpression (Plt00001)

4 Discussion

We established an orthotopic lung cancer model through avisible transthoracic injection with A549 cells and demon-strated that baicalein was effective in this model We iden-tified Id1 as the pivotal respondent to baicalein in itsanti-tumor effect The downstream molecules of Id1 (N-cadherin vimentin and VEGF-A) were further suppressedand epithelial marker (E-cadherin) was increased We alsodemonstrated that baicalein might function through inhibit-ing the phosphorylation of Src as it was the upstreamof Id1 In light of our results we propose baicalein as apromising adjuvant therapy phytochemical and Id1 and its

6 Evidence-Based Complementary and Alternative Medicine

Id1

Relat

ive e

xpre

ssio

n le

vel

Src00

05

10

15

5

10

15

0 h12 h

24 h36 h

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

-actin

VEGF-A

Vimentin

N-Cadherin

E-Cadherin

p-Src(Tyr416)

Src

0 hBaicalein 12 h 24 h 36 h

p-Src(T

yr416

)

E-Cad

herin

N-Cad

herin

Vimentin

VEGF-A Id 1

Figure 5 Baicalein regulated SrcId1 signaling pathway A549 cells were treated with baicalein (10120583M) for 0 h 12 h 24 h or 36 h and westernblot was performed to determine expressions of p-Src (Tyr416) Id1 E-cadherin N-cadherin vimentin and VEGF-A Data was shown asmean plusmn SEM lowastPlt005 lowastlowastPlt001

mediators as candidates for targeted anti-tumor therapeuticstrategies

Nowadays lung cancer is the most lethal cancer type inwhich NSCLC accounts for approximately 85 of all lungcancers [22 23] Except for chemotherapy drugs a varietyof other medicines for instance EGFR inhibitors ALKinhibitors angiogenesis inhibitors and immune checkpointinhibitors are available in the market [24] However despitethe advances in therapies the five-year survival of NSCLCremains lowThis highlights the necessity for alternative treat-ments for unresectable NSCLC bearing patients Baicaleinis a main component of Scutellaria baicalensis which hasbeen used to treat diseases for thousands of years in ChinaProperties of its effectiveness and low toxicity have gainedadmiration of patients The anticancer potential of baicaleinwas observed in lung cancer bladder cancer breast cancerovarian cancer etc [25] Nevertheless most of these studiesobtained the results through experiments in vitro or subcu-taneous xenografts in vivo model Our study firstly demon-strates its anticancer potential by using orthotopic lungcancer model which mimics the clinical situation preferably(Figure 2) This is of significant importance for preclinicaldrug research and provides more powerful evidence forbaicalein of being a promising anticancer therapeutic drug

Researchers have shown that baicalein may functionthrough apoptosis induction autophagy triggering cell cyclearrest or inhibition of 12-lipoxygenase (LOX) [12] Ourstudy identified Id1 as a critical respondent to baicaleinfor the first time Id1 is a member of HLH family whichfunctions as a differentiation inhibitor and it is overexpressedin many cancers and facilitates the growth and metastasisof cancer [5] In our study tumor burden increased the

expression of Id1 and baicalein significantly inhibited itsexpression time dependently (Figures 3 and 5) and thusproliferation and metastasis of tumor cells were suppressedWe also showed that mesenchymal markers (vimentin andN-cadherin) and angiogenesis marker (VEGFA) increasedand epithelial markers (E-cadherin) significantly decreasedin lungs of tumor bearing mice and baicalein regressedthese effects in vivo and in vitro This indicated that EMTprocedure and angiogenesis of lung cancer were abrogated bybaicalein Studies have shown that Src is the upstream of Id1We observed that baicalein could inhibit the p-Src (Tyr416)expression which indicated that its effect to Id1 might bethrough inhibiting Src phosphorylation (Figures 4 and 5)

In conclusion our data demonstrated the antitumoreffect of baicalein in orthotopic NSCLC model for the firsttime and clarified that SrcId1 pathway was involved inthe baicalein-induced inhibition of tumor growth providingfurther insight into the therapeutic strategies

Data Availability

All data generated or analyzed during this study are includedin this article

Conflicts of Interest

The authors declare no conflicts of interest in relation to thework described

Acknowledgments

This study was funded by grants from National Natu-ral Science Foundation of China [Grant nos 81403148

Evidence-Based Complementary and Alternative Medicine 7

81673916 8150150396 and 81703829] Shanghai MunicipalCommission of Health and Family Planning [Grant noZYKC201602001] and Development Project of ShanghaiPeak Disciplines Integrative Medicine [Grant no 20150407]

References

[1] L A Torre F Bray R L Siegel J Ferlay and J Lortet-Tieulent ldquoGlobal cancer statistics 2012rdquo CA A Cancer Journalfor Clinicians vol 65 no 2 pp 87ndash108 2015

[2] C Gridelli A Rossi D P Carbone et al ldquoNon-small-cell lungcancerrdquo Nature Reviews Disease Primers vol 1 article 150092015

[3] C Liam S Andarini P Lee J C Ho N Q Chau and JTscheikuna ldquoLung cancer staging now and in the futurerdquoRespirology vol 1 pp 526ndash534 2015

[4] R Benezra R L Davis D Lockshon D L Turner and HWeintraub ldquoThe protein Id a negative regulator of helix-loop-helix DNA binding proteinsrdquoCell vol 61 no 1 pp 49ndash59 1990

[5] J Perk A Iavarone and R Benezra ldquoId family of helix-loop-helix proteins in cancerrdquo Nature Reviews Cancer vol 5 no 8pp 603ndash614 2005

[6] S I Rothschild A Kappeler D Ratschiller et al ldquoThe stem cellgene ldquoinhibitor of differentiation 1rdquo (ID1) is frequently expressedin non-small cell lung cancerrdquo Lung Cancer vol 71 no 3 pp306ndash311 2011

[7] J G Trevino S Pillai S Kunigal et al ldquoNicotine inducesinhibitor of differentiation-1 in a Src-dependent pathway pro-motingmetastasis and chemoresistance in pancreatic adenocar-cinomardquo Neoplasia vol 14 no 12 pp 1102ndash1114 2012

[8] R Bhattacharya J Kowalski A R Larson M Brock and RM Alani ldquoId1 promotes tumor cell migration in nonsmall celllung cancersrdquo Journal of Oncology vol 2010 Article ID 8561058 pages 2010

[9] J Li Y Li B Wang Y Ma and P Chen ldquoId-1 promotes migra-tion and invasion of non-small cell lung cancer cells throughactivating NF-120581B signaling pathwayrdquo Journal of BiomedicalScience vol 24 article 95 2017

[10] E Castanon A Soltermann I Lopez et al ldquoThe inhibitor ofdifferentiation-1 (Id1) enables lung cancer liver colonizationthrough activation of an EMT program in tumor cells andestablishment of the pre-metastatic nicherdquo Cancer Letters vol402 pp 43ndash51 2017

[11] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009

[12] W-Y Gong Z-X Zhao B-J Liu L-W Lu and J-C DongldquoExploring the chemopreventive properties and perspectives ofbaicalin and its aglycone baicalein in solid tumorsrdquo EuropeanJournal of Medicinal Chemistry vol 126 pp 844ndash852 2017

[13] C Naveenkumar S Asokkumar S Raghunandhakumar et alldquoPotent antitumor and antineoplastic efficacy of baicalein onbenzo(a)pyrene-induced experimental pulmonary tumorigen-esisrdquo Fundamental amp Clinical Pharmacology vol 26 no 2 pp259ndash270 2012

[14] W-Y I Gong J-F Wu B-J Liu et al ldquoFlavonoid componentsin Scutellaria baicalensis inhibit nicotine-induced proliferationmetastasis and lung cancer-associated inflammation in vitrordquoInternational Journal of Oncology vol 44 no 5 pp 1561ndash15702014

[15] J Gao W A Morgan A Sanchez-Medina and O CorcoranldquoThe ethanol extract of Scutellaria baicalensis and the activecompounds induce cell cycle arrest and apoptosis includingupregulation of p53 and Bax in human lung cancer cellsrdquoToxicology and Applied Pharmacology vol 254 no 3 pp 221ndash228 2011

[16] G Su H Chen and X Sun ldquoBaicalein suppresses non smallcell lung cancer cell proliferation invasion and Notch signalingpathwayrdquo Cancer Biomarkers vol 22 no 1 Article ID 170673pp 13ndash18 2018

[17] C Roschger and C Cabrele ldquoThe Id-protein family in develop-mental and cancer-associated pathwaysrdquo Cell Communicationand Signaling vol 15 article 26 2017

[18] E Cubillo A Diaz-Lopez E P Cuevas et al ldquoE47 and Id1interplay in epithelial-mesenchymal transitionrdquo PLoS One vol8 article e59948 2013

[19] M-T Ling T C M Lau C Zhou et al ldquoOverexpression ofId-1 in prostate cancer cells promotes angiogenesis throughthe activation of vascular endothelial growth factor (VEGF)rdquoCarcinogenesis vol 26 no 10 pp 1668ndash1676 2005

[20] V J Young S F Ahmad J K Brown W C Duncan and AWHorne ldquoPeritoneal VEGF-A expression is regulated by TGF-beta1 through an ID1 pathway in women with endometriosisrdquoScientific Reports vol 5 article 16859 2015

[21] S I Rothschild M P Tschan E A Federzoni et alldquoMicroRNA-29b is involved in the Src-ID1 signaling pathwayand is dysregulated in human lung adenocarcinomardquoOncogenevol 31 no 38 pp 4221ndash4232 2012

[22] World Health Organization ldquoGLOBOCAN 2012 Estimatedcancer incidence mortality and prevalence worldwiderdquo 2012httpglobocaniarcfrPagesfact sheets canceraspx

[23] D S Ettinger W Akerley and G Bepler ldquoNonsmall cell lungcancerrdquo Journal of the National Comprehensive Cancer Networkvol 8 pp 740ndash801 2010

[24] J Minguet K H Smith and P Bramlage ldquoTargeted therapiesfor treatment of non-small cell lung cancer - Recent advancesand future perspectivesrdquo International Journal of Cancer vol138 no 11 pp 2549ndash2561 2016

[25] H Liu Y Dong Y Gao et al ldquoThe fascinating effects ofbaicalein on cancer a reviewrdquo International Journal ofMolecularSciences vol 17 article 168110 2016

4 Evidence-Based Complementary and Alternative Medicine

1 1 12 2 2

Normal Control Baicalein

Id1

-actin

lowastlowastlowastlowast lowastlowastlowastlowast

Normal

Baicale

in

Control

0

20

40

60

Relat

ive e

xpre

ssio

n le

vel o

f Id1

(a)

Normal Control Baicaleinlowastlowast lowastlowast

Normal

Baicale

in

Control

0

20

40

60

80

o

f id1

pos

itive

cells

(b)

Figure 3 Baicalein inhibited Id1 expression of A549 orthotopic NSCLC xenografts (a)Western blot analysis was performed to determine theId1 protein Two representative mice were presented (b) Immunohistochemical analysis for Id1 of normal lung or xenograft tissues The totalnumber of Id1 positive cells (brown-stained nuclei regardless of staining intensity were counted as positive) in three random microscopicfields was counted by Image-Pro Plus 60 Data was shown as mean plusmn SEM (lowastlowastPlt 001 lowast lowast lowastlowastPlt 00001 compared with the control group)

baicalein we built an orthotopic lung cancer model in Balbcnude mice by A549 cells implantation Except for normalgroup mice were injected with 1x106 A549 cells into the leftlungs Thirty days following inoculation mice in baicaleingroupwere intragastrically administeredwith baicalein (05CMC-Na solution 40mgkg) Control group was intra-gastrically administered with CMC-Na (05 02mL permouse) and normal group was with 09 physiologicalsaline After 28 days of treatment mice were scanned withmicro-CT and sacrificed Lungs of mice were harvested graynodules could be seen in control group (Figure 2(a) whitearrows) In control and baicalein groups HE staining oflungs showed heterogeneous cells with larger eosinophilicnucleoli and vacuoles in the cytoplasm Visible glandularcavities could be seen among these cells (Figure 2(b) blackarrows) which indicated the success of orthotopic lungcancer model Micro-CT scanning exhibited much smallernodules in left lung of baicalein group comparing to con-trol group (Figure 2(c) yellow arrows) and the differenceswere significant (Plt001) We also measured the weightof lungs in each group Results showed that the lungs incontrol group were heavier than the other two groupsbut significant difference was not observed (Figure 2(d))Mice weight in normal group increased gradually over timeand was higher than that of control and baicalein-treatedgroups after 20th day of inoculation the difference between

normal group and control group was significant (Plt001)(Figure 2(e))

32 Baicalein Inhibited Id1 Expression To exploremechanismof the anti-tumor effect of baicalein we detected Id1 expres-sion in each group as Id1 is an essential tumor promoterwhich promotes the proliferation of tumor cells and facilitatestumor growth [17] When compared with lungs of normalmice lungs of tumor bearing mice expressed higher Id1protein (Plt00001) and baicalein significantly inhibited theId1 expression level (Plt00001) (Figure 3(a)) The immuno-histochemistry also showed much more Id1 positive cells incontrol group (Plt001) baicalein significantly reduced thenumber of Id1 positive cells (Plt001) (Figure 3(b))

33 Baicalein Abrogated Epithelial-Mesenchymal Transition(EMT) and Angiogenesis through SrcId1 Signaling PathwayTissue microarrays containing 532 NSCLC patientsrsquo samplesshow that Id1 is significantly correlated with EMT-relatedproteins [10]The loss of Id1 reduces the levels of EMT-relatedproteins [18] which indicate that EMT-related proteins maybe the downstream of Id1 in NSCLC Here we obtained theinhibiting effect of baicalein to Id1 protein thus we examinedthe expression of EMT-associated proteins (vimentin E-cadherin and N-cadherin) Results showed baicalein abro-gated the increase of N-cadherin (Plt001) and vimentin

Evidence-Based Complementary and Alternative Medicine 5

-actin

1 1 12 2 2

Normal Control Baicalein

VEGF-A

Vimentin

N-Cadherin

E-Cadherin

000510152025 lowastlowastlowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n of

Vim

entin

Normal

Control

Baicale

in

Normal

Control

Baicale

in

Relat

ive e

xpre

ssio

n of

E-C

adhe

rin

Relat

ive e

xpre

ssio

n of

N-C

adhe

rin

Normal

Control

Baicale

in

Normal

Control

Baicale

in00

05

10

15lowastlowast lowastlowast

0

1

2

3

4 lowastlowast lowastlowast

Relat

ive e

xpre

ssio

n of

VEG

F-A

000510152025 lowastlowast lowastlowast

(a)

-actin

p-Src(Tyr416)

Src

1 1 12 2 2Normal Control Baicalein

Normal

Control

Baicale

in

Normal

Control

Baicale

in

Relat

ive e

xpre

ssio

n le

vel o

f Src

00

05

10

15

Relat

ive e

xpre

ssio

n le

vel o

f p-S

rc (T

yr41

6)

00

05

10

15

20

25 lowastlowastlowastlowastlowastlowastlowastlowast

(b)

Figure 4 Baicalein suppressed the EMTprocedure VEGF-A andphosphorylation of SrcWestern blot was performed to analyze the (a) EMTrelated markers VEGF-A and (b) Src p-Src (Tyr416) Two representative mice were presented Data was shown as mean plusmn SEM lowastPlt005lowastlowastPlt001 lowast lowast lowastlowast Plt00001

(Plt001) and the decrease of E-cadherin in tumor bearinglung tissue (Plt001) (Figure 4(a)) A study showed that Id1has been implicated in VEGF-A regulation during tumorangiogenesis [19] and peritoneal expression of VEGF-A isregulated by TGF-1205731 through the ID1 pathway in womenwith endometriosis [20] In our research we observed thatVEGF-A was overexpressed in control group and baicaleinsignificantly inhibited the expression of VEGF-A (Plt001)(Figure 4(a)) To assess how baicalein inhibits the expressionof Id1 protein we analyzed the effect of baicalein to Srcand its phosphorylation as studies show that Src regulatesthe expression of Id1 in human lung adenocarcinoma andpancreatic adenocarcinoma [7 21] Our results showed thatbaicalein significantly reversed high phosphorylation of Srcin tumor bearing mice indicating that baicalein inhibits Id1in an Src dependent manner (Plt00001) (Figure 4(b))

34 Regulation of Baicalein on SrcId1 Signaling PathwayWas Verified In Vitro To verify the effect of baicalein toSrcId1 signaling pathway A549 cells were treated withbaicalein (10120583M) in different time points and expressions ofp-Src (Tyr416) Id1 E-cadherin N-cadherin vimentin and

VEGF-Awere determined by using western blot As shown inFigure 5 baicalein inhibited Id1 expression time dependentlyWhen A549 cells were treated with baicalein for 24 h or 36 hthe phosphorylation of Src and expressions of N-cadherinand vimentin were significantly suppressed (Plt001) Whentreated for 36 h VEGF-A was also reduced significantlyAs for E-Cadherin baicalein significantly upregulated itsexpression (Plt00001)

4 Discussion

We established an orthotopic lung cancer model through avisible transthoracic injection with A549 cells and demon-strated that baicalein was effective in this model We iden-tified Id1 as the pivotal respondent to baicalein in itsanti-tumor effect The downstream molecules of Id1 (N-cadherin vimentin and VEGF-A) were further suppressedand epithelial marker (E-cadherin) was increased We alsodemonstrated that baicalein might function through inhibit-ing the phosphorylation of Src as it was the upstreamof Id1 In light of our results we propose baicalein as apromising adjuvant therapy phytochemical and Id1 and its

6 Evidence-Based Complementary and Alternative Medicine

Id1

Relat

ive e

xpre

ssio

n le

vel

Src00

05

10

15

5

10

15

0 h12 h

24 h36 h

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

-actin

VEGF-A

Vimentin

N-Cadherin

E-Cadherin

p-Src(Tyr416)

Src

0 hBaicalein 12 h 24 h 36 h

p-Src(T

yr416

)

E-Cad

herin

N-Cad

herin

Vimentin

VEGF-A Id 1

Figure 5 Baicalein regulated SrcId1 signaling pathway A549 cells were treated with baicalein (10120583M) for 0 h 12 h 24 h or 36 h and westernblot was performed to determine expressions of p-Src (Tyr416) Id1 E-cadherin N-cadherin vimentin and VEGF-A Data was shown asmean plusmn SEM lowastPlt005 lowastlowastPlt001

mediators as candidates for targeted anti-tumor therapeuticstrategies

Nowadays lung cancer is the most lethal cancer type inwhich NSCLC accounts for approximately 85 of all lungcancers [22 23] Except for chemotherapy drugs a varietyof other medicines for instance EGFR inhibitors ALKinhibitors angiogenesis inhibitors and immune checkpointinhibitors are available in the market [24] However despitethe advances in therapies the five-year survival of NSCLCremains lowThis highlights the necessity for alternative treat-ments for unresectable NSCLC bearing patients Baicaleinis a main component of Scutellaria baicalensis which hasbeen used to treat diseases for thousands of years in ChinaProperties of its effectiveness and low toxicity have gainedadmiration of patients The anticancer potential of baicaleinwas observed in lung cancer bladder cancer breast cancerovarian cancer etc [25] Nevertheless most of these studiesobtained the results through experiments in vitro or subcu-taneous xenografts in vivo model Our study firstly demon-strates its anticancer potential by using orthotopic lungcancer model which mimics the clinical situation preferably(Figure 2) This is of significant importance for preclinicaldrug research and provides more powerful evidence forbaicalein of being a promising anticancer therapeutic drug

Researchers have shown that baicalein may functionthrough apoptosis induction autophagy triggering cell cyclearrest or inhibition of 12-lipoxygenase (LOX) [12] Ourstudy identified Id1 as a critical respondent to baicaleinfor the first time Id1 is a member of HLH family whichfunctions as a differentiation inhibitor and it is overexpressedin many cancers and facilitates the growth and metastasisof cancer [5] In our study tumor burden increased the

expression of Id1 and baicalein significantly inhibited itsexpression time dependently (Figures 3 and 5) and thusproliferation and metastasis of tumor cells were suppressedWe also showed that mesenchymal markers (vimentin andN-cadherin) and angiogenesis marker (VEGFA) increasedand epithelial markers (E-cadherin) significantly decreasedin lungs of tumor bearing mice and baicalein regressedthese effects in vivo and in vitro This indicated that EMTprocedure and angiogenesis of lung cancer were abrogated bybaicalein Studies have shown that Src is the upstream of Id1We observed that baicalein could inhibit the p-Src (Tyr416)expression which indicated that its effect to Id1 might bethrough inhibiting Src phosphorylation (Figures 4 and 5)

In conclusion our data demonstrated the antitumoreffect of baicalein in orthotopic NSCLC model for the firsttime and clarified that SrcId1 pathway was involved inthe baicalein-induced inhibition of tumor growth providingfurther insight into the therapeutic strategies

Data Availability

All data generated or analyzed during this study are includedin this article

Conflicts of Interest

The authors declare no conflicts of interest in relation to thework described

Acknowledgments

This study was funded by grants from National Natu-ral Science Foundation of China [Grant nos 81403148

Evidence-Based Complementary and Alternative Medicine 7

81673916 8150150396 and 81703829] Shanghai MunicipalCommission of Health and Family Planning [Grant noZYKC201602001] and Development Project of ShanghaiPeak Disciplines Integrative Medicine [Grant no 20150407]

References

[1] L A Torre F Bray R L Siegel J Ferlay and J Lortet-Tieulent ldquoGlobal cancer statistics 2012rdquo CA A Cancer Journalfor Clinicians vol 65 no 2 pp 87ndash108 2015

[2] C Gridelli A Rossi D P Carbone et al ldquoNon-small-cell lungcancerrdquo Nature Reviews Disease Primers vol 1 article 150092015

[3] C Liam S Andarini P Lee J C Ho N Q Chau and JTscheikuna ldquoLung cancer staging now and in the futurerdquoRespirology vol 1 pp 526ndash534 2015

[4] R Benezra R L Davis D Lockshon D L Turner and HWeintraub ldquoThe protein Id a negative regulator of helix-loop-helix DNA binding proteinsrdquoCell vol 61 no 1 pp 49ndash59 1990

[5] J Perk A Iavarone and R Benezra ldquoId family of helix-loop-helix proteins in cancerrdquo Nature Reviews Cancer vol 5 no 8pp 603ndash614 2005

[6] S I Rothschild A Kappeler D Ratschiller et al ldquoThe stem cellgene ldquoinhibitor of differentiation 1rdquo (ID1) is frequently expressedin non-small cell lung cancerrdquo Lung Cancer vol 71 no 3 pp306ndash311 2011

[7] J G Trevino S Pillai S Kunigal et al ldquoNicotine inducesinhibitor of differentiation-1 in a Src-dependent pathway pro-motingmetastasis and chemoresistance in pancreatic adenocar-cinomardquo Neoplasia vol 14 no 12 pp 1102ndash1114 2012

[8] R Bhattacharya J Kowalski A R Larson M Brock and RM Alani ldquoId1 promotes tumor cell migration in nonsmall celllung cancersrdquo Journal of Oncology vol 2010 Article ID 8561058 pages 2010

[9] J Li Y Li B Wang Y Ma and P Chen ldquoId-1 promotes migra-tion and invasion of non-small cell lung cancer cells throughactivating NF-120581B signaling pathwayrdquo Journal of BiomedicalScience vol 24 article 95 2017

[10] E Castanon A Soltermann I Lopez et al ldquoThe inhibitor ofdifferentiation-1 (Id1) enables lung cancer liver colonizationthrough activation of an EMT program in tumor cells andestablishment of the pre-metastatic nicherdquo Cancer Letters vol402 pp 43ndash51 2017

[11] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009

[12] W-Y Gong Z-X Zhao B-J Liu L-W Lu and J-C DongldquoExploring the chemopreventive properties and perspectives ofbaicalin and its aglycone baicalein in solid tumorsrdquo EuropeanJournal of Medicinal Chemistry vol 126 pp 844ndash852 2017

[13] C Naveenkumar S Asokkumar S Raghunandhakumar et alldquoPotent antitumor and antineoplastic efficacy of baicalein onbenzo(a)pyrene-induced experimental pulmonary tumorigen-esisrdquo Fundamental amp Clinical Pharmacology vol 26 no 2 pp259ndash270 2012

[14] W-Y I Gong J-F Wu B-J Liu et al ldquoFlavonoid componentsin Scutellaria baicalensis inhibit nicotine-induced proliferationmetastasis and lung cancer-associated inflammation in vitrordquoInternational Journal of Oncology vol 44 no 5 pp 1561ndash15702014

[15] J Gao W A Morgan A Sanchez-Medina and O CorcoranldquoThe ethanol extract of Scutellaria baicalensis and the activecompounds induce cell cycle arrest and apoptosis includingupregulation of p53 and Bax in human lung cancer cellsrdquoToxicology and Applied Pharmacology vol 254 no 3 pp 221ndash228 2011

[16] G Su H Chen and X Sun ldquoBaicalein suppresses non smallcell lung cancer cell proliferation invasion and Notch signalingpathwayrdquo Cancer Biomarkers vol 22 no 1 Article ID 170673pp 13ndash18 2018

[17] C Roschger and C Cabrele ldquoThe Id-protein family in develop-mental and cancer-associated pathwaysrdquo Cell Communicationand Signaling vol 15 article 26 2017

[18] E Cubillo A Diaz-Lopez E P Cuevas et al ldquoE47 and Id1interplay in epithelial-mesenchymal transitionrdquo PLoS One vol8 article e59948 2013

[19] M-T Ling T C M Lau C Zhou et al ldquoOverexpression ofId-1 in prostate cancer cells promotes angiogenesis throughthe activation of vascular endothelial growth factor (VEGF)rdquoCarcinogenesis vol 26 no 10 pp 1668ndash1676 2005

[20] V J Young S F Ahmad J K Brown W C Duncan and AWHorne ldquoPeritoneal VEGF-A expression is regulated by TGF-beta1 through an ID1 pathway in women with endometriosisrdquoScientific Reports vol 5 article 16859 2015

[21] S I Rothschild M P Tschan E A Federzoni et alldquoMicroRNA-29b is involved in the Src-ID1 signaling pathwayand is dysregulated in human lung adenocarcinomardquoOncogenevol 31 no 38 pp 4221ndash4232 2012

[22] World Health Organization ldquoGLOBOCAN 2012 Estimatedcancer incidence mortality and prevalence worldwiderdquo 2012httpglobocaniarcfrPagesfact sheets canceraspx

[23] D S Ettinger W Akerley and G Bepler ldquoNonsmall cell lungcancerrdquo Journal of the National Comprehensive Cancer Networkvol 8 pp 740ndash801 2010

[24] J Minguet K H Smith and P Bramlage ldquoTargeted therapiesfor treatment of non-small cell lung cancer - Recent advancesand future perspectivesrdquo International Journal of Cancer vol138 no 11 pp 2549ndash2561 2016

[25] H Liu Y Dong Y Gao et al ldquoThe fascinating effects ofbaicalein on cancer a reviewrdquo International Journal ofMolecularSciences vol 17 article 168110 2016

Evidence-Based Complementary and Alternative Medicine 5

-actin

1 1 12 2 2

Normal Control Baicalein

VEGF-A

Vimentin

N-Cadherin

E-Cadherin

000510152025 lowastlowastlowastlowast

lowastlowast

Relat

ive e

xpre

ssio

n of

Vim

entin

Normal

Control

Baicale

in

Normal

Control

Baicale

in

Relat

ive e

xpre

ssio

n of

E-C

adhe

rin

Relat

ive e

xpre

ssio

n of

N-C

adhe

rin

Normal

Control

Baicale

in

Normal

Control

Baicale

in00

05

10

15lowastlowast lowastlowast

0

1

2

3

4 lowastlowast lowastlowast

Relat

ive e

xpre

ssio

n of

VEG

F-A

000510152025 lowastlowast lowastlowast

(a)

-actin

p-Src(Tyr416)

Src

1 1 12 2 2Normal Control Baicalein

Normal

Control

Baicale

in

Normal

Control

Baicale

in

Relat

ive e

xpre

ssio

n le

vel o

f Src

00

05

10

15

Relat

ive e

xpre

ssio

n le

vel o

f p-S

rc (T

yr41

6)

00

05

10

15

20

25 lowastlowastlowastlowastlowastlowastlowastlowast

(b)

Figure 4 Baicalein suppressed the EMTprocedure VEGF-A andphosphorylation of SrcWestern blot was performed to analyze the (a) EMTrelated markers VEGF-A and (b) Src p-Src (Tyr416) Two representative mice were presented Data was shown as mean plusmn SEM lowastPlt005lowastlowastPlt001 lowast lowast lowastlowast Plt00001

(Plt001) and the decrease of E-cadherin in tumor bearinglung tissue (Plt001) (Figure 4(a)) A study showed that Id1has been implicated in VEGF-A regulation during tumorangiogenesis [19] and peritoneal expression of VEGF-A isregulated by TGF-1205731 through the ID1 pathway in womenwith endometriosis [20] In our research we observed thatVEGF-A was overexpressed in control group and baicaleinsignificantly inhibited the expression of VEGF-A (Plt001)(Figure 4(a)) To assess how baicalein inhibits the expressionof Id1 protein we analyzed the effect of baicalein to Srcand its phosphorylation as studies show that Src regulatesthe expression of Id1 in human lung adenocarcinoma andpancreatic adenocarcinoma [7 21] Our results showed thatbaicalein significantly reversed high phosphorylation of Srcin tumor bearing mice indicating that baicalein inhibits Id1in an Src dependent manner (Plt00001) (Figure 4(b))

34 Regulation of Baicalein on SrcId1 Signaling PathwayWas Verified In Vitro To verify the effect of baicalein toSrcId1 signaling pathway A549 cells were treated withbaicalein (10120583M) in different time points and expressions ofp-Src (Tyr416) Id1 E-cadherin N-cadherin vimentin and

VEGF-Awere determined by using western blot As shown inFigure 5 baicalein inhibited Id1 expression time dependentlyWhen A549 cells were treated with baicalein for 24 h or 36 hthe phosphorylation of Src and expressions of N-cadherinand vimentin were significantly suppressed (Plt001) Whentreated for 36 h VEGF-A was also reduced significantlyAs for E-Cadherin baicalein significantly upregulated itsexpression (Plt00001)

4 Discussion

We established an orthotopic lung cancer model through avisible transthoracic injection with A549 cells and demon-strated that baicalein was effective in this model We iden-tified Id1 as the pivotal respondent to baicalein in itsanti-tumor effect The downstream molecules of Id1 (N-cadherin vimentin and VEGF-A) were further suppressedand epithelial marker (E-cadherin) was increased We alsodemonstrated that baicalein might function through inhibit-ing the phosphorylation of Src as it was the upstreamof Id1 In light of our results we propose baicalein as apromising adjuvant therapy phytochemical and Id1 and its

6 Evidence-Based Complementary and Alternative Medicine

Id1

Relat

ive e

xpre

ssio

n le

vel

Src00

05

10

15

5

10

15

0 h12 h

24 h36 h

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

-actin

VEGF-A

Vimentin

N-Cadherin

E-Cadherin

p-Src(Tyr416)

Src

0 hBaicalein 12 h 24 h 36 h

p-Src(T

yr416

)

E-Cad

herin

N-Cad

herin

Vimentin

VEGF-A Id 1

Figure 5 Baicalein regulated SrcId1 signaling pathway A549 cells were treated with baicalein (10120583M) for 0 h 12 h 24 h or 36 h and westernblot was performed to determine expressions of p-Src (Tyr416) Id1 E-cadherin N-cadherin vimentin and VEGF-A Data was shown asmean plusmn SEM lowastPlt005 lowastlowastPlt001

mediators as candidates for targeted anti-tumor therapeuticstrategies

Nowadays lung cancer is the most lethal cancer type inwhich NSCLC accounts for approximately 85 of all lungcancers [22 23] Except for chemotherapy drugs a varietyof other medicines for instance EGFR inhibitors ALKinhibitors angiogenesis inhibitors and immune checkpointinhibitors are available in the market [24] However despitethe advances in therapies the five-year survival of NSCLCremains lowThis highlights the necessity for alternative treat-ments for unresectable NSCLC bearing patients Baicaleinis a main component of Scutellaria baicalensis which hasbeen used to treat diseases for thousands of years in ChinaProperties of its effectiveness and low toxicity have gainedadmiration of patients The anticancer potential of baicaleinwas observed in lung cancer bladder cancer breast cancerovarian cancer etc [25] Nevertheless most of these studiesobtained the results through experiments in vitro or subcu-taneous xenografts in vivo model Our study firstly demon-strates its anticancer potential by using orthotopic lungcancer model which mimics the clinical situation preferably(Figure 2) This is of significant importance for preclinicaldrug research and provides more powerful evidence forbaicalein of being a promising anticancer therapeutic drug

Researchers have shown that baicalein may functionthrough apoptosis induction autophagy triggering cell cyclearrest or inhibition of 12-lipoxygenase (LOX) [12] Ourstudy identified Id1 as a critical respondent to baicaleinfor the first time Id1 is a member of HLH family whichfunctions as a differentiation inhibitor and it is overexpressedin many cancers and facilitates the growth and metastasisof cancer [5] In our study tumor burden increased the

expression of Id1 and baicalein significantly inhibited itsexpression time dependently (Figures 3 and 5) and thusproliferation and metastasis of tumor cells were suppressedWe also showed that mesenchymal markers (vimentin andN-cadherin) and angiogenesis marker (VEGFA) increasedand epithelial markers (E-cadherin) significantly decreasedin lungs of tumor bearing mice and baicalein regressedthese effects in vivo and in vitro This indicated that EMTprocedure and angiogenesis of lung cancer were abrogated bybaicalein Studies have shown that Src is the upstream of Id1We observed that baicalein could inhibit the p-Src (Tyr416)expression which indicated that its effect to Id1 might bethrough inhibiting Src phosphorylation (Figures 4 and 5)

In conclusion our data demonstrated the antitumoreffect of baicalein in orthotopic NSCLC model for the firsttime and clarified that SrcId1 pathway was involved inthe baicalein-induced inhibition of tumor growth providingfurther insight into the therapeutic strategies

Data Availability

All data generated or analyzed during this study are includedin this article

Conflicts of Interest

The authors declare no conflicts of interest in relation to thework described

Acknowledgments

This study was funded by grants from National Natu-ral Science Foundation of China [Grant nos 81403148

Evidence-Based Complementary and Alternative Medicine 7

81673916 8150150396 and 81703829] Shanghai MunicipalCommission of Health and Family Planning [Grant noZYKC201602001] and Development Project of ShanghaiPeak Disciplines Integrative Medicine [Grant no 20150407]

References

[1] L A Torre F Bray R L Siegel J Ferlay and J Lortet-Tieulent ldquoGlobal cancer statistics 2012rdquo CA A Cancer Journalfor Clinicians vol 65 no 2 pp 87ndash108 2015

[2] C Gridelli A Rossi D P Carbone et al ldquoNon-small-cell lungcancerrdquo Nature Reviews Disease Primers vol 1 article 150092015

[3] C Liam S Andarini P Lee J C Ho N Q Chau and JTscheikuna ldquoLung cancer staging now and in the futurerdquoRespirology vol 1 pp 526ndash534 2015

[4] R Benezra R L Davis D Lockshon D L Turner and HWeintraub ldquoThe protein Id a negative regulator of helix-loop-helix DNA binding proteinsrdquoCell vol 61 no 1 pp 49ndash59 1990

[5] J Perk A Iavarone and R Benezra ldquoId family of helix-loop-helix proteins in cancerrdquo Nature Reviews Cancer vol 5 no 8pp 603ndash614 2005

[6] S I Rothschild A Kappeler D Ratschiller et al ldquoThe stem cellgene ldquoinhibitor of differentiation 1rdquo (ID1) is frequently expressedin non-small cell lung cancerrdquo Lung Cancer vol 71 no 3 pp306ndash311 2011

[7] J G Trevino S Pillai S Kunigal et al ldquoNicotine inducesinhibitor of differentiation-1 in a Src-dependent pathway pro-motingmetastasis and chemoresistance in pancreatic adenocar-cinomardquo Neoplasia vol 14 no 12 pp 1102ndash1114 2012

[8] R Bhattacharya J Kowalski A R Larson M Brock and RM Alani ldquoId1 promotes tumor cell migration in nonsmall celllung cancersrdquo Journal of Oncology vol 2010 Article ID 8561058 pages 2010

[9] J Li Y Li B Wang Y Ma and P Chen ldquoId-1 promotes migra-tion and invasion of non-small cell lung cancer cells throughactivating NF-120581B signaling pathwayrdquo Journal of BiomedicalScience vol 24 article 95 2017

[10] E Castanon A Soltermann I Lopez et al ldquoThe inhibitor ofdifferentiation-1 (Id1) enables lung cancer liver colonizationthrough activation of an EMT program in tumor cells andestablishment of the pre-metastatic nicherdquo Cancer Letters vol402 pp 43ndash51 2017

[11] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009

[12] W-Y Gong Z-X Zhao B-J Liu L-W Lu and J-C DongldquoExploring the chemopreventive properties and perspectives ofbaicalin and its aglycone baicalein in solid tumorsrdquo EuropeanJournal of Medicinal Chemistry vol 126 pp 844ndash852 2017

[13] C Naveenkumar S Asokkumar S Raghunandhakumar et alldquoPotent antitumor and antineoplastic efficacy of baicalein onbenzo(a)pyrene-induced experimental pulmonary tumorigen-esisrdquo Fundamental amp Clinical Pharmacology vol 26 no 2 pp259ndash270 2012

[14] W-Y I Gong J-F Wu B-J Liu et al ldquoFlavonoid componentsin Scutellaria baicalensis inhibit nicotine-induced proliferationmetastasis and lung cancer-associated inflammation in vitrordquoInternational Journal of Oncology vol 44 no 5 pp 1561ndash15702014

[15] J Gao W A Morgan A Sanchez-Medina and O CorcoranldquoThe ethanol extract of Scutellaria baicalensis and the activecompounds induce cell cycle arrest and apoptosis includingupregulation of p53 and Bax in human lung cancer cellsrdquoToxicology and Applied Pharmacology vol 254 no 3 pp 221ndash228 2011

[16] G Su H Chen and X Sun ldquoBaicalein suppresses non smallcell lung cancer cell proliferation invasion and Notch signalingpathwayrdquo Cancer Biomarkers vol 22 no 1 Article ID 170673pp 13ndash18 2018

[17] C Roschger and C Cabrele ldquoThe Id-protein family in develop-mental and cancer-associated pathwaysrdquo Cell Communicationand Signaling vol 15 article 26 2017

[18] E Cubillo A Diaz-Lopez E P Cuevas et al ldquoE47 and Id1interplay in epithelial-mesenchymal transitionrdquo PLoS One vol8 article e59948 2013

[19] M-T Ling T C M Lau C Zhou et al ldquoOverexpression ofId-1 in prostate cancer cells promotes angiogenesis throughthe activation of vascular endothelial growth factor (VEGF)rdquoCarcinogenesis vol 26 no 10 pp 1668ndash1676 2005

[20] V J Young S F Ahmad J K Brown W C Duncan and AWHorne ldquoPeritoneal VEGF-A expression is regulated by TGF-beta1 through an ID1 pathway in women with endometriosisrdquoScientific Reports vol 5 article 16859 2015

[21] S I Rothschild M P Tschan E A Federzoni et alldquoMicroRNA-29b is involved in the Src-ID1 signaling pathwayand is dysregulated in human lung adenocarcinomardquoOncogenevol 31 no 38 pp 4221ndash4232 2012

[22] World Health Organization ldquoGLOBOCAN 2012 Estimatedcancer incidence mortality and prevalence worldwiderdquo 2012httpglobocaniarcfrPagesfact sheets canceraspx

[23] D S Ettinger W Akerley and G Bepler ldquoNonsmall cell lungcancerrdquo Journal of the National Comprehensive Cancer Networkvol 8 pp 740ndash801 2010

[24] J Minguet K H Smith and P Bramlage ldquoTargeted therapiesfor treatment of non-small cell lung cancer - Recent advancesand future perspectivesrdquo International Journal of Cancer vol138 no 11 pp 2549ndash2561 2016

[25] H Liu Y Dong Y Gao et al ldquoThe fascinating effects ofbaicalein on cancer a reviewrdquo International Journal ofMolecularSciences vol 17 article 168110 2016

6 Evidence-Based Complementary and Alternative Medicine

Id1

Relat

ive e

xpre

ssio

n le

vel

Src00

05

10

15

5

10

15

0 h12 h

24 h36 h

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

-actin

VEGF-A

Vimentin

N-Cadherin

E-Cadherin

p-Src(Tyr416)

Src

0 hBaicalein 12 h 24 h 36 h

p-Src(T

yr416

)

E-Cad

herin

N-Cad

herin

Vimentin

VEGF-A Id 1

Figure 5 Baicalein regulated SrcId1 signaling pathway A549 cells were treated with baicalein (10120583M) for 0 h 12 h 24 h or 36 h and westernblot was performed to determine expressions of p-Src (Tyr416) Id1 E-cadherin N-cadherin vimentin and VEGF-A Data was shown asmean plusmn SEM lowastPlt005 lowastlowastPlt001

mediators as candidates for targeted anti-tumor therapeuticstrategies

Nowadays lung cancer is the most lethal cancer type inwhich NSCLC accounts for approximately 85 of all lungcancers [22 23] Except for chemotherapy drugs a varietyof other medicines for instance EGFR inhibitors ALKinhibitors angiogenesis inhibitors and immune checkpointinhibitors are available in the market [24] However despitethe advances in therapies the five-year survival of NSCLCremains lowThis highlights the necessity for alternative treat-ments for unresectable NSCLC bearing patients Baicaleinis a main component of Scutellaria baicalensis which hasbeen used to treat diseases for thousands of years in ChinaProperties of its effectiveness and low toxicity have gainedadmiration of patients The anticancer potential of baicaleinwas observed in lung cancer bladder cancer breast cancerovarian cancer etc [25] Nevertheless most of these studiesobtained the results through experiments in vitro or subcu-taneous xenografts in vivo model Our study firstly demon-strates its anticancer potential by using orthotopic lungcancer model which mimics the clinical situation preferably(Figure 2) This is of significant importance for preclinicaldrug research and provides more powerful evidence forbaicalein of being a promising anticancer therapeutic drug

Researchers have shown that baicalein may functionthrough apoptosis induction autophagy triggering cell cyclearrest or inhibition of 12-lipoxygenase (LOX) [12] Ourstudy identified Id1 as a critical respondent to baicaleinfor the first time Id1 is a member of HLH family whichfunctions as a differentiation inhibitor and it is overexpressedin many cancers and facilitates the growth and metastasisof cancer [5] In our study tumor burden increased the

expression of Id1 and baicalein significantly inhibited itsexpression time dependently (Figures 3 and 5) and thusproliferation and metastasis of tumor cells were suppressedWe also showed that mesenchymal markers (vimentin andN-cadherin) and angiogenesis marker (VEGFA) increasedand epithelial markers (E-cadherin) significantly decreasedin lungs of tumor bearing mice and baicalein regressedthese effects in vivo and in vitro This indicated that EMTprocedure and angiogenesis of lung cancer were abrogated bybaicalein Studies have shown that Src is the upstream of Id1We observed that baicalein could inhibit the p-Src (Tyr416)expression which indicated that its effect to Id1 might bethrough inhibiting Src phosphorylation (Figures 4 and 5)

In conclusion our data demonstrated the antitumoreffect of baicalein in orthotopic NSCLC model for the firsttime and clarified that SrcId1 pathway was involved inthe baicalein-induced inhibition of tumor growth providingfurther insight into the therapeutic strategies

Data Availability

All data generated or analyzed during this study are includedin this article

Conflicts of Interest

The authors declare no conflicts of interest in relation to thework described

Acknowledgments

This study was funded by grants from National Natu-ral Science Foundation of China [Grant nos 81403148

Evidence-Based Complementary and Alternative Medicine 7

81673916 8150150396 and 81703829] Shanghai MunicipalCommission of Health and Family Planning [Grant noZYKC201602001] and Development Project of ShanghaiPeak Disciplines Integrative Medicine [Grant no 20150407]

References

[1] L A Torre F Bray R L Siegel J Ferlay and J Lortet-Tieulent ldquoGlobal cancer statistics 2012rdquo CA A Cancer Journalfor Clinicians vol 65 no 2 pp 87ndash108 2015

[2] C Gridelli A Rossi D P Carbone et al ldquoNon-small-cell lungcancerrdquo Nature Reviews Disease Primers vol 1 article 150092015

[3] C Liam S Andarini P Lee J C Ho N Q Chau and JTscheikuna ldquoLung cancer staging now and in the futurerdquoRespirology vol 1 pp 526ndash534 2015

[4] R Benezra R L Davis D Lockshon D L Turner and HWeintraub ldquoThe protein Id a negative regulator of helix-loop-helix DNA binding proteinsrdquoCell vol 61 no 1 pp 49ndash59 1990

[5] J Perk A Iavarone and R Benezra ldquoId family of helix-loop-helix proteins in cancerrdquo Nature Reviews Cancer vol 5 no 8pp 603ndash614 2005

[6] S I Rothschild A Kappeler D Ratschiller et al ldquoThe stem cellgene ldquoinhibitor of differentiation 1rdquo (ID1) is frequently expressedin non-small cell lung cancerrdquo Lung Cancer vol 71 no 3 pp306ndash311 2011

[7] J G Trevino S Pillai S Kunigal et al ldquoNicotine inducesinhibitor of differentiation-1 in a Src-dependent pathway pro-motingmetastasis and chemoresistance in pancreatic adenocar-cinomardquo Neoplasia vol 14 no 12 pp 1102ndash1114 2012

[8] R Bhattacharya J Kowalski A R Larson M Brock and RM Alani ldquoId1 promotes tumor cell migration in nonsmall celllung cancersrdquo Journal of Oncology vol 2010 Article ID 8561058 pages 2010

[9] J Li Y Li B Wang Y Ma and P Chen ldquoId-1 promotes migra-tion and invasion of non-small cell lung cancer cells throughactivating NF-120581B signaling pathwayrdquo Journal of BiomedicalScience vol 24 article 95 2017

[10] E Castanon A Soltermann I Lopez et al ldquoThe inhibitor ofdifferentiation-1 (Id1) enables lung cancer liver colonizationthrough activation of an EMT program in tumor cells andestablishment of the pre-metastatic nicherdquo Cancer Letters vol402 pp 43ndash51 2017

[11] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009

[12] W-Y Gong Z-X Zhao B-J Liu L-W Lu and J-C DongldquoExploring the chemopreventive properties and perspectives ofbaicalin and its aglycone baicalein in solid tumorsrdquo EuropeanJournal of Medicinal Chemistry vol 126 pp 844ndash852 2017

[13] C Naveenkumar S Asokkumar S Raghunandhakumar et alldquoPotent antitumor and antineoplastic efficacy of baicalein onbenzo(a)pyrene-induced experimental pulmonary tumorigen-esisrdquo Fundamental amp Clinical Pharmacology vol 26 no 2 pp259ndash270 2012

[14] W-Y I Gong J-F Wu B-J Liu et al ldquoFlavonoid componentsin Scutellaria baicalensis inhibit nicotine-induced proliferationmetastasis and lung cancer-associated inflammation in vitrordquoInternational Journal of Oncology vol 44 no 5 pp 1561ndash15702014

[15] J Gao W A Morgan A Sanchez-Medina and O CorcoranldquoThe ethanol extract of Scutellaria baicalensis and the activecompounds induce cell cycle arrest and apoptosis includingupregulation of p53 and Bax in human lung cancer cellsrdquoToxicology and Applied Pharmacology vol 254 no 3 pp 221ndash228 2011

[16] G Su H Chen and X Sun ldquoBaicalein suppresses non smallcell lung cancer cell proliferation invasion and Notch signalingpathwayrdquo Cancer Biomarkers vol 22 no 1 Article ID 170673pp 13ndash18 2018

[17] C Roschger and C Cabrele ldquoThe Id-protein family in develop-mental and cancer-associated pathwaysrdquo Cell Communicationand Signaling vol 15 article 26 2017

[18] E Cubillo A Diaz-Lopez E P Cuevas et al ldquoE47 and Id1interplay in epithelial-mesenchymal transitionrdquo PLoS One vol8 article e59948 2013

[19] M-T Ling T C M Lau C Zhou et al ldquoOverexpression ofId-1 in prostate cancer cells promotes angiogenesis throughthe activation of vascular endothelial growth factor (VEGF)rdquoCarcinogenesis vol 26 no 10 pp 1668ndash1676 2005

[20] V J Young S F Ahmad J K Brown W C Duncan and AWHorne ldquoPeritoneal VEGF-A expression is regulated by TGF-beta1 through an ID1 pathway in women with endometriosisrdquoScientific Reports vol 5 article 16859 2015

[21] S I Rothschild M P Tschan E A Federzoni et alldquoMicroRNA-29b is involved in the Src-ID1 signaling pathwayand is dysregulated in human lung adenocarcinomardquoOncogenevol 31 no 38 pp 4221ndash4232 2012

[22] World Health Organization ldquoGLOBOCAN 2012 Estimatedcancer incidence mortality and prevalence worldwiderdquo 2012httpglobocaniarcfrPagesfact sheets canceraspx

[23] D S Ettinger W Akerley and G Bepler ldquoNonsmall cell lungcancerrdquo Journal of the National Comprehensive Cancer Networkvol 8 pp 740ndash801 2010

[24] J Minguet K H Smith and P Bramlage ldquoTargeted therapiesfor treatment of non-small cell lung cancer - Recent advancesand future perspectivesrdquo International Journal of Cancer vol138 no 11 pp 2549ndash2561 2016

[25] H Liu Y Dong Y Gao et al ldquoThe fascinating effects ofbaicalein on cancer a reviewrdquo International Journal ofMolecularSciences vol 17 article 168110 2016

Evidence-Based Complementary and Alternative Medicine 7

81673916 8150150396 and 81703829] Shanghai MunicipalCommission of Health and Family Planning [Grant noZYKC201602001] and Development Project of ShanghaiPeak Disciplines Integrative Medicine [Grant no 20150407]

References

[1] L A Torre F Bray R L Siegel J Ferlay and J Lortet-Tieulent ldquoGlobal cancer statistics 2012rdquo CA A Cancer Journalfor Clinicians vol 65 no 2 pp 87ndash108 2015

[2] C Gridelli A Rossi D P Carbone et al ldquoNon-small-cell lungcancerrdquo Nature Reviews Disease Primers vol 1 article 150092015

[3] C Liam S Andarini P Lee J C Ho N Q Chau and JTscheikuna ldquoLung cancer staging now and in the futurerdquoRespirology vol 1 pp 526ndash534 2015

[4] R Benezra R L Davis D Lockshon D L Turner and HWeintraub ldquoThe protein Id a negative regulator of helix-loop-helix DNA binding proteinsrdquoCell vol 61 no 1 pp 49ndash59 1990

[5] J Perk A Iavarone and R Benezra ldquoId family of helix-loop-helix proteins in cancerrdquo Nature Reviews Cancer vol 5 no 8pp 603ndash614 2005

[6] S I Rothschild A Kappeler D Ratschiller et al ldquoThe stem cellgene ldquoinhibitor of differentiation 1rdquo (ID1) is frequently expressedin non-small cell lung cancerrdquo Lung Cancer vol 71 no 3 pp306ndash311 2011

[7] J G Trevino S Pillai S Kunigal et al ldquoNicotine inducesinhibitor of differentiation-1 in a Src-dependent pathway pro-motingmetastasis and chemoresistance in pancreatic adenocar-cinomardquo Neoplasia vol 14 no 12 pp 1102ndash1114 2012

[8] R Bhattacharya J Kowalski A R Larson M Brock and RM Alani ldquoId1 promotes tumor cell migration in nonsmall celllung cancersrdquo Journal of Oncology vol 2010 Article ID 8561058 pages 2010

[9] J Li Y Li B Wang Y Ma and P Chen ldquoId-1 promotes migra-tion and invasion of non-small cell lung cancer cells throughactivating NF-120581B signaling pathwayrdquo Journal of BiomedicalScience vol 24 article 95 2017

[10] E Castanon A Soltermann I Lopez et al ldquoThe inhibitor ofdifferentiation-1 (Id1) enables lung cancer liver colonizationthrough activation of an EMT program in tumor cells andestablishment of the pre-metastatic nicherdquo Cancer Letters vol402 pp 43ndash51 2017

[11] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009

[12] W-Y Gong Z-X Zhao B-J Liu L-W Lu and J-C DongldquoExploring the chemopreventive properties and perspectives ofbaicalin and its aglycone baicalein in solid tumorsrdquo EuropeanJournal of Medicinal Chemistry vol 126 pp 844ndash852 2017

[13] C Naveenkumar S Asokkumar S Raghunandhakumar et alldquoPotent antitumor and antineoplastic efficacy of baicalein onbenzo(a)pyrene-induced experimental pulmonary tumorigen-esisrdquo Fundamental amp Clinical Pharmacology vol 26 no 2 pp259ndash270 2012

[14] W-Y I Gong J-F Wu B-J Liu et al ldquoFlavonoid componentsin Scutellaria baicalensis inhibit nicotine-induced proliferationmetastasis and lung cancer-associated inflammation in vitrordquoInternational Journal of Oncology vol 44 no 5 pp 1561ndash15702014

[15] J Gao W A Morgan A Sanchez-Medina and O CorcoranldquoThe ethanol extract of Scutellaria baicalensis and the activecompounds induce cell cycle arrest and apoptosis includingupregulation of p53 and Bax in human lung cancer cellsrdquoToxicology and Applied Pharmacology vol 254 no 3 pp 221ndash228 2011

[16] G Su H Chen and X Sun ldquoBaicalein suppresses non smallcell lung cancer cell proliferation invasion and Notch signalingpathwayrdquo Cancer Biomarkers vol 22 no 1 Article ID 170673pp 13ndash18 2018

[17] C Roschger and C Cabrele ldquoThe Id-protein family in develop-mental and cancer-associated pathwaysrdquo Cell Communicationand Signaling vol 15 article 26 2017

[18] E Cubillo A Diaz-Lopez E P Cuevas et al ldquoE47 and Id1interplay in epithelial-mesenchymal transitionrdquo PLoS One vol8 article e59948 2013

[19] M-T Ling T C M Lau C Zhou et al ldquoOverexpression ofId-1 in prostate cancer cells promotes angiogenesis throughthe activation of vascular endothelial growth factor (VEGF)rdquoCarcinogenesis vol 26 no 10 pp 1668ndash1676 2005

[20] V J Young S F Ahmad J K Brown W C Duncan and AWHorne ldquoPeritoneal VEGF-A expression is regulated by TGF-beta1 through an ID1 pathway in women with endometriosisrdquoScientific Reports vol 5 article 16859 2015

[21] S I Rothschild M P Tschan E A Federzoni et alldquoMicroRNA-29b is involved in the Src-ID1 signaling pathwayand is dysregulated in human lung adenocarcinomardquoOncogenevol 31 no 38 pp 4221ndash4232 2012

[22] World Health Organization ldquoGLOBOCAN 2012 Estimatedcancer incidence mortality and prevalence worldwiderdquo 2012httpglobocaniarcfrPagesfact sheets canceraspx

[23] D S Ettinger W Akerley and G Bepler ldquoNonsmall cell lungcancerrdquo Journal of the National Comprehensive Cancer Networkvol 8 pp 740ndash801 2010

[24] J Minguet K H Smith and P Bramlage ldquoTargeted therapiesfor treatment of non-small cell lung cancer - Recent advancesand future perspectivesrdquo International Journal of Cancer vol138 no 11 pp 2549ndash2561 2016

[25] H Liu Y Dong Y Gao et al ldquoThe fascinating effects ofbaicalein on cancer a reviewrdquo International Journal ofMolecularSciences vol 17 article 168110 2016

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