Ginkgo biloba Extract Decreases Non-Small Cell Lung Cancer Cell Migration by Downregulating...

Ginkgo biloba Extract Decreases Non-Small Cell LungCancer Cell Migration by Downregulating Metastasis-Associated Factor Heat-Shock Protein 27Jong-Rung Tsai1,2, Po-Len Liu1, Yung-Hsiang Chen3, Shah-Hwa Chou1,4, Ming-Chan Yang5, Yu-

Jen Cheng6, Jhi-Jhu Hwang1,2, Wei-Hsian Yin7, Inn-Wen Chong1,2*

1 Department of Respiratory Therapy, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, 2 Department of Internal Medicine, Kaohsiung Medical

University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 3 Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan,

4 Department of Surgery, Division of Chest Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 5 Department of Surgery,

Yuan’s General Hospital, Kaohsiung, Taiwan, 6 Department of Health Management, Division of Thoracic Surgery, Department of Surgery, E-Da Hospital, I-Shou University,

Kaohsiung, Taiwan, 7 Division of Cardiology, Cheng-Hsin General Hospital, Faculty of Medicine, and Cardiovascular Research Centre, School of Medicine, National Yang-

Ming University, Taipei, Taiwan

Abstract

Heat-shock proteins (HSPs) are molecular chaperones that protect proteins from damage. HSP27 expression is associatedwith cancer transformation and invasion. Ginkgo biloba extract (EGb761), the most widely sold herbal supplement, hasantiangiogenic effects and induces tumor apoptosis. Data regarding the effect of EGb761 on HSP expression is limited,particularly in cancer. HSP27 expression in paired tumors and normal lung tissues of 64 patients with non-small cell lungcancer (NSCLC) were detected by real-time PCR, western blotting, and immunohistochemistry. NSCLC cell lines (A549/H441)were used to examine the migratory abilities in vitro. NSCLC tissue showed higher HSP27 expression than normal lungtissue. Kaplan–Meier survival analysis showed that NSCLC patients with low HSP27 expression ratio (,1) had significantlylonger survival time than those with a high expression ratio (.1) (p = 0.04). EGb761 inhibited HSP27 expression andmigratory ability of A549/H441 cells, which is the same as HSP27-siRNA transfection effect. Moreover, EGb761 treatmentactivated the AKT and p38 pathways and did not affect the expression of PI3K, ERK, and JNK pathways. HSP27 is a poorprognostic indicator of NSCLC. EGb761 can decrease the migration ability of A549/H441 by inhibiting HSP27 expressionmost likely through AKT and p38 MAPK pathways activation.

Citation: Tsai J-R, Liu P-L, Chen Y-H, Chou S-H, Yang M-C, et al. (2014) Ginkgo biloba Extract Decreases Non-Small Cell Lung Cancer Cell Migration byDownregulating Metastasis-Associated Factor Heat-Shock Protein 27. PLoS ONE 9(3): e91331. doi:10.1371/journal.pone.0091331

Editor: Prasad S. Adusumilli, Memorial Sloan-Kettering Cancer Center, United States of America

Received October 29, 2013; Accepted February 9, 2014; Published March 11, 2014

Copyright: � 2014 Tsai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported in part by grant KMU-Q098022 from the Kaohsiung Medical University, and grant NSC102-2314-B-037-067 from NationalScience Council, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Lung cancer is the leading cause of cancer mortality worldwide.

Even with improvements in both diagnostic and therapeutic

techniques, the overall 5-year survival rate is still poor. Poor

prognosis of lung cancer is primarily caused by early relapse,

metastasis, and poor response to treatments such as surgery,

chemotherapy, and radiotherapy [1–2]. Lack of good prognostic

biomarkers, which can predict treatment response and prognosis,

also affects treatment plans and patient outcomes.

Heat-shock proteins (HSPs) are a large family of proteins that

have essential hemostatic functions in cells under physiological

conditions. According to molecular weights, HSPs are grouped

into several subfamilies: small (HSP 20–30 kDa), HSP40, HSP60,

HSP70, HSP90, and HSP100. The main function of HSP is to

protect cells against damage in stressful conditions [3]. In addition

to their cytoprotective effects, HSPs can promote carcinogenesis

by inhibiting apoptosis [4–8] and by enhancing resistance to

treatment [9–10]. However, the role of HSPs in different tumors is

complicated [4,9].

HSP27 is a cytoplasmic protein that is constitutively expressed

in several normal tissues and malignancies. Its expression has been

associated with poor prognosis in ovarian [11], breast [9,12],

gastric [13–14], liver [15], and prostate cancers [16] as well as

osteoscarcomas [7]. In head and neck cancer [3] and genitouri-

nary tract tumor [17], HSP27 expression has no effect on

prognosis. However, the prognostic role of HSP27 expression in

lung cancer is under debate. Zimmermann et al. [18] reported that

serum HSP27 level was positively correlated with advanced lung

cancer stages. HSP27 expression can increase the chemoresistance

of non-small cell lung cancer (NSCLC) and is a poor indicator of

prognosis [10,19]. In contrast, HSP27 overexpression is associated

with better survival in patients with NSCLC according to a study

by Malusecka et al. [20]. Immunohistochemical staining showed

that HSP27 expression in lung cancer tissue is not correlated with

T (primary tumor) N (lymph nodes) M (metastasis) and stages [21].

Extracts from the leaves of Ginkgo biloba have been used in China

and Western countries for centuries due to their antioxidant

properties [22]. A standard G. biloba extract, EGb761 (commercial

name), contains 22%–27% flavonoids and 5%–7% terpenoids,

PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e91331

http://creativecommons.org/licenses/by/4.0/

which are the most important active substances [23]. EGb761 can

scavenge free radicals and neutralize ferry ion-induced peroxidants

[24]. Therefore, EGb761 is helpful for the prevention and

treatment of degenerative processes associated with oxidative

stress [25–26].

Although chemotherapy is still the primary treatment used for

lung cancer, the adverse side effects related to this treatment limit

its use. Complementary medications such as herbal medication

have become more popular in recent decades. Moreover, several

experiments have reported that EGb761 has antitumor effects.

The anticancer properties of EGb761 are attributed to its

antioxidant, antiangiogenic, and gene-regulatory effects [24].

EGb761 can inhibit tumor proliferation via apoptosis in colon

cancer [27] and oral cavity cancer [28]. Phase II combined

treatment involving 5-fluorouracil (5-FU) and EGb761 has been

tested in patients with pancreatic or colorectal cancer [29–30] and

has shown promising results. In this study, we investigated HSP27

expression in patients with NSCLC and analyzed the relationship

between HSP27 expression and clinical outcomes. In addition, the

effects of EGb761 on HSP27 expression were explored. We hope

that the results of this study will provide clinicians with a novel

combination of drug regimens and serve as a predictor to improve

NSCLC prognosis.

Results

Demographic data and HSP27 expression in patientswith NSCLC

In total, 64 patients with NSCLC were included in this study.

Of these, 47 (73%) were histologically identified as having

adenocarcinoma and 17 (27%) patients were identified as having

squamous cell carcinoma. The average age of patients was

61.269.5 years (range, 36–78 years). The TNM staging and

HSP27 expression status in NSCLC patients are summarized in

Table 1. The average HSP27 expression ratio was 2.0561.95.

Patients with NSCLC with metastasis and advanced stage (stage

IIIb-IV) cancers had higher HSP27 expression ratio than those

Table 1. Relationship between HSP27 expression ratio, demographic characteristic in non-small cell lung cancer (NSCLC) patients.

Number Mean ± SD P value

STAGE 0.009*

1 (1-3a) 37 1.5261.49

2 (3b-4) 27 2.7762.33

Tumor 0.28

1 16 1.6161.35

2 33 2.0062.05

3 3 1.2461.52

4 12 2.9662.26

Node 0.39

0 35 1.7861.65

1 10 2.4062.44

2 14 2.0061.62

3 5 3.3063.70

Metastasis 0.03*

0 41 1.6661.50

1 23 2.7462.42

Pathology 0.99

SQ 17 2.0462.28

AD 47 2.0461.84

Sex 0.34

Male 42 1.8861.70

Female 22 2.3762.36

SQ: squamous cell carcinoma, AD: adenocarcinoma (*p,0.05).doi:10.1371/journal.pone.0091331.t001

Figure 1. Survival curve of non-small cell lung cancer patientsin high and low HSP27 expression groups using the Kaplan-Meier method.doi:10.1371/journal.pone.0091331.g001

EGb761 Decreases Cancer Metastasis via HSP27


with no metastasis and early stage cancers (p = 0.03 and p = 0.009,

respectively). Kaplan–Meier survival curve showed NSCLC

patients with a low HSP27 expression ratio had significantly

better survival time than those with a high expression ratio (p,

0.05; Fig. 1). The multivariate-adjusted risk ratios were computed

using Cox regression with additional variables of gender (male

versus female), age (years), metastasis, tumor, and lymph node

involvement (Table 2). By doing so, we found that patients with

high HSP27 expression had a 2.30-times higher mortality risk

(p = 0.04) than patients with low HSP27 expression.

HSP27 expression in patients with NSCLCHSP27 expression in patients with NSCLC was analyzed

(Fig. 2). Immunohistochemical staining and western blot analysis

of lung cancer tissue showed higher HSP27 expression in lung

cancer tissue than in the normal lung tissue (Fig. 2A–B).

Effect of EGb761 on cytotoxicity and HSP27 expression inBEAS-2B and NSCLC cell lines (A549 and H441)

We treated 3 cell lines (BEAS-2B, A549, and H441) with

different concentrations of EGb761. The MTT assay showed that

EGb761 did not have cytotoxic effect on these 3 cell lines even at

higher concentrations (Fig. 3A). The DNA fragmentation assay

also showed that EGb761 did not induce apoptosis in BEAS-2B,

A549, and H441 cell lines at different concentrations (Fig. 3B).

HSP27 expression in A549 and H441 cell lines significantly

decreased in a dose-dependent manner with an increase in

EGb761 concentration, as determined by western blot analysis

(Fig. 3C). However, HSP27 expression in normal bronchial

epithelial cells (BEAS-2B) did not change when the EGb761

concentrations was below 500 ug/mL.

Effect of EGb761 and HSP27-siRNA transfection on HSP27expression

We transfected the A549/H441 cells with HSP27-siRNA

plasmid. The HSP27-siRNA plasmid transfection significantly

decreased HSP27 expression in A549/H441 cells, which was

confirmed using real-time PCR and western blotting (Fig. 4A–B).

EGb761 also had the same effect as HSP27-siRNA transfection in

significantly decreasing HSP27 mRNA and protein expression in

A549/H441 (Fig. 4A–B). Although both HSP27-siRNA and

EGb761 treatment can downregulate HSP27 expression, it does

not mean that there is an effector relationship between them.

Additional studies are needed to further examine this relationship.

Effects of EGb761 and HSP27-siRNA transfection on themigratory ability of A549/H441

A549/H441 cells were used to evaluate the effect of HSP27 on

NSCLC cell migratory ability in vitro. Cellular migration was

analyzed using wound scratch assay. Compared to the migratory

ability of the control group, the migratory activity of the A549/

H441 cells was inhibited significantly after HSP27-siRNA

transfection or EGb761 treatment. (p,0.05) (Fig. 5A–B)

The regulatory effect of EGb761 on HSP27 expressionthrough AKT and p38 MAPK pathway

The intracellular signaling pathways such as MAPKs and

PI3K/AKT are important determinants of cancer migration [31–

33]. However, the intracellular regulatory signaling pathways

between EGb761 and HSP27 expression are still unclear. We

analyzed the expression of different pathway proteins after the

treatment of A549/H441 cells with different EGb761 concentra-

tions. The expression of p-AKT and p-P38 was significantly

increased after EGb761 treatment compared with the control

group, and the expression of PI3K, ERK, and JNK did not change

after EGb761 treatment (Fig. 6A, D) We further treated the A549/

H441 cell line with AKT inhibitor (API-59, 3 mM) and p38

MAPK inhibitor (SB203580, 10 mM). The inhibitory effect of

EGb761 on HSP27 expression was blocked by the AKT or p38

MAPK inhibitor (Fig. 6B, E). Furthermore, the migration ability of

A549/H441 cells also recovered after AKT or p38 inhibitor

treatment even in the presence of EGb761 (Fig. 6C, F). These

results suggested that EGb761 regulated HSP27 expression, most

likely through the AKT and p38 MAPK signaling pathways.

Discussion

HSPs are ubiquitous molecular chaperones present in all living

cells. They protect cells from environmental stress damage [34].

The small HSP27 plays a role in signal transduction, growth

regulation, development, differentiation, and tumorigenesis [35].

Increasing levels of HSP27 have been observed in several

malignancies such as breast cancer [36], colon cancer [37],

prostate cancer [16], and lung cancer [10,21]. In our study, we

Table 2. Multivariate Cox regression analysis of mortality.

Term Risk Ratio Lower 95% Upper 95% P value

HSP27 ratio .1 2.30 1.03 5.38 0.04*

Age 1.04 1.00 1.08 0.05

Tumor 2 vs.1 1.81 0.75 4.75 0.59

3 vs.1 2.02 0.31 10.54

4 vs.1 1.37 0.40 4.81

Node 1 vs.0 1.17 0.42 2.99 0.57

2 vs.0 0.96 0.31 2.75

3 vs.0 2.72 0.63 10.21

Metastasis 1.18 0.51 2.61 0.70

Cell type SQ vs. AD 0.86 0.35 2.14 0.74

Gender M vs. F 1.74 0.71 4.29 0.22

SQ: squamous cell carcinoma, AD: adenocarcinoma (*p,0.05).doi:10.1371/journal.pone.0091331.t002



screened the expression of HSPs, including HSP10, HSP27,

HSP30, HSP60, HSP70, and HSP90 in NSCLC tissue samples

(data not shown). Only HSP27 was found to be overexpressed in

NSCLC tissue. Coordinating with patient clinical parameters,

overexpression of HSP27 seems to increase the metastatic

potential of NSCLC and is a poor prognostic predictor. Blocking

HSP27 expression by EGb761 decreased the migratory ability of

NSCLC cell lines.

HSP27 has attracted a lot of attention in recent decades due to

its role in tumor carcinogenesis, prognostic, predictive, and

treatment implications [4]. In esophageal cancer, HSP27 expres-

sion decreases during carcinogenesis to adenocarcinomas but

increases during carcinogenesis to squamous carcinomas [38].

HSP27 expression is correlated with the degree of differentiation

in skin [39], endometrial [40], and uterine cancers [41]. HSP27

overexpression also induces chemoresistance in prostate cancer

[42] and esophageal cancer [43] but enhanced response to

treatment in head and neck cancer [44]. In summary, the role of

HSP27 is variable and is dependent on different tumor types.

In our study, HSP27 was a poor prognostic indicator in patients

with NSCLC after adjusting for other factors. Higher expression

ratio of HSP27 was observed in patients with NSCLC with an

advanced cancer, which was same as that reported in a study by

Zimmermann et al. [18]. Serum HSP27 level is a useful biomarker

to discriminate healthy smokers and patients with cancer with

early and advanced stage NSCLC [18]. However, a report by

Malusecka et al. showed that HSP27 expression may be a

favorable prognostic factor in NSCLC [20]. Subgroup analysis

showed that HSP27 retained its prognostic significance in

squamous cell carcinoma but not in adenocarcinoma. Two third

of patients had squamous cell carcinoma in the Malusecka study,

but the percentage of squamous cell carcinoma was only 27% in

our study. Our subgroup analysis showed no prognostic difference

between patients with adenocarcinoma and squamous cell

carcinoma. Small sample sizes and heterogeneity are our

limitations. Furthermore, the migratory ability of A549 and

H441 in vitro was significantly decreased after silencing HSP27

expression, which was consistent with our clinical observation that

HSP27 expression may increase the metastatic potential. Although

HSP27 had been identified as a metastasis-associated protein in

NSCLC [45–46], the exact association between HSP27 and the

metastatic mechanism requires further investigation.

Chemotherapy drugs usually affect both pathological tumor

cells and normal cells and cause serious complications and toxicity.

Cisplatin, an effective antineoplastic agent, is the main element in

lung cancer chemotherapy regimen. Sensorineural hearing loss

and nephrotoxicity are the major adverse effects of cisplatin, which

may involve excessive formation of free radicals [24]. Through

scavenging and preventing the formation of free radicals, EGb761

may induce protective effects against cisplatin-induced toxicity. In

an experiment in rats, EGb761 successfully decreased cisplatin-

associated toxicity without attenuating its antitumor activity [47].

Unresponsiveness or survival of cancer stem cells after treatment

may be responsible for the recurrence and resistance of lung

cancer to modern therapy [48]. HSP27 activation has been

observed in chemoresistant lung cancer stem cell-like cells [10].

Our study showed that EGb761 inhibited HSP27 expression in

NSCLC cells. Therefore, EGb761 may have great potential in

lung cancer therapy because of its inhibitory effect on HSP27

expression.

Cellular heat-shock response usually develops quickly, which is

related to the activation of major signaling transduction pathways

involving mitogen-activated protein (MAP) kinases, extracellular

signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK),

and p38 [49]. These signaling cascades play a central role in

regulating and determining cell fate such as growth, differentia-

tion, or apoptosis in numerous physiological as well as stress

conditions [50]. AKT/PI3K-dependent signal transduction path-

ways are cell survival signals stimulated by growth factors,

cytokines, and oncoproteins. Overactive AKT/PI3K pathway

Figure 2. Expression of heat shock protein 27 in NSCLC patients. (A) Lung samples (lung cancer and corresponding normal adjacent lungtissues) were analyzed with antibody to heat shock protein 27 (HSP27) in immunohistochemical staining (DAB staining and hematoxylincounterstaining). For negative controls, the antibody was replaced by control IgG. HSP 27 was overexpressed in NSCLC tissue than normal lung tissue.(B) Western blotting analysis of HSP 27 expression in NSCLC tissue and normal tissue. Representative data from four different patients with NSCLC areshown (T = tumor; N = normal). The expression of HSP27 protein expression was significantly increased in NSCLC tissue as compared to normal lungtissue (*p,0.05).doi:10.1371/journal.pone.0091331.g002



can reduce tumor cell apoptosis and augment proliferation [51].

However, the regulatory relationship between HSP27, PI3K/

AKT, and MAP kinase pathways is complex. For example, the

p38MAPK-MAPKAPK2-HSP27 pathway will be activated by

chemotherapy agent ciplastin plus gemcitabin in lung cancer stem

cells [10]. In contrast, HSP27 induces cisplatin resistance by

depressing p38 activation and enhancing AKT activation in lung

cancer cells [52]. This difference may be due to different cell types

and the study design. Guo et al. showed that an early, transient

activation of JNK or p38 MAPK (or both) is usually associated

with cell survival or differentiation, whereas a late, sustained

activation of these kinases is generally associated with apoptosis

[53]. In colorectal cancers, HSP27 overexpression is KRAS

mutation dependent [54], but both A549 and H441 are KRAS

mutation cell lines. The relationship of KRAS mutation and

HSP27 expression in lung cancer needs further investigation. In

our report, EGb761 attenuated HSP27 expression in A549/H441

cell lines by activating p38 MAPK and AKT pathways but not

ERK or JNK pathway. Because the combination of AKT and p38

inhibitor treatment may decrease cellular viability, the synergistic

effects of the combination treatment on the expression of HSP27

may need further analysis. Moreover, because the activation

(phosphorylation) status is unstable and dephosphorylation

occurred rapidly under physiological conditions, it is difficult to

observe the true results of AKT and p38 activation in vivo in the

low HSP27 expression patient group.

In conclusion, HSP27 is a poor prognostic factor of NSCLC,

and EGb761 has great potential to being used as a complement

therapy for the treatment of NSCLC due to its inhibitory effect on

HSP27 expression.

Materials and Methods

Tumor sample collectionNSCLC and corresponding normal tissues were collected from

64 patients who underwent surgical resection at the Division of

Figure 3. Effect of EGb761 in cell cytotoxicity and heat shock protein 27 expression of BEAS-2B, A549 and H441. (A) EGb761 did nothave obvious cytotoxic effect on lung cancer cell lines (A549 and H441) and normal bronchial epithelial cells (BEAS-2B). (B) DNA fragment assayshowed EGb761 did not induce BEAS-2B, A549 and H441 cell apoptosis. (C) The HSP27 mRNA/protein expression of BEAS-2B cells did not changesignificantly when the concentrations of EGb761 was below 500[0-9][A-z]ug/mL. In addition, the HSP27 mRNA/protein expression of A549/H441 cellscan be decreasing significantly with increasing EGb761 concentration in a dose dependent manner. Representative photos of three independentexperiments. *P,0.05 versus control. Assays were carried out in triplicate.doi:10.1371/journal.pone.0091331.g003



Thoracic Surgery, Department of Surgery, Kaohsiung Medical

University Hospital, from 2004 to 2010. Tissue samples were

immediately placed in liquid nitrogen for shipment to the

laboratory, and then stored in 280uC freezers until RNA isolation

and protein extraction were conducted. Complete staging

procedures, including chest radiography, bronchoscopy, brain

and thoracic computed tomography, sonography, and bone

scintigraphy were performed to precisely determine the charac-

teristics of TNM in patients with NSCLC according to the TNM

International Staging System for Lung cancer [55]. All patients

were followed up until March 2011, and details of their

demographic and survival data were updated.

Ethics statementThe study was approved by the Ethical Review Board for

Research (KMUH-IRB-990358) of the Kaohsiung Medical

University Hospital, Kaohsiung, Taiwan. The participants provide

their written informed consent to participate in this study.

RNA extraction and real-time polymerase chain reaction(PCR)

Total RNA was isolated from frozen lung tumor tissues of

patients with NSCLC and corresponding normal adjacent lung

tissues. RNA from normal lung tissue, lung tumor tissue, and lung

cancer cells was analyzed using real-time PCR. Total RNA was

isolated using an RNeasy Mini kit and an RNase-free DNase Set

Figure 4. EGb761 and HSP27-siRNA transfection effect on the expression of HSP27 in A549/H441. (A) The HSP27 mRNA expression ofA549/H441 cells was decreased with EGb761 treatment or HSP27-siRNA transfection 24 hours later as compared to control group. (B) The HSP27protein expression of A549/H441 cells was also decreased with EGb761 treatment and HSP27-siRNA transfection 24 hours later as compared tocontrol group. *P,0.05 versus control. Assays were carried out in triplicate. NC-siRNA is the negative control.doi:10.1371/journal.pone.0091331.g004



(Qiagen, Valencia, CA, USA). Total RNA (2 mg) was reverse-

transcribed using SuperScriptTM and First-Strand Synthesis

System for RT-PCR Kit (Invitrogen, Carlsbad, CA, USA). A

1:5 dilution of the resulting cDNA was used as the standard, and a

1:10 template dilution of the resulting cDNA was used as the

standard to quantify the relative content of mRNA by using real-

time PCR (LightCycler FastStart DNA Master SYBR Green I,

Roche, Mannheim, Germany). The following primers for real-

time PCR were designed using the Primer Express software

(RealQuant, Roche, Mannheim, Germany) by using published

sequences: human HSP27 (GenBank: accession number:

AB020027.1) sense primer: 59-GTC CCA CGA GAT CAC

CAT-39, human HSP27 antisense primer: 59-GGT GGT TGC

TTT GAA CTT TAT T-39, human GAPDH sense primer: 59-

AGC CAC ATC GCT CAG ACA-39, and GAPDH antisense

primer: 59-GCC CAA TAC GAC CAA ATC C-39. Fluorescence

data were acquired at the end of extension. Melt analysis was

performed for all products to determine the specificity of the

Figure 5. EGb761 and HSP27-siRNA transfection effect on the cellular migratory ability of A549/H441. (A) Cellular migration wasdetermined by monolayer denudation assay and analyzed by the Wimasis WimScratch software. The migratory ability of A549/H441 cells wasinhibited with EGb761 (100 ug/mL) treatment as compared to control group. Silencing the HSP27 expression by HSP27-siRNA transfection, themigratory ability also decreased in A549/H441. NC-siRNA is the negative control. Representative photos of three independent experiments. Data arefrom three independent experiments. *P,0.05 versus control. Assays were carried out in triplicate.doi:10.1371/journal.pone.0091331.g005



amplification. In addition, PCR products were electrophoresed on

1% agarose gels to confirm whether the correct band sizes were

present. Relative expression was calculated as a ratio of the

expression in the tumor compared with the expression in the

normal adjacent tissue (high expression: tumor lesion/normal

tissue .1; low expression: tumor lesion/normal tissue ,1) [56–

57].

Immunohistochemical stainingTumor specimens were dissected from human lung tissue, fixed

in 4% buffered formalin solution overnight, embedded in paraffin,

and sectioned in 5-mm thicknesses. The paraffin sections were

deparaffinized with xylene and stained with antihuman-HSP27

(Santa Cruz, Dallas, TX, USA) antibody. After PBS washing, the

sections were incubated with horseradish peroxidase-conjugated

secondary antibody for 1 h at room temperature. For color

reactions, diaminobenzidine (DAB) was used and counter stained

with hematoxylin. For negative controls, the antibody was

replaced by control IgG.

Western blot analysisCells were lysed with lysis buffer [0.5 M NaCl, 50 mM Tris,

1 mM EDTA, 0.05% SDS, 0.5% Triton X-100, 1 mM phenyl-

methylsulfonyl fluoride (PMSF), pH 7.4] for 30 min at 4uC, and

the cell lysates were centrifuged at 4,000 g for 30 min at 4uC.

Protein concentrations in the supernatants were measured using a

BioRad protein determination kit (BioRad, Hercules, CA, USA).

The supernatants were subjected to 12% SDS-PAGE and then

transferred to polyvinylidene difluoride (PVDF) membranes (NEN)

for 1 h at room temperature. The membranes were then treated

with PBS containing 0.05% Tween 20 and 2% skimmed milk for

1 h at room temperature and incubated separately with mouse

antihuman HSP27, AKT/pAKT, P38/pP38, PI3K/pPI3K,

ERK/pERK, JNK/pJNK or a-tubulin (Abcam, Cambridge,

MA, USA) for 1 h at room temperature as internal control. After

washing, the membranes were incubated for with horseradish

peroxidase-conjugated rabbit antigoat or mouse IgG at room

temperature. Immunodetection was performed using chemilumi-

nescence reagent plus NEN and exposure to Biomax MR Film

(Kodak, Rochester, NY, USA).

Culture of NSCLC cellsLung adenocarcinoma A549/H441 cells (ATCC CCL-185/

ATCC HTB-174) were cultured in flasks in F12K growth medium

supplemented with 5% fetal bovine serum (FBS), 100 units/mL

penicillin, and 100 pg/mL streptomycin. The cells were cultured

at 37uC in a humidified atmosphere of 95% air and 5% CO2.

BEAS-2B cells (ATCC CRL- 9609), which are normal bronchial

epithelial cells, were used as control.

HSP27 silencing in A549/H441 cellsSmall interfering RNA (siRNA), a specific double-stranded 21-

nucleotide RNA sequence homologous to the target gene, was

used to silence HSP27 expression. Human HSP27 siRNA sense

primer: 59-GCG UGU CCC UGG AUG UCA ATT-39 and

antisense primer: 59-UUG ACA UCC AGG GAC ACG CGC-39.

SiRNA for HSP27 and negative control (NC-siRNA) were

designed and synthesized using a computer software from Ambion

(Austin, TX, USA) and SilencerTM siRNA construction kit from

Ambion, according to the manufacturer’s instructions. Inhibition

of HSP27 mRNA and protein expression was assessed using real-

time PCR and immunoblot analysis after transfection of A549/

H441 with HSP27-siRNA. Briefly, cells were grown in 6 wells and

transiently transfected with 20 nM siRNA by using 8 mL siPORT

Amine (Ambion, Austin, TX, USA) in a total transfection volume

of 0.5 mL of the medium. After incubation at 37uC, 5% CO2 for

5 h, 1.5 mL of the normal growth medium was added, and the

cells were incubated for 48 h.

MTT assay for cell viabilityThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT; Sigma, Louis, MO, USA) assay was used to measure

cell viability[58]. The principle of this assay is that mitochondrial

dehydrogenase in viable cells reduces MTT to a blue formazan.

Briefly, cells were grown in 96-well plates and incubated with

various concentrations of EGb761. After washing the cells twice

with PBS, 100 mL of the medium containing MTT (0.5 mg/mL)

was added to each well, and the cells were incubated at 37uC for

an additional 4 h. The medium was then carefully removed so as

to not disturb the formazan crystals formed. Next 100 mL DMSO,

which solubilizes the formazan crystals, was added to each well,

and the absorbance of the solubilized blue formazan was read at

540 nm by using a microplate reader (Multiskan Ex, Thermo-

Labsystems), with DMSO as the blank. The reduction in optical

density by the drugs was used as the measurement of cell viability,

normalized to cells incubated in control medium, which were

considered 100% viable.

Quantification of DNA fragmentationCellular DNA fragmentation ELISA assays were performed

using a kit according to the manufacturer’s protocol (Roche

Molecular Biochemicals, Mannheim, Germany). Cells were

incubated for 12 h at 37uC with a non-radioactive thymidine

analog 5-bromo-2-deoxyuridine (BrdU), which can be incorporat-

ed into genomic DNA. Subsequently, cells were incubated with or

without EGb761 for 6 h. Cells were then lysed, transferred to a

microtiter plate coated with an anti-DNA antibody, and incubated

for 90 min at room temperature. The plate was washed 3 times

and heated for 5 min in a microwave oven. Anti-BrdU-peroxidase

conjugate solution was added to each well, and the plate was

incubated for 90 min at room temperature. After the plate was

washed 3 times, substrate solution was added, and the plate was

incubated in the dark on a shaker until color development was

sufficient. The reaction was then stopped by adding 25 mL 0.56 M

H2SO4. A microplate reader (BioRad, Hercules, CA, USA) was

used to measure absorbance at 450 and 655 nm for each well.

Figure 6. The regulation pathways of EGb761 in HSP27 expression. (A, D) The expressions of AKT/pAKT, P38/pP38, PI3K/pPI3K, ERK/pERK,JNK/pJNK of A549/H441 were analyzed by western blotting after treatment with EGb761(100 ug/mL). The expression of pAKT and p-p38 wereenhanced by EGb761 treatment significantly (p,0.05)(B, E). A549/H441 cells line were treated with inhibitors of AKT (API-59) (3 mM), p38 MAPK(SB203580) (10 mM) for 1 hour and EGb761 (100 ug/mL) were treated later. The AKT inhibitor or p38 inhibitor can block the inhibitory effect ofEGb761 in HSP27 protein expression. (C, F) Cellular migration was determined by monolayer denudation assay and analyzed by the WimasisWimScratch software. With AKT inhibitor or p38 inhibitor, the cellular migratory ability of A549/H441 was not attenuated by EGb761. Representativephotos of three independent experiments. Data are from three independent experiments. *P,0.05 versus control. Assays were carried out intriplicate.doi:10.1371/journal.pone.0091331.g006



In vitro migration analysesThe migratory ability of cells were assayed in a monolayer

denudation assay, as described previously [59]. The confluent cells

were wounded by scraping with a 100-mL pipette tip, which

denuded a strip of the monolayer that was 300 mm in diameter.

The cultures were washed twice with PBS; then a medium

supplemented with 5% FBS was added, and the rate of wound

closure was observed after 24 h. The cells that migrated into the

denuded area were photographed, and the areas were analyzed

using the Wimasis WimScratch software. Wimasis WimScratch is

a new generation web-based image tool for cell migration analysis.

Edge detection techniques can easily recognize the leading edge

and the gap area. Users can upload the images and analysis will

start automatically.

EGb761 and pathway inhibitorsThe AKT and p38 MAPK inhibitors, API-59 (Santa Cruz,

Dallas, TX, USA) and SB203580 (Selleckchem, Radnor, PA,

USA), respectively, were used in our study. The EGb761 used in

our study were pursued from Dr. Willmar Schwabe GmbH & Co.,

Karlsruhe, (Germany).

Statistical analysesStatistical differences in HSP27 expression ratio and clinical

parameters were tested using student’s t-test or one-way analysis of

variance (ANOVA) test. Survival curves were drawn using the

Kaplan–Meier method. Results are expressed as mean 6 SEM.

Data was analyzed using the JMP software (SAS, JMP, Version

8.0, Cary, NC). The chi-square test was used for statistical analysis.

One-way ANOVA was applied to assess the differences among

groups. A p-value of ,0.05 was considered statistically significant.

Author Contributions

Conceived and designed the experiments: JRT PLL IWC. Performed the

experiments: PLL SHC MCY YJC JJH WHY. Analyzed the data: JRT

PLL YHC. Contributed reagents/materials/analysis tools: PLL SHC

MCY YJC JJH WHY IWC. Wrote the paper: JRT PLL IWC.

References

1. Rozsas A, Berta J, Rojko L, Horvath LZ, Keszthelyi M, et al. (2013)

Erythropoietin receptor expression is a potential prognostic factor in human

lung adenocarcinoma. PLoS One 8: e77459.

2. Chen X, Liu Y, Roe OD, Qian Y, Guo R, et al. (2013) Gefitinib or erlotinib as

maintenance therapy in patients with advanced stage non-small cell lung cancer:

a systematic review. PLoS One 8: e59314.

3. Gandour-Edwards R, Trock BJ, Gumerlock P, Donald PJ (1998) Heat shock

protein and p53 expression in head and neck squamous cell carcinoma.

Otolaryngol Head Neck Surg 118: 610–615.

4. Ciocca DR, Calderwood SK (2005) Heat shock proteins in cancer: diagnostic,

prognostic, predictive, and treatment implications. Cell Stress Chaperones 10:

86–103.

5. Erkizan O, Kirkali G, Yorukoglu K, Kirkali Z (2004) Significance of heat shock

protein-27 expression in patients with renal cell carcinoma. Urology 64: 474–

478.

6. Eskenazi AE, Powers J, Pinkas J, Oesterreich S, Fuqua SA, et al. (1998)

Induction of heat shock protein 27 by hydroxyurea and its relationship to

experimental metastasis. Clin Exp Metastasis 16: 283–290.

7. Moon A, Bacchini P, Bertoni F, Olvi LG, Santini-Araujo E, et al. (2010)

Expression of heat shock proteins in osteosarcomas. Pathology 42: 421–425.

8. Bausero MA, Page DT, Osinaga E, Asea A (2004) Surface expression of Hsp25

and Hsp72 differentially regulates tumor growth and metastasis. Tumour Biol

25: 243–251.

9. Conroy SE, Sasieni PD, Amin V, Wang DY, Smith P, et al. (1998) Antibodies to

heat-shock protein 27 are associated with improved survival in patients with

breast cancer. Br J Cancer 77: 1875–1879.

10. Hsu HS, Lin JH, Huang WC, Hsu TW, Su K, et al. (2011) Chemoresistance of

lung cancer stemlike cells depends on activation of Hsp27. Cancer.

11. Arts HJ, Hollema H, Lemstra W, Willemse PH, De Vries EG, et al. (1999) Heat-

shock-protein-27 (hsp27) expression in ovarian carcinoma: relation in response

to chemotherapy and prognosis. Int J Cancer 84: 234–238.

12. Lemieux P, Oesterreich S, Lawrence JA, Steeg PS, Hilsenbeck SG, et al. (1997)

The small heat shock protein hsp27 increases invasiveness but decreases motility

of breast cancer cells. Invasion Metastasis 17: 113–123.

13. Kapranos N, Kominea A, Konstantinopoulos PA, Savva S, Artelaris S, et al.

(2002) Expression of the 27-kDa heat shock protein (HSP27) in gastric

carcinomas and adjacent normal, metaplastic, and dysplastic gastric mucosa,

and its prognostic significance. J Cancer Res Clin Oncol 128: 426–432.

14. Takeno S, Noguchi T, Kikuchi R, Sato T, Uchida Y, et al. (2001) Analysis of the

survival period in resectable stage IV gastric cancer. Ann Surg Oncol 8: 215–

221.

15. King KL, Li AF, Chau GY, Chi CW, Wu CW, et al. (2000) Prognostic

significance of heat shock protein-27 expression in hepatocellular carcinoma and

its relation to histologic grading and survival. Cancer 88: 2464–2470.

16. Cornford PA, Dodson AR, Parsons KF, Desmond AD, Woolfenden A, et al.

(2000) Heat shock protein expression independently predicts clinical outcome in

prostate cancer. Cancer Res 60: 7099–7105.

17. Atkins D, Lichtenfels R, Seliger B (2005) Heat shock proteins in renal cell

carcinomas. Contrib Nephrol 148: 35–56.

18. Zimmermann M, Nickl S, Lambers C, Hacker S, Mitterbauer A, et al. (2012)

Discrimination of clinical stages in non-small cell lung cancer patients by serum

HSP27 and HSP70: a multi-institutional case-control study. Clin Chim Acta

413: 1115–1120.

19. Berrieman HK, Cawkwell L, O’Kane SL, Smith L, Lind MJ (2006) Hsp27 may

allow prediction of the response to single-agent vinorelbine chemotherapy innon-small cell lung cancer. Oncol Rep 15: 283–286.

20. Malusecka E, Krzyzowska-Gruca S, Gawrychowski J, Fiszer-Kierzkowska A,

Kolosza Z, et al. (2008) Stress proteins HSP27 and HSP70i predict survival in

non-small cell lung carcinoma. Anticancer Res 28: 501–506.

21. Wang W, Xu X, Wang W, Shao W, Li L, et al. (2011) The expression andclinical significance of CLIC1 and HSP27 in lung adenocarcinoma. Tumour

Biol 32: 1199–1208.

22. Pietri S, Seguin JR, d’Arbigny P, Drieu K, Culcasi M (1997) Ginkgo bilobaextract (EGb 761) pretreatment limits free radical-induced oxidative stress in

patients undergoing coronary bypass surgery. Cardiovasc Drugs Ther 11: 121–

131.

23. Chao JC, Chu CC (2004) Effects of Ginkgo biloba extract on cell proliferation

and cytotoxicity in human hepatocellular carcinoma cells. World J Gastroenterol

10: 37–41.

24. DeFeudis FV, Papadopoulos V, Drieu K (2003) Ginkgo biloba extracts andcancer: a research area in its infancy. Fundam Clin Pharmacol 17: 405–417.

25. Onen A, Deveci E, Inaloz SS, Isik B, Kilinc M (1999) Histopathological

assessment of the prophylactic effect of gingko-biloba extract on intestinalischemia-reperfusion injury. Acta Gastroenterol Belg 62: 386–389.

26. Oken BS, Storzbach DM, Kaye JA (1998) The efficacy of Ginkgo biloba on

cognitive function in Alzheimer disease. Arch Neurol 55: 1409–1415.

27. Chen XH, Miao YX, Wang XJ, Yu Z, Geng MY, et al. (2011) Effects of Ginkgo

biloba extract EGb761 on human colon adenocarcinoma cells. Cell PhysiolBiochem 27: 227–232.

28. Kim KS, Rhee KH, Yoon JH, Lee JG, Lee JH, et al. (2005) Ginkgo biloba

extract (EGb 761) induces apoptosis by the activation of caspase-3 in oral cavitycancer cells. Oral Oncol 41: 383–389.

29. Hauns B, Haring B, Kohler S, Mross K, Robben-Bathe P, et al. (1999) Phase II

study with 5-fluorouracil and ginkgo biloba extract (GBE 761 ONC) in patientswith pancreatic cancer. Arzneimittelforschung 49: 1030–1034.

30. Nano M, Lanfranco G, Dal Corso H, Ferronato M, Solej M (2000) The lateral

ligaments of the rectum: myth or reality? Chir Ital 52: 313–321.

31. Wang G, Reed E, Li QQ (2004) Molecular basis of cellular response to cisplatin

chemotherapy in non-small cell lung cancer (Review). Oncol Rep 12: 955–965.

32. Grabinski N, Bartkowiak K, Grupp K, Brandt B, Pantel K, et al. (2011) Distinctfunctional roles of Akt isoforms for proliferation, survival, migration and EGF-

mediated signalling in lung cancer derived disseminated tumor cells. Cell Signal.

33. Mansi L, Viel E, Curtit E, Medioni J, Le Tourneau C (2011) Targeting the RASsignalling pathway in cancer. Bull Cancer.

34. Hightower LE (1991) Heat shock, stress proteins, chaperones, and proteotoxi-

city. Cell 66: 191–197.

35. Aldrian S, Trautinger F, Frohlich I, Berger W, Micksche M, et al. (2002)Overexpression of Hsp27 affects the metastatic phenotype of human melanoma

cells in vitro. Cell Stress Chaperones 7: 177–185.

36. Storm FK, Mahvi DM, Gilchrist KW (1996) Heat shock protein 27

overexpression in breast cancer lymph node metastasis. Ann Surg Oncol 3:570–573.

37. Tsuruta M, Nishibori H, Hasegawa H, Ishii Y, Endo T, et al. (2008) Heat shock

protein 27, a novel regulator of 5-fluorouracil resistance in colon cancer. OncolRep 20: 1165–1172.

38. Soldes OS, Kuick RD, Thompson IA 2nd, Hughes SJ, Orringer MB, et al.

(1999) Differential expression of Hsp27 in normal oesophagus, Barrett’smetaplasia and oesophageal adenocarcinomas. Br J Cancer 79: 595–603.



39. Kanitakis J, Zambruno G, Viac J, Tommaselli L, Thivolet J (1989) Expression of

an estrogen receptor-associated protein (p29) in epithelial tumors of the skin.J Cutan Pathol 16: 272–276.

40. Ciocca DR, Puy LA, Fasoli LC (1989) Study of estrogen receptor, progesterone

receptor, and the estrogen-regulated Mr 24,000 protein in patients withcarcinomas of the endometrium and cervix. Cancer Res 49: 4298–4304.

41. Puy LA, Lo Castro G, Olcese JE, Lotfi HO, Brandi HR, et al. (1989) Analysis ofa 24-kilodalton (KD) protein in the human uterine cervix during abnormal

growth. Cancer 64: 1067–1073.

42. Bubendorf L, Kolmer M, Kononen J, Koivisto P, Mousses S, et al. (1999)Hormone therapy failure in human prostate cancer: analysis by complementary

DNA and tissue microarrays. J Natl Cancer Inst 91: 1758–1764.43. Takeno S, Noguchi T, Takahashi Y, Kikuchi R, Uchida Y, et al. (2001)

Immunohistochemical and clinicopathologic analysis of response to neoadjuvanttherapy for esophageal squamous cell carcinoma. Dis Esophagus 14: 149–154.

44. Hadchity E, Aloy MT, Paulin C, Armandy E, Watkin E, et al. (2009) Heat shock

protein 27 as a new therapeutic target for radiation sensitization of head andneck squamous cell carcinoma. Mol Ther 17: 1387–1394.

45. Yao H, Zhang Z, Xiao Z, Chen Y, Li C, et al. (2009) Identification of metastasisassociated proteins in human lung squamous carcinoma using two-dimensional

difference gel electrophoresis and laser capture microdissection. Lung Cancer

65: 41–48.46. Tian T, Hao J, Xu A, Hao J, Luo C, et al. (2007) Determination of metastasis-

associated proteins in non-small cell lung cancer by comparative proteomicanalysis. Cancer Sci 98: 1265–1274.

47. Fukaya H, Kanno H (1999) [Experimental studies of the protective effect ofginkgo biloba extract (GBE) on cisplatin-induced toxicity in rats]. Nihon

Jibiinkoka Gakkai Kaiho 102: 907–917.

48. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, et al. (2006) Glioma stem cellspromote radioresistance by preferential activation of the DNA damage response.

Nature 444: 756–760.49. Dorion S, Landry J (2002) Activation of the mitogen-activated protein kinase

pathways by heat shock. Cell Stress Chaperones 7: 200–206.

50. Chretien P, Landry J (1988) Enhanced constitutive expression of the 27-kDa

heat shock proteins in heat-resistant variants from Chinese hamster cells. J Cell

Physiol 137: 157–166.

51. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME (1995) Opposing

effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326–

1331.

52. Zhang Y, Shen X (2007) Heat shock protein 27 protects L929 cells from

cisplatin-induced apoptosis by enhancing Akt activation and abating suppression

of thioredoxin reductase activity. Clin Cancer Res 13: 2855–2864.

53. Guo YL, Baysal K, Kang B, Yang LJ, Williamson JR (1998) Correlation

between sustained c-Jun N-terminal protein kinase activation and apoptosis

induced by tumor necrosis factor-alpha in rat mesangial cells. J Biol Chem 273:

4027–4034.

54. Ghosh A, Lai C, McDonald S, Suraweera N, Sengupta N, et al. (2012) HSP27

expression in primary colorectal cancers is dependent on mutation of KRAS and

PI3K/AKT activation status and is independent of TP53. Exp Mol Pathol 94:

103–108.

55. Mountain CF (1997) Revisions in the International System for Staging Lung

Cancer. Chest 111: 1710–1717.

56. Sarhadi VK, Wikman H, Salmenkivi K, Kuosma E, Sioris T, et al. (2006)

Increased expression of high mobility group A proteins in lung cancer. J Pathol

209: 206–212.

57. Wikman H, Kettunen E, Seppanen JK, Karjalainen A, Hollmen J, et al. (2002)

Identification of differentially expressed genes in pulmonary adenocarcinoma by

using cDNA array. Oncogene 21: 5804–5813.

58. Weiss A, Wiskocil RL, Stobo JD (1984) The role of T3 surface molecules in the

activation of human T cells: a two-stimulus requirement for IL 2 production

reflects events occurring at a pre-translational level. J Immunol 133: 123–128.

59. Tang S, Morgan KG, Parker C, Ware JA (1997) Requirement for protein kinase

C theta for cell cycle progression and formation of actin stress fibers and

filopodia in vascular endothelial cells. J Biol Chem 272: 28704–28711.



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