research article extracts from curcuma zedoaria inhibit...

Research ArticleExtracts from Curcuma zedoaria Inhibit Proliferation ofHuman Breast Cancer Cell MDA-MB-231 In Vitro

Xiu-fei Gao1 Qing-lin Li2 Hai-long Li1 Hong-yan Zhang2 Jian-ying Su1

Bei Wang1 Pei Liu1 and Ai-qin Zhang2

1 The First Affiliated Hospital of Zhejiang Chinese Medical University Hangzhou Zhejiang 310006 China2 Zhejiang Cancer Hospital Zhejiang 310022 China

Correspondence should be addressed to Ai-qin Zhang zhanghaojianbb163com

Received 14 February 2014 Revised 3 April 2014 Accepted 7 April 2014 Published 5 May 2014

Academic Editor Lourdes Dıaz-Rodrıguez

Copyright copy 2014 Xiu-fei Gao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective To evaluate the effect of petroleum ether extracts of Curcuma zedoaria on the proliferation of human triple negativebreast cancer cell lineMDA-MB-231MethodsThereagentswere isolated fromCurcuma zedoaria by petroleumether fraction It wasassayed byCCK8 forMDA-MB-231 cellular viability with various concentrations and days cell cycle analysesWestern Blot analysisand Realtime Reverse Transcriptase PCR analyses for chemokines molecules including E-cadherin and E-selectin and adhesionmolecules including CCR7 SLC SDF-1 and CXCR4 Epirubicin was used as control in the study ResultsMDA-MB-231 cells wereinhibited by petroleum ether extracts of Curcuma zedoaria (119875 lt 005) and the inhibition rate was dependent on concentrationsand time Petroleum ether extracts of Curcuma zedoaria as well as Epirubicin produce a significant G0G1 cell cycle arrest Thelevel of expression of proteins E-cadherin and E-cadherin mRNA was significantly increased while proteins SDF-1 CCR7 andCXCR4 mRNA were decreased after being incubated with petroleum ether extracts of Curcuma zedoaria at the concentrations of300 120583gmL than control (119875 lt 005) The differences were that the protein CXCR4 mRNA expression level was higher than vehicleConclusionsMDA-MB-231 cells were inhibited by petroleum ether extracts of Curcuma zedoaria

1 Introduction

Breast cancer (BC) is one of the most common humanmalignancies accounting for 22 of all cancers diagnosedin women BC represents a complex and heterogeneousdisease comprising distinct pathologies with specific histo-logical features therapeutic responses metastatic dissem-ination patterns and patient outcomes In recent yearsglobal molecular analyses have revealed four main distinctsubgroups in human breast tumors luminal A and luminalB (LA and LB) human epidermal growth factor receptor2-overexpressing (Her2) and triple-negative breast cancer(TNBC) [1] Of all the BC subgroups TNBC is accountingfor 10sim208 [2] TNBC represent the greatest clinicalchallenge because these tumors are prevalent in youngerwomen associatedwith theworst prognosis and often relapserapidly [3] In contrast to nontriple-negative breast cancerTNBC are often highly histological grade and malignancy

and preferentially metastasize to lung liver and brain [4]TNBC is one of the worst prognosis in BC subgroups [5] ER-positive luminal tumors and Her2 carcinomas which can betreated with targeted therapies such as Tamoxifen (estrogenantagonist) aromatase inhibitors or anti-Her2 monoclonalantibodies [6] there is no available targeted therapy forTNBC Patients with TNBC are treated exclusively withconventional chemotherapy While they show high rates ofobjective initial response themajority of patients do not havea complete and prolonged response and are at high risk forrelapse and death within the first 3sim5 years of diagnosis [7]Epirubicin is extensively used in chemotherapy for patientswith breast cancer In spite of its excellent antitumor activitythe associated acute and chronic toxicities lead to a relativelylow therapeutic efficacy Whereas some molecules are inclinical trials in patients with TNBC such as dasatinib(Src inhibitor) cetuximab (EGFR inhibitor) bevacizumab(vascular endothelial growth factor inhibitor) or olaparib

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 730678 9 pageshttpdxdoiorg1011552014730678

2 Evidence-Based Complementary and Alternative Medicine

(Poly [ADP-ribose] polymerase inhibitor) [8] identificationof relevant molecular targets in TNBC remains a criticalchallenge

Accumulated data have demonstrated that complemen-tary and alternativemedicine (CAM) showbeneficial effect inthe treatment of several kinds of cancers [9ndash11] Curcuma is acommonly prescribed Chinese herb with anticancer poten-tials Curcuma extracts and active ingredients have beenidentified as its main bioactive components with anticancereffect both in vitro and in vivo [12 13] Furanodiene signif-icantly inhibited cancer cell proliferation while germacroneand curdione showed no effect Germacrone enhanced fura-nodienersquos antiproliferative effect curdione showed no effecton furanodienersquos antiproliferative effect but partly reversedthe antiproliferative effect of germacrone and furanodienecombinedThere are unpredictable and complex interactionsamong different components in curcuma phaeocaulisThere-fore in this study petroleum ether extracts of this plantnot isolated sesquiterpene has effect on the proliferation ofhuman triple negative breast cancer cell line MDA-MB-231in vitro

2 Materials and Methods

21 Chemicals RPMI 1640 fetal bovine serum (FBS)phosphate-buffered saline (PBS) TRIzol SuperScript reversetranscriptase enzyme and buffer were purchased fromGibco-BRL Company (USA) Dimethyl sulfoxide (DMSO)DEPC and propidium iodide (PI) were purchased fromSigma-Aldrich Chemical Company (USA) CCK-8 Kit waspurchased from DOJINDO Company (Japan) Ethanolpetroleum ether ethyl acetate n-butanol and all otherchemicals were of analytical grade Target gene primers andprobes were synthesized from Takar Company Taq DNApolymerase and buffer were purchased from Roche Com-pany The antibodies for detecting E-cadherin E-selectinCCR7 SLC SDF-1 and CXCR4 were purchased from CellSignaling Technology

22 Cell Culture MBA-MB-231 cells were cultured in RPMI1640 medium supplemented with 10 FBS and 40mgLgentamicine Cells were maintained at 37∘C in a humidifiedatmosphere containing 5 CO

2

23 Plant Material and Extraction Procedure Curcumazedoaria were collected from Guangxi province (China) inSeptember 2012 They were deposited in the First AffiliatedHospital of ZhejiangChineseMedicalUniversityThe identityof the plant was confirmed by morphological examination incomparison to the herbarium specimens

The sample was pulverized to 40 meshes The sample(40 g) was weighed accurately and placed into a 100mLflask containing 400mL 70 ethanol then soaked overnightThe sample was extracted two times through heating refluxThe ethanol extracts were filtered and concentrated underreduced pressure to give a residue Then the residue wasextracted with petroleum etherThe petroleum ether extractswere dried with rotary evaporators to obtain the dried

petroleum ether extracts The 5mg of petroleum etherextracts was dissolved in 500 120583L DMSO to prepare a stocksolution of 10mgmLThe stock solution was stored at minus20∘Cuntil use

24 In Vitro CCK8 Assay for Cellular Viability Cell countingkit-8 (CCK-8 reagent) can be used for simple and accuratecell viability assay The basic principle is the reagent contain-ing WST-8 (chemical name 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(24-disulfonic acid phenyl)-2H-tetra-zoli-um monosodium salt) is reduced to Formazan dyeby a dehydrogenase enzyme of cell mitochondria throughelectron carrier 1-Methoxy PMSMDA-MB-231 cells whichwere seeded at a density of 1 times 104 cells per 200120583L perwell in 96-well microtiter plates (Promega Corporation)24 h after seeding the cultures were washed twice with PBSand then exposed to various concentrations of petroleumether extracts of Curcuma zedoaria (100 120583gmL 200120583gmL300 120583gmL 400 120583gmL 500 120583gmL) various concentrationsof Epirubicin (025 120583gmL 05120583gmL 1120583gmL 2 120583gmL4 120583gmL) and DSMO as vehicle for 24 h or 5 days Perconcentration was performed in triplicate Then 10120583L ofCCK8 solution was added to each well and the plates wereincubated for an additional 3 hours at 37∘C Cell viabilitywas measured as the absorbance at 450 nm with a microplatereader (Synergy 2 Multimode Microplate Reader) BioTekWinooski VT (USA) and expressed as a percentage of thecontrol level The mean optical density (OD) values fromtriplicate wells for each treatment were used as the index ofcell viability

25 Cell Cycle Analyses MDA-MB-231 cells were plated in24-well plates and maintained in 10 FBS RPMI1640 ata density of 1 times 106 cells for 24 hours Cells were thenexposed to petroleum ether extracts of Curcuma zedoaria(300 120583gmL) Epirubicin (1 120583gmL) DSMO as control groupgrown another 24 hours Cells were harvested by trypsiniza-tion and pelleted by centrifugation The pellets were thenresuspended in PBS containing 50 120583gmL propidium iodide01TritonX-100 and 01 sodium citrate Propidium iodidefluorescence was measured by fluorescence-activated cellsorting by flow cytometry (FACSCanto II BectonDickinsonUSA) using the Multicyclersquos cycle analysis software

26 Western Blot Analysis MDA-MB-231 cells were washedonce in PBS supplemented with complete protease inhibitor(Roche Mannheim Germany) Washed cell pellets (3 times105 cells) were resuspended in protease buffer containing

10mM Tris-buffer (pH 76) 15mM MgCl2 1 mM EDTA

10mM KCl 1mM phenylmethylsulphonyl fluoride (PMSF)and protease inhibitor tablets (contain 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) E-64 bestatin leupeptinaprotinin and EDTA for inhibition of serine cysteineand metalloproteases) Whole cell extracts were preparedaccording to the published methods [14] Briefly MDA-MB-231 cells were washed once in cold PBS followed bythe cell pellets which were lysed by a single freeze-thawcycle in the presence of protease inhibitors and whole cell

Evidence-Based Complementary and Alternative Medicine 3

extracts were obtained by centrifugation at 14000 rpm for40min after extraction with 05M NaCl Protein concen-trations were determined using the Bio-Rad microproteinassay using bovine serum albumin as the standard Twenty-five microgram of each protein sample was resolved by 10or 12 SDS-PAGE and electroblotted onto nitrocellulosemembranes (Bio-Rad Hercules CA USA) The membraneswere blocked for 1 h at room temperature in PBS containing5 skim milk plus 01 Tween-20 (PBST) and incubatedovernight at 4∘C with different first antibodies followed byincubation with a horseradish-peroxidase-conjugated sec-ondary antibody (Jackson ImmunoResearch West GrovePA 1 10000 dilution) for 1 hour at room temperature

27 Realtime Reverse Transcriptase PCR Analyses Cellulartotal RNA was extracted with TRIzol reagent (Invitrogen)The RNA concentration and purity were measured using aspectrophotometer cDNA was synthesized from total RNAby reverse transcription The primer sequences and productsize were as follows E-cadherin forward 51015840-CGG GAA TGCAGT TGA GGA TC-31015840 reverse 51015840-AGG ATG GTG TAAGCG ATG GC-31015840 E-selectin forward 51015840-TGC ATG GAGGGT TGT TAA TGG-31015840 reverse 51015840-GGA TGA A AG TGATTA AAT TGT GCA TAG-31015840 CCR7 forward 51015840-TGC CATCTA CAA GAT GA GCT-31015840 reverse 51015840-GGT GCT ACTGGT GAT GTT GA-31015840 CXCR4 forward 51015840-GCT GTT GGCTGA AAAGGT GGT C-31015840 reverse 51015840-CAC CTC GCT TTCCTT TGG AGA-31015840 SDF-1 forward 51015840-CAG CCG TGC AACAAT CTG AAG-31015840 reverse 51015840-CTG CAT CAG TGA CGGTAA ACC-31015840 SLC forward 51015840-TCC CGG CAA TCC TGTTCTC-31015840 reverse 51015840-CCTTCCTCAGGGTTTGCACA-31015840GAPDH forward 51015840-CAGCCTCAAGATCATCAGCA-31015840reverse 51015840-ACA GTC TTC TGG GTG GCA GT-31015840 GAPDHwas used as an internal control The products were checkedby agarose electrophoresis and analyzed using Fast RealtimePCR system (ABI USA)

28 Statistical Analysis Petroleum ether extracts ofCurcumazedoaria were observed object DMSO as vehicle and Epiru-bicin as positive control during all experiments Each viabilityvalue represents the mean plusmn SD From three determinationsIC50 values were calculated from the log-log plot betweenthe percentages of viable cells Subsequently each experimentwas performed in triplicatemeasurements Statistical analysisof data was carried out using a one-way ANOVA followed byHolm-Sidak pairwise multiple comparison test (Sigma PlotSystat Software Inc) and a probability value of less than005 (lowast119875 lt 005 lowastlowast119875 lt 001) was accepted as a significantdifference

3 Results

31 In Vitro CCK8 Assay for Cellular Viability The resultsfrom the in vitro CCK8 assay of cellular viability in vari-ous concentrations of petroleum ether extracts of Curcumazedoaria as well as the Epirubicin are listed in Table 1 MDA-MB-231 cells were inhibited by petroleum ether extracts ofCurcuma zedoaria (119875 lt 00) and the inhibition rate was

Table 1 The various concentrations of petroleum ether extracts ofCurcuma zedoaria in vitro CCK8 assay of cellular viability

Groups Concentrations(120583gmL) 119899

OD value(mean plusmn SD)

Inhibition rate()

Vehicle mdash 3 149 plusmn 022 0

Epirubicin

025 3 085 plusmn 012lowast 49205 3 074 plusmn 013lowastlowast 58310 3 067 plusmn 010lowastlowast 63820 3 059 plusmn 002lowastlowast 69540 3 039 plusmn 001lowastlowast 858

Curcumazedoaria


Values are average of triplicate experiment and are represented as mean plusmnSD lowast119875 lt 005 lowastlowast119875 lt 001 compared with vehicle

higher with increasing concentration (Figure 2(a)) The inhi-bition rate of petroleum ether extracts of Curcuma zedoaria(500120583gmL) is about 90 and there are little viable cellsin electron microscope These relationships were measuredgraphically by plotting concentrations versus percentageof inhibition According to Table 1 the inhibition rate ofpetroleum ether extracts of Curcuma zedoaria (300 120583gmL)is more than 50 then the following experiments were per-formed with petroleum ether extracts of Curcuma zedoaria(300 120583gmL) and Epirubicin (10 120583gmL) of middle concen-tration The cell grow curve shows that the inhibition of cellproliferation is higher with time grew by petroleum etherextracts of Curcuma zedoaria on 300 120583gmL concentration(Figure 2(b))Under themicroscope onfirst day the cells hadmicroscopic contour increase and continued to proliferateOn the second and third days cell proliferation was slowercell outline was clear and cytoplasm was rough there wereparticle accumulations On the fourth day the cytoplasmwasextremely rough with much debris inside On the fifth daycell accumulated and many cells were necrotic (Figure 2(c))

32 Cell Cycle Analyses The analysis of cell cycle phasedistribution demonstrated that petroleum ether extracts ofCurcuma zedoaria as well as Epirubicin produce a significantincrease in the number of MDA-MB-231 cells in G1 phaseat 24 h after treatment clearly demonstrating a significantG0G1 cell cycle arrest (Table 2) A consequent decrease incells in S and G2 phase versus control was also observed(Figures 3(a) and 3(b))

33 Western Blot Analysis It is known that chemokinesand adhesion molecules family are strongly linked to theprocess of tumor recurrence and metastasis Some membersof the family are chemokines molecules such as E-cadherinand E-selectin and adhesion molecules such as CCR7 SLCSDF-1 and CXCR4 We determined the expression of theseproteins byWestern blotWe showed that 24 h after treatment


(a) (b)

Figure 1 Curcuma zedoaria of the plant and the plant root sample

Table 2 Effect of petroleum ether extracts of Curcuma zedoaria invitro cell cycle on progression

Group Cell cycle pase ()G1

S G2

Vehicle 5823 plusmn 202 3139 plusmn 178 1037 plusmn 094

Epirubicin 7660 plusmn 121lowastlowast

2011 plusmn 140lowastlowast


Petroleum etherextracts ofCurcuma zedoaria




Values are average of triplicate experiment and are represented asmeanplusmn SDlowastlowast

119875 lt 001 compared with control

with petroleum ether extracts of Curcuma zedoaria thelevel of expression of protein E-cadherin and CXCR4 weresignificantly increased while protein SDF-1 was decreased inrelation to control at the same time The level of expressionof protein E-selectin was slightly increased and proteinCCR7 was slightly decreased although this increase was notstatistically significant The difference is that the level ofexpression of proteins E-cadherin E-selectin and SDF-1 wassignificantly decreased while protein CXCR4 was increasedin relation to control at the same time after treatment withEpirubicin (Figures 4(a) and 4(b))

34 Realtime Reverse Transcriptase PCR Analyses Reversetranscriptase-polymerase chain reaction (RT-PCR) analysisdemonstrated that the E-cadherin mRNA expression levelwas higher and theCCR7 andCXCR4mRNAexpression levelwere lower 24 h after incubated with petroleum ether extractsofCurcuma zedoaria at the concentrations of 300 120583gmL thancontrol (119875 lt 005) The level of expression of protein E-cadherin was significantly increased while protein CXCR7was decreased in relation to control at the same time aftertreatment with Epirubicin On the other hand the differenceis that the CXCR4 mRNA expression level was higher and

Table 3 Effect of petroleum ether extracts of Curcuma zedoaria(300 120583gmL) on expression level of E-cadherin E-selectin CCR7SLC SDF-1 and CXCR4 mRNA after 24 hours

mRNAs The relative expression of mRNA (compared to control)Vehicle Epirubicin Curcuma zedoaria

E-cadherin 1 plusmn 0 300 plusmn 108

lowast

1455 plusmn 334lowast

E-selectin 1 plusmn 0 547 plusmn 313 269 plusmn 164

CCR7 1 plusmn 0 045 plusmn 017lowast


SDF-1 1 plusmn 0 045 plusmn 010lowast

108 plusmn 014

CXCR4 1 plusmn 0 6708plusmn3264lowast


Values are average of triplicate experiment and are represented as mean plusmnSD lowast119875 lt 001 compared with control lowastcompared with Epirubicin

the SDF-1mRNA expression level was lower (Table 3 Figures5(a) and 5(b))

4 Discussion

Many previous studies support the use of Curcuma zedoariarhizomes in traditional medicine for the treatment of cancer-related diseases especially breast cervical and colon cancersAs the rhizomes are also widely consumed as salad in foodwithout any known undesirable side effect it can be assumedthat the plant is safe for consumption at the normal doseas food Curcuma zedoaria is therefore a promising dietaryagent that holds great promise for use in chemopreventiveand chemotherapeutic strategiesWe have previously demon-strated that MDA-MB-231 triple-negative breast cancer cellswere inhibited by different extracts of Curcuma zedoaria invitroCCK8 assay of cellular viability Curcuma zedoaria plantsample was extracted successively with petroleum ether (PE)ethyl acetate (EA) n-butanol (NB) and water (see Figure 1)The result from assay of cellular viability is that petroleumether extract of Curcuma zedoaria is the strongest in allextracts (Figure 5) In this paper we analyzed the effect on


0

20

40

60

80

Inhi

bitio

n (

)

025 05 10 20 40Epirubicin (120583gmL)

(a)

100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)

Curcuma zedoaria (120583gmL)

(b)

1 2 3 4 5

02

04

06

08

10

OD

Days

VehicleCurcuma zedoariaEpirubicin

(c)

Figure 2 The inhibition rate of the various concentrations of epirubicin (a) and petroleum ether extracts of Curcuma zedoaria (b) weredetermined by CCK8 assays The values were reported as (c) The inhibition rate of different days was determined by CCK8 assay asdescribed compared with vehicle Values are average of triplicate experiment and are represented as mean plusmn SD

MDA-MB-231 cells of petroleum ether extracts of Curcumazedoaria

This study affirms that CCK8 assay has shown thatMDA-MB-231 cell viability was significantly affected by exposure topetroleum ether extracts ofCurcuma zedoariaThe inhibitionrate of cell viability was correlated positively with concentra-tion and timeThe inhibition rate of petroleum ether extractsofCurcuma zedoaria (300 120583gmL concentration) ismore than50 The analysis of cell cycle progression indicated thatpetroleum ether extracts of Curcuma zedoaria produce anarrest in the G0G1 phase of cell cycle after 24 h of treatment

Consistent with these observations petroleum ether extractsof Curcuma zedoaria inhibited the active DNA synthesis ofMDA-MB-231 cells From above studies a similar result wasalso observed in Epirubicin (see Figure 6)

Why wereMDA-MB-231 cell viability andDNA synthesisinhibited by petroleum ether extracts of Curcuma zedoariaFurther studies that provide data leading to mechanismsof antitumor are now underway In breast cancer patientsmetastases remain a major cause of disease morbidity andmortality Breast cancer metastases frequently follow a pat-tern of dissemination in humans that results in the formation


Vehicle Curcuma zedoaria Epirubicin

100

00 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150

200

300

400

500

100

0 0

200

200

300

400

400

600

800500600

Dip G2

Dip G1

Dip G2 Dip G2

Dip G1 Dip G1Dip S

DebrisAggregates Dip S


DebrisAggregates

Channels (PI-A) Channels (PI-A) Channels (PI-A)

Num

ber

Num

ber

Num

ber

(a)

Vehicle Curcuma zedoaria Epirubicin0

25

50

75

100

Cel

l cyc

le p

ase (

)

G2SG1

(b)

Figure 3 Synchronized MDA-MB-231 cells were treated with petroleum ether extracts of Curcuma zedoaria or Epirubicin for 24 hours andthe fraction of cells in each phase of cell cycle was evaluated by flow cytometry

of lesions in the lymph nodes lungs liver and bone marrow[15 16] Cross talk between cancer cells and their microen-vironment is considered an essential event in tumorigenesisinvasion and metastasis [17 18] Specifically interactionsbetween transformed epithelial cells and their surroundingstroma may decide the fate of evolving cancers [19] sincesignals from the microenvironment profoundly influence thesurvival and migration of cancer cells [20] We investigatedthe effects of petroleum ether extracts of Curcuma zedoariaon some cell chemokines and adhesion molecules

Multivariate analysis showed that the combination of E-cadherin-negative and Ki67-positive expression was stronglypredictive of poor overall survival in TNBCpatients receivingadjuvant chemotherapy [21] The mechanisms responsiblefor the chemosensitivity of TNBC with E-cadherin-negativeand Ki67-positive expression remain to be determined Lossof E-cadherin induces epithelial-to-mesenchymal transition

(EMT) EMT is a key step toward cancer metastasis Ahmedet al reported the close relationship between EMT and thecancer stem cell-like phenotype in response to chemore-sistance [22] Also other studies have shown that SnailSlug and Notch signaling as EMT markers were correlatedwith chemoresistance These findings suggested that oneof the possible mechanisms by which chemosensitivity isreduced in patients with TNBC with loss of E-cadherinexpression may involve EMT signaling [23 24] In contrastseveral studies have reported that E-cadherin-dependentintercellular adhesion enhances chemoresistance [25ndash27]Fortunately the protein andmRNA expression of E-cadherinwere significantly increased after treatment with petroleumether extracts of Curcuma zedoaria from our results

Increasing evidence shows that CXCR4 and its ligandstromal-derived factor-1 (SDF-1120572 also known as CXCL12)may play a critical role in the organ-selective process of


VehicleCurcuma zedoaria Epirubicin

E-cadherin

E-selectin

CCR7

SDF1

CXCR4

120573-Actin

(a)

E-cadherin E-selectin CCR7 SDF-1 CXCR40

1

2

3

45

6


Prot

ein

expr

essio

n (fo

ld o

f veh

icle

)

lowast

lowastlowast

lowast lowast

lowast

lowast

(b)

Figure 4 Effect of petroleum ether extracts of Curcuma zedoaria (300 120583gmL) and Epirubicin (1 120583gmL) on protein E-cadherin E-selectinCCR7 SLC SDF-1 and CXCR4 expression after 24 hours Bars represent mean plusmn SD of three individual experiments lowast119875 lt 005 Beta-actinwas used as a loading control

E-cadherin E-selectin0

4

8

12

16

20


mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowast

lowast

(a)


CCR7 SDF-1 CXCR400

04

08

12

16

206080

100

mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowastlowast lowast

lowast

lowast

(b)

Figure 5 The level of adhesion molecules E-cadherin and E-selectin mRNA expression (a) and chemokines molecules CCR7 SLC SDF-1and CXCR4 (b) after 24 hours in different groups by using RT-PCR analysis lowast119875 lt 001 compared with control

tumorigenesis and metastasis including those observed inbreast cancers [28ndash30] CXCR4 expression has been estab-lished as a prognostic marker in many cancer cell typesincluding breast carcinomas [31ndash33] and the SDF-1120572-CXCR4signaling axis has been associatedwith breast cancermetasta-sis [34 35] The SDF-1120572-CXCR4 interaction promotes tumorprogression by several possible mechanisms [17 36 37] Sev-eral novel CXCR4 antagonists have shown promising in vitro

anticancer activity in several tumor cell types includingthose derived from breast Furthermore using animal tumormodels CXCR4 antagonists have in vivo anticancer activityas well [38 39]The results are in agreement with the reportsshowing that the expression of proteins SDF-1 CCR7mRNAand CXCR4 mRNA was significantly decreased after treat-ment with petroleum ether extracts ofCurcuma zedoaria Butthe expression of protein CXCR4 was increased in Western


100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)

Petroleum ether extracts N-butanol extractsEthyl acetate extracts Water extracts

Concentrations (120583gmL)

Figure 6 The inhibition rate of the different extracts of Curcumazedoaria was determined by CCK8 assay as described comparedwith Epirubicin The values were reported as

blot assays The absence of mRNA-protein correlation fora subset of investigated genes suggests that the relationbetween mRNA and protein is not strictly linear but it hasa more intrinsic and complex dependence The reason maybe that mRNA levels have come down after interventionexpression of constitutive protein is regulated higher byintracellular activation factor or the expression of protein islag

As we know triple-negative breast cancer (TNBC) whichis characterized by negativity for estrogen receptor pro-gesterone receptor and human epidermal growth factorreceptor 2 (HER2) is a high risk breast cancer that lacksspecific targets for treatment selection Chemotherapy istherefore the primary systemic modality used in the treat-ment of this disease but reliable parameters to predictthe chemosensitivity of TNBC have not been clinicallyavailable Therefore combination treatment with a nontoxicdrug which can improve the TNBC prognosis would beadvantageous Data obtained in our experiments indicate thatpetroleum ether extracts of Curcuma zedoaria are possiblyone of antineoplastic parts of the plant Nevertheless furtherinvestigations are necessary to validate its therapeutic claimsand to determine the active ingredient of Curcuma zedoariaIt needs to be further investigated both in vivo and invitro

5 Conclusion

From above studies results showed that MDA-MB-231 cellswere inhibited by petroleum ether extracts of Curcumazedoaria This preliminary study and its data persuade us tofocus on inhibition TNBC cell of petroleum ether extractsof Curcuma zedoaria and investigating further on animalmodels for in vivo evaluation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Xiu-fei Gao Qing-lin Li Hai-long Li and Hong-yan Zhangcontributed equally to the project and are considered cofirstauthors

Acknowledgments

The authors wish to acknowledge the Zhejiang ProvincialNatural Science Foundation of China (no LQ12H27002)ldquoIntegrative Medicinerdquo key discipline of Zhejiang UniversityProgram and The First Affiliated Hospital of Zhejiang Chi-nese Medical University Eagles Program for the Cultivationof Talents

References

[1] C Fan D S Oh L Wessels et al ldquoConcordance among gene-expression-based predictors for breast cancerrdquoTheNewEnglandJournal of Medicine vol 355 no 6 pp 560ndash569 2006

[2] K C Amirikia P Mills J Bush and L A Newman ldquoHigherpopulation-based incidence rates of triple-negative breast can-cer among young African-American womenrdquo Cancer vol 117no 12 pp 2747ndash2753 2011

[3] E A Rakha J S Reis-Filho and I O Ellis ldquoBasal-like breastcancer a critical reviewrdquo Journal of Clinical Oncology vol 26no 15 pp 2568ndash2581 2008

[4] M Smid Y Wang Y Zhang et al ldquoSubtypes of breast cancershow preferential site of relapserdquoCancer Research vol 68 no 9pp 3108ndash3114 2008

[5] W J Yi J S Lu G H GH et al ldquoClinicopathological featuresof the triple-negative tumors in Chinese breast cancer patientsrdquoBreast Cancer Research and Treatment vol 115 no 2 pp 325ndash333 2009

[6] B P Schneider E PWinerWD Foulkes et al ldquoTriple-negativebreast cancer risk factors to potential targetsrdquo Clinical CancerResearch vol 14 no 24 pp 8010ndash8018 2008

[7] C Liedtke C Mazouni K R Hess et al ldquoResponse toneoadjuvant therapy and long-term survival in patients withtriple-negative breast cancerrdquo Journal of Clinical Oncology vol26 no 8 pp 1275ndash1281 2008

[8] D J Toft and V L Cryns ldquoMinireview basal-like breast can-cer from molecular profiles to targeted therapiesrdquo MolecularEndocrinology vol 25 no 2 pp 199ndash211 2011

[9] E Ernst ldquoComplementary and alternativemedicine (CAM) andcancer the kind face of complementary medicinerdquo Interna-tional Journal of Surgery vol 7 no 6 pp 499ndash500 2009

[10] A Wanchai J M Armer and B R Stewart ldquoComplementaryand alternative medicine use among women with breast cancera systematic reviewrdquo Clinical Journal of Oncology Nursing vol14 no 4 pp E45ndashE55 2010

[11] M J Sewitch and Y Rajput ldquoA literature review of complemen-tary and alternative medicine use by colorectal cancer patientsrdquoComplementary Therapies in Clinical Practice vol 16 no 1 pp52ndash56 2010


[12] Q Kong F Sun and X Chen ldquoImpact of fixed-dose combina-tion of germacrone curdione and furanodiene on breast cancercell proliferationrdquo Cell Journal vol 15 pp 160ndash165 2013

[13] Z Zhong Y Dang X Yuan et al ldquoFuranodiene a naturalproduct inhibits breast cancer growth both in vitro and in vivordquoCellular Physiology and Biochemistry vol 30 pp 778ndash790 2012

[14] H Naranmandura X Chen M Tanaka et al ldquoRelease of apop-totic cytochrome c from mitochondria by dimethylarsinousacid occurs through interaction with voltagedependent anionchannel in vitrordquo Toxicological Sciences vol 128 pp 137ndash1462012

[15] M Konopleva Y Tabe Z Zeng and M Andreeff ldquoTherapeu-tic targeting of microenvironmental interactions in leukemiaMechanisms and approachesrdquo Drug Resistance Updates vol 12no 4-5 pp 103ndash113 2009

[16] D X Nguyen and J Massague ldquoGenetic determinants of cancermetastasisrdquo Nature Reviews Genetics vol 8 no 5 pp 341ndash3522007

[17] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006

[18] A Patocs L Zhang Y Xu et al ldquoBreast-cancer stromal cellswith TP53 mutations and nodal metastasesrdquo The New EnglandJournal of Medicine vol 357 no 25 pp 2543ndash2551 2007

[19] F Mannello ldquoMultipotent mesenchymal stromal cell recruit-ment migration and differentiation what have matrix metal-loproteinases got to do with itrdquo Stem Cells vol 24 no 8 pp1904ndash1907 2006

[20] B Tilton L Ho E Oberlin et al ldquoSignal transduction by CXCchemokine receptor 4 stromal cell-derived factor 1 stimulatesprolonged protein kinase B and extracellular signal-regulatedkinase 2 activation in T lymphocytesrdquo Journal of ExperimentalMedicine vol 192 no 3 pp 313ndash324 2000

[21] S Kashiwagi M Yashiro T Takashima et al ldquoAdvantages ofadjuvant chemotherapy for patients with triple-negative breastcancer at Stage II usefulness of prognostic markers E-cadherinand Ki67rdquo Breast Cancer Research vol 13 no 6 article R1222011

[22] N Ahmed K Abubaker J Findlay and M Quinn ldquoEpithelialmesenchymal transition and cancer stem cell-like phenotypesfacilitate chemoresistance in recurrent ovarian cancerrdquo CurrentCancer Drug Targets vol 10 no 3 pp 268ndash278 2010

[23] B Mahler-Araujo K Savage S Parry and J S Reis-FilholdquoReduction of E-cadherin expression is associated with non-lobular breast carcinomas of basal-like and triple negativephenotyperdquo Journal of Clinical Pathology vol 61 no 5 pp 615ndash620 2008

[24] S Kashiwagi M Yashiro T Takashima et al ldquoSignificance ofE-cadherin expression in triple-negative breast cancerrdquo BritishJournal of Cancer vol 103 no 2 pp 249ndash255 2010

[25] T Nakamura Y Kato H Fuji T Horiuchi Y Chibaand K Tanaka ldquoE-cadherin-dependent intercellular adhesionenhances chemoresistancerdquo International journal of molecularmedicine vol 12 no 5 pp 693ndash700 2003

[26] H-G Kang J M Jenabi J Zhang et al ldquoE-cadherin cell-cell adhesion in Ewing tumor cells mediates suppression ofanoikis through activation of the ErbB4 tyrosine kinaserdquoCancerResearch vol 67 no 7 pp 3094ndash3105 2007

[27] S K Green G Francia C Isidoro and R S Kerbel ldquoAntiad-hesive antibodies targeting E-cadherin sensitize multicellulartumor spheroids to chemotherapy in vitrordquo Molecular CancerTherapeutics vol 3 no 2 pp 149ndash159 2004

[28] R E Bachelder M A Wendt and A M Mercurio ldquoVascularendothelial growth factor promotes breast carcinoma invasionin an autocrine manner by regulating the chemokine receptorCXCR4rdquo Cancer Research vol 62 no 24 pp 7203ndash7206 2002

[29] A Muller B Homey H Soto et al ldquoInvolvement of chemokinereceptors in breast cancermetastasisrdquoNature vol 410 no 6824pp 50ndash56 2001

[30] B S Wiseman and Z Werb ldquoDevelopment stromal effects onmammary gland development and breast cancerrdquo Science vol296 no 5570 pp 1046ndash1049 2002

[31] M Kim Y J Koh K E Kim et al ldquoCXCR4 signaling regulatesmetastasis of chemoresistant melanoma cells by a lymphaticmetastatic nicherdquo Cancer Research vol 70 no 24 pp 10411ndash10421 2010

[32] S Konoplev J L Jorgensen D A Thomas et al ldquoPhospho-rylated CXCR4 is associated with poor survival in adults withB-acute lymphoblastic leukemiardquo Cancer vol 117 no 20 pp4689ndash4695 2011

[33] L V Rhodes S P Short N F Neel V A Salvo Y Zhuand S Elliott ldquoCytokine receptor CXCR4 mediates estrogen-independent tumorigenesis metastasis and resistance toendocrine therapy in human breast cancerrdquo Cancer Researchvol 71 no 9 pp 603ndash613 2011

[34] F Jin U Brockmeier F Otterbach and E Metzen ldquoNewinsight into the SDF-1CXCR4 axis in a breast carcinomamodelhypoxia-induced endothelial SDF-1 and tumor cell CXCR4are required for tumor cell intravasationrdquo Molecular CancerResearch vol 10 pp 1021ndash1031 2012

[35] M Zhang H X Liu X D Teng H B Wang J Cui et alldquoThe differences in CXCR4 protein expression are significantfor the five molecular subtypes of breast cancerrdquoUltrastructuralPathology vol 36 pp 381ndash386 2012

[36] M Darash-Yahana E Pikarsky R Abramovitch et al ldquoRole ofhigh expression levels of CXCR4 in tumor growth vascular-ization and metastasisrdquo The FASEB Journal vol 18 no 11 pp1240ndash1242 2004

[37] J Hesselgesser H P Ng M Liang et al ldquoIdentification andcharacterization of small molecule functional antagonists of theCCR1 chemokine receptorrdquo Journal of Biological Chemistry vol273 no 25 pp 15687ndash15692 1998

[38] Y Yoon Z Liang X Zhang et al ldquoCXC chemokine receptor-4antagonist blocks both growth of primary tumor andmetastasisof head and neck cancer in xenograft mouse modelsrdquo CancerResearch vol 67 no 15 pp 7518ndash7524 2007

[39] Z Zeng I J Samudio M Munsell et al ldquoInhibition of CXCR4with the novel RCP168 peptide overcomes stroma-mediatedchemoresistance in chronic and acute leukemiasrdquo MolecularCancer Therapeutics vol 5 no 12 pp 3113ndash3121 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


(Poly [ADP-ribose] polymerase inhibitor) [8] identificationof relevant molecular targets in TNBC remains a criticalchallenge

Accumulated data have demonstrated that complemen-tary and alternativemedicine (CAM) showbeneficial effect inthe treatment of several kinds of cancers [9ndash11] Curcuma is acommonly prescribed Chinese herb with anticancer poten-tials Curcuma extracts and active ingredients have beenidentified as its main bioactive components with anticancereffect both in vitro and in vivo [12 13] Furanodiene signif-icantly inhibited cancer cell proliferation while germacroneand curdione showed no effect Germacrone enhanced fura-nodienersquos antiproliferative effect curdione showed no effecton furanodienersquos antiproliferative effect but partly reversedthe antiproliferative effect of germacrone and furanodienecombinedThere are unpredictable and complex interactionsamong different components in curcuma phaeocaulisThere-fore in this study petroleum ether extracts of this plantnot isolated sesquiterpene has effect on the proliferation ofhuman triple negative breast cancer cell line MDA-MB-231in vitro

2 Materials and Methods

21 Chemicals RPMI 1640 fetal bovine serum (FBS)phosphate-buffered saline (PBS) TRIzol SuperScript reversetranscriptase enzyme and buffer were purchased fromGibco-BRL Company (USA) Dimethyl sulfoxide (DMSO)DEPC and propidium iodide (PI) were purchased fromSigma-Aldrich Chemical Company (USA) CCK-8 Kit waspurchased from DOJINDO Company (Japan) Ethanolpetroleum ether ethyl acetate n-butanol and all otherchemicals were of analytical grade Target gene primers andprobes were synthesized from Takar Company Taq DNApolymerase and buffer were purchased from Roche Com-pany The antibodies for detecting E-cadherin E-selectinCCR7 SLC SDF-1 and CXCR4 were purchased from CellSignaling Technology

22 Cell Culture MBA-MB-231 cells were cultured in RPMI1640 medium supplemented with 10 FBS and 40mgLgentamicine Cells were maintained at 37∘C in a humidifiedatmosphere containing 5 CO

2

23 Plant Material and Extraction Procedure Curcumazedoaria were collected from Guangxi province (China) inSeptember 2012 They were deposited in the First AffiliatedHospital of ZhejiangChineseMedicalUniversityThe identityof the plant was confirmed by morphological examination incomparison to the herbarium specimens

The sample was pulverized to 40 meshes The sample(40 g) was weighed accurately and placed into a 100mLflask containing 400mL 70 ethanol then soaked overnightThe sample was extracted two times through heating refluxThe ethanol extracts were filtered and concentrated underreduced pressure to give a residue Then the residue wasextracted with petroleum etherThe petroleum ether extractswere dried with rotary evaporators to obtain the dried

petroleum ether extracts The 5mg of petroleum etherextracts was dissolved in 500 120583L DMSO to prepare a stocksolution of 10mgmLThe stock solution was stored at minus20∘Cuntil use

24 In Vitro CCK8 Assay for Cellular Viability Cell countingkit-8 (CCK-8 reagent) can be used for simple and accuratecell viability assay The basic principle is the reagent contain-ing WST-8 (chemical name 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(24-disulfonic acid phenyl)-2H-tetra-zoli-um monosodium salt) is reduced to Formazan dyeby a dehydrogenase enzyme of cell mitochondria throughelectron carrier 1-Methoxy PMSMDA-MB-231 cells whichwere seeded at a density of 1 times 104 cells per 200120583L perwell in 96-well microtiter plates (Promega Corporation)24 h after seeding the cultures were washed twice with PBSand then exposed to various concentrations of petroleumether extracts of Curcuma zedoaria (100 120583gmL 200120583gmL300 120583gmL 400 120583gmL 500 120583gmL) various concentrationsof Epirubicin (025 120583gmL 05120583gmL 1120583gmL 2 120583gmL4 120583gmL) and DSMO as vehicle for 24 h or 5 days Perconcentration was performed in triplicate Then 10120583L ofCCK8 solution was added to each well and the plates wereincubated for an additional 3 hours at 37∘C Cell viabilitywas measured as the absorbance at 450 nm with a microplatereader (Synergy 2 Multimode Microplate Reader) BioTekWinooski VT (USA) and expressed as a percentage of thecontrol level The mean optical density (OD) values fromtriplicate wells for each treatment were used as the index ofcell viability

25 Cell Cycle Analyses MDA-MB-231 cells were plated in24-well plates and maintained in 10 FBS RPMI1640 ata density of 1 times 106 cells for 24 hours Cells were thenexposed to petroleum ether extracts of Curcuma zedoaria(300 120583gmL) Epirubicin (1 120583gmL) DSMO as control groupgrown another 24 hours Cells were harvested by trypsiniza-tion and pelleted by centrifugation The pellets were thenresuspended in PBS containing 50 120583gmL propidium iodide01TritonX-100 and 01 sodium citrate Propidium iodidefluorescence was measured by fluorescence-activated cellsorting by flow cytometry (FACSCanto II BectonDickinsonUSA) using the Multicyclersquos cycle analysis software

26 Western Blot Analysis MDA-MB-231 cells were washedonce in PBS supplemented with complete protease inhibitor(Roche Mannheim Germany) Washed cell pellets (3 times105 cells) were resuspended in protease buffer containing

10mM Tris-buffer (pH 76) 15mM MgCl2 1 mM EDTA

10mM KCl 1mM phenylmethylsulphonyl fluoride (PMSF)and protease inhibitor tablets (contain 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) E-64 bestatin leupeptinaprotinin and EDTA for inhibition of serine cysteineand metalloproteases) Whole cell extracts were preparedaccording to the published methods [14] Briefly MDA-MB-231 cells were washed once in cold PBS followed bythe cell pellets which were lysed by a single freeze-thawcycle in the presence of protease inhibitors and whole cell





3 Results





Inhibition rate()


Epirubicin


Curcumazedoaria







(a) (b)




S G2
















lowast






108 plusmn 014





4 Discussion



0

20

40

60

80

Inhi

bitio

n (

)

025 05 10 20 40Epirubicin (120583gmL)

(a)

100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)


(b)

1 2 3 4 5

02

04

06

08

10

OD

Days


(c)








100

00 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150

200

300

400

500

100

0 0

200

200

300

400

400

600

800500600

Dip G2

Dip G1

Dip G2 Dip G2

Dip G1 Dip G1Dip S



DebrisAggregates


Num

ber

Num

ber

Num

ber

(a)


25

50

75

100

Cel

l cyc

le p

ase (

)

G2SG1

(b)








E-cadherin

E-selectin

CCR7

SDF1

CXCR4

120573-Actin

(a)


1

2

3

45

6


Prot

ein

expr

essio

n (fo

ld o

f veh

icle

)

lowast

lowastlowast

lowast lowast

lowast

lowast

(b)



4

8

12

16

20


mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowast

lowast

(a)


CCR7 SDF-1 CXCR400

04

08

12

16

206080

100

mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowastlowast lowast

lowast

lowast

(b)





100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)






5 Conclusion






Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results





Inhibition rate()


Epirubicin


Curcumazedoaria







(a) (b)




S G2
















lowast






108 plusmn 014





4 Discussion



0

20

40

60

80

Inhi

bitio

n (

)

025 05 10 20 40Epirubicin (120583gmL)

(a)

100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)


(b)

1 2 3 4 5

02

04

06

08

10

OD

Days


(c)








100

00 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150

200

300

400

500

100

0 0

200

200

300

400

400

600

800500600

Dip G2

Dip G1

Dip G2 Dip G2

Dip G1 Dip G1Dip S



DebrisAggregates


Num

ber

Num

ber

Num

ber

(a)


25

50

75

100

Cel

l cyc

le p

ase (

)

G2SG1

(b)








E-cadherin

E-selectin

CCR7

SDF1

CXCR4

120573-Actin

(a)


1

2

3

45

6


Prot

ein

expr

essio

n (fo

ld o

f veh

icle

)

lowast

lowastlowast

lowast lowast

lowast

lowast

(b)



4

8

12

16

20


mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowast

lowast

(a)


CCR7 SDF-1 CXCR400

04

08

12

16

206080

100

mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowastlowast lowast

lowast

lowast

(b)





100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)






5 Conclusion






Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)




S G2
















lowast






108 plusmn 014





4 Discussion



0

20

40

60

80

Inhi

bitio

n (

)

025 05 10 20 40Epirubicin (120583gmL)

(a)

100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)


(b)

1 2 3 4 5

02

04

06

08

10

OD

Days


(c)








100

00 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150

200

300

400

500

100

0 0

200

200

300

400

400

600

800500600

Dip G2

Dip G1

Dip G2 Dip G2

Dip G1 Dip G1Dip S



DebrisAggregates


Num

ber

Num

ber

Num

ber

(a)


25

50

75

100

Cel

l cyc

le p

ase (

)

G2SG1

(b)








E-cadherin

E-selectin

CCR7

SDF1

CXCR4

120573-Actin

(a)


1

2

3

45

6


Prot

ein

expr

essio

n (fo

ld o

f veh

icle

)

lowast

lowastlowast

lowast lowast

lowast

lowast

(b)



4

8

12

16

20


mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowast

lowast

(a)


CCR7 SDF-1 CXCR400

04

08

12

16

206080

100

mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowastlowast lowast

lowast

lowast

(b)





100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)






5 Conclusion






Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

Inhi

bitio

n (

)

025 05 10 20 40Epirubicin (120583gmL)

(a)

100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)


(b)

1 2 3 4 5

02

04

06

08

10

OD

Days


(c)








100

00 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150

200

300

400

500

100

0 0

200

200

300

400

400

600

800500600

Dip G2

Dip G1

Dip G2 Dip G2

Dip G1 Dip G1Dip S



DebrisAggregates


Num

ber

Num

ber

Num

ber

(a)


25

50

75

100

Cel

l cyc

le p

ase (

)

G2SG1

(b)








E-cadherin

E-selectin

CCR7

SDF1

CXCR4

120573-Actin

(a)


1

2

3

45

6


Prot

ein

expr

essio

n (fo

ld o

f veh

icle

)

lowast

lowastlowast

lowast lowast

lowast

lowast

(b)



4

8

12

16

20


mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowast

lowast

(a)


CCR7 SDF-1 CXCR400

04

08

12

16

206080

100

mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowastlowast lowast

lowast

lowast

(b)





100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)






5 Conclusion






Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















100

00 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150

200

300

400

500

100

0 0

200

200

300

400

400

600

800500600

Dip G2

Dip G1

Dip G2 Dip G2

Dip G1 Dip G1Dip S



DebrisAggregates


Num

ber

Num

ber

Num

ber

(a)


25

50

75

100

Cel

l cyc

le p

ase (

)

G2SG1

(b)








E-cadherin

E-selectin

CCR7

SDF1

CXCR4

120573-Actin

(a)


1

2

3

45

6


Prot

ein

expr

essio

n (fo

ld o

f veh

icle

)

lowast

lowastlowast

lowast lowast

lowast

lowast

(b)



4

8

12

16

20


mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowast

lowast

(a)


CCR7 SDF-1 CXCR400

04

08

12

16

206080

100

mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowastlowast lowast

lowast

lowast

(b)





100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)






5 Conclusion






Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















E-cadherin

E-selectin

CCR7

SDF1

CXCR4

120573-Actin

(a)


1

2

3

45

6


Prot

ein

expr

essio

n (fo

ld o

f veh

icle

)

lowast

lowastlowast

lowast lowast

lowast

lowast

(b)



4

8

12

16

20


mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowast

lowast

(a)


CCR7 SDF-1 CXCR400

04

08

12

16

206080

100

mRN

As e

xpre

ssio

n (fo

ld o

f veh

icle

)

lowastlowast lowast

lowast

lowast

(b)





100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)






5 Conclusion






Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100 200 300 400 5000

20

40

60

80

100

Inhi

bitio

n (

)






5 Conclusion






Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article extracts from curcuma zedoaria inhibit...

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