Research Article Saussurea lappa Clarke-Derived Costunolide ...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 936257 10 pageshttpdxdoiorg1011552013936257

Research ArticleSaussurea lappa Clarke-Derived Costunolide PreventsTNF120572-Induced Breast Cancer Cell Migration and Invasion byInhibiting NF-120581B Activity

Youn Kyung Choi Sung-Gook Cho Sang-Mi Woo Yee Jin Yun Jeakyung JoWooyoung Kim Yong Cheol Shin and Seong-Gyu Ko

Laboratory of Clinical Biology and Pharmacogenomics Center for Clinical Research and GenomicsDepartment of PreventiveMedicine College of KoreanMedicine Kyung Hee University 1 Hoegi-dong Seoul 130-701 Republic of Korea

Correspondence should be addressed to Seong-Gyu Ko epikokhuackr

Received 5 April 2013 Accepted 3 May 2013

Academic Editor Bo-Hyoung Jang

Copyright copy 2013 Youn Kyung Choi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Saussurea lappa Clarke (SLC) has been used as a traditional medicine in Korea China and Japan for the treatment of abdominalpain and tenesmus Costunolide a sesquiterpene lactone isolated from SLC has diverse medicinal effects However the anticancereffects of costunolide are still unclear in breast cancer In this study we demonstrate that costunolide suppresses tumor growthand metastases of MDA-MB-231 highly metastatic human breast cancer cells via inhibiting TNF120572-induced NF-120581B activationCostunolide inhibited MDA-MB-231 tumor growth and metastases without affecting body weights in the in vivomouse orthotopictumor growth assays In addition costunolide inhibited in vitro TNF120572-induced invasion and migration of MDA-MB-231 cellsCostunolide further suppressed TNF120572-induced NF-120581B signaling activation resulting in a reduced expression of MMP-9 a well-known NF-120581B-dependent gene to mediate breast cancer cell growth and metastases Therefore we conclude that SLC and itsderivative costunolide suppress breast cancer growth and metastases by inhibiting TNF120572-induced NF-120581B activation suggestingthat costunolide as well as SLC may be promising anticancer drugs especially for metastatic breast cancer

1 Introduction

Most breast cancer is an epithelial tumor that develops frommammary gland tissue and the inner lining of milk ducts[1] Metastatic breast cancer is not well cured by surgeryradiotherapy and chemotherapy [2ndash4] Cancer metastasis isthe spread of tumor cells from an original site to distantparts of the body This event consists of multistep processeswhich includes tumor cell dissemination extracellularmatrix(ECM) degradation tumor cell invasion into the ECMangiogenesis and secondary metastatic tumor growth [5ndash7]Interestingly primary tumors metastasize to specific organsfor example aggressive breast cancers selectively metastasizeto lung bone and brain tissue This organ tropism seems tobe related to different gene expression patterns [8ndash10]

TNF120572 is frequently detected in many human cancertissues including breast ovarian and renal cancers [11 12]

In addition tumor cells producing TNF120572 are correlatedwith poor prognoses [11] TNF120572 signaling activation throughTNF receptor leads to promoting a recruitment of adaptorproteins and to activating signal cascades including NF-120581Bpathway [13 14] NF-120581B regulates diverse physiological andpathological processes including development metabolisminflammation and tissue homeostasis by regulating expres-sion of various genes In particular genes regulated by NF-120581B play roles such as development proliferation survivaland metastasis in cancer [15ndash17] NF-120581B protein bound toI120581B120572 in the cytoplasm is maintained as an inactive state [18]In response to NF-120581B activation signals IKK120572120573 complexis activated resulting in phosphorylation of I120581B120572 on serineresidues 32 and 35 Phosphorylated I120581B120572 is then ubiqui-tinated and polyubiquitinated I120581B120572 is degraded throughproteasomal pathway As a result free NF-120581B translocatesfrom the cytoplasm to the nucleus and binds to specific DNA

2 Evidence-Based Complementary and Alternative Medicine

sequences to regulate expression of target genes which arerelated to tumor development and metastases [19ndash21]

The dried root of Saussurea lappa Clarke (SLC) hastransitionally been used as an ingredient in Korea China andJapan for the treatment of either abdominal pain or tenesmusSeveral earlier studies indicated that the root of SLC hasanticancer effect in gastric cancer cells [22 23] Costunolide(C15H20O2) a sesquiterpene lactone that is a major com-

ponent of the root of SLC [24] has been reported to havediverse effects such as anti-inflammatory [25] anti-viral [26]and -fungal [27] effects Furthermore costunolide affectedanti various cancers includingmelanoma [28] intestinal [29]leukemia [30] prostate [31] and breast cancers [32]

While anti-cancer effects of either SLC or costunolidehave been reported as mentioned befor antimetastatic effectsof either SLC or costunolide on metastatic breast cancerare still poorly understood In this study we found thatSLC and costunolide inhibit TNF120572-mediated breast cancercell migration and invasion by inhibiting NF-120581B activationthereby suggesting the antimetastatic property of costunolideusing highly metastatic MDA-MB-231 breast cancer cells

2 Materials and Methods

21 Reagents and Cell Lines Costunolide (molecular weightof 23232 purity gt 99 see Figure 2(a)) was purchasedfromWako (Wako Pure Chemical Industries Osaka Japan)RPMI 1640 fetal bovine serum (FBS) antibiotic-antimycoticand phosphate-buffered Saline (PBS) were purchased fromGibco-BRL (Rockville MD USA) EZ-western detection kitwas obtained from Daeillab (Daeillab service Co SeoulKorea) TNF120572was purchased fromRampDsystems (Minneapo-lis MNUSA)

22 Preparation of Saussurea lappa Clarke (SLC) ExtractSaussurea lappa Clarke was purchased from Omniherb(Gyeong Buk Korea) The 100 g of root of SLC was dipped in1 L of 80 ethanol and sonicated by using an ultrasonicator(Branson MO USA) for 30min at room temperature Thesonicated extract was filtered through a 022mm filter andconcentrated The ethanol extracts were dried in a 42∘C byusing a vacuum pump evaporator (Eyela Tokyo Japan) The285 g of concentrated extract was dissolved in DMSO toprepare a stock solution of 100mgmLThe stock solutionwasstored at minus80∘C until use

23 Cell Migration and Invasion Cell migration was mea-sured by wound healing assays Cells were seeded in 6-wellplates and scratched with a 200120583L pipette tip 24 hoursafter treatments with Saussurea lappaClarke and costunolidemigrated cell numbers were counted For invasion assay cellswere seeded in the upper chambers precoated with Matrigeland treated with SLC and costunolide Low chambers werefilled with 10 FBS or TNF120572-contained medium and inva-sive cells were stainedwith hematoxylin and eosin to visualizeand count All experiments were performed in triplicateand studentrsquos t-test was performed to determine statistics119875 values below 005 and 0001 were considered statistically

significant All data was represented as the mean plusmn standarddeviation

24 Immunofluorescence Assays Immunofluorescence as-says were used for p-NF120581B nuclear translocation in MDA-MB-231 cell After treatment with SLC and costunolide for6 hours cells were fixed with 4 paraformaldehyde for15min and then permeabilized with 05 Triton X-100 for10min The cells were washed with PBS blocked with 5FBS in PBS for 30min and then incubated with anti-p-NF-120581B antibody overnight at 4∘C and with anti-Alexa Fluor-488secondary antibody (Invitrogen Eugene Oregon USA) for1 hour Phalloidin (Sigma) and TO-PRO-3 (Invitrogen) wereused to contain F-actin and the nucleus respectively Imageswere obtained with Olympus FV10i Self-Contained ConfocalLaser SystemThe object was 20x and scale bars on the imageindicate 50120583m

25 Luciferase Assays Cells were seeded in 24-well platesand NF-120581B-luc plasmid (Stratagene La Jolla CA USA)transfected in MDA-MB-231 cells by using Lipofectaminereagent (Invitrogen Carlsbad CA USA) Cells were treatedwith SLC and costunolide for 6 hours and then the luciferaseassays were done by using dual-luciferase reporter assay(Promega Madison WI USA) All transfections includedthe RLTK-Luc (kindly provided by Sang Hoon Kim) fortransfection efficiency All experiments were performed intriplicate and studentrsquos t-test was performed to determinestatistics 119875 values below 005 and 0001 were consideredstatistically significant All data was represented as the meanplusmn standard deviation

26 Western Blot Total protein (30 120583g) was separated bySDS-PAGE After electrophoresis the proteins were trans-ferred to a nitrocellulose membrane The membrane wasblocked incubated overnight at 4∘Cwith primary antibodieswashedwith PBS-T (PBSwith 01Tween-20) and incubatedwith appropriate HRP-conjugated secondary antibodies atroom temperature for 1 hour Immunoreactive protein wasdeveloped using an EZ-western detection kit (Daeillab ser-vice Co Seoul Korea) Anti-MMP-9 -p-IKK -IKK -p-I120581B -I120581B -p-NF-120581B and -NF-120581B were purchased from CellSignaling (Danvers MA USA) Anti-Tubulin was purchasedfrom Sigma (Louis MO USA)

27 RNA Extraction and RT-PCR Cellular total RNA wasextractedwithTRIzol reagent (Invitrogen)TheRNAconcen-tration and purity weremeasured using a spectrophotometercDNAwas synthesized from total RNA (1120583g) by reverse tran-scriptionThe primer sequences and product size were as fol-lows MMP-9 (262 bp) forward 51015840-CACTGTCCACCCCTC-AGAGC-31015840 reverse 51015840-GCCACTTGTCGGCGATAAGG-31015840 GAPDH (300 bp) forward 51015840-CGTCTTCACCACCAT-GGAG-A-31015840 reverse 51015840-CGGCCATCACGCCACAGTTT-31015840 The products were checked by agarose electrophoresisand analyzed using ChemiDoc imaging system (BioRadHercules CA USA)

Evidence-Based Complementary and Alternative Medicine 3

0

100

200

None TNF120572

(a)

TNF120572 + SLC

Cel

l mig

ratio

n (

)lowast

(b)

TNF120572 TNF120572 + SLC0

5

10

None

Cell

inva

sion

(fold

) lowastlowast

(c)

None

p-NF-120581B Phalloidin

TO-PRO-3 Merged

TNF120572


TO-PRO-3 Merged

TNF120572 + SLC


TO-PRO-3 Merged

TNF+minus +SLC

MMP-9

Actin

+minus minus

(e)

(d)

0

2

4

6

NF-120581

B-de

pend

ent r

epor

ter a

ctiv

ity (f

old)

lowast

None TNF120572 TNF120572 + SLC

Figure 1 SLC inhibits TNF120572-induced MDA-MB-231 cell migration and invasion by inhibiting NF-120581B activation (a) Cell migration wasmeasured by wound healing assay MDA-MB-231 cells were seeded and scratched pretreated with SLC for 1 hour and then exposed to TNF120572for 24 hours Cell migration was determined by counting cell numbersmigrated from the wound healing region lowast119875 lt 005 (b)MDA-MB-231cells were seeded on the upper chambers and pretreated with SLC for 1 hour and then exposed to TNF120572 for 24 hours Invading cells werestained with hematoxylin and eosin and the cell numbers were measured lowastlowast119875 lt 0001 (c) MDA-MB-231 cells were pretreated with SLCfor 1 hour then exposed to TNF120572 for 6 hours and stained with p-NF-120581B antibody Phalloidin and TO-PRO-3 were for staining F-actin andthe nucleus respectively The object was 20x and scale bars on the image indicate 50 120583m (d) MDA-MB-231 cells were transfected with theNF-120581B-dependent luciferase reporter pretreated with SLC for 1 hour and then exposed to TNF120572 for 6 hours Luciferase assays were done byusing dual-luciferase reporter assay All transfections included the RLTK-Luc for transfection efficiency lowast119875 lt 005 (e) MDA-MB-231 cellswere pretreated with SLC for 1 hour and then exposed to TNF120572 for 24 hours MMP-9 protein was measured by western blotting Tubulin wasused for the loading control


H

H

Abs OO

(a)

Cel

l mig

ratio

n (

)

0

50

100

150

Control DMSO CTL

lowast

(b)

Cell

inva

sion

()

0

50

100

150

Control DMSO CTL

lowast

(c)

Cel

l mig

ratio

n (

)

0

100

200

Control TNF120572 TNF120572 + CTL

lowast

(d)

None TNF120572 TNF120572 + CTL

Cel

l inv

asio

n (fo

ld)

0

5

10

lowastlowast

(e)

Figure 2 Costunolide inhibits TNF120572-induced MDA-MB-231 cell migration and invasion (a) Structure of costunolide (b) MDA-MB-231cells were seeded and scratched Treatment with costunolide for 24 hours and counted lowast119875 lt 005 (c) MDA-MB-231 cells were seeded on theupper chambers and treated with costunolide in 0 serum Low chamber filed with 10 serum lowast119875 lt 005 (d) Pretreated with costunolidefor 1 hour and then exposed to TNF120572 for 24 hours Cell migration was determined by counting numbers of cells migrated from the woundhealing region lowast119875 lt 005 (e) MDA-MB-231 cells were seeded on the upper chambers and treated with costunolide Low chamber filed withTNF120572 lowast119875 lt 0001

28 Gelatin Zymography Assay Conditioned medium washarvested concentrated mixed with nonreducing samplebuffer and separated by SDS-PAGE electrophoresis contain-ing 01 gelatin After electrophoresis the gel was washedwith washing buffer (25 Triton X-100 in reaction buffer)and then incubated in reaction buffer (50mM Tris-HCl5mM CaCL

2 1 120583M ZnCl

2 and pH 74) for 18 h at 37∘C To

visualize the gel was stained with Coomassie brilliant blueR-250 and destained in 50 methanol 40 distilled waterand 10 acetic acid

29 In Vivo Studies Animal studies were approved by KyungHee University Institutional Animal Care and Use Commit-tee (KHU-IACUC) Six-week-old nude (NuNu) mice were


2412 Treatment (h)CTL++

p-IKK

IKK

p-I120581B120572

I120581B120572

Tubulin

minus

(a)

2412 Treatment (h)CTL++minus

Lamin B

Nuclear p65

Tubulin

Cytosol p65

(b)

0 3015

p-IKK

IKK

I120581B120572

Tubulin

(min)(min)

p-IKK

IKK

I120581B120572

Tubulin

TNF1205720 3015

TNF120572 + CTL

(c)

None


TO-PRO-3 Merged

TNF120572


TO-PRO-3 Merged

TNF120572 + CTL


TO-PRO-3 Merged

(d)

Figure 3 Costunolide inhibits TNF120572-induced NF-120581B pathway in MDA-MB-231 cells (a) MDA-MB-231 cell were treated with costunolidefor indicated time periodsWhole lysates were analyzed by western blotting with anti-pIKK -IKK -pI120581B120572 -I120581B120572 and Tubulin (b) Cells werefractionated into cytoplasmic and nuclear compartment and western blotting for NF-120581B Tubulin and LaminB were used as loading control(c) MDA-MB-231 cells were treated with TNF120572 and cotreated with TNF120572 and costunolide for 15 to 30min Whole lysates were analyzed bywestern blotting with anti-pIKK -IKK -I120581B120572 and Tubulin (d) MDA-MB-231 cells were pretreated with costunolide for 1 hour then exposedto TNF120572 for 6 hours and stained with p-NF-Κb antibody Phalloidin and TO-PRO-3 were for staining with F-actin and nucleus respectivelyThe object was 20x and the scale bars on the image indicate 50 120583m

purchased from Oriental Science and injected orthotopicallyinto the 4th mammary fat fads with MDA-MB-231 cells (1 times106 resuspended in a 1 1 mixture of PBS and growth factor-reduced matrigel (BD Biosciences San Jose CA USA)) Aday after tumor cell injection 20120583M of costunolide wasinjected into the mammary fat fads three times a week for30 days Tumor volumes were measured using calipers andcalculated using the following formula tumor volume (cubicmillimeters) = width2 times length2 In addition body weightwas monitored

210 Immunohistochemistry Tumors were fixed with 4formaldehyde for further analyses Tumor tissues wereembedded in paraffin dissected with 5120583m and deparaf-finized in 100 xylene and ethanol series (100 95and70) Heat-induced antigen retrieval was 10mM sodiumcitrate buffer for 5min Endogenous peroxidase was blockedwith peroxidase blocking reagent containing 35 hydro-gen peroxide Nonspecific antigen was blocked with serum

containing PBS followed by incubation with human ki-67(5 120583gmL) (Abcam MA USA) and MMP-9 antibody (1 100)(Cell Signaling Beverly MA USA) overnight at 4∘C It wasincubated with biotin-labeled rabbit antibody for 1 hourat room temperature and incubated with ABC and DABbuffer substrate Sections were visualized with DAB andhematoxylin mounted and analyzed using a bright fieldmicroscope The object was 20x and the scale bars on theimage indicate 10 120583m

211 Statistics Data were shown as the means and standarddeviations 119875 values less than 005 in the two-tailed Studentrsquost-test or one-way ANOVA were considered statistically sig-nificant

3 Results

31 Saussurea lappa Clarke Suppresses TNF120572-Induced BreastCancer Cell Migration and Invasion via an Inhibition of NF-120581B


0

2

4

6

Non

e

TNF 120572

TNFR

I

TNFR

I + C

TL

TNF 120572

+ C

TL

lowastlowast lowastlowast

NF-120581

B-de

pend

ent r

epor

ter a

ctiv

ity (f

old)

(a)

Immunoblot

Zymography

MMP-9 (secreted form)

MMP-9 (cell)

Tubulin

GAPDH

MMP-9

MMP-9

RT-PCR

CTL+TNF120572minusminus

(b)

Figure 4 Costunolide inhibits TNF120572-induced NF-120581B activity and MMP-9 expression (a) MDA-MB-231 cells were transfected with theNF-120581B-dependent luciferase reporter pretreated with costunolide for 1 hour and then exposed to TNF120572 for 6 hours In addition aftercotransfected with TNFRI and NF-120581B-dependent luciferase reporter treated with costunolide for 6 hours Luciferase assay were done byusing dual-luciferase reporter assays All transfections included the RLTK-Luc for transfection efficiency lowastlowast119875 lt 0001 (b)MDA-MB-231 cellswere pretreated with costunolide for 1 hour and then exposed to TNF120572 for 6 hours MMP-9 expression was analyzed by RT-PCR westernblotting and zymography

Activation Because TNF120572 expression is abundant in tumormicroenvironment and its expression is correlated with poorprognoses [14 15] we investigate effects of Saussurea lappaClarke (SLC) on highly metastatic MDA-MB-231 cells Innormal culture condition SLC treatment (50120583gmL) inhib-ited MDA-MB-231 cell migration (data not shown) NextTNF120572 increased the MDA-MB-231 cells migration comparedto nontreated cells and 50 120583gmL of SLC suppressed TNF120572-inducedMDA-MB-231 cells migration by approximately 63(Figure 1(a)) In addition 50120583gmL of SLC significantlyinhibited TNF120572-induced cell invasion (Figure 1(b)) Next toclarify the mechanism of SLC to inhibit cell migration andinvasion we performed the immunofluorescence assays toexamine NF-120581B pathway As shown in Figure 1(c) TNF120572induced nuclear translocation of phosphorylated NF-120581Bwhich was blocked by SLC In the luciferase assay SLC inhib-ited TNF120572-induced NF-120581B-dependent transcriptional activ-ity (Figure 1(d)) Accordingly 50120583gmL of SLC suppressedTNF120572-inducedmRNAandprotein expression ofMMP-9 thatis well known as NF-120581B-dependent gene (Figure 1(e)) Thusour data indicate that SLC inhibits TNF120572-induced highlymetastaticMDA-MB-231 humanbreast cancer cellmigrationinvasion and NF-120581B activation

32 Saussurea lappa Clarke-Derived Costunolide SuppressesCell Migration and Invasion in Breast Cancer Cells SinceCostunolide (C

15H20O2) is a major component of SLC

[22] we examined whether SLC-derived costunolide inhibitsmetastatic properties of breast cancer cells Costunolide(20120583M) blocked cells migration in normal serum conditionsby approximately 45 (Figure 2(a)) Next to examine cos-tunolide effect on cells invasion MDA-MB-231 cells wereseeded in the upper chambers precoated with matrigel and

treated with costunolide in 1 serum contained media andthe low chambers were filled with 10 serum containedmedia As shown in Figure 2(b) a treatment of breast cancercells with 20120583M of costunolide for 24 hours reduced cellsinvasion

Next we performed experiments to determine whethercostunolide inhibits TNF120572-induced cells migration and inva-sion 20120583Mof costunolide suppressed TNF120572-inducedMDA-MB-231 cell migration by approximately 62 (Figure 2(c))In addition whereas TNF120572 increased an invasiveness ofMDA-MD-231 cells by approximately five folds costuno-lide significantly inhibited TNF120572-induced cell invasion byapproximately five folds (Figure 2(d))

33 Costunolide Inhibits NF-120581B Pathway in Breast CancerCells Next we examined costunolide effect on NF-120581B signal-ing pathway in MDA-MB-231 cells As shown in Figure 3(a)costunolide inhibited phosphorylation of IKK and I120581B120572resulting in blocking I120581B120572 degradation in a time-dependentmanner Accordingly a treatment of the MDA-MB-231 cellswith costunolide inhibited the nuclear translocation of p65NF-120581B subunit (Figure 3(b))

Next in order to examinewhether costunolide suppressesTNF120572-induced NF-120581B pathway we stimulated cells withTNF120572 for 15 to 30min in the presence or absence ofcostunolide As shown in Figure 3(c) TNF120572-induced IKKphosphorylation was prolonged until 30min which wasblocked by costunolide Furthermore while TNF120572 inducedI120581B degradation costunolide slowly recovered I120581B120572 expres-sion at 15min

To confirm costunolide suppression of NF-120581B nucleartranslocation we performed immunofluorescence assayusing the anti-pNF-120581B antibody As shown in Figure 3(d)


Days after injection

0

50

100

150

200

1 2 5 8 11 14 18 21 24 27 30

CTLControl

Tum

or v

olum

e (m

m3)

(a)


Body

wei

ght (

g)

0

10

20

1 2 5 8 11 14 18 21 24 27 30

CTLControl

(b)

Tum

or

Control CTL

Lung

Live

r

H and E

(c)

Tum

orControl CTL

Lung

Live

r

ki-67

(d)

Control CTL

MMP-9

(e)

Figure 5 Costunolide inhibits orthotopically tumor growth and metastasis (a) 1 times 106 MDA-MB-231 cells were orthotopically injected innude mice (119899 = 5group) Costunolide was injected into the mammary fat fad and repeated every three days for 30 days Tumor volumeswere measured using calipers Tumor volume (cubic millimeters) = width2 times length2 (b) Body weight measured three times a week (c)Tumor tissues were stained with hematoxylin and eosin Photo images were taken at 20x magnification (d) Tumor tissues were stained withanti-ki-67 antibody (e) Tumor tissues were stained with anti-MMP-9 antibody The object was 20x and scale bars on the image indicate10120583m


TRAD

IKK

NF-120581B

NF-120581B

I120581B120572 Proteasomal degradation of I120581B120572

Nucleus

MMP-9 transcriptional

TNF120572

TNFR

CostunolideSaussurea lappa Clarke

H

H

Abs OO

Figure 6 Schematic representation of the mechanism where cos-tunolide inhibits TNF120572-induced breast cancer cell migration andinvasion by inhibiting NF-120581B activity

NF-120581B was observed in the cytosol of the cells treatedwith costunolide Thus our data indicate that SLC-derivedcostunolide inhibits NF-120581B pathway

34 Costunolide Inhibits NF-120581B Transcriptional Activity andMMP-9 To confirm the inhibition of NF-120581B pathway bycostunolide we performed the transcriptional activation ofNF-120581B by using the luciferase assay As shown in Figure 4(a)costunolide reduced TNF120572-induced NF-120581B transcriptionalactivation by 5-fold inMDA-MB-231 cellsWe next examinedwhether costunolide affects upstream of IKK in TNF120572-induced NF-120581B pathway MDA-MB-231 cells were cotrans-fected with NF-120581B reporter gene and TNFRI and then cul-tured in the presence or absence of costunolide Costunolidereduced TNFRI-induced NF-120581B transcriptional activity byapproximately 25-fold in MDA-MB-231 cells (Figure 4(a))

It is known that MMP-9 is regulated by NF-120581B andthe promoter region of MMP-9 gene contains binding sitesfor NF-120581B Thus we examined whether costunolide inhibitsMMP-9 we checked MMP-9 by using RT-RCR westernblot and zymography As shown in Figure 4(b) Costuno-lide inhibited TNF120572-induced MMP-9 mRNA protein andenzyme activity when cells were treated with costunolide for24 hours

35 Costunolide Inhibits Tumor Growth and Metastasis Toexamine costunolide effect on breast cancer growth andmetastases in vivo MDA-MB-231 cells were orthotopicallyinjected into the 4th mammary fat fads A day after tumor

cell injection costunolide at 20120583M was injected into themammary fat fad three times a week for 30 days In additiontumor volume and body weight of mice were also measuredthree times a week As shown in Figure 5(a) costunolidereduced tumor volume (119875 = 0007628) and no significantweight loss in mice treated with either costunolide or vehiclewas observed (Figure 5(b))When tumor tissues were stainedwith hematoxylin and eosin we found that tumor cohorttreated with costunolide compared to that with controlwas well differentiated (Figure 1(c)) In addition tumor andorgan (lung and liver) tissues were stained with anti-ki-67Costunolide compared to control reduced ki-67 positive cellin tumor lung and liver (Figure 1(d)) When tumor tissueswere stained with MMP-9 antibody costunolide inhibiteda number of MMP-9 positive cells (Figure 1(e)) Thus ourdata indicate that costunolide inhibits tumor growth andmetastasis

4 Discussion

TNF120572-induced NF-120581B pathway is a well-known moleculartarget for cancer therapy Tumor cells released NF-120581B-dependent MMPs by NF-120581B-mediated TNF120572 productionof immune cells in tumor microenvironment [33] In thisstudy we found that Saussurea lappaClarke-derived costuno-lide suppressed TNF120572-induced MDA-MB-231 breast cancercell migration and invasion by inhibiting NF-120581B activity(Figure 5(c)) Thus SLC as well as costunolide appears to beuseful for treating highly metastatic breast cancer

Matrix metalloproteinases (MMPs) a family of zinc-dependent endoproteinase is necessary for extracellularmatrix (ECM) degradation amongmetastasis process MMPsalso affect many biological processes such as normal tissueremodeling wound healing angiogenesis embryogenesisand many diseases including cancer atheroma and arthritis[34] MMP-9 is frequently overexpressed in many cancersand correlates with poor prognosis and survival in can-cer patients [35ndash37] In addition MMP-9 is important fortumor metastasis by cleaving basement membranes whichallows migratory phenotype cells to be more invasive andmotile [38ndash40] MMP-9 is regulated by stimulators (phorbol12-myristate 13-acetate PMA transforming necrosis factoralpha TNF120572 growth factor UV and stress) and transcriptionfactors (nuclear factor kappaB NF-120581B and activator protein-1 AP-1) [39 40] In addition MMP-9 is important for tumormetastasis by cleaving basement membranes which allowsmigratory phenotype cells to be more invasive and motileThus TNF120572-inducedMMP-9 expression viaNF-120581B is impor-tant for cancer growth and metastasis In our study TNF120572-induced cell migration and invasion were inhibited by eitherSLC or costunolide SLC and costunolide suppressed TNF120572-induced NF-120581B translocation to nucleus and transcriptionalactivity In addition costunolide specifically inhibited IKKphosphorylation and I120581B120572 degradation Those inhibitionsfurther reduced NF-120581B-dependent MMP-9 expression Asa result costunolide suppressed in vivo tumor growth andmetastasis

This study concludes that (a) SLC suppresses TNF120572-induced MDA-MB-231 cell migration and invasion by


inhibiting NF-120581B-dependent MMP-9 expression (b) SLC-derived costunolide inhibited serum or TNF120572-inducedMDA-MB-231 cell migration and invasion (c) costunolideinhibited TNF120572-induced NF-120581B translocation resulting fromthe suppression phosphorylation and I120581B120572 degradation (d)costunolide blocked TNF120572-induced NF-120581B transcriptionactivity and TNF120572-induced MMP-9 expression and (e)costunolide decreased in vivo tumor growth and metastasiswithout weight loss (Figure 6) In sum we provide evidencethat the anti-cancer effect of both SLC and its componentcostunolide on MDA-MB-231 result from the inhibition ofTNF120572-induced NF-120581B activation Therefore SLC-derivedcostunolide could be useful for treating highly metastaticbreast cancer growth and metastases

Acknowledgments

The authors thank Dr Sang Hoon Kim (Department ofBiology Kyung Hee University) for sharing the luminometerand ChemiDoc imaging system This study was supportedby the National Research Foundation of Korea (NRF) grantfunded by theKorea government (MSIP) (No 2007-0054931)and by a Grant from the KoreanMedicine RampDProject of theMinistry of Health and Welfare (B110043 and B120014)

References

[1] J Sariego ldquoBreast cancer in the young patientrdquo The AmericanSurgeon vol 76 no 12 pp 1397ndash1400 2010

[2] K I Block C Gyllenhaal D Tripathy et al ldquoSurvival impactof integrative cancer care in advanced metastatic breast cancerrdquoBreast Journal vol 15 no 4 pp 357ndash366 2009

[3] P A C Greenberg G N Hortobagyi T L Smith L D ZieglerD K Frye and A U Buzdar ldquoLong-term follow-up of patientswith complete remission following combination chemotherapyfor metastatic breast cancerrdquo Journal of Clinical Oncology vol14 no 8 pp 2197ndash2205 1996

[4] E-F Solomayer I J Diel G C Meyberg C Gollan and GBastert ldquoMetastatic breast cancer clinical course prognosis andtherapy related to the first site of metastasisrdquo Breast CancerResearch and Treatment vol 59 no 3 pp 271ndash278 2000

[5] DHanahan andRAWeinberg ldquoThehallmarks of cancerrdquoCellvol 100 no 1 pp 57ndash70 2000

[6] K Pantel and R H Brakenhoff ldquoDissecting the metastaticcascaderdquo Nature Reviews Cancer vol 4 no 6 pp 448ndash4562004

[7] P S Steeg ldquoTumormetastasis mechanistic insights and clinicalchallengesrdquo Nature Medicine vol 12 no 8 pp 895ndash904 2006

[8] R O Hynes ldquoMetastatic potential generic predisposition of theprimary tumor or rare metastatic variants or bothrdquo Cell vol113 no 7 pp 821ndash823 2003

[9] Y Kang P M Siegel W Shu et al ldquoA multigenic programmediating breast cancer metastasis to bonerdquo Cancer Cell vol3 no 6 pp 537ndash549 2003

[10] A J Minn G P Gupta P M Siegel et al ldquoGenes that mediatebreast cancer metastasis to lungrdquo Nature vol 436 no 7050 pp518ndash524 2005

[11] F Balkwill ldquoTumor necrosis factor or tumor promoting factorrdquoCytokine and Growth Factor Reviews vol 13 no 2 pp 135ndash1412002

[12] M S Naylor G W H Stamp W D Foulkes D Eccles and FR Balkwill ldquoTumor necrosis factor and its receptors in humanovarian cancer potential role in disease progressionrdquo Journal ofClinical Investigation vol 91 no 5 pp 2194ndash2206 1993

[13] T Hehlgans and K Pfeffer ldquoThe intriguing biology of thetumour necrosis factortumour necrosis factor receptor super-family players rules and the gamesrdquo Immunology vol 115 no1 pp 1ndash20 2005

[14] E E Varfolomeev andA Ashkenazi ldquoTumor necrosis factor anapoptosis JuNKierdquo Cell vol 116 no 4 pp 491ndash497 2004

[15] S Shishodia and B B Aggarwal ldquoNuclear factor-120581B activationa question of life or deathrdquo Journal of Biochemistry and Molecu-lar Biology vol 35 no 1 pp 28ndash40 2002

[16] M K Pandey B Sung A B Kunnumakkara G Sethi M MChaturvedi and B B Aggarwal ldquoBerberine modifies cysteine179 of I120581B120572 kinase suppresses nuclear factor-120581B-regulatedantiapoptotic gene products and potentiates apoptosisrdquoCancerResearch vol 68 no 13 pp 5370ndash5379 2008

[17] G Helbig K W Christopherson P Bhat-Nakshatri et al ldquoNF-120581B promotes breast cancer cell migration and metastasis byinducing the expression of the chemokine receptor CXCR4rdquoJournal of Biological Chemistry vol 278 no 24 pp 21631ndash216382003

[18] S Ghosh M J May and E B Kopp ldquoNF-120581B and rel proteinsevolutionarily conserved mediators of immune responsesrdquoAnnual Review of Immunology vol 16 pp 225ndash260 1998

[19] S Janssens A Tinel S Lippens and J Tschopp ldquoPIDDMediates NF-120581B activation in response to DNA damagerdquo Cellvol 123 no 6 pp 1079ndash1092 2005

[20] F Balkwill ldquoTNF-120572 in promotion and progression of cancerrdquoCancer andMetastasis Reviews vol 25 no 3 pp 409ndash416 2006

[21] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[22] S G Ko H-P Kim D-H Jin et al ldquoSaussurea lappa inducesG2-growth arrest and apoptosis in AGS gastric cancer cellsrdquoCancer Letters vol 220 no 1 pp 11ndash19 2005

[23] S-G Ko S-H Koh C-Y Jun C-G Nam H-S Bae andM-KShin ldquoInduction of apoptosis by Saussurea lappa and Pharbitisnil on AGS gastric cancer cellsrdquo Biological and PharmaceuticalBulletin vol 27 no 10 pp 1604ndash1610 2004

[24] A Robinson T V Kumar E Sreedhar et al ldquoA new sesquiter-pene lactone from the roots of Saussurea lappa structure-anticancer activity studyrdquo Bioorganic and Medicinal ChemistryLetters vol 18 no 14 pp 4015ndash4017 2008

[25] C A L Kassuya A Cremoneze L F L Barros et alldquoAntipyretic and anti-inflammatory properties of the ethanolicextract dichloromethane fraction and costunolide from Mag-nolia ovata (Magnoliaceae)rdquo Journal of Ethnopharmacology vol124 no 3 pp 369ndash376 2009

[26] H-C Chen C-K Chou S-D Lee J-C Wang and S-F YehldquoActive compounds from Saussurea lappa Clarks that suppresshepatitis B virus surface antigen gene expression in humanhepatoma cellsrdquo Antiviral Research vol 27 no 1-2 pp 99ndash1091995

[27] D E Wedge J C G Galindo and F A Macıas ldquoFungicidalactivity of natural and synthetic sesquiterpene lactone analogsrdquoPhytochemistry vol 53 no 7 pp 747ndash757 2000

[28] C-N Chen H-H Huang C-L Wu et al ldquoIsocostunolidea sesquiterpene lactone induces mitochondrial membranedepolarization and caspase-dependent apoptosis in human


melanoma cellsrdquo Cancer Letters vol 246 no 1-2 pp 237ndash2522007

[29] H Mori T Kawamori T Tanaka M Ohnishi and J YamaharaldquoChemopreventive effect of costunolide a constituent of orien-tal medicine on azoxymethane-induced intestinal carcinogen-esis in ratsrdquo Cancer Letters vol 83 no 1-2 pp 171ndash175 1994

[30] J-H Choi J Ha J-H Park et al ldquoCostunolide triggers apop-tosis in human leukemia U937 cells by depleting intracellularthiolsrdquo Japanese Journal of Cancer Research vol 93 no 12 pp1327ndash1333 2002

[31] J-L Hsu S-L Pan Y-F Ho T-L Hwang F-L Kung andJ-H Guh ldquoCostunolide induces apoptosis through nuclearcalcium2+ overload and DNA damage response in humanprostate cancerrdquo Journal ofUrology vol 185 no 5 pp 1967ndash19742011

[32] Y K Choi H S Seo H S Choi et al ldquoInduction of Fas-mediated extrinsic apoptosis p21WAF1-related G2M cell cyclearrest and ROS generation by costunolide in estrogen receptor-negative breast cancer cells MDA-MB-231rdquo Molecular andCellular Biochemistry vol 363 no 1-2 pp 119ndash128 2012

[33] P Nyberg T Salo and R Kalluri ldquoTumor microenvironmentand angiogenesisrdquo Frontiers in Bioscience vol 13 no 17 pp6537ndash6553 2008

[34] C M Overall and C Lopez-Otın ldquoStrategies for MMP inhi-bition in cancer innovations for the post-trial erardquo NatureReviews Cancer vol 2 no 9 pp 657ndash672 2002

[35] L M Coussens and Z Werb ldquoMatrix metalloproteinases andthe development of cancerrdquo Chemistry and Biology vol 3 no11 pp 895ndash904 1996

[36] E I Deryugina A Zijlstra J J Partridge et al ldquoUnex-pected effect of matrix metalloproteinase down-regulation onvascular intravasation and metastasis of human fibrosarcomacells selected in vivo for high rates of disseminationrdquo CancerResearch vol 65 no 23 pp 10959ndash10969 2005

[37] M E Kupferman M E Fini W J Muller R Weber Y Chengand R J Muschel ldquoMatrix metalloproteinase 9 promoteractivity is induced coincident with invasion during tumorprogressionrdquoThe American Journal of Pathology vol 157 no 6pp 1777ndash1783 2000

[38] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[39] M Egeblad and Z Werb ldquoNew functions for the matrix met-alloproteinases in cancer progressionrdquo Nature Reviews Cancervol 2 no 3 pp 161ndash174 2002

[40] H Sato and M Seiki ldquoRegulatory mechanism of 92 kDatype IV collagenase gene expression which is associated withinvasiveness of tumor cellsrdquoOncogene vol 8 no 2 pp 395ndash4051993

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


sequences to regulate expression of target genes which arerelated to tumor development and metastases [19ndash21]

The dried root of Saussurea lappa Clarke (SLC) hastransitionally been used as an ingredient in Korea China andJapan for the treatment of either abdominal pain or tenesmusSeveral earlier studies indicated that the root of SLC hasanticancer effect in gastric cancer cells [22 23] Costunolide(C15H20O2) a sesquiterpene lactone that is a major com-

ponent of the root of SLC [24] has been reported to havediverse effects such as anti-inflammatory [25] anti-viral [26]and -fungal [27] effects Furthermore costunolide affectedanti various cancers includingmelanoma [28] intestinal [29]leukemia [30] prostate [31] and breast cancers [32]

While anti-cancer effects of either SLC or costunolidehave been reported as mentioned befor antimetastatic effectsof either SLC or costunolide on metastatic breast cancerare still poorly understood In this study we found thatSLC and costunolide inhibit TNF120572-mediated breast cancercell migration and invasion by inhibiting NF-120581B activationthereby suggesting the antimetastatic property of costunolideusing highly metastatic MDA-MB-231 breast cancer cells

2 Materials and Methods

21 Reagents and Cell Lines Costunolide (molecular weightof 23232 purity gt 99 see Figure 2(a)) was purchasedfromWako (Wako Pure Chemical Industries Osaka Japan)RPMI 1640 fetal bovine serum (FBS) antibiotic-antimycoticand phosphate-buffered Saline (PBS) were purchased fromGibco-BRL (Rockville MD USA) EZ-western detection kitwas obtained from Daeillab (Daeillab service Co SeoulKorea) TNF120572was purchased fromRampDsystems (Minneapo-lis MNUSA)

22 Preparation of Saussurea lappa Clarke (SLC) ExtractSaussurea lappa Clarke was purchased from Omniherb(Gyeong Buk Korea) The 100 g of root of SLC was dipped in1 L of 80 ethanol and sonicated by using an ultrasonicator(Branson MO USA) for 30min at room temperature Thesonicated extract was filtered through a 022mm filter andconcentrated The ethanol extracts were dried in a 42∘C byusing a vacuum pump evaporator (Eyela Tokyo Japan) The285 g of concentrated extract was dissolved in DMSO toprepare a stock solution of 100mgmLThe stock solutionwasstored at minus80∘C until use

23 Cell Migration and Invasion Cell migration was mea-sured by wound healing assays Cells were seeded in 6-wellplates and scratched with a 200120583L pipette tip 24 hoursafter treatments with Saussurea lappaClarke and costunolidemigrated cell numbers were counted For invasion assay cellswere seeded in the upper chambers precoated with Matrigeland treated with SLC and costunolide Low chambers werefilled with 10 FBS or TNF120572-contained medium and inva-sive cells were stainedwith hematoxylin and eosin to visualizeand count All experiments were performed in triplicateand studentrsquos t-test was performed to determine statistics119875 values below 005 and 0001 were considered statistically

significant All data was represented as the mean plusmn standarddeviation

24 Immunofluorescence Assays Immunofluorescence as-says were used for p-NF120581B nuclear translocation in MDA-MB-231 cell After treatment with SLC and costunolide for6 hours cells were fixed with 4 paraformaldehyde for15min and then permeabilized with 05 Triton X-100 for10min The cells were washed with PBS blocked with 5FBS in PBS for 30min and then incubated with anti-p-NF-120581B antibody overnight at 4∘C and with anti-Alexa Fluor-488secondary antibody (Invitrogen Eugene Oregon USA) for1 hour Phalloidin (Sigma) and TO-PRO-3 (Invitrogen) wereused to contain F-actin and the nucleus respectively Imageswere obtained with Olympus FV10i Self-Contained ConfocalLaser SystemThe object was 20x and scale bars on the imageindicate 50120583m

25 Luciferase Assays Cells were seeded in 24-well platesand NF-120581B-luc plasmid (Stratagene La Jolla CA USA)transfected in MDA-MB-231 cells by using Lipofectaminereagent (Invitrogen Carlsbad CA USA) Cells were treatedwith SLC and costunolide for 6 hours and then the luciferaseassays were done by using dual-luciferase reporter assay(Promega Madison WI USA) All transfections includedthe RLTK-Luc (kindly provided by Sang Hoon Kim) fortransfection efficiency All experiments were performed intriplicate and studentrsquos t-test was performed to determinestatistics 119875 values below 005 and 0001 were consideredstatistically significant All data was represented as the meanplusmn standard deviation

26 Western Blot Total protein (30 120583g) was separated bySDS-PAGE After electrophoresis the proteins were trans-ferred to a nitrocellulose membrane The membrane wasblocked incubated overnight at 4∘Cwith primary antibodieswashedwith PBS-T (PBSwith 01Tween-20) and incubatedwith appropriate HRP-conjugated secondary antibodies atroom temperature for 1 hour Immunoreactive protein wasdeveloped using an EZ-western detection kit (Daeillab ser-vice Co Seoul Korea) Anti-MMP-9 -p-IKK -IKK -p-I120581B -I120581B -p-NF-120581B and -NF-120581B were purchased from CellSignaling (Danvers MA USA) Anti-Tubulin was purchasedfrom Sigma (Louis MO USA)

27 RNA Extraction and RT-PCR Cellular total RNA wasextractedwithTRIzol reagent (Invitrogen)TheRNAconcen-tration and purity weremeasured using a spectrophotometercDNAwas synthesized from total RNA (1120583g) by reverse tran-scriptionThe primer sequences and product size were as fol-lows MMP-9 (262 bp) forward 51015840-CACTGTCCACCCCTC-AGAGC-31015840 reverse 51015840-GCCACTTGTCGGCGATAAGG-31015840 GAPDH (300 bp) forward 51015840-CGTCTTCACCACCAT-GGAG-A-31015840 reverse 51015840-CGGCCATCACGCCACAGTTT-31015840 The products were checked by agarose electrophoresisand analyzed using ChemiDoc imaging system (BioRadHercules CA USA)


0

100

200

None TNF120572

(a)

TNF120572 + SLC

Cel

l mig

ratio

n (

)lowast

(b)

TNF120572 TNF120572 + SLC0

5

10

None

Cell

inva

sion

(fold

) lowastlowast

(c)

None


TO-PRO-3 Merged

TNF120572


TO-PRO-3 Merged

TNF120572 + SLC


TO-PRO-3 Merged

TNF+minus +SLC

MMP-9

Actin

+minus minus

(e)

(d)

0

2

4

6

NF-120581

B-de

pend

ent r

epor

ter a

ctiv

ity (f

old)

lowast




H

H

Abs OO

(a)

Cel

l mig

ratio

n (

)

0

50

100

150

Control DMSO CTL

lowast

(b)

Cell

inva

sion

()

0

50

100

150

Control DMSO CTL

lowast

(c)

Cel

l mig

ratio

n (

)

0

100

200


lowast

(d)


Cel

l inv

asio

n (fo

ld)

0

5

10

lowastlowast

(e)



2 1 120583M ZnCl






p-IKK

IKK

p-I120581B120572

I120581B120572

Tubulin

minus

(a)


Lamin B

Nuclear p65

Tubulin

Cytosol p65

(b)

0 3015

p-IKK

IKK

I120581B120572

Tubulin

(min)(min)

p-IKK

IKK

I120581B120572

Tubulin

TNF1205720 3015

TNF120572 + CTL

(c)

None


TO-PRO-3 Merged

TNF120572


TO-PRO-3 Merged

TNF120572 + CTL


TO-PRO-3 Merged

(d)






3 Results



0

2

4

6

Non

e

TNF 120572

TNFR

I

TNFR

I + C

TL

TNF 120572

+ C

TL


NF-120581

B-de

pend

ent r

epor

ter a

ctiv

ity (f

old)

(a)

Immunoblot

Zymography


MMP-9 (cell)

Tubulin

GAPDH

MMP-9

MMP-9

RT-PCR


(b)













0

50

100

150

200

1 2 5 8 11 14 18 21 24 27 30

CTLControl

Tum

or v

olum

e (m

m3)

(a)


Body

wei

ght (

g)

0

10

20

1 2 5 8 11 14 18 21 24 27 30

CTLControl

(b)

Tum

or

Control CTL

Lung

Live

r

H and E

(c)

Tum

orControl CTL

Lung

Live

r

ki-67

(d)

Control CTL

MMP-9

(e)



TRAD

IKK

NF-120581B

NF-120581B


Nucleus


TNF120572

TNFR


H

H

Abs OO







4 Discussion






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

None TNF120572

(a)

TNF120572 + SLC

Cel

l mig

ratio

n (

)lowast

(b)

TNF120572 TNF120572 + SLC0

5

10

None

Cell

inva

sion

(fold

) lowastlowast

(c)

None


TO-PRO-3 Merged

TNF120572


TO-PRO-3 Merged

TNF120572 + SLC


TO-PRO-3 Merged

TNF+minus +SLC

MMP-9

Actin

+minus minus

(e)

(d)

0

2

4

6

NF-120581

B-de

pend

ent r

epor

ter a

ctiv

ity (f

old)

lowast




H

H

Abs OO

(a)

Cel

l mig

ratio

n (

)

0

50

100

150

Control DMSO CTL

lowast

(b)

Cell

inva

sion

()

0

50

100

150

Control DMSO CTL

lowast

(c)

Cel

l mig

ratio

n (

)

0

100

200


lowast

(d)


Cel

l inv

asio

n (fo

ld)

0

5

10

lowastlowast

(e)



2 1 120583M ZnCl






p-IKK

IKK

p-I120581B120572

I120581B120572

Tubulin

minus

(a)


Lamin B

Nuclear p65

Tubulin

Cytosol p65

(b)

0 3015

p-IKK

IKK

I120581B120572

Tubulin

(min)(min)

p-IKK

IKK

I120581B120572

Tubulin

TNF1205720 3015

TNF120572 + CTL

(c)

None


TO-PRO-3 Merged

TNF120572


TO-PRO-3 Merged

TNF120572 + CTL


TO-PRO-3 Merged

(d)






3 Results



0

2

4

6

Non

e

TNF 120572

TNFR

I

TNFR

I + C

TL

TNF 120572

+ C

TL


NF-120581

B-de

pend

ent r

epor

ter a

ctiv

ity (f

old)

(a)

Immunoblot

Zymography


MMP-9 (cell)

Tubulin

GAPDH

MMP-9

MMP-9

RT-PCR


(b)













0

50

100

150

200

1 2 5 8 11 14 18 21 24 27 30

CTLControl

Tum

or v

olum

e (m

m3)

(a)


Body

wei

ght (

g)

0

10

20

1 2 5 8 11 14 18 21 24 27 30

CTLControl

(b)

Tum

or

Control CTL

Lung

Live

r

H and E

(c)

Tum

orControl CTL

Lung

Live

r

ki-67

(d)

Control CTL

MMP-9

(e)



TRAD

IKK

NF-120581B

NF-120581B


Nucleus


TNF120572

TNFR


H

H

Abs OO







4 Discussion






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















H

H

Abs OO

(a)

Cel

l mig

ratio

n (

)

0

50

100

150

Control DMSO CTL

lowast

(b)

Cell

inva

sion

()

0

50

100

150

Control DMSO CTL

lowast

(c)

Cel

l mig

ratio

n (

)

0

100

200


lowast

(d)


Cel

l inv

asio

n (fo

ld)

0

5

10

lowastlowast

(e)



2 1 120583M ZnCl






p-IKK

IKK

p-I120581B120572

I120581B120572

Tubulin

minus

(a)


Lamin B

Nuclear p65

Tubulin

Cytosol p65

(b)

0 3015

p-IKK

IKK

I120581B120572

Tubulin

(min)(min)

p-IKK

IKK

I120581B120572

Tubulin

TNF1205720 3015

TNF120572 + CTL

(c)

None


TO-PRO-3 Merged

TNF120572


TO-PRO-3 Merged

TNF120572 + CTL


TO-PRO-3 Merged

(d)






3 Results



0

2

4

6

Non

e

TNF 120572

TNFR

I

TNFR

I + C

TL

TNF 120572

+ C

TL


NF-120581

B-de

pend

ent r

epor

ter a

ctiv

ity (f

old)

(a)

Immunoblot

Zymography


MMP-9 (cell)

Tubulin

GAPDH

MMP-9

MMP-9

RT-PCR


(b)













0

50

100

150

200

1 2 5 8 11 14 18 21 24 27 30

CTLControl

Tum

or v

olum

e (m

m3)

(a)


Body

wei

ght (

g)

0

10

20

1 2 5 8 11 14 18 21 24 27 30

CTLControl

(b)

Tum

or

Control CTL

Lung

Live

r

H and E

(c)

Tum

orControl CTL

Lung

Live

r

ki-67

(d)

Control CTL

MMP-9

(e)



TRAD

IKK

NF-120581B

NF-120581B


Nucleus


TNF120572

TNFR


H

H

Abs OO







4 Discussion






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















p-IKK

IKK

p-I120581B120572

I120581B120572

Tubulin

minus

(a)


Lamin B

Nuclear p65

Tubulin

Cytosol p65

(b)

0 3015

p-IKK

IKK

I120581B120572

Tubulin

(min)(min)

p-IKK

IKK

I120581B120572

Tubulin

TNF1205720 3015

TNF120572 + CTL

(c)

None


TO-PRO-3 Merged

TNF120572


TO-PRO-3 Merged

TNF120572 + CTL


TO-PRO-3 Merged

(d)






3 Results



0

2

4

6

Non

e

TNF 120572

TNFR

I

TNFR

I + C

TL

TNF 120572

+ C

TL


NF-120581

B-de

pend

ent r

epor

ter a

ctiv

ity (f

old)

(a)

Immunoblot

Zymography


MMP-9 (cell)

Tubulin

GAPDH

MMP-9

MMP-9

RT-PCR


(b)













0

50

100

150

200

1 2 5 8 11 14 18 21 24 27 30

CTLControl

Tum

or v

olum

e (m

m3)

(a)


Body

wei

ght (

g)

0

10

20

1 2 5 8 11 14 18 21 24 27 30

CTLControl

(b)

Tum

or

Control CTL

Lung

Live

r

H and E

(c)

Tum

orControl CTL

Lung

Live

r

ki-67

(d)

Control CTL

MMP-9

(e)



TRAD

IKK

NF-120581B

NF-120581B


Nucleus


TNF120572

TNFR


H

H

Abs OO







4 Discussion






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

Non

e

TNF 120572

TNFR

I

TNFR

I + C

TL

TNF 120572

+ C

TL


NF-120581

B-de

pend

ent r

epor

ter a

ctiv

ity (f

old)

(a)

Immunoblot

Zymography


MMP-9 (cell)

Tubulin

GAPDH

MMP-9

MMP-9

RT-PCR


(b)













0

50

100

150

200

1 2 5 8 11 14 18 21 24 27 30

CTLControl

Tum

or v

olum

e (m

m3)

(a)


Body

wei

ght (

g)

0

10

20

1 2 5 8 11 14 18 21 24 27 30

CTLControl

(b)

Tum

or

Control CTL

Lung

Live

r

H and E

(c)

Tum

orControl CTL

Lung

Live

r

ki-67

(d)

Control CTL

MMP-9

(e)



TRAD

IKK

NF-120581B

NF-120581B


Nucleus


TNF120572

TNFR


H

H

Abs OO







4 Discussion






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

50

100

150

200

1 2 5 8 11 14 18 21 24 27 30

CTLControl

Tum

or v

olum

e (m

m3)

(a)


Body

wei

ght (

g)

0

10

20

1 2 5 8 11 14 18 21 24 27 30

CTLControl

(b)

Tum

or

Control CTL

Lung

Live

r

H and E

(c)

Tum

orControl CTL

Lung

Live

r

ki-67

(d)

Control CTL

MMP-9

(e)



TRAD

IKK

NF-120581B

NF-120581B


Nucleus


TNF120572

TNFR


H

H

Abs OO







4 Discussion






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TRAD

IKK

NF-120581B

NF-120581B


Nucleus


TNF120572

TNFR


H

H

Abs OO







4 Discussion






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















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 Saussurea lappa Clarke-Derived Costunolide ...

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