Research ArticleOroxylin A Suppresses the Cell Proliferation Migrationand EMT via NF-120581B Signaling Pathway in Human BreastCancer Cells

XiaoHu Sun 123 Xinzhong Chang123 YunhuaWang4 Boyang Xu4 and Xuchen Cao123

1Tianjin Medical University Cancer Institute and Hospital National Clinical Research Center for Cancer Tianjin China2Key Laboratory of Cancer Prevention anderapy Tianjin China3Key Laboratory of Breast Cancer Prevention anderapy Tianjin Medical University Ministry of Education Tianjin China4Tianjin University of Traditional Chinese Medicine China

Correspondence should be addressed to XiaoHu Sun tiger sun1986126com

Received 29 April 2019 Accepted 29 May 2019 Published 23 June 2019

Academic Editor Nadia M Hamdy

Copyright copy 2019 XiaoHu Sun 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

Oroxylin A is a natural extract and has been reported to have a remarkable anticancer function However the mechanism of itsanticancer activity remains not quite clear In this study we examined the inhibiting effects of Oroxylin A on breast cancer cellproliferation migration and epithelial-mesenchymal transition (EMT) and its possible molecular mechanismThe cytoactive andinflammatory factors were analyzed via Cell Counting Kit-8 assay and ELISA assay respectively Flow cytometry and westernblotting were used to assess the cell proliferation In addition a wound healing assay and transwell assay were used to detectcell invasion and migration qRT-PCR and western blot were employed to determine the effect of Oroxylin A on the EMTformation Moreover expression level of protein related to NF-120581B signaling pathway was determined by western blot The resultsrevealed that Oroxylin A attenuated the cytoactivity of MDA-MB-231 cells in a dose- and a time-dependent manner Moreover cellproliferation invasion and migration of breast cancer cells were inhibited by Oroxylin A compared to the control The mRNA andprotein expression levels of E-cadherin were remarkably increased while N-cadherin and Vimentin remarkably decreased BesidesOroxylin A suppressed the expression of inflammatory factors and NF-120581B activation Furthermore we also found that supplementof TNF-120572 reversed the effects of Oroxylin A on the cell proliferation invasion migration and EMT in breast cancer cells Takentogether our results suggested that Oroxylin A inhibited the cell proliferation invasion migration and EMT through inactivatingNF-120581B signaling pathway in human breast cancer cells These findings strongly suggest that Oroxylin A could be a therapeuticpotential candidate for the treatment of breast cancer

1 Introduction

Breast cancer is one of the most common cancers and thesecond leading cause of cancer death in women Even ifadvances in inchoate diagnosis and treatment of breast cancerhave been made approximately 30 of patients with breastcancer will develop cureless metastatic disease[1 2] in whichthe median survival times range from 1 to 4 years [3] Triplenegative (TN) breast cancer is a particular type of breastcancer which lacks the expression of oestrogen receptor(ER) progesterone receptor (PgR) and ERBB2 The featurescontribute to biological aggressiveness worse prognosis andlack of a therapeutic target compared to other subtypes [4]

Thus triple negative breast cancer is a huge challenge formodern medicine

The relationship between cancer and inflammation hasbeen studied for many years Inflammation in the tumormicroenvironment plays an extremely important role intumor progress [5] In cancer-related inflammation majorinflammatory cytokines (such as IL-1120573 IL-6 and TNF-120572) and transcription factors (such as NF-120581B and STAT3)are recognized as key endogenous factors [6] It has beenproved that NF-120581B participates in initiation and progres-sion of tumor in tissues where cancer-related inflammationeasily occurs [7] NF-120581B is not only a key coordinator ofinflammation and innate immunity but also regarded as an

HindawiBioMed Research InternationalVolume 2019 Article ID 9241769 10 pageshttpsdoiorg10115520199241769

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important endogenous tumor promoter In tumor cells NF-120581B induces the expression of antiapoptotic genes to acceleratecell growth and activates relative inflammatory cytokinesadhesion molecules and angiogenic factors to expedite theproliferation of tumor cells [8ndash11]

Oroxylin A (OA) is natural compound extracted fromScutellariae radix It has been proved that the com-pound exerts broad functions including anticancer anti-inflammation neuroprotective and anticoagulation [12]Thefunctions have attracted much attention wisely especiallythe antitumor activity OA performs a strong anticanceractivity by inducing apoptosis inhibiting metastasis andinvasion reversing multidrug resistance suppressing angio-genesis and so on [13] In a previous study OA couldsuppress the expression of Bcl-2 and procaspase-3 proteinin HepG2 cell significantly to exert proapoptotic effect [14]OA suppressed cell invasion and had antimetastatic effectthrough downregulating the expression ofMMP2 andMMP9in MDA-MB-435 human breast cancer cells [15] MoreoverOA prevented inflammation-related tumor through down-regulation of inflammatory gene expression by inhibitingNF-120581B signaling pathway [5] Further OA inhibited activatedtranscription factor NF-120581B reducing the expression of LPS-induced iNOS and COX-2 in RAW 2647 cells [16] A largenumber of researches about the anticancer effects of OA havebeen studied However it is unclear whether and howOA canexert the anticancer effects on TN breast cancer by NF-120581Bsignaling pathway

Thus we explored the inhibiting effects of OA on theproliferation migration and EMT on MDA-MB-231 humanbreast cancer cells in this study and reported the protectiveeffects of OA via suppressing NF-120581B signaling pathway

2 Materials and Methods21 Reagents and Antibodies Oroxylin A was bought fromNovartis Pharmaceuticals (Basel Switzerland) and dissolvedin dimethyl sulfoxide (DMSO) as a stock solution Primaryantibodies against CDK2 CyclinE p27 p-p65 p65 andCOX-2 were obtained from Cell Signaling (Beverly MAUSA) Primary antibodies against E-cad N-cad Vimentinp-I120581B I120581B and GAPDH were obtained from Cell SignalingTechnology (Danvers MA) Secondary antibodies conju-gated with horseradish peroxidase were obtained from SantaCruz Biotechnology All other reagents were purchased fromSigma-Aldrich (Louis MO USA)

22 Cell Culture The human breast cancer cells MDA-MB-231 were obtained from the American Type CultureCollection (ATCC Manassas VA) and maintained in Dul-beccorsquos modified Eaglersquos medium (DMEM Gibco Gaithers-burg) supplemented with 10 fetal bovine serum penicillin100Uml streptomycin 100 120583gml and incubated in a humid-ified incubator at 37∘C and 5 CO

2 Cells were divided into

four groups the control (treated with PBS) TNF120572 (treatedwith TNF-120572) OA20 (treated with 20120583M Oroxylin A) andOA20+TNF120572 (treated with 20120583MOroxylin A+TNF-120572)

23 Cell Viability Assay The effect of OA on MDA-MB-231 cell growth was measured by CCK-8 assay Cells were

seeded into 96-well plates and treated with OA at variousconcentrations (0120583M 10 120583M 20 120583M and 40 120583M) Eachconcentration was repeated five times After 24 and 48 hours10 120583l CCK-8 (Dojindo Kumamoto Japan) reagent was addedto each well Plates were then estimated on a microplatereader after 2 h incubation Absorbance was measured at 450nm using microplate spectrophotometry

24 Flow Cytometric Analysis Flow cytometry was used toexplore the cell proliferation Treated with OA (20 120583M)orand TNF-120572 for 24 h the cells were harvested and cen-trifuged at 300 g for 5 minThen the cells were stained with aCell CycleDetectionKit (KeyGeneHolland) according to themanufacturerrsquos instructions Finally the samples were ana-lyzed with the FACSCalibur Flow Cytometer (BD BioscienceUSA) and BD CellQuest software (BD Bioscience USA)

25 Western Blotting MDA-MB-231 cells were treated withOA (20 120583M) orand TNF-120572 for 24h harvested and homog-enized in 200 120583L RIPA lysis buffer The concentration ofprotein was determined by the protein quantitative kit Thenthe proteins samples were separated by in SDS-PAGE andtransferred to PVDF membrane After blocked with 5 ofskimmed milk powder for 2 h the PVDF membranes wereincubated with the primary antibodies (CDK2 11000 2546CyclinE 11000 4129 p27 11000 3688 E-cadherin 1100014472 N-cadherin 11000 13116 Vimentin 11000 5741p-p65 11000 3036 p65 11000 8242 p-I120581B 11000 2859I120581B 11000 4814 COX-2 11000 12282 GAPDH 110005174) overnight at 4∘C Then the membranes were culturedwith secondary horseradish peroxidase-labeled antibody at37∘C for 1 h The target proteins on the membranes wereanalyzed using ECL Blotting Detection Reagents

26 Wound Healing Assay Cell migration capacity wascalculated by a wound healing assay Cells were seeded in6-well plates and allowed to growth to 80ndash90 confluenceThe cell monolayers were then wounded with white pipettetips and washed four times with phosphate-buffered saline toremove cell debris The cells were then incubated in mediumwith OA (20 120583M) orand TNF-120572 for 24 h Cell migrationinto the wound surface and number of migrated cells weredetermined under an inverted microscopy Five randomlychosen fields were analyzed in each well

27 Cell Invasion Assay An invasion assay was implementedto examine tumor invasion using transwell chamber (65 mmin diameter 8 120583m pore-size Corning Costar CambridgeMA) Firstly the transwell chambers were first loaded with01mL of matrigel (Becton Dickinson Bedford MA) at37∘C for 1 h MDA-MB-231 cells were incubated in DMEMcontaining 1FBS and treatedwithOA (20120583M)orandTNF-120572 for 24h Then cells were trypsinized and suspended at afinal concentration of 5times105 cellsmL in DMEM containing1 FBS Cell suspensions were then loaded into the uppercompartment and medium with 10 fetal bovine serum wasadded in the lower compartment Incubated at 37∘C in 5CO2for 24 h cells on the upper surface were wiped off

with a cotton swab Then invaded cells on the lower surface

BioMed Research International 3

were fixed stained and counted under a microscope Fiverandomly chosen fields were counted for each group

28 Quantitative Real-Time PCR Cells were pretreatedwith 20120583M OA for 24 h Total RNA was extracted byTRIzol (Invitrogen Carlsbad CA) reagent The purity andquality of RNA were examined by NanoDrop2000 (ThermoScientific Wilmington DE) Total RNA was reverselytranscribed into cDNA using the Reverse TranscriptionSystem Kit (Takara Dalian China) Then Quantitativereal-time PCR was carried out using SYBR green qPCRkit (TakaraBio Inc) on an ABI PRISM 7900 Real-Timesystem (Applied Biosystems Foster City CA USA) ThePCR amplification program was the following 95∘C for 20sec followed by 40 cycles of 95∘C for 1 sec and 60∘C for 20sec The sequences of PCR primers were listed as follows E-cadherin (forward 51015840-CCACCAAAGTCACGCTGAAT-31015840reverse 51015840-GGAGTTGGGAAATGTGAGC-31015840) N-cadherin(forward 51015840-GTGCCATTAGCCAAGGAATTCAGC-31015840reverse 51015840-GCGTTCCTGTTCCACTCATAGGAGG-31015840)Vimentin (forward 51015840-ATGAAGGTGCTGCAAAAC-31015840 reverse 51015840-GTGACTGCACCTGTCTCCGGTA-31015840)GAPDH (forward 51015840-ATG AGCCCCAGCCTTCTCCAT-31015840reverse 51015840-GGTCGGAGTCAACGGATTTG-31015840) mRNAlevel was expressed as the relative change after normalizedversus GAPDH Relative expression was quantified by the2minusΔΔCq method [17] The expression levels were relative tothe fold change of the controls which were defined as 10

29 ELISA Assay IL-6 IL-8 and TNF-120572 secretion in cellsupernatants was measured by ELISA according to themanufacturerrsquos instructions 6 replicates were established foreach group and results were from triplicate experiments

210 Statistical Analysis SPSS 200 statistical analysis soft-ware was used to analyze the experimental data The resultswere expressed as mean plusmn SD Statistical comparisons weremade by two-tailed Studentrsquos t-test or one-way analysis ofvariance (ANOVA) P lt 005 was considered statisticallysignificant

3 Results

31 Effect of OA on Cell Viability of Breast Cancer Cells Toinvestigate the effect of OA on the growth of MDA-MB-231 cells CCK-8 assay was performed The results showedthat OA inhibited the growth of MDA-MB-231 cells in adose- and a time-dependent manner (Figure 1) Based onthe above-mentioned result a 24-hour treatment of OA atthe concentration of 20 120583M was applied to the subsequentexperiments

32 Effect of OA on the Proliferation of Breast Cancer CellsTo determine whether OA would influence the proliferationof MDA-MB-231 cells flow cytometry and western blottingwere usedAs shown in Figures 2(a) and 2(b)OAhadobviouseffects on the cell cycle OA treatment increased the G1phase population while decreasing the S phase population inMDA-MB-231 cells Furthermore the results of western blot

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Figure 1 Effect of Oroxylin A on growth of MDA-MB-231 cells atdifferent time points Data are expressed as mean plusmn SDlowastP lt 005lowastlowastP lt 001 lowastlowastlowastP lt 0001 versus control

indicated that the protein expression of CDK2 and Cyclin Ewas extremely decreased compared to the control while p27had the opposite results (Figure 2(c))

33 Effect of OA on the Migration and Invasion of BreastCancer Cells Wound healing assay and transwell assay wereapplied for investigating the effect of OA on the migrationand invasion of MDA-MB-231 cells respectively The resultsshowed that cells in the control rapidly spread to the woundarea after 24 h incubation whereas very few cells inOA groupspread forward (Figure 3(a)) Additionally the results frominvasion assay revealed that numerous cells invaded throughthematrigel in the control while the number of cells invadingto the lower side significantly decreased in the treatment ofOA (Figure 3(c)) And the relative cell invasive andmigrationrates in OA group were both dramatically lower than thecontrol (Figures 3(b) and 3(d))

34 Effect of OA on EMT Formation To examine the effectof OA on the formation of EMT the expression of E-cadherin N-cadherin and Vimentin in MDA-MB-231cellswas detected by qRT-PCR and western blotting analysis Itwas shown that the mRNA and protein expression levels ofE-cadherin remarkably elevated whereas the expression ofN-cadherin and Vimentin was considerably decreased whencompared to the control (Figure 4)These results suggest thatEMT formation could be suppressed by OA

35 Effect of OA on the Expression of Inflammatory CytokinesTo explore whether inflammatory cytokines were inhibited inbreast cancer in response to OA the levels of IL-6 IL-8 andTNF- 120572 were detected by ELISAThe results showed that OAmarkedly suppressed the protein expressions of IL-6 IL-8

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Figure 2 Effect of Oroxylin A on proliferation of MDA-MB-231 cells (a-b) Flow cytometry assays were performed to analyze the cell cycleprogression when MDA-MB-231 cells were treated with OA 24 h later (c) The expressions of proliferation-related protein CDK2 CyclinEand p27 were detected by western blot analysis Data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt 001 versus control

and TNF-120572 in MDA-MB-231cells compared with the control(Figure 5)

36 Inhibitory Effect of OA on NF-120581B Signaling Pathway NF-120581B signaling pathway has been recognized to play vital rolesin the regulating of inflammatory pathways in the tumormicroenvironmentThus the effects of OA onNF-120581B activitywere assessed by western blotting As shown in Figure 6 OAdid not affect the total amounts of p65 and I120581B but signifi-cantly inhibited the phosphorylation of them Furthermorethe protein level of COX-2 was markedly decreased aftertreatment with OA compared to the control These resultsindicated that OA effectively attenuated activation of the NF-120581B pathway with the repression of COX-2 in breast cancercells

37 TNF-120572 Reverses the Functions of OA on TN BreastCancer Cells TNF stimulates the growth of normal mam-mary epithelial cells and the mammary cancer cells [18]thus we studied the effects of TNF-120572 on the proliferationmigration and EMT of breast cancer cells with or without

OA Flow cytometry demonstrated that OA suppressed andTNF-120572 promoted cell proliferation compared to the controlwhile the OA +TNF120572 group showed that OA-inhibited cellproliferation was promoted by TNF-120572 (Figures 7(a) and7(b)) In addition the results from wound healing assayand transwell assay showed that OA suppressed and TNF-120572promoted cell migration and invasion while treatment of OA+TNF120572 promoted OA-inhibited cell migration and invasion(Figures 7(c) and 7(d)) than the OA group Furthermorethe results from a western blot revealed that OA inhibitedand TNF-120572 enhance EMT phenotype formation of MDA-MB-231 cells while the OA +TNF120572 group reversed the EMTformation inhibited by OA (Figure 8) Taken together thesefindings suggest that the effects of OA on the suppressionof proliferation migration and EMT formation on breastcancer cells could be reversed by TNF-120572

4 Discussion

OA one of themain bioactive flavonoids of Scutellariae radixexerts obvious antitumor activities [19] It has been proved

BioMed Research International 5

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Figure 3 Effect of Oroxylin A onMDA-MB-435 cell migration and invasion in vitro (a) Oroxylin A inhibits the cell migrationThe cells wereseeded in a 6-well plate and cell monolayer was wounded and washed by PBS and then incubated with 20 120583MOroxylin A for 24 h Migrationwas assessed by microscope with a camera Image magnification 100times (c) Oroxylin A inhibits the cell invasion Cells were cultured in thepresence of 20 120583M Oroxylin A for 24 h and seeded in the upside of transwell with matrigel for 24 h The lower side of the membrane wasstained and counted under a microscope Image magnification 100times Data are expressed as mean plusmn SD lowastlowastlowastP lt 0001 versus control

that OA inhibited breast cancer progression by proapoptosisantiproliferation antiangiogenesis and so on [15 20ndash22]However the mechanism of OA defenses against breastcancer remains not to be fully elucidated

In the present study we found that OA significantlyreduced the cytoactivity of MDA-MB-231 cells in a dose-dependent manner Moreover compared with the controlOA inhibited proliferation migration and EMT formationand the expression of inflammatory cytokines of breastcancer cells obviously The experiments also showed that OAinhibited NF-120581B activation by suppressing the phosphory-lation of p65 and I120581B as well as the expression of COX-2Furthermore it was found that TNF-120572 reversed the effects ofOA on breast cancer cells

The inflammatory antitumor response plays a critical rolein inhibiting tumor growth in human cancers [23] NF-120581Bis one of the most important regulators of proinflammatory

gene expression [24] contributing to the progress of tumorRecent studies have revealed that NF-120581B is involved inregulation of expression of COX-2 [25] Cyclooxygenase-2(COX-2) a rate-limiting enzyme regulates the formation ofcarcinogens apoptosis and angiogenesis in tumor progres-sion [26] Previous study showed that Oroxylin A modulatedNF-120581B signaling to hold back inflammation related tumor[5] Moreover it is reported that Oroxylin A suppressedLPS-induced expression of iNOS and COX-2 related toinflammatory processes by inhibition of NF-120581B activation inmacrophages [27] In the present study we found that OAsignificantly suppressed the activation of NF-120581B signalingin MDA-MB-231cells by suppressing phosphorylation of I120581Band p-65 which are important steps in NF-120581B activationWhat is more the protein expression of COX-2 was foundto be decreased which may be associated with inhibition ofNF-120581B signaling

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Figure 4 Effect of Oroxylin A on EMT formation in MDA-MB-231 cells Cells were treated with 20 120583M Oroxylin A for 24 h E-cadherin(a) N-cadherin (b) and Vimentin (c) mRNAs were measured with qRT-PCR (d) The expression of E-cadherin N-cadherin and Vimentinproteins in the cells was analyzed by western blotting using specific antibodies Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt0001 versus control

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Figure 5 Effect of Oroxylin A on IL-6 IL-8 and TNF-120572 levels in MDA-MB-231 cells The expression of IL-6 IL-8 and TNF-120572 was detectedby ELISA assay from the supernatants of cells cultured with 20 120583MOroxylin A for 24 h Data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt001 versus control

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Figure 6 Effect of Oroxylin A on NF-120581B activation and the expression of COX-2 in MDA-MB-231 cells Phosphorylation of p65 (a) and I120581B(b) and COX-2 (c) expression were analyzed by western blot GAPDH was used as an internal control Data are expressed as mean plusmn SDlowastlowastP lt 001 versus control

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8 BioMed Research International

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Figure 7 TNF-120572 reverses the function of OA in breast cancer cells (a-b) Flow cytometry (c) wound healing assay and (d) transwell assaywere performed in MDA-MB-231 cells treated with control TNF-120572 OA and OA + TNF120572 Image magnification 100times Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 versus control P lt 005 P lt 001 P lt 0001 versus the OA control

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Figure 8 TNF-120572 reverses the effect of Oroxylin A on EMT formation in MDA-MB-231 cells The protein expressions of E-cadherin N-cadherin and Vimentin in the cells were analyzed by western blotting Data are expressed as mean plusmn SD lowastlowastP lt 001 lowastlowastlowastP lt 0001 versuscontrol P lt 005 P lt 0001 versus the OA control

BioMed Research International 9

Chronic inflammation is a main activator in themetastatic cascade [28] Epithelial-mesenchymal transition(EMT) is a complex process losing cell-cell adhesioncontributing to cancer invasion and metastasis The resultsof the present study indicated that OA inhibited the EMTformation in breast cancer cells by upregulation of E-cadherin levels and downregulation of N-cadherin andVimentin levels EMT programs stimulate the productionof proinflammatory factors in cancer environment whileinflammation is a potent inducement for EMT in tumors[9] What is more NF-120581B activity promotes the collectionof inflammatory cells and secretion of proinflammatorycytokines like IL-1 IL-6 IL-8 and TNF-120572 [29] The presentresults demonstrated that OA markedly suppressed thesecretion of IL-6 IL-8 and TNF-120572 protein in MDA-MB-231cellsThese findings indicate OAhas a repressive influenceon EMT and inflammation progress in breast cancercells

TNF-120572 has been proved to have pleiotropic effects oncells death growth or differentiation under different con-centrations and context [30 31] TNF has anticancer andanti-infection effects in high concentrations because of itscytotoxic effects on the tumor vessel [32] However it isshown that TNF can promote the proliferation andmigrationof normal epithelial cell and breast cancer cells both atlow doses [33 34] One study demonstrated that TNF-120572secreted by tumor cells stimulated the growth of malignantmammary epithelial cells which contributed to mammarytumorigenesis in neuerbB2 transgenic mice [18] Anotherstudy showed that TNF-120572 can promote transendothelialmigration of breast cancer via upregulation of LOX [35]In the present study we found OA-suppressed cell prolif-eration migration invasion and EMT phenotype forma-tion were promoted by TNF-120572 leading to aggravation ofbreast cancer process which is consistent with previousreports On these bases we hypothesized that the blockadeof TNF-120572 may enhance the antitumor effects of OA forbreast cancer patients and this speculation warrants furtherinvestigation

5 Conclusion

In summary the present study showed that OA couldsuppress cell proliferation migration invasion and EMTformation and downregulate the expressions of inflammatorycytokines Furthermore OA exerted protective effect onbreast cancer by inhibiting NF-120581B signaling pathway Theresults provided a theoretical foundation for Oroxylin A asa potential drug for the treatment of breast cancer

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

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

Acknowledgments

This work was supported by Funding from Science andTechnology Development of Universities and Colleges ofTianjin (No 20130120)

References

[1] A Ugolkov I Gaisina J Zhang et al ldquoGSK-3 inhibitionovercomes chemoresistance in human breast cancerrdquo CancerLetters vol 380 no 2 pp 384ndash392 2016

[2] C A Santa-Maria and W J Gradishar ldquoChanging treatmentparadigms in metastatic breast cancerrdquo JAMA Oncology vol 1no 4 pp 528ndash534 2015

[3] J OrsquoShaughnessy ldquoExtending survival with chemotherapy inmetastatic breast cancerrdquo e Oncologist vol 10 no 3 pp 20ndash29 2005

[4] A Bosch P Eroles R Zaragoza et al ldquoTriple-negative breastcancer molecular features pathogenesis treatment and currentlines of researchrdquo Cancer Treatment Reviews vol 36 no 3 pp206ndash215 2010

[5] J Yao R Hu J Sun et al ldquoOroxylin a prevents inflammation-related tumor through down-regulation of inflammatory geneexpression by inhibiting NF-120581B signalingrdquo Molecular Carcino-genesis vol 53 no 2 pp 145ndash158 2014

[6] A Mantovani P Allavena A Sica and F Balkwill ldquoCancer-related inflammationrdquo Nature vol 454 no 7203 pp 436ndash4442008

[7] M Karin ldquoNF-kappaB as a critical link between inflammationand cancerrdquo Cold Spring Harbor Perspectives in Biology vol 1no 5 Article ID a000141 2009

[8] X Cui D Shen C Kong et al ldquoNF-120581B suppresses apoptosisand promotes bladder cancer cell proliferation by upregulatingsurvivin expression in vitro and in vivordquo Scientific Reports vol7 no 1 Article ID 40723 2017

[9] M Suarez-Carmona J Lesage D Cataldo and C Gilles ldquoEMTand inflammation inseparable actors of cancer progressionrdquoMolecular Oncology vol 11 no 7 pp 805ndash823 2017

[10] H Kiefel S Bondong N Erbe-Hoffmann et al ldquoL1CAM-integrin interaction induces constitutive NF-120581B activation inpancreatic adenocarcinoma cells by enhancing IL-1Β expres-sionrdquo Oncogene vol 29 no 34 pp 4766ndash4778 2010

[11] R A Stoltz N G Abraham and M Laniado-SchwartzmanldquoThe role of NF-kappaB in the angiogenic response of coro-nary microvessel endothelial cellsrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 93 no7 pp 2832ndash2837 1996

[12] Z Lu N Lu C Li et al ldquoOroxylin A inhibits matrixmetalloproteinase-29 expression and activation by up-regulating tissue inhibitor of metalloproteinase-2 andsuppressing the ERK12 signaling pathwayrdquo ToxicologyLetters vol 209 no 3 pp 211ndash220 2012

[13] L Lu Q Guo and L Zhao ldquoOverview of Oroxylin A apromising flavonoid compoundrdquo Phytotherapy Research vol30 no 11 pp 1765ndash1774 2016

[14] Y Hu Y Yang Q-D You et al ldquoOroxylin A induced apop-tosis of human hepatocellular carcinoma cell line HepG2 wasinvolved in its antitumor activityrdquo Biochemical and BiophysicalResearch Communications vol 351 no 2 pp 521ndash527 2006

[15] Y Sun N Lu Y Ling et al ldquoOroxylin A suppressesinvasion through down-regulating the expression of matrix

10 BioMed Research International

metalloproteinase-29 in MDA-MB-435 human breast cancercellsrdquo European Journal of Pharmacology vol 603 no 1-3 pp22ndash28 2009

[16] T Tseng M Chen M Tsai et al ldquoOroxylin-A rescues LPS-induced acute lung injury via regulation of NF-120581B signalingpathway in rodentsrdquo PLoS ONE vol 7 no 10 Article ID e474032012

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[18] M A Warren S F Shoemaker D J Shealy W Bshara andM M Ip ldquoTumor necrosis factor deficiency inhibits mam-mary tumorigenesis and a tumor necrosis factor neutralizingantibody decreases mammary tumor growth in neuerbB2transgenic micerdquo Molecular Cancer erapeutics vol 8 no 9pp 2655ndash2663 2009

[19] T Ni Z He Y Dai J Yao Q Guo and L Wei ldquoOrox-ylin A suppresses the development and growth of colorectalcancer through reprogram of HIF1120572-modulated fatty acidmetabolismrdquo Cell Death amp Disease vol 8 no 6 Article IDe2865 2017

[20] L Wei Y Zhou C Qiao et al ldquoOroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promotingSIRT3-mediated SOD2 transcription and HIF1120572 destabiliza-tionrdquo Cell Death amp Disease vol 6 no 4 Article ID e1714 2015

[21] Y Gao N Lu Y Ling et al ldquoOroxylin A inhibits angiogenesisthrough blocking vascular endothelial growth factor-inducedKDRFlk-1 phosphorylationrdquo Journal of Cancer Research andClinical Oncology vol 136 no 5 pp 667ndash675 2010

[22] R Mu Q Qi H Gu et al ldquoInvolvement of p53 in oroxylinA-induced apoptosis in cancer cellsrdquoMolecular Carcinogenesisvol 48 no 12 pp 1159ndash1169 2009

[23] H Ohshima H Tazawa B S Sylla and T Sawa ldquoPreventionof human cancer by modulation of chronic inflammatoryprocessesrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 591 no 1-2 pp 110ndash122 2005

[24] P P Tak and G S Firestein ldquoNF-kappaB a key role ininflammatory diseasesrdquoe Journal of Clinical Investigation vol107 pp 7ndash11 2001

[25] E Heiss C Herhaus K Klimo H Bartsch and C GerhauserldquoNuclear factor 120581B is a molecular target for sulforaphane-mediated anti-inflammatory mechanismsrdquo e Journal of Bio-logical Chemistry vol 276 no 34 pp 32008ndash32015 2001

[26] A J Dannenberg N K Altorki J O Boyle et al ldquoCyclo-oxygenase 2 a pharmacological target for the prevention ofcancerrdquoe Lancet Oncology vol 2 no 9 pp 544ndash551 2001

[27] Y-C Chen L-L Yang and T J-F Lee ldquoOroxylin A inhi-bition of lipopolysaccharide-induced iNOS and COX-2 geneexpression via suppression of nuclear factor-120581B activationrdquoBiochemical Pharmacology vol 59 no 11 pp 1445ndash1457 2000

[28] D-F Lee andM-CHung ldquoAdvances in targeting IKK and IKK-related kinases for cancer therapyrdquoClinical Cancer Research vol14 no 18 pp 5656ndash5662 2008

[29] Z Han D L Boyle A M Manning and G S Firestein ldquoAP-1and NF-kappaB regulation in rheumatoid arthritis and murinecollagen-induced arthritisrdquo Autoimmunity vol 28 no 4 pp197ndash208 1998

[30] D Liu X Wang and Z Chen ldquoTumor Necrosis Factor-alphaa regulator and therapeutic agent on breast cancerrdquo CurrentPharmaceutical Biotechnology vol 17 no 6 pp 486ndash494 2016

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

[32] R van Horssen T L M ten Hagen and A M M EggermontldquoTNF-120572 in cancer treatment molecular insights antitumoreffects and clinical utilityrdquoeOncologist vol 11 no 4 pp 397ndash408 2006

[33] M M Ip S F Shoemaker and K M Darcy ldquoRegulation ofrat mammary epithelial cell proliferation and differentiation bytumor necrosis factor-alphardquo Endocrinology vol 130 no 5 pp2833ndash2844 1992

[34] J Zhang M A Warren S F Shoemaker and M MIp ldquoNF120581B1p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells impli-cations for NF120581B2p52 and RelBrdquo Endocrinology vol 148 no 1pp 268ndash278 2007

[35] M Liang P Zhang and J Fu ldquoUp-regulation of LOX-1expression by TNF-120572 promotes trans-endothelial migration ofMDA-MB-231 breast cancer cellsrdquo Cancer Letters vol 258 no1 pp 31ndash37 2007

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

BioMed Research International 3

were fixed stained and counted under a microscope Fiverandomly chosen fields were counted for each group

28 Quantitative Real-Time PCR Cells were pretreatedwith 20120583M OA for 24 h Total RNA was extracted byTRIzol (Invitrogen Carlsbad CA) reagent The purity andquality of RNA were examined by NanoDrop2000 (ThermoScientific Wilmington DE) Total RNA was reverselytranscribed into cDNA using the Reverse TranscriptionSystem Kit (Takara Dalian China) Then Quantitativereal-time PCR was carried out using SYBR green qPCRkit (TakaraBio Inc) on an ABI PRISM 7900 Real-Timesystem (Applied Biosystems Foster City CA USA) ThePCR amplification program was the following 95∘C for 20sec followed by 40 cycles of 95∘C for 1 sec and 60∘C for 20sec The sequences of PCR primers were listed as follows E-cadherin (forward 51015840-CCACCAAAGTCACGCTGAAT-31015840reverse 51015840-GGAGTTGGGAAATGTGAGC-31015840) N-cadherin(forward 51015840-GTGCCATTAGCCAAGGAATTCAGC-31015840reverse 51015840-GCGTTCCTGTTCCACTCATAGGAGG-31015840)Vimentin (forward 51015840-ATGAAGGTGCTGCAAAAC-31015840 reverse 51015840-GTGACTGCACCTGTCTCCGGTA-31015840)GAPDH (forward 51015840-ATG AGCCCCAGCCTTCTCCAT-31015840reverse 51015840-GGTCGGAGTCAACGGATTTG-31015840) mRNAlevel was expressed as the relative change after normalizedversus GAPDH Relative expression was quantified by the2minusΔΔCq method [17] The expression levels were relative tothe fold change of the controls which were defined as 10

29 ELISA Assay IL-6 IL-8 and TNF-120572 secretion in cellsupernatants was measured by ELISA according to themanufacturerrsquos instructions 6 replicates were established foreach group and results were from triplicate experiments

210 Statistical Analysis SPSS 200 statistical analysis soft-ware was used to analyze the experimental data The resultswere expressed as mean plusmn SD Statistical comparisons weremade by two-tailed Studentrsquos t-test or one-way analysis ofvariance (ANOVA) P lt 005 was considered statisticallysignificant

3 Results

31 Effect of OA on Cell Viability of Breast Cancer Cells Toinvestigate the effect of OA on the growth of MDA-MB-231 cells CCK-8 assay was performed The results showedthat OA inhibited the growth of MDA-MB-231 cells in adose- and a time-dependent manner (Figure 1) Based onthe above-mentioned result a 24-hour treatment of OA atthe concentration of 20 120583M was applied to the subsequentexperiments

32 Effect of OA on the Proliferation of Breast Cancer CellsTo determine whether OA would influence the proliferationof MDA-MB-231 cells flow cytometry and western blottingwere usedAs shown in Figures 2(a) and 2(b)OAhadobviouseffects on the cell cycle OA treatment increased the G1phase population while decreasing the S phase population inMDA-MB-231 cells Furthermore the results of western blot

0h 12h 24h 48h0

50

100

150

Control

OA10

Cel

l via

bilit

y (

)

OA20

OA40

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 1 Effect of Oroxylin A on growth of MDA-MB-231 cells atdifferent time points Data are expressed as mean plusmn SDlowastP lt 005lowastlowastP lt 001 lowastlowastlowastP lt 0001 versus control

indicated that the protein expression of CDK2 and Cyclin Ewas extremely decreased compared to the control while p27had the opposite results (Figure 2(c))

33 Effect of OA on the Migration and Invasion of BreastCancer Cells Wound healing assay and transwell assay wereapplied for investigating the effect of OA on the migrationand invasion of MDA-MB-231 cells respectively The resultsshowed that cells in the control rapidly spread to the woundarea after 24 h incubation whereas very few cells inOA groupspread forward (Figure 3(a)) Additionally the results frominvasion assay revealed that numerous cells invaded throughthematrigel in the control while the number of cells invadingto the lower side significantly decreased in the treatment ofOA (Figure 3(c)) And the relative cell invasive andmigrationrates in OA group were both dramatically lower than thecontrol (Figures 3(b) and 3(d))

34 Effect of OA on EMT Formation To examine the effectof OA on the formation of EMT the expression of E-cadherin N-cadherin and Vimentin in MDA-MB-231cellswas detected by qRT-PCR and western blotting analysis Itwas shown that the mRNA and protein expression levels ofE-cadherin remarkably elevated whereas the expression ofN-cadherin and Vimentin was considerably decreased whencompared to the control (Figure 4)These results suggest thatEMT formation could be suppressed by OA

35 Effect of OA on the Expression of Inflammatory CytokinesTo explore whether inflammatory cytokines were inhibited inbreast cancer in response to OA the levels of IL-6 IL-8 andTNF- 120572 were detected by ELISAThe results showed that OAmarkedly suppressed the protein expressions of IL-6 IL-8

4 BioMed Research International

Control OA20

B2-HPerCP-Vio700-H B2-HPerCP-Vio700-H

Cou

nt

Cou

nt

lt2N 5232N 693 [ 59840 cv 794]S 1784N 51 [ 113408 cv 137]Ratio 190gt4N 083

50

40

30

20

10

00 50k 100k 150k 200k 250k

50

40

30

20

10

00 50k 100k 150k 200k 250k

lt2N 6332N 725 [ 59840 cv 794]S 1434N1005 [ 115648 cv 480]Ratio 193gt4N 018

(a)

G0G1 S G20

20

40

60

80

ControlOA 20

Cel

l cyc

le d

istrib

utio

n (

)

lowastlowast

(b)

CDK2

Con

trol

OA

20

Con

trol

OA

20

Con

trol

OA

20

00

05

10

15CyclinE

00

05

10

15p27

00

05

10

15

20

CDK2

GAPDH

CyclinE p27

GAPDH GAPDH

Control OA20 Control OA20 Control OA20

lowastlowastlowastlowast

lowastlowast

Relat

ive C

DK2

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive C

yclin

E ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

Relat

ive p

27 ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(c)

Figure 2 Effect of Oroxylin A on proliferation of MDA-MB-231 cells (a-b) Flow cytometry assays were performed to analyze the cell cycleprogression when MDA-MB-231 cells were treated with OA 24 h later (c) The expressions of proliferation-related protein CDK2 CyclinEand p27 were detected by western blot analysis Data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt 001 versus control

and TNF-120572 in MDA-MB-231cells compared with the control(Figure 5)

36 Inhibitory Effect of OA on NF-120581B Signaling Pathway NF-120581B signaling pathway has been recognized to play vital rolesin the regulating of inflammatory pathways in the tumormicroenvironmentThus the effects of OA onNF-120581B activitywere assessed by western blotting As shown in Figure 6 OAdid not affect the total amounts of p65 and I120581B but signifi-cantly inhibited the phosphorylation of them Furthermorethe protein level of COX-2 was markedly decreased aftertreatment with OA compared to the control These resultsindicated that OA effectively attenuated activation of the NF-120581B pathway with the repression of COX-2 in breast cancercells

37 TNF-120572 Reverses the Functions of OA on TN BreastCancer Cells TNF stimulates the growth of normal mam-mary epithelial cells and the mammary cancer cells [18]thus we studied the effects of TNF-120572 on the proliferationmigration and EMT of breast cancer cells with or without

OA Flow cytometry demonstrated that OA suppressed andTNF-120572 promoted cell proliferation compared to the controlwhile the OA +TNF120572 group showed that OA-inhibited cellproliferation was promoted by TNF-120572 (Figures 7(a) and7(b)) In addition the results from wound healing assayand transwell assay showed that OA suppressed and TNF-120572promoted cell migration and invasion while treatment of OA+TNF120572 promoted OA-inhibited cell migration and invasion(Figures 7(c) and 7(d)) than the OA group Furthermorethe results from a western blot revealed that OA inhibitedand TNF-120572 enhance EMT phenotype formation of MDA-MB-231 cells while the OA +TNF120572 group reversed the EMTformation inhibited by OA (Figure 8) Taken together thesefindings suggest that the effects of OA on the suppressionof proliferation migration and EMT formation on breastcancer cells could be reversed by TNF-120572

4 Discussion

OA one of themain bioactive flavonoids of Scutellariae radixexerts obvious antitumor activities [19] It has been proved

BioMed Research International 5

0h

24h

Control OA20

(a)

00

05

10

15

Relat

ive c

ell m

igra

tion

rate

Con

trol

OA

20

lowastlowastlowast

(b)

Control OA20

(c)

00

05

10

15

Relat

ive c

ell i

nvas

ive t

ate

Con

trol

OA

20

lowastlowastlowast

(d)

Figure 3 Effect of Oroxylin A onMDA-MB-435 cell migration and invasion in vitro (a) Oroxylin A inhibits the cell migrationThe cells wereseeded in a 6-well plate and cell monolayer was wounded and washed by PBS and then incubated with 20 120583MOroxylin A for 24 h Migrationwas assessed by microscope with a camera Image magnification 100times (c) Oroxylin A inhibits the cell invasion Cells were cultured in thepresence of 20 120583M Oroxylin A for 24 h and seeded in the upside of transwell with matrigel for 24 h The lower side of the membrane wasstained and counted under a microscope Image magnification 100times Data are expressed as mean plusmn SD lowastlowastlowastP lt 0001 versus control

that OA inhibited breast cancer progression by proapoptosisantiproliferation antiangiogenesis and so on [15 20ndash22]However the mechanism of OA defenses against breastcancer remains not to be fully elucidated

In the present study we found that OA significantlyreduced the cytoactivity of MDA-MB-231 cells in a dose-dependent manner Moreover compared with the controlOA inhibited proliferation migration and EMT formationand the expression of inflammatory cytokines of breastcancer cells obviously The experiments also showed that OAinhibited NF-120581B activation by suppressing the phosphory-lation of p65 and I120581B as well as the expression of COX-2Furthermore it was found that TNF-120572 reversed the effects ofOA on breast cancer cells

The inflammatory antitumor response plays a critical rolein inhibiting tumor growth in human cancers [23] NF-120581Bis one of the most important regulators of proinflammatory

gene expression [24] contributing to the progress of tumorRecent studies have revealed that NF-120581B is involved inregulation of expression of COX-2 [25] Cyclooxygenase-2(COX-2) a rate-limiting enzyme regulates the formation ofcarcinogens apoptosis and angiogenesis in tumor progres-sion [26] Previous study showed that Oroxylin A modulatedNF-120581B signaling to hold back inflammation related tumor[5] Moreover it is reported that Oroxylin A suppressedLPS-induced expression of iNOS and COX-2 related toinflammatory processes by inhibition of NF-120581B activation inmacrophages [27] In the present study we found that OAsignificantly suppressed the activation of NF-120581B signalingin MDA-MB-231cells by suppressing phosphorylation of I120581Band p-65 which are important steps in NF-120581B activationWhat is more the protein expression of COX-2 was foundto be decreased which may be associated with inhibition ofNF-120581B signaling

6 BioMed Research International

0

1

2

3

4Re

lativ

e E-c

ad m

RNA

expr

essio

n

Con

trol

OA

20

lowastlowastlowast

(a)

00

05

10

15

Relat

ive N

-cad

mRN

A ex

pres

sion

Con

trol

OA

20

lowastlowast

(b)

00

05

10

15

Relat

ive V

imen

tin m

RNA

expr

essio

n

Con

trol

OA

20

lowastlowast

(c)

E-cad

N-cad

Vimentin

GAPDH

Control OA20

E-cad

00

05

10

15

20

N-cad

00

05

10

15Vimentin

00

05

10

15C

ontro

l

OA

20

Con

trol

OA

20

Con

trol

OA

20

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive V

imen

tin ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

lowastlowast

lowastlowastlowast

(d)

Figure 4 Effect of Oroxylin A on EMT formation in MDA-MB-231 cells Cells were treated with 20 120583M Oroxylin A for 24 h E-cadherin(a) N-cadherin (b) and Vimentin (c) mRNAs were measured with qRT-PCR (d) The expression of E-cadherin N-cadherin and Vimentinproteins in the cells was analyzed by western blotting using specific antibodies Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt0001 versus control

15

Relat

ive e

xpre

ssio

n of

IL-6

00

05

10

Con

trol

OA

20

lowastlowast

(a)

Relat

ive e

xpre

ssio

n of

IL-8 15

00

05

10

Con

trol

OA

20

lowastlowast

(b)

Relat

ive e

xpre

ssio

n of

TN

F- 15

00

05

10

Con

trol

OA

20

lowast

(c)

Figure 5 Effect of Oroxylin A on IL-6 IL-8 and TNF-120572 levels in MDA-MB-231 cells The expression of IL-6 IL-8 and TNF-120572 was detectedby ELISA assay from the supernatants of cells cultured with 20 120583MOroxylin A for 24 h Data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt001 versus control

BioMed Research International 7

p-p65

p65

GAPDH

p-p65

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive p

-p65

expr

essio

nin

diff

eren

t gro

ups (

fold

)

(a)

p-IB

IB

GAPDH

p-IB

00

05

10

15

OA20Control

Con

trol

OA

20

lowastlowast

Rela

tive p

-I

B ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(b)

GAPDH

COX-2

COX-2

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive C

OX-

2 ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(c)

Figure 6 Effect of Oroxylin A on NF-120581B activation and the expression of COX-2 in MDA-MB-231 cells Phosphorylation of p65 (a) and I120581B(b) and COX-2 (c) expression were analyzed by western blot GAPDH was used as an internal control Data are expressed as mean plusmn SDlowastlowastP lt 001 versus control

Control

B2-HPerCP-Vio700-H

Cou

nt

OA20

B2-HPerCP-Vio700-H

Cou

nt

TNF-

B2-HPerCP-Vio700-H B2-HPerCP-Vio700-H

Cou

nt

OA20+TNF

Cou

nt

50

40

30

20

10

00 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k

50

40

30

20

10

0

50

40

30

20

10

0

40

30

20

10

00 50k 100k 150k 200k 250k

lt2N 7582N 640 [ 60800 cv 663]S 1734N 113 [ 116608 cv 930]Ratio 192gt4N 0

lt2N 762N 507 [ 60800 cv 619]S 2084N 191 [ 115776 cv 137]Ratio 190gt4N 057

lt2N 1532N 697 [ 50561 cv 585]S 1654N 122 [ 95297 cv 107]Ratio 188gt4N 030

lt2N 7692N 593 [ 60800 cv 648]S 1744N 185 [ 115264 cv 127]Ratio 190gt4N 100

(a)

G0G1 S G20

20

40

60

80

ControlTNF-

OA 20

Cell

cycle

dist

ribut

ion

()

OA 20+TNF

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Control OA20TNF- OA20+TNF

0h

24h

(c)

Control OA20 OA20+TNFTNF-

(d)

Figure 7 Continued

8 BioMed Research International

05

10

15

00

Con

trol

OA

20

TNF-

a

OA

20+

TNFa

Relat

ive c

ell m

igra

tion

rate

lowastlowastlowast

(e)

00

10

15

05

Con

trol

OA

20

TNF-

OA

20+

TNF

Relat

ive c

ell i

nvas

ive t

ate

lowastlowast

lowastlowastlowast

(f)

Figure 7 TNF-120572 reverses the function of OA in breast cancer cells (a-b) Flow cytometry (c) wound healing assay and (d) transwell assaywere performed in MDA-MB-231 cells treated with control TNF-120572 OA and OA + TNF120572 Image magnification 100times Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 versus control P lt 005 P lt 001 P lt 0001 versus the OA control

E-cad

N-cad

Vimentin

GAPDH

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

E-cad

Con

trol

TNF-

OA

20

OA

20+T

NF

00

05

10

15

20

N-cad

00

05

10

15

20

Vimentin

00

05

10

15

20

lowastlowast

lowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowast

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)Re

lativ

e Vim

entin

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Figure 8 TNF-120572 reverses the effect of Oroxylin A on EMT formation in MDA-MB-231 cells The protein expressions of E-cadherin N-cadherin and Vimentin in the cells were analyzed by western blotting Data are expressed as mean plusmn SD lowastlowastP lt 001 lowastlowastlowastP lt 0001 versuscontrol P lt 005 P lt 0001 versus the OA control

BioMed Research International 9

Chronic inflammation is a main activator in themetastatic cascade [28] Epithelial-mesenchymal transition(EMT) is a complex process losing cell-cell adhesioncontributing to cancer invasion and metastasis The resultsof the present study indicated that OA inhibited the EMTformation in breast cancer cells by upregulation of E-cadherin levels and downregulation of N-cadherin andVimentin levels EMT programs stimulate the productionof proinflammatory factors in cancer environment whileinflammation is a potent inducement for EMT in tumors[9] What is more NF-120581B activity promotes the collectionof inflammatory cells and secretion of proinflammatorycytokines like IL-1 IL-6 IL-8 and TNF-120572 [29] The presentresults demonstrated that OA markedly suppressed thesecretion of IL-6 IL-8 and TNF-120572 protein in MDA-MB-231cellsThese findings indicate OAhas a repressive influenceon EMT and inflammation progress in breast cancercells

TNF-120572 has been proved to have pleiotropic effects oncells death growth or differentiation under different con-centrations and context [30 31] TNF has anticancer andanti-infection effects in high concentrations because of itscytotoxic effects on the tumor vessel [32] However it isshown that TNF can promote the proliferation andmigrationof normal epithelial cell and breast cancer cells both atlow doses [33 34] One study demonstrated that TNF-120572secreted by tumor cells stimulated the growth of malignantmammary epithelial cells which contributed to mammarytumorigenesis in neuerbB2 transgenic mice [18] Anotherstudy showed that TNF-120572 can promote transendothelialmigration of breast cancer via upregulation of LOX [35]In the present study we found OA-suppressed cell prolif-eration migration invasion and EMT phenotype forma-tion were promoted by TNF-120572 leading to aggravation ofbreast cancer process which is consistent with previousreports On these bases we hypothesized that the blockadeof TNF-120572 may enhance the antitumor effects of OA forbreast cancer patients and this speculation warrants furtherinvestigation

5 Conclusion

In summary the present study showed that OA couldsuppress cell proliferation migration invasion and EMTformation and downregulate the expressions of inflammatorycytokines Furthermore OA exerted protective effect onbreast cancer by inhibiting NF-120581B signaling pathway Theresults provided a theoretical foundation for Oroxylin A asa potential drug for the treatment of breast cancer

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

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

Acknowledgments

This work was supported by Funding from Science andTechnology Development of Universities and Colleges ofTianjin (No 20130120)

References

[1] A Ugolkov I Gaisina J Zhang et al ldquoGSK-3 inhibitionovercomes chemoresistance in human breast cancerrdquo CancerLetters vol 380 no 2 pp 384ndash392 2016

[2] C A Santa-Maria and W J Gradishar ldquoChanging treatmentparadigms in metastatic breast cancerrdquo JAMA Oncology vol 1no 4 pp 528ndash534 2015

[3] J OrsquoShaughnessy ldquoExtending survival with chemotherapy inmetastatic breast cancerrdquo e Oncologist vol 10 no 3 pp 20ndash29 2005

[4] A Bosch P Eroles R Zaragoza et al ldquoTriple-negative breastcancer molecular features pathogenesis treatment and currentlines of researchrdquo Cancer Treatment Reviews vol 36 no 3 pp206ndash215 2010

[5] J Yao R Hu J Sun et al ldquoOroxylin a prevents inflammation-related tumor through down-regulation of inflammatory geneexpression by inhibiting NF-120581B signalingrdquo Molecular Carcino-genesis vol 53 no 2 pp 145ndash158 2014

[6] A Mantovani P Allavena A Sica and F Balkwill ldquoCancer-related inflammationrdquo Nature vol 454 no 7203 pp 436ndash4442008

[7] M Karin ldquoNF-kappaB as a critical link between inflammationand cancerrdquo Cold Spring Harbor Perspectives in Biology vol 1no 5 Article ID a000141 2009

[8] X Cui D Shen C Kong et al ldquoNF-120581B suppresses apoptosisand promotes bladder cancer cell proliferation by upregulatingsurvivin expression in vitro and in vivordquo Scientific Reports vol7 no 1 Article ID 40723 2017

[9] M Suarez-Carmona J Lesage D Cataldo and C Gilles ldquoEMTand inflammation inseparable actors of cancer progressionrdquoMolecular Oncology vol 11 no 7 pp 805ndash823 2017

[10] H Kiefel S Bondong N Erbe-Hoffmann et al ldquoL1CAM-integrin interaction induces constitutive NF-120581B activation inpancreatic adenocarcinoma cells by enhancing IL-1Β expres-sionrdquo Oncogene vol 29 no 34 pp 4766ndash4778 2010

[11] R A Stoltz N G Abraham and M Laniado-SchwartzmanldquoThe role of NF-kappaB in the angiogenic response of coro-nary microvessel endothelial cellsrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 93 no7 pp 2832ndash2837 1996

[12] Z Lu N Lu C Li et al ldquoOroxylin A inhibits matrixmetalloproteinase-29 expression and activation by up-regulating tissue inhibitor of metalloproteinase-2 andsuppressing the ERK12 signaling pathwayrdquo ToxicologyLetters vol 209 no 3 pp 211ndash220 2012

[13] L Lu Q Guo and L Zhao ldquoOverview of Oroxylin A apromising flavonoid compoundrdquo Phytotherapy Research vol30 no 11 pp 1765ndash1774 2016

[14] Y Hu Y Yang Q-D You et al ldquoOroxylin A induced apop-tosis of human hepatocellular carcinoma cell line HepG2 wasinvolved in its antitumor activityrdquo Biochemical and BiophysicalResearch Communications vol 351 no 2 pp 521ndash527 2006

[15] Y Sun N Lu Y Ling et al ldquoOroxylin A suppressesinvasion through down-regulating the expression of matrix

10 BioMed Research International

metalloproteinase-29 in MDA-MB-435 human breast cancercellsrdquo European Journal of Pharmacology vol 603 no 1-3 pp22ndash28 2009

[16] T Tseng M Chen M Tsai et al ldquoOroxylin-A rescues LPS-induced acute lung injury via regulation of NF-120581B signalingpathway in rodentsrdquo PLoS ONE vol 7 no 10 Article ID e474032012

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[18] M A Warren S F Shoemaker D J Shealy W Bshara andM M Ip ldquoTumor necrosis factor deficiency inhibits mam-mary tumorigenesis and a tumor necrosis factor neutralizingantibody decreases mammary tumor growth in neuerbB2transgenic micerdquo Molecular Cancer erapeutics vol 8 no 9pp 2655ndash2663 2009

[19] T Ni Z He Y Dai J Yao Q Guo and L Wei ldquoOrox-ylin A suppresses the development and growth of colorectalcancer through reprogram of HIF1120572-modulated fatty acidmetabolismrdquo Cell Death amp Disease vol 8 no 6 Article IDe2865 2017

[20] L Wei Y Zhou C Qiao et al ldquoOroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promotingSIRT3-mediated SOD2 transcription and HIF1120572 destabiliza-tionrdquo Cell Death amp Disease vol 6 no 4 Article ID e1714 2015

[21] Y Gao N Lu Y Ling et al ldquoOroxylin A inhibits angiogenesisthrough blocking vascular endothelial growth factor-inducedKDRFlk-1 phosphorylationrdquo Journal of Cancer Research andClinical Oncology vol 136 no 5 pp 667ndash675 2010

[22] R Mu Q Qi H Gu et al ldquoInvolvement of p53 in oroxylinA-induced apoptosis in cancer cellsrdquoMolecular Carcinogenesisvol 48 no 12 pp 1159ndash1169 2009

[23] H Ohshima H Tazawa B S Sylla and T Sawa ldquoPreventionof human cancer by modulation of chronic inflammatoryprocessesrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 591 no 1-2 pp 110ndash122 2005

[24] P P Tak and G S Firestein ldquoNF-kappaB a key role ininflammatory diseasesrdquoe Journal of Clinical Investigation vol107 pp 7ndash11 2001

[25] E Heiss C Herhaus K Klimo H Bartsch and C GerhauserldquoNuclear factor 120581B is a molecular target for sulforaphane-mediated anti-inflammatory mechanismsrdquo e Journal of Bio-logical Chemistry vol 276 no 34 pp 32008ndash32015 2001

[26] A J Dannenberg N K Altorki J O Boyle et al ldquoCyclo-oxygenase 2 a pharmacological target for the prevention ofcancerrdquoe Lancet Oncology vol 2 no 9 pp 544ndash551 2001

[27] Y-C Chen L-L Yang and T J-F Lee ldquoOroxylin A inhi-bition of lipopolysaccharide-induced iNOS and COX-2 geneexpression via suppression of nuclear factor-120581B activationrdquoBiochemical Pharmacology vol 59 no 11 pp 1445ndash1457 2000

[28] D-F Lee andM-CHung ldquoAdvances in targeting IKK and IKK-related kinases for cancer therapyrdquoClinical Cancer Research vol14 no 18 pp 5656ndash5662 2008

[29] Z Han D L Boyle A M Manning and G S Firestein ldquoAP-1and NF-kappaB regulation in rheumatoid arthritis and murinecollagen-induced arthritisrdquo Autoimmunity vol 28 no 4 pp197ndash208 1998

[30] D Liu X Wang and Z Chen ldquoTumor Necrosis Factor-alphaa regulator and therapeutic agent on breast cancerrdquo CurrentPharmaceutical Biotechnology vol 17 no 6 pp 486ndash494 2016

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

[32] R van Horssen T L M ten Hagen and A M M EggermontldquoTNF-120572 in cancer treatment molecular insights antitumoreffects and clinical utilityrdquoeOncologist vol 11 no 4 pp 397ndash408 2006

[33] M M Ip S F Shoemaker and K M Darcy ldquoRegulation ofrat mammary epithelial cell proliferation and differentiation bytumor necrosis factor-alphardquo Endocrinology vol 130 no 5 pp2833ndash2844 1992

[34] J Zhang M A Warren S F Shoemaker and M MIp ldquoNF120581B1p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells impli-cations for NF120581B2p52 and RelBrdquo Endocrinology vol 148 no 1pp 268ndash278 2007

[35] M Liang P Zhang and J Fu ldquoUp-regulation of LOX-1expression by TNF-120572 promotes trans-endothelial migration ofMDA-MB-231 breast cancer cellsrdquo Cancer Letters vol 258 no1 pp 31ndash37 2007

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

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ObesityJournal of

Hindawiwwwhindawicom Volume 2018

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Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

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Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

4 BioMed Research International

Control OA20

B2-HPerCP-Vio700-H B2-HPerCP-Vio700-H

Cou

nt

Cou

nt

lt2N 5232N 693 [ 59840 cv 794]S 1784N 51 [ 113408 cv 137]Ratio 190gt4N 083

50

40

30

20

10

00 50k 100k 150k 200k 250k

50

40

30

20

10

00 50k 100k 150k 200k 250k

lt2N 6332N 725 [ 59840 cv 794]S 1434N1005 [ 115648 cv 480]Ratio 193gt4N 018

(a)

G0G1 S G20

20

40

60

80

ControlOA 20

Cel

l cyc

le d

istrib

utio

n (

)

lowastlowast

(b)

CDK2

Con

trol

OA

20

Con

trol

OA

20

Con

trol

OA

20

00

05

10

15CyclinE

00

05

10

15p27

00

05

10

15

20

CDK2

GAPDH

CyclinE p27

GAPDH GAPDH

Control OA20 Control OA20 Control OA20

lowastlowastlowastlowast

lowastlowast

Relat

ive C

DK2

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive C

yclin

E ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

Relat

ive p

27 ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(c)

Figure 2 Effect of Oroxylin A on proliferation of MDA-MB-231 cells (a-b) Flow cytometry assays were performed to analyze the cell cycleprogression when MDA-MB-231 cells were treated with OA 24 h later (c) The expressions of proliferation-related protein CDK2 CyclinEand p27 were detected by western blot analysis Data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt 001 versus control

and TNF-120572 in MDA-MB-231cells compared with the control(Figure 5)

36 Inhibitory Effect of OA on NF-120581B Signaling Pathway NF-120581B signaling pathway has been recognized to play vital rolesin the regulating of inflammatory pathways in the tumormicroenvironmentThus the effects of OA onNF-120581B activitywere assessed by western blotting As shown in Figure 6 OAdid not affect the total amounts of p65 and I120581B but signifi-cantly inhibited the phosphorylation of them Furthermorethe protein level of COX-2 was markedly decreased aftertreatment with OA compared to the control These resultsindicated that OA effectively attenuated activation of the NF-120581B pathway with the repression of COX-2 in breast cancercells

37 TNF-120572 Reverses the Functions of OA on TN BreastCancer Cells TNF stimulates the growth of normal mam-mary epithelial cells and the mammary cancer cells [18]thus we studied the effects of TNF-120572 on the proliferationmigration and EMT of breast cancer cells with or without

OA Flow cytometry demonstrated that OA suppressed andTNF-120572 promoted cell proliferation compared to the controlwhile the OA +TNF120572 group showed that OA-inhibited cellproliferation was promoted by TNF-120572 (Figures 7(a) and7(b)) In addition the results from wound healing assayand transwell assay showed that OA suppressed and TNF-120572promoted cell migration and invasion while treatment of OA+TNF120572 promoted OA-inhibited cell migration and invasion(Figures 7(c) and 7(d)) than the OA group Furthermorethe results from a western blot revealed that OA inhibitedand TNF-120572 enhance EMT phenotype formation of MDA-MB-231 cells while the OA +TNF120572 group reversed the EMTformation inhibited by OA (Figure 8) Taken together thesefindings suggest that the effects of OA on the suppressionof proliferation migration and EMT formation on breastcancer cells could be reversed by TNF-120572

4 Discussion

OA one of themain bioactive flavonoids of Scutellariae radixexerts obvious antitumor activities [19] It has been proved

BioMed Research International 5

0h

24h

Control OA20

(a)

00

05

10

15

Relat

ive c

ell m

igra

tion

rate

Con

trol

OA

20

lowastlowastlowast

(b)

Control OA20

(c)

00

05

10

15

Relat

ive c

ell i

nvas

ive t

ate

Con

trol

OA

20

lowastlowastlowast

(d)

Figure 3 Effect of Oroxylin A onMDA-MB-435 cell migration and invasion in vitro (a) Oroxylin A inhibits the cell migrationThe cells wereseeded in a 6-well plate and cell monolayer was wounded and washed by PBS and then incubated with 20 120583MOroxylin A for 24 h Migrationwas assessed by microscope with a camera Image magnification 100times (c) Oroxylin A inhibits the cell invasion Cells were cultured in thepresence of 20 120583M Oroxylin A for 24 h and seeded in the upside of transwell with matrigel for 24 h The lower side of the membrane wasstained and counted under a microscope Image magnification 100times Data are expressed as mean plusmn SD lowastlowastlowastP lt 0001 versus control

that OA inhibited breast cancer progression by proapoptosisantiproliferation antiangiogenesis and so on [15 20ndash22]However the mechanism of OA defenses against breastcancer remains not to be fully elucidated

In the present study we found that OA significantlyreduced the cytoactivity of MDA-MB-231 cells in a dose-dependent manner Moreover compared with the controlOA inhibited proliferation migration and EMT formationand the expression of inflammatory cytokines of breastcancer cells obviously The experiments also showed that OAinhibited NF-120581B activation by suppressing the phosphory-lation of p65 and I120581B as well as the expression of COX-2Furthermore it was found that TNF-120572 reversed the effects ofOA on breast cancer cells

The inflammatory antitumor response plays a critical rolein inhibiting tumor growth in human cancers [23] NF-120581Bis one of the most important regulators of proinflammatory

gene expression [24] contributing to the progress of tumorRecent studies have revealed that NF-120581B is involved inregulation of expression of COX-2 [25] Cyclooxygenase-2(COX-2) a rate-limiting enzyme regulates the formation ofcarcinogens apoptosis and angiogenesis in tumor progres-sion [26] Previous study showed that Oroxylin A modulatedNF-120581B signaling to hold back inflammation related tumor[5] Moreover it is reported that Oroxylin A suppressedLPS-induced expression of iNOS and COX-2 related toinflammatory processes by inhibition of NF-120581B activation inmacrophages [27] In the present study we found that OAsignificantly suppressed the activation of NF-120581B signalingin MDA-MB-231cells by suppressing phosphorylation of I120581Band p-65 which are important steps in NF-120581B activationWhat is more the protein expression of COX-2 was foundto be decreased which may be associated with inhibition ofNF-120581B signaling

6 BioMed Research International

0

1

2

3

4Re

lativ

e E-c

ad m

RNA

expr

essio

n

Con

trol

OA

20

lowastlowastlowast

(a)

00

05

10

15

Relat

ive N

-cad

mRN

A ex

pres

sion

Con

trol

OA

20

lowastlowast

(b)

00

05

10

15

Relat

ive V

imen

tin m

RNA

expr

essio

n

Con

trol

OA

20

lowastlowast

(c)

E-cad

N-cad

Vimentin

GAPDH

Control OA20

E-cad

00

05

10

15

20

N-cad

00

05

10

15Vimentin

00

05

10

15C

ontro

l

OA

20

Con

trol

OA

20

Con

trol

OA

20

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive V

imen

tin ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

lowastlowast

lowastlowastlowast

(d)

Figure 4 Effect of Oroxylin A on EMT formation in MDA-MB-231 cells Cells were treated with 20 120583M Oroxylin A for 24 h E-cadherin(a) N-cadherin (b) and Vimentin (c) mRNAs were measured with qRT-PCR (d) The expression of E-cadherin N-cadherin and Vimentinproteins in the cells was analyzed by western blotting using specific antibodies Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt0001 versus control

15

Relat

ive e

xpre

ssio

n of

IL-6

00

05

10

Con

trol

OA

20

lowastlowast

(a)

Relat

ive e

xpre

ssio

n of

IL-8 15

00

05

10

Con

trol

OA

20

lowastlowast

(b)

Relat

ive e

xpre

ssio

n of

TN

F- 15

00

05

10

Con

trol

OA

20

lowast

(c)

Figure 5 Effect of Oroxylin A on IL-6 IL-8 and TNF-120572 levels in MDA-MB-231 cells The expression of IL-6 IL-8 and TNF-120572 was detectedby ELISA assay from the supernatants of cells cultured with 20 120583MOroxylin A for 24 h Data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt001 versus control

BioMed Research International 7

p-p65

p65

GAPDH

p-p65

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive p

-p65

expr

essio

nin

diff

eren

t gro

ups (

fold

)

(a)

p-IB

IB

GAPDH

p-IB

00

05

10

15

OA20Control

Con

trol

OA

20

lowastlowast

Rela

tive p

-I

B ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(b)

GAPDH

COX-2

COX-2

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive C

OX-

2 ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(c)

Figure 6 Effect of Oroxylin A on NF-120581B activation and the expression of COX-2 in MDA-MB-231 cells Phosphorylation of p65 (a) and I120581B(b) and COX-2 (c) expression were analyzed by western blot GAPDH was used as an internal control Data are expressed as mean plusmn SDlowastlowastP lt 001 versus control

Control

B2-HPerCP-Vio700-H

Cou

nt

OA20

B2-HPerCP-Vio700-H

Cou

nt

TNF-

B2-HPerCP-Vio700-H B2-HPerCP-Vio700-H

Cou

nt

OA20+TNF

Cou

nt

50

40

30

20

10

00 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k

50

40

30

20

10

0

50

40

30

20

10

0

40

30

20

10

00 50k 100k 150k 200k 250k

lt2N 7582N 640 [ 60800 cv 663]S 1734N 113 [ 116608 cv 930]Ratio 192gt4N 0

lt2N 762N 507 [ 60800 cv 619]S 2084N 191 [ 115776 cv 137]Ratio 190gt4N 057

lt2N 1532N 697 [ 50561 cv 585]S 1654N 122 [ 95297 cv 107]Ratio 188gt4N 030

lt2N 7692N 593 [ 60800 cv 648]S 1744N 185 [ 115264 cv 127]Ratio 190gt4N 100

(a)

G0G1 S G20

20

40

60

80

ControlTNF-

OA 20

Cell

cycle

dist

ribut

ion

()

OA 20+TNF

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Control OA20TNF- OA20+TNF

0h

24h

(c)

Control OA20 OA20+TNFTNF-

(d)

Figure 7 Continued

8 BioMed Research International

05

10

15

00

Con

trol

OA

20

TNF-

a

OA

20+

TNFa

Relat

ive c

ell m

igra

tion

rate

lowastlowastlowast

(e)

00

10

15

05

Con

trol

OA

20

TNF-

OA

20+

TNF

Relat

ive c

ell i

nvas

ive t

ate

lowastlowast

lowastlowastlowast

(f)

Figure 7 TNF-120572 reverses the function of OA in breast cancer cells (a-b) Flow cytometry (c) wound healing assay and (d) transwell assaywere performed in MDA-MB-231 cells treated with control TNF-120572 OA and OA + TNF120572 Image magnification 100times Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 versus control P lt 005 P lt 001 P lt 0001 versus the OA control

E-cad

N-cad

Vimentin

GAPDH

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

E-cad

Con

trol

TNF-

OA

20

OA

20+T

NF

00

05

10

15

20

N-cad

00

05

10

15

20

Vimentin

00

05

10

15

20

lowastlowast

lowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowast

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)Re

lativ

e Vim

entin

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Figure 8 TNF-120572 reverses the effect of Oroxylin A on EMT formation in MDA-MB-231 cells The protein expressions of E-cadherin N-cadherin and Vimentin in the cells were analyzed by western blotting Data are expressed as mean plusmn SD lowastlowastP lt 001 lowastlowastlowastP lt 0001 versuscontrol P lt 005 P lt 0001 versus the OA control

BioMed Research International 9

Chronic inflammation is a main activator in themetastatic cascade [28] Epithelial-mesenchymal transition(EMT) is a complex process losing cell-cell adhesioncontributing to cancer invasion and metastasis The resultsof the present study indicated that OA inhibited the EMTformation in breast cancer cells by upregulation of E-cadherin levels and downregulation of N-cadherin andVimentin levels EMT programs stimulate the productionof proinflammatory factors in cancer environment whileinflammation is a potent inducement for EMT in tumors[9] What is more NF-120581B activity promotes the collectionof inflammatory cells and secretion of proinflammatorycytokines like IL-1 IL-6 IL-8 and TNF-120572 [29] The presentresults demonstrated that OA markedly suppressed thesecretion of IL-6 IL-8 and TNF-120572 protein in MDA-MB-231cellsThese findings indicate OAhas a repressive influenceon EMT and inflammation progress in breast cancercells

TNF-120572 has been proved to have pleiotropic effects oncells death growth or differentiation under different con-centrations and context [30 31] TNF has anticancer andanti-infection effects in high concentrations because of itscytotoxic effects on the tumor vessel [32] However it isshown that TNF can promote the proliferation andmigrationof normal epithelial cell and breast cancer cells both atlow doses [33 34] One study demonstrated that TNF-120572secreted by tumor cells stimulated the growth of malignantmammary epithelial cells which contributed to mammarytumorigenesis in neuerbB2 transgenic mice [18] Anotherstudy showed that TNF-120572 can promote transendothelialmigration of breast cancer via upregulation of LOX [35]In the present study we found OA-suppressed cell prolif-eration migration invasion and EMT phenotype forma-tion were promoted by TNF-120572 leading to aggravation ofbreast cancer process which is consistent with previousreports On these bases we hypothesized that the blockadeof TNF-120572 may enhance the antitumor effects of OA forbreast cancer patients and this speculation warrants furtherinvestigation

5 Conclusion

In summary the present study showed that OA couldsuppress cell proliferation migration invasion and EMTformation and downregulate the expressions of inflammatorycytokines Furthermore OA exerted protective effect onbreast cancer by inhibiting NF-120581B signaling pathway Theresults provided a theoretical foundation for Oroxylin A asa potential drug for the treatment of breast cancer

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

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

Acknowledgments

This work was supported by Funding from Science andTechnology Development of Universities and Colleges ofTianjin (No 20130120)

References

[1] A Ugolkov I Gaisina J Zhang et al ldquoGSK-3 inhibitionovercomes chemoresistance in human breast cancerrdquo CancerLetters vol 380 no 2 pp 384ndash392 2016

[2] C A Santa-Maria and W J Gradishar ldquoChanging treatmentparadigms in metastatic breast cancerrdquo JAMA Oncology vol 1no 4 pp 528ndash534 2015

[3] J OrsquoShaughnessy ldquoExtending survival with chemotherapy inmetastatic breast cancerrdquo e Oncologist vol 10 no 3 pp 20ndash29 2005

[4] A Bosch P Eroles R Zaragoza et al ldquoTriple-negative breastcancer molecular features pathogenesis treatment and currentlines of researchrdquo Cancer Treatment Reviews vol 36 no 3 pp206ndash215 2010

[5] J Yao R Hu J Sun et al ldquoOroxylin a prevents inflammation-related tumor through down-regulation of inflammatory geneexpression by inhibiting NF-120581B signalingrdquo Molecular Carcino-genesis vol 53 no 2 pp 145ndash158 2014

[6] A Mantovani P Allavena A Sica and F Balkwill ldquoCancer-related inflammationrdquo Nature vol 454 no 7203 pp 436ndash4442008

[7] M Karin ldquoNF-kappaB as a critical link between inflammationand cancerrdquo Cold Spring Harbor Perspectives in Biology vol 1no 5 Article ID a000141 2009

[8] X Cui D Shen C Kong et al ldquoNF-120581B suppresses apoptosisand promotes bladder cancer cell proliferation by upregulatingsurvivin expression in vitro and in vivordquo Scientific Reports vol7 no 1 Article ID 40723 2017

[9] M Suarez-Carmona J Lesage D Cataldo and C Gilles ldquoEMTand inflammation inseparable actors of cancer progressionrdquoMolecular Oncology vol 11 no 7 pp 805ndash823 2017

[10] H Kiefel S Bondong N Erbe-Hoffmann et al ldquoL1CAM-integrin interaction induces constitutive NF-120581B activation inpancreatic adenocarcinoma cells by enhancing IL-1Β expres-sionrdquo Oncogene vol 29 no 34 pp 4766ndash4778 2010

[11] R A Stoltz N G Abraham and M Laniado-SchwartzmanldquoThe role of NF-kappaB in the angiogenic response of coro-nary microvessel endothelial cellsrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 93 no7 pp 2832ndash2837 1996

[12] Z Lu N Lu C Li et al ldquoOroxylin A inhibits matrixmetalloproteinase-29 expression and activation by up-regulating tissue inhibitor of metalloproteinase-2 andsuppressing the ERK12 signaling pathwayrdquo ToxicologyLetters vol 209 no 3 pp 211ndash220 2012

[13] L Lu Q Guo and L Zhao ldquoOverview of Oroxylin A apromising flavonoid compoundrdquo Phytotherapy Research vol30 no 11 pp 1765ndash1774 2016

[14] Y Hu Y Yang Q-D You et al ldquoOroxylin A induced apop-tosis of human hepatocellular carcinoma cell line HepG2 wasinvolved in its antitumor activityrdquo Biochemical and BiophysicalResearch Communications vol 351 no 2 pp 521ndash527 2006

[15] Y Sun N Lu Y Ling et al ldquoOroxylin A suppressesinvasion through down-regulating the expression of matrix

10 BioMed Research International

metalloproteinase-29 in MDA-MB-435 human breast cancercellsrdquo European Journal of Pharmacology vol 603 no 1-3 pp22ndash28 2009

[16] T Tseng M Chen M Tsai et al ldquoOroxylin-A rescues LPS-induced acute lung injury via regulation of NF-120581B signalingpathway in rodentsrdquo PLoS ONE vol 7 no 10 Article ID e474032012

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[18] M A Warren S F Shoemaker D J Shealy W Bshara andM M Ip ldquoTumor necrosis factor deficiency inhibits mam-mary tumorigenesis and a tumor necrosis factor neutralizingantibody decreases mammary tumor growth in neuerbB2transgenic micerdquo Molecular Cancer erapeutics vol 8 no 9pp 2655ndash2663 2009

[19] T Ni Z He Y Dai J Yao Q Guo and L Wei ldquoOrox-ylin A suppresses the development and growth of colorectalcancer through reprogram of HIF1120572-modulated fatty acidmetabolismrdquo Cell Death amp Disease vol 8 no 6 Article IDe2865 2017

[20] L Wei Y Zhou C Qiao et al ldquoOroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promotingSIRT3-mediated SOD2 transcription and HIF1120572 destabiliza-tionrdquo Cell Death amp Disease vol 6 no 4 Article ID e1714 2015

[21] Y Gao N Lu Y Ling et al ldquoOroxylin A inhibits angiogenesisthrough blocking vascular endothelial growth factor-inducedKDRFlk-1 phosphorylationrdquo Journal of Cancer Research andClinical Oncology vol 136 no 5 pp 667ndash675 2010

[22] R Mu Q Qi H Gu et al ldquoInvolvement of p53 in oroxylinA-induced apoptosis in cancer cellsrdquoMolecular Carcinogenesisvol 48 no 12 pp 1159ndash1169 2009

[23] H Ohshima H Tazawa B S Sylla and T Sawa ldquoPreventionof human cancer by modulation of chronic inflammatoryprocessesrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 591 no 1-2 pp 110ndash122 2005

[24] P P Tak and G S Firestein ldquoNF-kappaB a key role ininflammatory diseasesrdquoe Journal of Clinical Investigation vol107 pp 7ndash11 2001

[25] E Heiss C Herhaus K Klimo H Bartsch and C GerhauserldquoNuclear factor 120581B is a molecular target for sulforaphane-mediated anti-inflammatory mechanismsrdquo e Journal of Bio-logical Chemistry vol 276 no 34 pp 32008ndash32015 2001

[26] A J Dannenberg N K Altorki J O Boyle et al ldquoCyclo-oxygenase 2 a pharmacological target for the prevention ofcancerrdquoe Lancet Oncology vol 2 no 9 pp 544ndash551 2001

[27] Y-C Chen L-L Yang and T J-F Lee ldquoOroxylin A inhi-bition of lipopolysaccharide-induced iNOS and COX-2 geneexpression via suppression of nuclear factor-120581B activationrdquoBiochemical Pharmacology vol 59 no 11 pp 1445ndash1457 2000

[28] D-F Lee andM-CHung ldquoAdvances in targeting IKK and IKK-related kinases for cancer therapyrdquoClinical Cancer Research vol14 no 18 pp 5656ndash5662 2008

[29] Z Han D L Boyle A M Manning and G S Firestein ldquoAP-1and NF-kappaB regulation in rheumatoid arthritis and murinecollagen-induced arthritisrdquo Autoimmunity vol 28 no 4 pp197ndash208 1998

[30] D Liu X Wang and Z Chen ldquoTumor Necrosis Factor-alphaa regulator and therapeutic agent on breast cancerrdquo CurrentPharmaceutical Biotechnology vol 17 no 6 pp 486ndash494 2016

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

[32] R van Horssen T L M ten Hagen and A M M EggermontldquoTNF-120572 in cancer treatment molecular insights antitumoreffects and clinical utilityrdquoeOncologist vol 11 no 4 pp 397ndash408 2006

[33] M M Ip S F Shoemaker and K M Darcy ldquoRegulation ofrat mammary epithelial cell proliferation and differentiation bytumor necrosis factor-alphardquo Endocrinology vol 130 no 5 pp2833ndash2844 1992

[34] J Zhang M A Warren S F Shoemaker and M MIp ldquoNF120581B1p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells impli-cations for NF120581B2p52 and RelBrdquo Endocrinology vol 148 no 1pp 268ndash278 2007

[35] M Liang P Zhang and J Fu ldquoUp-regulation of LOX-1expression by TNF-120572 promotes trans-endothelial migration ofMDA-MB-231 breast cancer cellsrdquo Cancer Letters vol 258 no1 pp 31ndash37 2007

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

BioMed Research International 5

0h

24h

Control OA20

(a)

00

05

10

15

Relat

ive c

ell m

igra

tion

rate

Con

trol

OA

20

lowastlowastlowast

(b)

Control OA20

(c)

00

05

10

15

Relat

ive c

ell i

nvas

ive t

ate

Con

trol

OA

20

lowastlowastlowast

(d)

Figure 3 Effect of Oroxylin A onMDA-MB-435 cell migration and invasion in vitro (a) Oroxylin A inhibits the cell migrationThe cells wereseeded in a 6-well plate and cell monolayer was wounded and washed by PBS and then incubated with 20 120583MOroxylin A for 24 h Migrationwas assessed by microscope with a camera Image magnification 100times (c) Oroxylin A inhibits the cell invasion Cells were cultured in thepresence of 20 120583M Oroxylin A for 24 h and seeded in the upside of transwell with matrigel for 24 h The lower side of the membrane wasstained and counted under a microscope Image magnification 100times Data are expressed as mean plusmn SD lowastlowastlowastP lt 0001 versus control

that OA inhibited breast cancer progression by proapoptosisantiproliferation antiangiogenesis and so on [15 20ndash22]However the mechanism of OA defenses against breastcancer remains not to be fully elucidated

In the present study we found that OA significantlyreduced the cytoactivity of MDA-MB-231 cells in a dose-dependent manner Moreover compared with the controlOA inhibited proliferation migration and EMT formationand the expression of inflammatory cytokines of breastcancer cells obviously The experiments also showed that OAinhibited NF-120581B activation by suppressing the phosphory-lation of p65 and I120581B as well as the expression of COX-2Furthermore it was found that TNF-120572 reversed the effects ofOA on breast cancer cells

The inflammatory antitumor response plays a critical rolein inhibiting tumor growth in human cancers [23] NF-120581Bis one of the most important regulators of proinflammatory

gene expression [24] contributing to the progress of tumorRecent studies have revealed that NF-120581B is involved inregulation of expression of COX-2 [25] Cyclooxygenase-2(COX-2) a rate-limiting enzyme regulates the formation ofcarcinogens apoptosis and angiogenesis in tumor progres-sion [26] Previous study showed that Oroxylin A modulatedNF-120581B signaling to hold back inflammation related tumor[5] Moreover it is reported that Oroxylin A suppressedLPS-induced expression of iNOS and COX-2 related toinflammatory processes by inhibition of NF-120581B activation inmacrophages [27] In the present study we found that OAsignificantly suppressed the activation of NF-120581B signalingin MDA-MB-231cells by suppressing phosphorylation of I120581Band p-65 which are important steps in NF-120581B activationWhat is more the protein expression of COX-2 was foundto be decreased which may be associated with inhibition ofNF-120581B signaling

6 BioMed Research International

0

1

2

3

4Re

lativ

e E-c

ad m

RNA

expr

essio

n

Con

trol

OA

20

lowastlowastlowast

(a)

00

05

10

15

Relat

ive N

-cad

mRN

A ex

pres

sion

Con

trol

OA

20

lowastlowast

(b)

00

05

10

15

Relat

ive V

imen

tin m

RNA

expr

essio

n

Con

trol

OA

20

lowastlowast

(c)

E-cad

N-cad

Vimentin

GAPDH

Control OA20

E-cad

00

05

10

15

20

N-cad

00

05

10

15Vimentin

00

05

10

15C

ontro

l

OA

20

Con

trol

OA

20

Con

trol

OA

20

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive V

imen

tin ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

lowastlowast

lowastlowastlowast

(d)

Figure 4 Effect of Oroxylin A on EMT formation in MDA-MB-231 cells Cells were treated with 20 120583M Oroxylin A for 24 h E-cadherin(a) N-cadherin (b) and Vimentin (c) mRNAs were measured with qRT-PCR (d) The expression of E-cadherin N-cadherin and Vimentinproteins in the cells was analyzed by western blotting using specific antibodies Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt0001 versus control

15

Relat

ive e

xpre

ssio

n of

IL-6

00

05

10

Con

trol

OA

20

lowastlowast

(a)

Relat

ive e

xpre

ssio

n of

IL-8 15

00

05

10

Con

trol

OA

20

lowastlowast

(b)

Relat

ive e

xpre

ssio

n of

TN

F- 15

00

05

10

Con

trol

OA

20

lowast

(c)

Figure 5 Effect of Oroxylin A on IL-6 IL-8 and TNF-120572 levels in MDA-MB-231 cells The expression of IL-6 IL-8 and TNF-120572 was detectedby ELISA assay from the supernatants of cells cultured with 20 120583MOroxylin A for 24 h Data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt001 versus control

BioMed Research International 7

p-p65

p65

GAPDH

p-p65

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive p

-p65

expr

essio

nin

diff

eren

t gro

ups (

fold

)

(a)

p-IB

IB

GAPDH

p-IB

00

05

10

15

OA20Control

Con

trol

OA

20

lowastlowast

Rela

tive p

-I

B ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(b)

GAPDH

COX-2

COX-2

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive C

OX-

2 ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(c)

Figure 6 Effect of Oroxylin A on NF-120581B activation and the expression of COX-2 in MDA-MB-231 cells Phosphorylation of p65 (a) and I120581B(b) and COX-2 (c) expression were analyzed by western blot GAPDH was used as an internal control Data are expressed as mean plusmn SDlowastlowastP lt 001 versus control

Control

B2-HPerCP-Vio700-H

Cou

nt

OA20

B2-HPerCP-Vio700-H

Cou

nt

TNF-

B2-HPerCP-Vio700-H B2-HPerCP-Vio700-H

Cou

nt

OA20+TNF

Cou

nt

50

40

30

20

10

00 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k

50

40

30

20

10

0

50

40

30

20

10

0

40

30

20

10

00 50k 100k 150k 200k 250k

lt2N 7582N 640 [ 60800 cv 663]S 1734N 113 [ 116608 cv 930]Ratio 192gt4N 0

lt2N 762N 507 [ 60800 cv 619]S 2084N 191 [ 115776 cv 137]Ratio 190gt4N 057

lt2N 1532N 697 [ 50561 cv 585]S 1654N 122 [ 95297 cv 107]Ratio 188gt4N 030

lt2N 7692N 593 [ 60800 cv 648]S 1744N 185 [ 115264 cv 127]Ratio 190gt4N 100

(a)

G0G1 S G20

20

40

60

80

ControlTNF-

OA 20

Cell

cycle

dist

ribut

ion

()

OA 20+TNF

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Control OA20TNF- OA20+TNF

0h

24h

(c)

Control OA20 OA20+TNFTNF-

(d)

Figure 7 Continued

8 BioMed Research International

05

10

15

00

Con

trol

OA

20

TNF-

a

OA

20+

TNFa

Relat

ive c

ell m

igra

tion

rate

lowastlowastlowast

(e)

00

10

15

05

Con

trol

OA

20

TNF-

OA

20+

TNF

Relat

ive c

ell i

nvas

ive t

ate

lowastlowast

lowastlowastlowast

(f)

Figure 7 TNF-120572 reverses the function of OA in breast cancer cells (a-b) Flow cytometry (c) wound healing assay and (d) transwell assaywere performed in MDA-MB-231 cells treated with control TNF-120572 OA and OA + TNF120572 Image magnification 100times Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 versus control P lt 005 P lt 001 P lt 0001 versus the OA control

E-cad

N-cad

Vimentin

GAPDH

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

E-cad

Con

trol

TNF-

OA

20

OA

20+T

NF

00

05

10

15

20

N-cad

00

05

10

15

20

Vimentin

00

05

10

15

20

lowastlowast

lowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowast

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)Re

lativ

e Vim

entin

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Figure 8 TNF-120572 reverses the effect of Oroxylin A on EMT formation in MDA-MB-231 cells The protein expressions of E-cadherin N-cadherin and Vimentin in the cells were analyzed by western blotting Data are expressed as mean plusmn SD lowastlowastP lt 001 lowastlowastlowastP lt 0001 versuscontrol P lt 005 P lt 0001 versus the OA control

BioMed Research International 9

Chronic inflammation is a main activator in themetastatic cascade [28] Epithelial-mesenchymal transition(EMT) is a complex process losing cell-cell adhesioncontributing to cancer invasion and metastasis The resultsof the present study indicated that OA inhibited the EMTformation in breast cancer cells by upregulation of E-cadherin levels and downregulation of N-cadherin andVimentin levels EMT programs stimulate the productionof proinflammatory factors in cancer environment whileinflammation is a potent inducement for EMT in tumors[9] What is more NF-120581B activity promotes the collectionof inflammatory cells and secretion of proinflammatorycytokines like IL-1 IL-6 IL-8 and TNF-120572 [29] The presentresults demonstrated that OA markedly suppressed thesecretion of IL-6 IL-8 and TNF-120572 protein in MDA-MB-231cellsThese findings indicate OAhas a repressive influenceon EMT and inflammation progress in breast cancercells

TNF-120572 has been proved to have pleiotropic effects oncells death growth or differentiation under different con-centrations and context [30 31] TNF has anticancer andanti-infection effects in high concentrations because of itscytotoxic effects on the tumor vessel [32] However it isshown that TNF can promote the proliferation andmigrationof normal epithelial cell and breast cancer cells both atlow doses [33 34] One study demonstrated that TNF-120572secreted by tumor cells stimulated the growth of malignantmammary epithelial cells which contributed to mammarytumorigenesis in neuerbB2 transgenic mice [18] Anotherstudy showed that TNF-120572 can promote transendothelialmigration of breast cancer via upregulation of LOX [35]In the present study we found OA-suppressed cell prolif-eration migration invasion and EMT phenotype forma-tion were promoted by TNF-120572 leading to aggravation ofbreast cancer process which is consistent with previousreports On these bases we hypothesized that the blockadeof TNF-120572 may enhance the antitumor effects of OA forbreast cancer patients and this speculation warrants furtherinvestigation

5 Conclusion

In summary the present study showed that OA couldsuppress cell proliferation migration invasion and EMTformation and downregulate the expressions of inflammatorycytokines Furthermore OA exerted protective effect onbreast cancer by inhibiting NF-120581B signaling pathway Theresults provided a theoretical foundation for Oroxylin A asa potential drug for the treatment of breast cancer

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

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

Acknowledgments

This work was supported by Funding from Science andTechnology Development of Universities and Colleges ofTianjin (No 20130120)

References

[1] A Ugolkov I Gaisina J Zhang et al ldquoGSK-3 inhibitionovercomes chemoresistance in human breast cancerrdquo CancerLetters vol 380 no 2 pp 384ndash392 2016

[2] C A Santa-Maria and W J Gradishar ldquoChanging treatmentparadigms in metastatic breast cancerrdquo JAMA Oncology vol 1no 4 pp 528ndash534 2015

[3] J OrsquoShaughnessy ldquoExtending survival with chemotherapy inmetastatic breast cancerrdquo e Oncologist vol 10 no 3 pp 20ndash29 2005

[4] A Bosch P Eroles R Zaragoza et al ldquoTriple-negative breastcancer molecular features pathogenesis treatment and currentlines of researchrdquo Cancer Treatment Reviews vol 36 no 3 pp206ndash215 2010

[5] J Yao R Hu J Sun et al ldquoOroxylin a prevents inflammation-related tumor through down-regulation of inflammatory geneexpression by inhibiting NF-120581B signalingrdquo Molecular Carcino-genesis vol 53 no 2 pp 145ndash158 2014

[6] A Mantovani P Allavena A Sica and F Balkwill ldquoCancer-related inflammationrdquo Nature vol 454 no 7203 pp 436ndash4442008

[7] M Karin ldquoNF-kappaB as a critical link between inflammationand cancerrdquo Cold Spring Harbor Perspectives in Biology vol 1no 5 Article ID a000141 2009

[8] X Cui D Shen C Kong et al ldquoNF-120581B suppresses apoptosisand promotes bladder cancer cell proliferation by upregulatingsurvivin expression in vitro and in vivordquo Scientific Reports vol7 no 1 Article ID 40723 2017

[9] M Suarez-Carmona J Lesage D Cataldo and C Gilles ldquoEMTand inflammation inseparable actors of cancer progressionrdquoMolecular Oncology vol 11 no 7 pp 805ndash823 2017

[10] H Kiefel S Bondong N Erbe-Hoffmann et al ldquoL1CAM-integrin interaction induces constitutive NF-120581B activation inpancreatic adenocarcinoma cells by enhancing IL-1Β expres-sionrdquo Oncogene vol 29 no 34 pp 4766ndash4778 2010

[11] R A Stoltz N G Abraham and M Laniado-SchwartzmanldquoThe role of NF-kappaB in the angiogenic response of coro-nary microvessel endothelial cellsrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 93 no7 pp 2832ndash2837 1996

[12] Z Lu N Lu C Li et al ldquoOroxylin A inhibits matrixmetalloproteinase-29 expression and activation by up-regulating tissue inhibitor of metalloproteinase-2 andsuppressing the ERK12 signaling pathwayrdquo ToxicologyLetters vol 209 no 3 pp 211ndash220 2012

[13] L Lu Q Guo and L Zhao ldquoOverview of Oroxylin A apromising flavonoid compoundrdquo Phytotherapy Research vol30 no 11 pp 1765ndash1774 2016

[14] Y Hu Y Yang Q-D You et al ldquoOroxylin A induced apop-tosis of human hepatocellular carcinoma cell line HepG2 wasinvolved in its antitumor activityrdquo Biochemical and BiophysicalResearch Communications vol 351 no 2 pp 521ndash527 2006

[15] Y Sun N Lu Y Ling et al ldquoOroxylin A suppressesinvasion through down-regulating the expression of matrix

10 BioMed Research International

metalloproteinase-29 in MDA-MB-435 human breast cancercellsrdquo European Journal of Pharmacology vol 603 no 1-3 pp22ndash28 2009

[16] T Tseng M Chen M Tsai et al ldquoOroxylin-A rescues LPS-induced acute lung injury via regulation of NF-120581B signalingpathway in rodentsrdquo PLoS ONE vol 7 no 10 Article ID e474032012

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[18] M A Warren S F Shoemaker D J Shealy W Bshara andM M Ip ldquoTumor necrosis factor deficiency inhibits mam-mary tumorigenesis and a tumor necrosis factor neutralizingantibody decreases mammary tumor growth in neuerbB2transgenic micerdquo Molecular Cancer erapeutics vol 8 no 9pp 2655ndash2663 2009

[19] T Ni Z He Y Dai J Yao Q Guo and L Wei ldquoOrox-ylin A suppresses the development and growth of colorectalcancer through reprogram of HIF1120572-modulated fatty acidmetabolismrdquo Cell Death amp Disease vol 8 no 6 Article IDe2865 2017

[20] L Wei Y Zhou C Qiao et al ldquoOroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promotingSIRT3-mediated SOD2 transcription and HIF1120572 destabiliza-tionrdquo Cell Death amp Disease vol 6 no 4 Article ID e1714 2015

[21] Y Gao N Lu Y Ling et al ldquoOroxylin A inhibits angiogenesisthrough blocking vascular endothelial growth factor-inducedKDRFlk-1 phosphorylationrdquo Journal of Cancer Research andClinical Oncology vol 136 no 5 pp 667ndash675 2010

[22] R Mu Q Qi H Gu et al ldquoInvolvement of p53 in oroxylinA-induced apoptosis in cancer cellsrdquoMolecular Carcinogenesisvol 48 no 12 pp 1159ndash1169 2009

[23] H Ohshima H Tazawa B S Sylla and T Sawa ldquoPreventionof human cancer by modulation of chronic inflammatoryprocessesrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 591 no 1-2 pp 110ndash122 2005

[24] P P Tak and G S Firestein ldquoNF-kappaB a key role ininflammatory diseasesrdquoe Journal of Clinical Investigation vol107 pp 7ndash11 2001

[25] E Heiss C Herhaus K Klimo H Bartsch and C GerhauserldquoNuclear factor 120581B is a molecular target for sulforaphane-mediated anti-inflammatory mechanismsrdquo e Journal of Bio-logical Chemistry vol 276 no 34 pp 32008ndash32015 2001

[26] A J Dannenberg N K Altorki J O Boyle et al ldquoCyclo-oxygenase 2 a pharmacological target for the prevention ofcancerrdquoe Lancet Oncology vol 2 no 9 pp 544ndash551 2001

[27] Y-C Chen L-L Yang and T J-F Lee ldquoOroxylin A inhi-bition of lipopolysaccharide-induced iNOS and COX-2 geneexpression via suppression of nuclear factor-120581B activationrdquoBiochemical Pharmacology vol 59 no 11 pp 1445ndash1457 2000

[28] D-F Lee andM-CHung ldquoAdvances in targeting IKK and IKK-related kinases for cancer therapyrdquoClinical Cancer Research vol14 no 18 pp 5656ndash5662 2008

[29] Z Han D L Boyle A M Manning and G S Firestein ldquoAP-1and NF-kappaB regulation in rheumatoid arthritis and murinecollagen-induced arthritisrdquo Autoimmunity vol 28 no 4 pp197ndash208 1998

[30] D Liu X Wang and Z Chen ldquoTumor Necrosis Factor-alphaa regulator and therapeutic agent on breast cancerrdquo CurrentPharmaceutical Biotechnology vol 17 no 6 pp 486ndash494 2016

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

[32] R van Horssen T L M ten Hagen and A M M EggermontldquoTNF-120572 in cancer treatment molecular insights antitumoreffects and clinical utilityrdquoeOncologist vol 11 no 4 pp 397ndash408 2006

[33] M M Ip S F Shoemaker and K M Darcy ldquoRegulation ofrat mammary epithelial cell proliferation and differentiation bytumor necrosis factor-alphardquo Endocrinology vol 130 no 5 pp2833ndash2844 1992

[34] J Zhang M A Warren S F Shoemaker and M MIp ldquoNF120581B1p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells impli-cations for NF120581B2p52 and RelBrdquo Endocrinology vol 148 no 1pp 268ndash278 2007

[35] M Liang P Zhang and J Fu ldquoUp-regulation of LOX-1expression by TNF-120572 promotes trans-endothelial migration ofMDA-MB-231 breast cancer cellsrdquo Cancer Letters vol 258 no1 pp 31ndash37 2007

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

6 BioMed Research International

0

1

2

3

4Re

lativ

e E-c

ad m

RNA

expr

essio

n

Con

trol

OA

20

lowastlowastlowast

(a)

00

05

10

15

Relat

ive N

-cad

mRN

A ex

pres

sion

Con

trol

OA

20

lowastlowast

(b)

00

05

10

15

Relat

ive V

imen

tin m

RNA

expr

essio

n

Con

trol

OA

20

lowastlowast

(c)

E-cad

N-cad

Vimentin

GAPDH

Control OA20

E-cad

00

05

10

15

20

N-cad

00

05

10

15Vimentin

00

05

10

15C

ontro

l

OA

20

Con

trol

OA

20

Con

trol

OA

20

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive V

imen

tin ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

lowastlowast

lowastlowastlowast

(d)

Figure 4 Effect of Oroxylin A on EMT formation in MDA-MB-231 cells Cells were treated with 20 120583M Oroxylin A for 24 h E-cadherin(a) N-cadherin (b) and Vimentin (c) mRNAs were measured with qRT-PCR (d) The expression of E-cadherin N-cadherin and Vimentinproteins in the cells was analyzed by western blotting using specific antibodies Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt0001 versus control

15

Relat

ive e

xpre

ssio

n of

IL-6

00

05

10

Con

trol

OA

20

lowastlowast

(a)

Relat

ive e

xpre

ssio

n of

IL-8 15

00

05

10

Con

trol

OA

20

lowastlowast

(b)

Relat

ive e

xpre

ssio

n of

TN

F- 15

00

05

10

Con

trol

OA

20

lowast

(c)

Figure 5 Effect of Oroxylin A on IL-6 IL-8 and TNF-120572 levels in MDA-MB-231 cells The expression of IL-6 IL-8 and TNF-120572 was detectedby ELISA assay from the supernatants of cells cultured with 20 120583MOroxylin A for 24 h Data are expressed as mean plusmn SD lowastP lt 005 lowastlowastP lt001 versus control

BioMed Research International 7

p-p65

p65

GAPDH

p-p65

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive p

-p65

expr

essio

nin

diff

eren

t gro

ups (

fold

)

(a)

p-IB

IB

GAPDH

p-IB

00

05

10

15

OA20Control

Con

trol

OA

20

lowastlowast

Rela

tive p

-I

B ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(b)

GAPDH

COX-2

COX-2

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive C

OX-

2 ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(c)

Figure 6 Effect of Oroxylin A on NF-120581B activation and the expression of COX-2 in MDA-MB-231 cells Phosphorylation of p65 (a) and I120581B(b) and COX-2 (c) expression were analyzed by western blot GAPDH was used as an internal control Data are expressed as mean plusmn SDlowastlowastP lt 001 versus control

Control

B2-HPerCP-Vio700-H

Cou

nt

OA20

B2-HPerCP-Vio700-H

Cou

nt

TNF-

B2-HPerCP-Vio700-H B2-HPerCP-Vio700-H

Cou

nt

OA20+TNF

Cou

nt

50

40

30

20

10

00 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k

50

40

30

20

10

0

50

40

30

20

10

0

40

30

20

10

00 50k 100k 150k 200k 250k

lt2N 7582N 640 [ 60800 cv 663]S 1734N 113 [ 116608 cv 930]Ratio 192gt4N 0

lt2N 762N 507 [ 60800 cv 619]S 2084N 191 [ 115776 cv 137]Ratio 190gt4N 057

lt2N 1532N 697 [ 50561 cv 585]S 1654N 122 [ 95297 cv 107]Ratio 188gt4N 030

lt2N 7692N 593 [ 60800 cv 648]S 1744N 185 [ 115264 cv 127]Ratio 190gt4N 100

(a)

G0G1 S G20

20

40

60

80

ControlTNF-

OA 20

Cell

cycle

dist

ribut

ion

()

OA 20+TNF

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Control OA20TNF- OA20+TNF

0h

24h

(c)

Control OA20 OA20+TNFTNF-

(d)

Figure 7 Continued

8 BioMed Research International

05

10

15

00

Con

trol

OA

20

TNF-

a

OA

20+

TNFa

Relat

ive c

ell m

igra

tion

rate

lowastlowastlowast

(e)

00

10

15

05

Con

trol

OA

20

TNF-

OA

20+

TNF

Relat

ive c

ell i

nvas

ive t

ate

lowastlowast

lowastlowastlowast

(f)

Figure 7 TNF-120572 reverses the function of OA in breast cancer cells (a-b) Flow cytometry (c) wound healing assay and (d) transwell assaywere performed in MDA-MB-231 cells treated with control TNF-120572 OA and OA + TNF120572 Image magnification 100times Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 versus control P lt 005 P lt 001 P lt 0001 versus the OA control

E-cad

N-cad

Vimentin

GAPDH

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

E-cad

Con

trol

TNF-

OA

20

OA

20+T

NF

00

05

10

15

20

N-cad

00

05

10

15

20

Vimentin

00

05

10

15

20

lowastlowast

lowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowast

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)Re

lativ

e Vim

entin

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Figure 8 TNF-120572 reverses the effect of Oroxylin A on EMT formation in MDA-MB-231 cells The protein expressions of E-cadherin N-cadherin and Vimentin in the cells were analyzed by western blotting Data are expressed as mean plusmn SD lowastlowastP lt 001 lowastlowastlowastP lt 0001 versuscontrol P lt 005 P lt 0001 versus the OA control

BioMed Research International 9

Chronic inflammation is a main activator in themetastatic cascade [28] Epithelial-mesenchymal transition(EMT) is a complex process losing cell-cell adhesioncontributing to cancer invasion and metastasis The resultsof the present study indicated that OA inhibited the EMTformation in breast cancer cells by upregulation of E-cadherin levels and downregulation of N-cadherin andVimentin levels EMT programs stimulate the productionof proinflammatory factors in cancer environment whileinflammation is a potent inducement for EMT in tumors[9] What is more NF-120581B activity promotes the collectionof inflammatory cells and secretion of proinflammatorycytokines like IL-1 IL-6 IL-8 and TNF-120572 [29] The presentresults demonstrated that OA markedly suppressed thesecretion of IL-6 IL-8 and TNF-120572 protein in MDA-MB-231cellsThese findings indicate OAhas a repressive influenceon EMT and inflammation progress in breast cancercells

TNF-120572 has been proved to have pleiotropic effects oncells death growth or differentiation under different con-centrations and context [30 31] TNF has anticancer andanti-infection effects in high concentrations because of itscytotoxic effects on the tumor vessel [32] However it isshown that TNF can promote the proliferation andmigrationof normal epithelial cell and breast cancer cells both atlow doses [33 34] One study demonstrated that TNF-120572secreted by tumor cells stimulated the growth of malignantmammary epithelial cells which contributed to mammarytumorigenesis in neuerbB2 transgenic mice [18] Anotherstudy showed that TNF-120572 can promote transendothelialmigration of breast cancer via upregulation of LOX [35]In the present study we found OA-suppressed cell prolif-eration migration invasion and EMT phenotype forma-tion were promoted by TNF-120572 leading to aggravation ofbreast cancer process which is consistent with previousreports On these bases we hypothesized that the blockadeof TNF-120572 may enhance the antitumor effects of OA forbreast cancer patients and this speculation warrants furtherinvestigation

5 Conclusion

In summary the present study showed that OA couldsuppress cell proliferation migration invasion and EMTformation and downregulate the expressions of inflammatorycytokines Furthermore OA exerted protective effect onbreast cancer by inhibiting NF-120581B signaling pathway Theresults provided a theoretical foundation for Oroxylin A asa potential drug for the treatment of breast cancer

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

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

Acknowledgments

This work was supported by Funding from Science andTechnology Development of Universities and Colleges ofTianjin (No 20130120)

References

[1] A Ugolkov I Gaisina J Zhang et al ldquoGSK-3 inhibitionovercomes chemoresistance in human breast cancerrdquo CancerLetters vol 380 no 2 pp 384ndash392 2016

[2] C A Santa-Maria and W J Gradishar ldquoChanging treatmentparadigms in metastatic breast cancerrdquo JAMA Oncology vol 1no 4 pp 528ndash534 2015

[3] J OrsquoShaughnessy ldquoExtending survival with chemotherapy inmetastatic breast cancerrdquo e Oncologist vol 10 no 3 pp 20ndash29 2005

[4] A Bosch P Eroles R Zaragoza et al ldquoTriple-negative breastcancer molecular features pathogenesis treatment and currentlines of researchrdquo Cancer Treatment Reviews vol 36 no 3 pp206ndash215 2010

[5] J Yao R Hu J Sun et al ldquoOroxylin a prevents inflammation-related tumor through down-regulation of inflammatory geneexpression by inhibiting NF-120581B signalingrdquo Molecular Carcino-genesis vol 53 no 2 pp 145ndash158 2014

[6] A Mantovani P Allavena A Sica and F Balkwill ldquoCancer-related inflammationrdquo Nature vol 454 no 7203 pp 436ndash4442008

[7] M Karin ldquoNF-kappaB as a critical link between inflammationand cancerrdquo Cold Spring Harbor Perspectives in Biology vol 1no 5 Article ID a000141 2009

[8] X Cui D Shen C Kong et al ldquoNF-120581B suppresses apoptosisand promotes bladder cancer cell proliferation by upregulatingsurvivin expression in vitro and in vivordquo Scientific Reports vol7 no 1 Article ID 40723 2017

[9] M Suarez-Carmona J Lesage D Cataldo and C Gilles ldquoEMTand inflammation inseparable actors of cancer progressionrdquoMolecular Oncology vol 11 no 7 pp 805ndash823 2017

[10] H Kiefel S Bondong N Erbe-Hoffmann et al ldquoL1CAM-integrin interaction induces constitutive NF-120581B activation inpancreatic adenocarcinoma cells by enhancing IL-1Β expres-sionrdquo Oncogene vol 29 no 34 pp 4766ndash4778 2010

[11] R A Stoltz N G Abraham and M Laniado-SchwartzmanldquoThe role of NF-kappaB in the angiogenic response of coro-nary microvessel endothelial cellsrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 93 no7 pp 2832ndash2837 1996

[12] Z Lu N Lu C Li et al ldquoOroxylin A inhibits matrixmetalloproteinase-29 expression and activation by up-regulating tissue inhibitor of metalloproteinase-2 andsuppressing the ERK12 signaling pathwayrdquo ToxicologyLetters vol 209 no 3 pp 211ndash220 2012

[13] L Lu Q Guo and L Zhao ldquoOverview of Oroxylin A apromising flavonoid compoundrdquo Phytotherapy Research vol30 no 11 pp 1765ndash1774 2016

[14] Y Hu Y Yang Q-D You et al ldquoOroxylin A induced apop-tosis of human hepatocellular carcinoma cell line HepG2 wasinvolved in its antitumor activityrdquo Biochemical and BiophysicalResearch Communications vol 351 no 2 pp 521ndash527 2006

[15] Y Sun N Lu Y Ling et al ldquoOroxylin A suppressesinvasion through down-regulating the expression of matrix

10 BioMed Research International

metalloproteinase-29 in MDA-MB-435 human breast cancercellsrdquo European Journal of Pharmacology vol 603 no 1-3 pp22ndash28 2009

[16] T Tseng M Chen M Tsai et al ldquoOroxylin-A rescues LPS-induced acute lung injury via regulation of NF-120581B signalingpathway in rodentsrdquo PLoS ONE vol 7 no 10 Article ID e474032012

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[18] M A Warren S F Shoemaker D J Shealy W Bshara andM M Ip ldquoTumor necrosis factor deficiency inhibits mam-mary tumorigenesis and a tumor necrosis factor neutralizingantibody decreases mammary tumor growth in neuerbB2transgenic micerdquo Molecular Cancer erapeutics vol 8 no 9pp 2655ndash2663 2009

[19] T Ni Z He Y Dai J Yao Q Guo and L Wei ldquoOrox-ylin A suppresses the development and growth of colorectalcancer through reprogram of HIF1120572-modulated fatty acidmetabolismrdquo Cell Death amp Disease vol 8 no 6 Article IDe2865 2017

[20] L Wei Y Zhou C Qiao et al ldquoOroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promotingSIRT3-mediated SOD2 transcription and HIF1120572 destabiliza-tionrdquo Cell Death amp Disease vol 6 no 4 Article ID e1714 2015

[21] Y Gao N Lu Y Ling et al ldquoOroxylin A inhibits angiogenesisthrough blocking vascular endothelial growth factor-inducedKDRFlk-1 phosphorylationrdquo Journal of Cancer Research andClinical Oncology vol 136 no 5 pp 667ndash675 2010

[22] R Mu Q Qi H Gu et al ldquoInvolvement of p53 in oroxylinA-induced apoptosis in cancer cellsrdquoMolecular Carcinogenesisvol 48 no 12 pp 1159ndash1169 2009

[23] H Ohshima H Tazawa B S Sylla and T Sawa ldquoPreventionof human cancer by modulation of chronic inflammatoryprocessesrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 591 no 1-2 pp 110ndash122 2005

[24] P P Tak and G S Firestein ldquoNF-kappaB a key role ininflammatory diseasesrdquoe Journal of Clinical Investigation vol107 pp 7ndash11 2001

[25] E Heiss C Herhaus K Klimo H Bartsch and C GerhauserldquoNuclear factor 120581B is a molecular target for sulforaphane-mediated anti-inflammatory mechanismsrdquo e Journal of Bio-logical Chemistry vol 276 no 34 pp 32008ndash32015 2001

[26] A J Dannenberg N K Altorki J O Boyle et al ldquoCyclo-oxygenase 2 a pharmacological target for the prevention ofcancerrdquoe Lancet Oncology vol 2 no 9 pp 544ndash551 2001

[27] Y-C Chen L-L Yang and T J-F Lee ldquoOroxylin A inhi-bition of lipopolysaccharide-induced iNOS and COX-2 geneexpression via suppression of nuclear factor-120581B activationrdquoBiochemical Pharmacology vol 59 no 11 pp 1445ndash1457 2000

[28] D-F Lee andM-CHung ldquoAdvances in targeting IKK and IKK-related kinases for cancer therapyrdquoClinical Cancer Research vol14 no 18 pp 5656ndash5662 2008

[29] Z Han D L Boyle A M Manning and G S Firestein ldquoAP-1and NF-kappaB regulation in rheumatoid arthritis and murinecollagen-induced arthritisrdquo Autoimmunity vol 28 no 4 pp197ndash208 1998

[30] D Liu X Wang and Z Chen ldquoTumor Necrosis Factor-alphaa regulator and therapeutic agent on breast cancerrdquo CurrentPharmaceutical Biotechnology vol 17 no 6 pp 486ndash494 2016

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

[32] R van Horssen T L M ten Hagen and A M M EggermontldquoTNF-120572 in cancer treatment molecular insights antitumoreffects and clinical utilityrdquoeOncologist vol 11 no 4 pp 397ndash408 2006

[33] M M Ip S F Shoemaker and K M Darcy ldquoRegulation ofrat mammary epithelial cell proliferation and differentiation bytumor necrosis factor-alphardquo Endocrinology vol 130 no 5 pp2833ndash2844 1992

[34] J Zhang M A Warren S F Shoemaker and M MIp ldquoNF120581B1p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells impli-cations for NF120581B2p52 and RelBrdquo Endocrinology vol 148 no 1pp 268ndash278 2007

[35] M Liang P Zhang and J Fu ldquoUp-regulation of LOX-1expression by TNF-120572 promotes trans-endothelial migration ofMDA-MB-231 breast cancer cellsrdquo Cancer Letters vol 258 no1 pp 31ndash37 2007

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

BioMed Research International 7

p-p65

p65

GAPDH

p-p65

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive p

-p65

expr

essio

nin

diff

eren

t gro

ups (

fold

)

(a)

p-IB

IB

GAPDH

p-IB

00

05

10

15

OA20Control

Con

trol

OA

20

lowastlowast

Rela

tive p

-I

B ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(b)

GAPDH

COX-2

COX-2

00

05

10

15

Control OA20

Con

trol

OA

20

lowastlowast

Relat

ive C

OX-

2 ex

pres

sion

in d

iffer

ent g

roup

s (fo

ld)

(c)

Figure 6 Effect of Oroxylin A on NF-120581B activation and the expression of COX-2 in MDA-MB-231 cells Phosphorylation of p65 (a) and I120581B(b) and COX-2 (c) expression were analyzed by western blot GAPDH was used as an internal control Data are expressed as mean plusmn SDlowastlowastP lt 001 versus control

Control

B2-HPerCP-Vio700-H

Cou

nt

OA20

B2-HPerCP-Vio700-H

Cou

nt

TNF-

B2-HPerCP-Vio700-H B2-HPerCP-Vio700-H

Cou

nt

OA20+TNF

Cou

nt

50

40

30

20

10

00 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k 0 50k 100k 150k 200k 250k

50

40

30

20

10

0

50

40

30

20

10

0

40

30

20

10

00 50k 100k 150k 200k 250k

lt2N 7582N 640 [ 60800 cv 663]S 1734N 113 [ 116608 cv 930]Ratio 192gt4N 0

lt2N 762N 507 [ 60800 cv 619]S 2084N 191 [ 115776 cv 137]Ratio 190gt4N 057

lt2N 1532N 697 [ 50561 cv 585]S 1654N 122 [ 95297 cv 107]Ratio 188gt4N 030

lt2N 7692N 593 [ 60800 cv 648]S 1744N 185 [ 115264 cv 127]Ratio 190gt4N 100

(a)

G0G1 S G20

20

40

60

80

ControlTNF-

OA 20

Cell

cycle

dist

ribut

ion

()

OA 20+TNF

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Control OA20TNF- OA20+TNF

0h

24h

(c)

Control OA20 OA20+TNFTNF-

(d)

Figure 7 Continued

8 BioMed Research International

05

10

15

00

Con

trol

OA

20

TNF-

a

OA

20+

TNFa

Relat

ive c

ell m

igra

tion

rate

lowastlowastlowast

(e)

00

10

15

05

Con

trol

OA

20

TNF-

OA

20+

TNF

Relat

ive c

ell i

nvas

ive t

ate

lowastlowast

lowastlowastlowast

(f)

Figure 7 TNF-120572 reverses the function of OA in breast cancer cells (a-b) Flow cytometry (c) wound healing assay and (d) transwell assaywere performed in MDA-MB-231 cells treated with control TNF-120572 OA and OA + TNF120572 Image magnification 100times Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 versus control P lt 005 P lt 001 P lt 0001 versus the OA control

E-cad

N-cad

Vimentin

GAPDH

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

E-cad

Con

trol

TNF-

OA

20

OA

20+T

NF

00

05

10

15

20

N-cad

00

05

10

15

20

Vimentin

00

05

10

15

20

lowastlowast

lowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowast

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)Re

lativ

e Vim

entin

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Figure 8 TNF-120572 reverses the effect of Oroxylin A on EMT formation in MDA-MB-231 cells The protein expressions of E-cadherin N-cadherin and Vimentin in the cells were analyzed by western blotting Data are expressed as mean plusmn SD lowastlowastP lt 001 lowastlowastlowastP lt 0001 versuscontrol P lt 005 P lt 0001 versus the OA control

BioMed Research International 9

Chronic inflammation is a main activator in themetastatic cascade [28] Epithelial-mesenchymal transition(EMT) is a complex process losing cell-cell adhesioncontributing to cancer invasion and metastasis The resultsof the present study indicated that OA inhibited the EMTformation in breast cancer cells by upregulation of E-cadherin levels and downregulation of N-cadherin andVimentin levels EMT programs stimulate the productionof proinflammatory factors in cancer environment whileinflammation is a potent inducement for EMT in tumors[9] What is more NF-120581B activity promotes the collectionof inflammatory cells and secretion of proinflammatorycytokines like IL-1 IL-6 IL-8 and TNF-120572 [29] The presentresults demonstrated that OA markedly suppressed thesecretion of IL-6 IL-8 and TNF-120572 protein in MDA-MB-231cellsThese findings indicate OAhas a repressive influenceon EMT and inflammation progress in breast cancercells

TNF-120572 has been proved to have pleiotropic effects oncells death growth or differentiation under different con-centrations and context [30 31] TNF has anticancer andanti-infection effects in high concentrations because of itscytotoxic effects on the tumor vessel [32] However it isshown that TNF can promote the proliferation andmigrationof normal epithelial cell and breast cancer cells both atlow doses [33 34] One study demonstrated that TNF-120572secreted by tumor cells stimulated the growth of malignantmammary epithelial cells which contributed to mammarytumorigenesis in neuerbB2 transgenic mice [18] Anotherstudy showed that TNF-120572 can promote transendothelialmigration of breast cancer via upregulation of LOX [35]In the present study we found OA-suppressed cell prolif-eration migration invasion and EMT phenotype forma-tion were promoted by TNF-120572 leading to aggravation ofbreast cancer process which is consistent with previousreports On these bases we hypothesized that the blockadeof TNF-120572 may enhance the antitumor effects of OA forbreast cancer patients and this speculation warrants furtherinvestigation

5 Conclusion

In summary the present study showed that OA couldsuppress cell proliferation migration invasion and EMTformation and downregulate the expressions of inflammatorycytokines Furthermore OA exerted protective effect onbreast cancer by inhibiting NF-120581B signaling pathway Theresults provided a theoretical foundation for Oroxylin A asa potential drug for the treatment of breast cancer

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

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

Acknowledgments

This work was supported by Funding from Science andTechnology Development of Universities and Colleges ofTianjin (No 20130120)

References

[1] A Ugolkov I Gaisina J Zhang et al ldquoGSK-3 inhibitionovercomes chemoresistance in human breast cancerrdquo CancerLetters vol 380 no 2 pp 384ndash392 2016

[2] C A Santa-Maria and W J Gradishar ldquoChanging treatmentparadigms in metastatic breast cancerrdquo JAMA Oncology vol 1no 4 pp 528ndash534 2015

[3] J OrsquoShaughnessy ldquoExtending survival with chemotherapy inmetastatic breast cancerrdquo e Oncologist vol 10 no 3 pp 20ndash29 2005

[4] A Bosch P Eroles R Zaragoza et al ldquoTriple-negative breastcancer molecular features pathogenesis treatment and currentlines of researchrdquo Cancer Treatment Reviews vol 36 no 3 pp206ndash215 2010

[5] J Yao R Hu J Sun et al ldquoOroxylin a prevents inflammation-related tumor through down-regulation of inflammatory geneexpression by inhibiting NF-120581B signalingrdquo Molecular Carcino-genesis vol 53 no 2 pp 145ndash158 2014

[6] A Mantovani P Allavena A Sica and F Balkwill ldquoCancer-related inflammationrdquo Nature vol 454 no 7203 pp 436ndash4442008

[7] M Karin ldquoNF-kappaB as a critical link between inflammationand cancerrdquo Cold Spring Harbor Perspectives in Biology vol 1no 5 Article ID a000141 2009

[8] X Cui D Shen C Kong et al ldquoNF-120581B suppresses apoptosisand promotes bladder cancer cell proliferation by upregulatingsurvivin expression in vitro and in vivordquo Scientific Reports vol7 no 1 Article ID 40723 2017

[9] M Suarez-Carmona J Lesage D Cataldo and C Gilles ldquoEMTand inflammation inseparable actors of cancer progressionrdquoMolecular Oncology vol 11 no 7 pp 805ndash823 2017

[10] H Kiefel S Bondong N Erbe-Hoffmann et al ldquoL1CAM-integrin interaction induces constitutive NF-120581B activation inpancreatic adenocarcinoma cells by enhancing IL-1Β expres-sionrdquo Oncogene vol 29 no 34 pp 4766ndash4778 2010

[11] R A Stoltz N G Abraham and M Laniado-SchwartzmanldquoThe role of NF-kappaB in the angiogenic response of coro-nary microvessel endothelial cellsrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 93 no7 pp 2832ndash2837 1996

[12] Z Lu N Lu C Li et al ldquoOroxylin A inhibits matrixmetalloproteinase-29 expression and activation by up-regulating tissue inhibitor of metalloproteinase-2 andsuppressing the ERK12 signaling pathwayrdquo ToxicologyLetters vol 209 no 3 pp 211ndash220 2012

[13] L Lu Q Guo and L Zhao ldquoOverview of Oroxylin A apromising flavonoid compoundrdquo Phytotherapy Research vol30 no 11 pp 1765ndash1774 2016

[14] Y Hu Y Yang Q-D You et al ldquoOroxylin A induced apop-tosis of human hepatocellular carcinoma cell line HepG2 wasinvolved in its antitumor activityrdquo Biochemical and BiophysicalResearch Communications vol 351 no 2 pp 521ndash527 2006

[15] Y Sun N Lu Y Ling et al ldquoOroxylin A suppressesinvasion through down-regulating the expression of matrix

10 BioMed Research International

metalloproteinase-29 in MDA-MB-435 human breast cancercellsrdquo European Journal of Pharmacology vol 603 no 1-3 pp22ndash28 2009

[16] T Tseng M Chen M Tsai et al ldquoOroxylin-A rescues LPS-induced acute lung injury via regulation of NF-120581B signalingpathway in rodentsrdquo PLoS ONE vol 7 no 10 Article ID e474032012

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[18] M A Warren S F Shoemaker D J Shealy W Bshara andM M Ip ldquoTumor necrosis factor deficiency inhibits mam-mary tumorigenesis and a tumor necrosis factor neutralizingantibody decreases mammary tumor growth in neuerbB2transgenic micerdquo Molecular Cancer erapeutics vol 8 no 9pp 2655ndash2663 2009

[19] T Ni Z He Y Dai J Yao Q Guo and L Wei ldquoOrox-ylin A suppresses the development and growth of colorectalcancer through reprogram of HIF1120572-modulated fatty acidmetabolismrdquo Cell Death amp Disease vol 8 no 6 Article IDe2865 2017

[20] L Wei Y Zhou C Qiao et al ldquoOroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promotingSIRT3-mediated SOD2 transcription and HIF1120572 destabiliza-tionrdquo Cell Death amp Disease vol 6 no 4 Article ID e1714 2015

[21] Y Gao N Lu Y Ling et al ldquoOroxylin A inhibits angiogenesisthrough blocking vascular endothelial growth factor-inducedKDRFlk-1 phosphorylationrdquo Journal of Cancer Research andClinical Oncology vol 136 no 5 pp 667ndash675 2010

[22] R Mu Q Qi H Gu et al ldquoInvolvement of p53 in oroxylinA-induced apoptosis in cancer cellsrdquoMolecular Carcinogenesisvol 48 no 12 pp 1159ndash1169 2009

[23] H Ohshima H Tazawa B S Sylla and T Sawa ldquoPreventionof human cancer by modulation of chronic inflammatoryprocessesrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 591 no 1-2 pp 110ndash122 2005

[24] P P Tak and G S Firestein ldquoNF-kappaB a key role ininflammatory diseasesrdquoe Journal of Clinical Investigation vol107 pp 7ndash11 2001

[25] E Heiss C Herhaus K Klimo H Bartsch and C GerhauserldquoNuclear factor 120581B is a molecular target for sulforaphane-mediated anti-inflammatory mechanismsrdquo e Journal of Bio-logical Chemistry vol 276 no 34 pp 32008ndash32015 2001

[26] A J Dannenberg N K Altorki J O Boyle et al ldquoCyclo-oxygenase 2 a pharmacological target for the prevention ofcancerrdquoe Lancet Oncology vol 2 no 9 pp 544ndash551 2001

[27] Y-C Chen L-L Yang and T J-F Lee ldquoOroxylin A inhi-bition of lipopolysaccharide-induced iNOS and COX-2 geneexpression via suppression of nuclear factor-120581B activationrdquoBiochemical Pharmacology vol 59 no 11 pp 1445ndash1457 2000

[28] D-F Lee andM-CHung ldquoAdvances in targeting IKK and IKK-related kinases for cancer therapyrdquoClinical Cancer Research vol14 no 18 pp 5656ndash5662 2008

[29] Z Han D L Boyle A M Manning and G S Firestein ldquoAP-1and NF-kappaB regulation in rheumatoid arthritis and murinecollagen-induced arthritisrdquo Autoimmunity vol 28 no 4 pp197ndash208 1998

[30] D Liu X Wang and Z Chen ldquoTumor Necrosis Factor-alphaa regulator and therapeutic agent on breast cancerrdquo CurrentPharmaceutical Biotechnology vol 17 no 6 pp 486ndash494 2016

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

[32] R van Horssen T L M ten Hagen and A M M EggermontldquoTNF-120572 in cancer treatment molecular insights antitumoreffects and clinical utilityrdquoeOncologist vol 11 no 4 pp 397ndash408 2006

[33] M M Ip S F Shoemaker and K M Darcy ldquoRegulation ofrat mammary epithelial cell proliferation and differentiation bytumor necrosis factor-alphardquo Endocrinology vol 130 no 5 pp2833ndash2844 1992

[34] J Zhang M A Warren S F Shoemaker and M MIp ldquoNF120581B1p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells impli-cations for NF120581B2p52 and RelBrdquo Endocrinology vol 148 no 1pp 268ndash278 2007

[35] M Liang P Zhang and J Fu ldquoUp-regulation of LOX-1expression by TNF-120572 promotes trans-endothelial migration ofMDA-MB-231 breast cancer cellsrdquo Cancer Letters vol 258 no1 pp 31ndash37 2007

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

8 BioMed Research International

05

10

15

00

Con

trol

OA

20

TNF-

a

OA

20+

TNFa

Relat

ive c

ell m

igra

tion

rate

lowastlowastlowast

(e)

00

10

15

05

Con

trol

OA

20

TNF-

OA

20+

TNF

Relat

ive c

ell i

nvas

ive t

ate

lowastlowast

lowastlowastlowast

(f)

Figure 7 TNF-120572 reverses the function of OA in breast cancer cells (a-b) Flow cytometry (c) wound healing assay and (d) transwell assaywere performed in MDA-MB-231 cells treated with control TNF-120572 OA and OA + TNF120572 Image magnification 100times Data are expressed asmean plusmn SD lowastP lt 005 lowastlowastP lt 001 lowastlowastlowastP lt 0001 versus control P lt 005 P lt 001 P lt 0001 versus the OA control

E-cad

N-cad

Vimentin

GAPDH

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

Con

trol

OA

20

TNF-

OA

20+T

NF

E-cad

Con

trol

TNF-

OA

20

OA

20+T

NF

00

05

10

15

20

N-cad

00

05

10

15

20

Vimentin

00

05

10

15

20

lowastlowast

lowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowast

Relat

ive N

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Relat

ive E

-cad

expr

essio

nin

diff

eren

t gro

ups (

fold

)Re

lativ

e Vim

entin

expr

essio

nin

diff

eren

t gro

ups (

fold

)

Figure 8 TNF-120572 reverses the effect of Oroxylin A on EMT formation in MDA-MB-231 cells The protein expressions of E-cadherin N-cadherin and Vimentin in the cells were analyzed by western blotting Data are expressed as mean plusmn SD lowastlowastP lt 001 lowastlowastlowastP lt 0001 versuscontrol P lt 005 P lt 0001 versus the OA control

BioMed Research International 9

Chronic inflammation is a main activator in themetastatic cascade [28] Epithelial-mesenchymal transition(EMT) is a complex process losing cell-cell adhesioncontributing to cancer invasion and metastasis The resultsof the present study indicated that OA inhibited the EMTformation in breast cancer cells by upregulation of E-cadherin levels and downregulation of N-cadherin andVimentin levels EMT programs stimulate the productionof proinflammatory factors in cancer environment whileinflammation is a potent inducement for EMT in tumors[9] What is more NF-120581B activity promotes the collectionof inflammatory cells and secretion of proinflammatorycytokines like IL-1 IL-6 IL-8 and TNF-120572 [29] The presentresults demonstrated that OA markedly suppressed thesecretion of IL-6 IL-8 and TNF-120572 protein in MDA-MB-231cellsThese findings indicate OAhas a repressive influenceon EMT and inflammation progress in breast cancercells

TNF-120572 has been proved to have pleiotropic effects oncells death growth or differentiation under different con-centrations and context [30 31] TNF has anticancer andanti-infection effects in high concentrations because of itscytotoxic effects on the tumor vessel [32] However it isshown that TNF can promote the proliferation andmigrationof normal epithelial cell and breast cancer cells both atlow doses [33 34] One study demonstrated that TNF-120572secreted by tumor cells stimulated the growth of malignantmammary epithelial cells which contributed to mammarytumorigenesis in neuerbB2 transgenic mice [18] Anotherstudy showed that TNF-120572 can promote transendothelialmigration of breast cancer via upregulation of LOX [35]In the present study we found OA-suppressed cell prolif-eration migration invasion and EMT phenotype forma-tion were promoted by TNF-120572 leading to aggravation ofbreast cancer process which is consistent with previousreports On these bases we hypothesized that the blockadeof TNF-120572 may enhance the antitumor effects of OA forbreast cancer patients and this speculation warrants furtherinvestigation

5 Conclusion

In summary the present study showed that OA couldsuppress cell proliferation migration invasion and EMTformation and downregulate the expressions of inflammatorycytokines Furthermore OA exerted protective effect onbreast cancer by inhibiting NF-120581B signaling pathway Theresults provided a theoretical foundation for Oroxylin A asa potential drug for the treatment of breast cancer

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

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

Acknowledgments

This work was supported by Funding from Science andTechnology Development of Universities and Colleges ofTianjin (No 20130120)

References

[1] A Ugolkov I Gaisina J Zhang et al ldquoGSK-3 inhibitionovercomes chemoresistance in human breast cancerrdquo CancerLetters vol 380 no 2 pp 384ndash392 2016

[2] C A Santa-Maria and W J Gradishar ldquoChanging treatmentparadigms in metastatic breast cancerrdquo JAMA Oncology vol 1no 4 pp 528ndash534 2015

[3] J OrsquoShaughnessy ldquoExtending survival with chemotherapy inmetastatic breast cancerrdquo e Oncologist vol 10 no 3 pp 20ndash29 2005

[4] A Bosch P Eroles R Zaragoza et al ldquoTriple-negative breastcancer molecular features pathogenesis treatment and currentlines of researchrdquo Cancer Treatment Reviews vol 36 no 3 pp206ndash215 2010

[5] J Yao R Hu J Sun et al ldquoOroxylin a prevents inflammation-related tumor through down-regulation of inflammatory geneexpression by inhibiting NF-120581B signalingrdquo Molecular Carcino-genesis vol 53 no 2 pp 145ndash158 2014

[6] A Mantovani P Allavena A Sica and F Balkwill ldquoCancer-related inflammationrdquo Nature vol 454 no 7203 pp 436ndash4442008

[7] M Karin ldquoNF-kappaB as a critical link between inflammationand cancerrdquo Cold Spring Harbor Perspectives in Biology vol 1no 5 Article ID a000141 2009

[8] X Cui D Shen C Kong et al ldquoNF-120581B suppresses apoptosisand promotes bladder cancer cell proliferation by upregulatingsurvivin expression in vitro and in vivordquo Scientific Reports vol7 no 1 Article ID 40723 2017

[9] M Suarez-Carmona J Lesage D Cataldo and C Gilles ldquoEMTand inflammation inseparable actors of cancer progressionrdquoMolecular Oncology vol 11 no 7 pp 805ndash823 2017

[10] H Kiefel S Bondong N Erbe-Hoffmann et al ldquoL1CAM-integrin interaction induces constitutive NF-120581B activation inpancreatic adenocarcinoma cells by enhancing IL-1Β expres-sionrdquo Oncogene vol 29 no 34 pp 4766ndash4778 2010

[11] R A Stoltz N G Abraham and M Laniado-SchwartzmanldquoThe role of NF-kappaB in the angiogenic response of coro-nary microvessel endothelial cellsrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 93 no7 pp 2832ndash2837 1996

[12] Z Lu N Lu C Li et al ldquoOroxylin A inhibits matrixmetalloproteinase-29 expression and activation by up-regulating tissue inhibitor of metalloproteinase-2 andsuppressing the ERK12 signaling pathwayrdquo ToxicologyLetters vol 209 no 3 pp 211ndash220 2012

[13] L Lu Q Guo and L Zhao ldquoOverview of Oroxylin A apromising flavonoid compoundrdquo Phytotherapy Research vol30 no 11 pp 1765ndash1774 2016

[14] Y Hu Y Yang Q-D You et al ldquoOroxylin A induced apop-tosis of human hepatocellular carcinoma cell line HepG2 wasinvolved in its antitumor activityrdquo Biochemical and BiophysicalResearch Communications vol 351 no 2 pp 521ndash527 2006

[15] Y Sun N Lu Y Ling et al ldquoOroxylin A suppressesinvasion through down-regulating the expression of matrix

10 BioMed Research International

metalloproteinase-29 in MDA-MB-435 human breast cancercellsrdquo European Journal of Pharmacology vol 603 no 1-3 pp22ndash28 2009

[16] T Tseng M Chen M Tsai et al ldquoOroxylin-A rescues LPS-induced acute lung injury via regulation of NF-120581B signalingpathway in rodentsrdquo PLoS ONE vol 7 no 10 Article ID e474032012

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[18] M A Warren S F Shoemaker D J Shealy W Bshara andM M Ip ldquoTumor necrosis factor deficiency inhibits mam-mary tumorigenesis and a tumor necrosis factor neutralizingantibody decreases mammary tumor growth in neuerbB2transgenic micerdquo Molecular Cancer erapeutics vol 8 no 9pp 2655ndash2663 2009

[19] T Ni Z He Y Dai J Yao Q Guo and L Wei ldquoOrox-ylin A suppresses the development and growth of colorectalcancer through reprogram of HIF1120572-modulated fatty acidmetabolismrdquo Cell Death amp Disease vol 8 no 6 Article IDe2865 2017

[20] L Wei Y Zhou C Qiao et al ldquoOroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promotingSIRT3-mediated SOD2 transcription and HIF1120572 destabiliza-tionrdquo Cell Death amp Disease vol 6 no 4 Article ID e1714 2015

[21] Y Gao N Lu Y Ling et al ldquoOroxylin A inhibits angiogenesisthrough blocking vascular endothelial growth factor-inducedKDRFlk-1 phosphorylationrdquo Journal of Cancer Research andClinical Oncology vol 136 no 5 pp 667ndash675 2010

[22] R Mu Q Qi H Gu et al ldquoInvolvement of p53 in oroxylinA-induced apoptosis in cancer cellsrdquoMolecular Carcinogenesisvol 48 no 12 pp 1159ndash1169 2009

[23] H Ohshima H Tazawa B S Sylla and T Sawa ldquoPreventionof human cancer by modulation of chronic inflammatoryprocessesrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 591 no 1-2 pp 110ndash122 2005

[24] P P Tak and G S Firestein ldquoNF-kappaB a key role ininflammatory diseasesrdquoe Journal of Clinical Investigation vol107 pp 7ndash11 2001

[25] E Heiss C Herhaus K Klimo H Bartsch and C GerhauserldquoNuclear factor 120581B is a molecular target for sulforaphane-mediated anti-inflammatory mechanismsrdquo e Journal of Bio-logical Chemistry vol 276 no 34 pp 32008ndash32015 2001

[26] A J Dannenberg N K Altorki J O Boyle et al ldquoCyclo-oxygenase 2 a pharmacological target for the prevention ofcancerrdquoe Lancet Oncology vol 2 no 9 pp 544ndash551 2001

[27] Y-C Chen L-L Yang and T J-F Lee ldquoOroxylin A inhi-bition of lipopolysaccharide-induced iNOS and COX-2 geneexpression via suppression of nuclear factor-120581B activationrdquoBiochemical Pharmacology vol 59 no 11 pp 1445ndash1457 2000

[28] D-F Lee andM-CHung ldquoAdvances in targeting IKK and IKK-related kinases for cancer therapyrdquoClinical Cancer Research vol14 no 18 pp 5656ndash5662 2008

[29] Z Han D L Boyle A M Manning and G S Firestein ldquoAP-1and NF-kappaB regulation in rheumatoid arthritis and murinecollagen-induced arthritisrdquo Autoimmunity vol 28 no 4 pp197ndash208 1998

[30] D Liu X Wang and Z Chen ldquoTumor Necrosis Factor-alphaa regulator and therapeutic agent on breast cancerrdquo CurrentPharmaceutical Biotechnology vol 17 no 6 pp 486ndash494 2016

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

[32] R van Horssen T L M ten Hagen and A M M EggermontldquoTNF-120572 in cancer treatment molecular insights antitumoreffects and clinical utilityrdquoeOncologist vol 11 no 4 pp 397ndash408 2006

[33] M M Ip S F Shoemaker and K M Darcy ldquoRegulation ofrat mammary epithelial cell proliferation and differentiation bytumor necrosis factor-alphardquo Endocrinology vol 130 no 5 pp2833ndash2844 1992

[34] J Zhang M A Warren S F Shoemaker and M MIp ldquoNF120581B1p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells impli-cations for NF120581B2p52 and RelBrdquo Endocrinology vol 148 no 1pp 268ndash278 2007

[35] M Liang P Zhang and J Fu ldquoUp-regulation of LOX-1expression by TNF-120572 promotes trans-endothelial migration ofMDA-MB-231 breast cancer cellsrdquo Cancer Letters vol 258 no1 pp 31ndash37 2007

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

BioMed Research International 9

Chronic inflammation is a main activator in themetastatic cascade [28] Epithelial-mesenchymal transition(EMT) is a complex process losing cell-cell adhesioncontributing to cancer invasion and metastasis The resultsof the present study indicated that OA inhibited the EMTformation in breast cancer cells by upregulation of E-cadherin levels and downregulation of N-cadherin andVimentin levels EMT programs stimulate the productionof proinflammatory factors in cancer environment whileinflammation is a potent inducement for EMT in tumors[9] What is more NF-120581B activity promotes the collectionof inflammatory cells and secretion of proinflammatorycytokines like IL-1 IL-6 IL-8 and TNF-120572 [29] The presentresults demonstrated that OA markedly suppressed thesecretion of IL-6 IL-8 and TNF-120572 protein in MDA-MB-231cellsThese findings indicate OAhas a repressive influenceon EMT and inflammation progress in breast cancercells

TNF-120572 has been proved to have pleiotropic effects oncells death growth or differentiation under different con-centrations and context [30 31] TNF has anticancer andanti-infection effects in high concentrations because of itscytotoxic effects on the tumor vessel [32] However it isshown that TNF can promote the proliferation andmigrationof normal epithelial cell and breast cancer cells both atlow doses [33 34] One study demonstrated that TNF-120572secreted by tumor cells stimulated the growth of malignantmammary epithelial cells which contributed to mammarytumorigenesis in neuerbB2 transgenic mice [18] Anotherstudy showed that TNF-120572 can promote transendothelialmigration of breast cancer via upregulation of LOX [35]In the present study we found OA-suppressed cell prolif-eration migration invasion and EMT phenotype forma-tion were promoted by TNF-120572 leading to aggravation ofbreast cancer process which is consistent with previousreports On these bases we hypothesized that the blockadeof TNF-120572 may enhance the antitumor effects of OA forbreast cancer patients and this speculation warrants furtherinvestigation

5 Conclusion

In summary the present study showed that OA couldsuppress cell proliferation migration invasion and EMTformation and downregulate the expressions of inflammatorycytokines Furthermore OA exerted protective effect onbreast cancer by inhibiting NF-120581B signaling pathway Theresults provided a theoretical foundation for Oroxylin A asa potential drug for the treatment of breast cancer

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

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

Acknowledgments

This work was supported by Funding from Science andTechnology Development of Universities and Colleges ofTianjin (No 20130120)

References

[1] A Ugolkov I Gaisina J Zhang et al ldquoGSK-3 inhibitionovercomes chemoresistance in human breast cancerrdquo CancerLetters vol 380 no 2 pp 384ndash392 2016

[2] C A Santa-Maria and W J Gradishar ldquoChanging treatmentparadigms in metastatic breast cancerrdquo JAMA Oncology vol 1no 4 pp 528ndash534 2015

[3] J OrsquoShaughnessy ldquoExtending survival with chemotherapy inmetastatic breast cancerrdquo e Oncologist vol 10 no 3 pp 20ndash29 2005

[4] A Bosch P Eroles R Zaragoza et al ldquoTriple-negative breastcancer molecular features pathogenesis treatment and currentlines of researchrdquo Cancer Treatment Reviews vol 36 no 3 pp206ndash215 2010

[5] J Yao R Hu J Sun et al ldquoOroxylin a prevents inflammation-related tumor through down-regulation of inflammatory geneexpression by inhibiting NF-120581B signalingrdquo Molecular Carcino-genesis vol 53 no 2 pp 145ndash158 2014

[6] A Mantovani P Allavena A Sica and F Balkwill ldquoCancer-related inflammationrdquo Nature vol 454 no 7203 pp 436ndash4442008

[7] M Karin ldquoNF-kappaB as a critical link between inflammationand cancerrdquo Cold Spring Harbor Perspectives in Biology vol 1no 5 Article ID a000141 2009

[8] X Cui D Shen C Kong et al ldquoNF-120581B suppresses apoptosisand promotes bladder cancer cell proliferation by upregulatingsurvivin expression in vitro and in vivordquo Scientific Reports vol7 no 1 Article ID 40723 2017

[9] M Suarez-Carmona J Lesage D Cataldo and C Gilles ldquoEMTand inflammation inseparable actors of cancer progressionrdquoMolecular Oncology vol 11 no 7 pp 805ndash823 2017

[10] H Kiefel S Bondong N Erbe-Hoffmann et al ldquoL1CAM-integrin interaction induces constitutive NF-120581B activation inpancreatic adenocarcinoma cells by enhancing IL-1Β expres-sionrdquo Oncogene vol 29 no 34 pp 4766ndash4778 2010

[11] R A Stoltz N G Abraham and M Laniado-SchwartzmanldquoThe role of NF-kappaB in the angiogenic response of coro-nary microvessel endothelial cellsrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 93 no7 pp 2832ndash2837 1996

[12] Z Lu N Lu C Li et al ldquoOroxylin A inhibits matrixmetalloproteinase-29 expression and activation by up-regulating tissue inhibitor of metalloproteinase-2 andsuppressing the ERK12 signaling pathwayrdquo ToxicologyLetters vol 209 no 3 pp 211ndash220 2012

[13] L Lu Q Guo and L Zhao ldquoOverview of Oroxylin A apromising flavonoid compoundrdquo Phytotherapy Research vol30 no 11 pp 1765ndash1774 2016

[14] Y Hu Y Yang Q-D You et al ldquoOroxylin A induced apop-tosis of human hepatocellular carcinoma cell line HepG2 wasinvolved in its antitumor activityrdquo Biochemical and BiophysicalResearch Communications vol 351 no 2 pp 521ndash527 2006

[15] Y Sun N Lu Y Ling et al ldquoOroxylin A suppressesinvasion through down-regulating the expression of matrix

10 BioMed Research International

metalloproteinase-29 in MDA-MB-435 human breast cancercellsrdquo European Journal of Pharmacology vol 603 no 1-3 pp22ndash28 2009

[16] T Tseng M Chen M Tsai et al ldquoOroxylin-A rescues LPS-induced acute lung injury via regulation of NF-120581B signalingpathway in rodentsrdquo PLoS ONE vol 7 no 10 Article ID e474032012

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[18] M A Warren S F Shoemaker D J Shealy W Bshara andM M Ip ldquoTumor necrosis factor deficiency inhibits mam-mary tumorigenesis and a tumor necrosis factor neutralizingantibody decreases mammary tumor growth in neuerbB2transgenic micerdquo Molecular Cancer erapeutics vol 8 no 9pp 2655ndash2663 2009

[19] T Ni Z He Y Dai J Yao Q Guo and L Wei ldquoOrox-ylin A suppresses the development and growth of colorectalcancer through reprogram of HIF1120572-modulated fatty acidmetabolismrdquo Cell Death amp Disease vol 8 no 6 Article IDe2865 2017

[20] L Wei Y Zhou C Qiao et al ldquoOroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promotingSIRT3-mediated SOD2 transcription and HIF1120572 destabiliza-tionrdquo Cell Death amp Disease vol 6 no 4 Article ID e1714 2015

[21] Y Gao N Lu Y Ling et al ldquoOroxylin A inhibits angiogenesisthrough blocking vascular endothelial growth factor-inducedKDRFlk-1 phosphorylationrdquo Journal of Cancer Research andClinical Oncology vol 136 no 5 pp 667ndash675 2010

[22] R Mu Q Qi H Gu et al ldquoInvolvement of p53 in oroxylinA-induced apoptosis in cancer cellsrdquoMolecular Carcinogenesisvol 48 no 12 pp 1159ndash1169 2009

[23] H Ohshima H Tazawa B S Sylla and T Sawa ldquoPreventionof human cancer by modulation of chronic inflammatoryprocessesrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 591 no 1-2 pp 110ndash122 2005

[24] P P Tak and G S Firestein ldquoNF-kappaB a key role ininflammatory diseasesrdquoe Journal of Clinical Investigation vol107 pp 7ndash11 2001

[25] E Heiss C Herhaus K Klimo H Bartsch and C GerhauserldquoNuclear factor 120581B is a molecular target for sulforaphane-mediated anti-inflammatory mechanismsrdquo e Journal of Bio-logical Chemistry vol 276 no 34 pp 32008ndash32015 2001

[26] A J Dannenberg N K Altorki J O Boyle et al ldquoCyclo-oxygenase 2 a pharmacological target for the prevention ofcancerrdquoe Lancet Oncology vol 2 no 9 pp 544ndash551 2001

[27] Y-C Chen L-L Yang and T J-F Lee ldquoOroxylin A inhi-bition of lipopolysaccharide-induced iNOS and COX-2 geneexpression via suppression of nuclear factor-120581B activationrdquoBiochemical Pharmacology vol 59 no 11 pp 1445ndash1457 2000

[28] D-F Lee andM-CHung ldquoAdvances in targeting IKK and IKK-related kinases for cancer therapyrdquoClinical Cancer Research vol14 no 18 pp 5656ndash5662 2008

[29] Z Han D L Boyle A M Manning and G S Firestein ldquoAP-1and NF-kappaB regulation in rheumatoid arthritis and murinecollagen-induced arthritisrdquo Autoimmunity vol 28 no 4 pp197ndash208 1998

[30] D Liu X Wang and Z Chen ldquoTumor Necrosis Factor-alphaa regulator and therapeutic agent on breast cancerrdquo CurrentPharmaceutical Biotechnology vol 17 no 6 pp 486ndash494 2016

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

[32] R van Horssen T L M ten Hagen and A M M EggermontldquoTNF-120572 in cancer treatment molecular insights antitumoreffects and clinical utilityrdquoeOncologist vol 11 no 4 pp 397ndash408 2006

[33] M M Ip S F Shoemaker and K M Darcy ldquoRegulation ofrat mammary epithelial cell proliferation and differentiation bytumor necrosis factor-alphardquo Endocrinology vol 130 no 5 pp2833ndash2844 1992

[34] J Zhang M A Warren S F Shoemaker and M MIp ldquoNF120581B1p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells impli-cations for NF120581B2p52 and RelBrdquo Endocrinology vol 148 no 1pp 268ndash278 2007

[35] M Liang P Zhang and J Fu ldquoUp-regulation of LOX-1expression by TNF-120572 promotes trans-endothelial migration ofMDA-MB-231 breast cancer cellsrdquo Cancer Letters vol 258 no1 pp 31ndash37 2007

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

10 BioMed Research International

metalloproteinase-29 in MDA-MB-435 human breast cancercellsrdquo European Journal of Pharmacology vol 603 no 1-3 pp22ndash28 2009

[16] T Tseng M Chen M Tsai et al ldquoOroxylin-A rescues LPS-induced acute lung injury via regulation of NF-120581B signalingpathway in rodentsrdquo PLoS ONE vol 7 no 10 Article ID e474032012

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[18] M A Warren S F Shoemaker D J Shealy W Bshara andM M Ip ldquoTumor necrosis factor deficiency inhibits mam-mary tumorigenesis and a tumor necrosis factor neutralizingantibody decreases mammary tumor growth in neuerbB2transgenic micerdquo Molecular Cancer erapeutics vol 8 no 9pp 2655ndash2663 2009

[19] T Ni Z He Y Dai J Yao Q Guo and L Wei ldquoOrox-ylin A suppresses the development and growth of colorectalcancer through reprogram of HIF1120572-modulated fatty acidmetabolismrdquo Cell Death amp Disease vol 8 no 6 Article IDe2865 2017

[20] L Wei Y Zhou C Qiao et al ldquoOroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promotingSIRT3-mediated SOD2 transcription and HIF1120572 destabiliza-tionrdquo Cell Death amp Disease vol 6 no 4 Article ID e1714 2015

[21] Y Gao N Lu Y Ling et al ldquoOroxylin A inhibits angiogenesisthrough blocking vascular endothelial growth factor-inducedKDRFlk-1 phosphorylationrdquo Journal of Cancer Research andClinical Oncology vol 136 no 5 pp 667ndash675 2010

[22] R Mu Q Qi H Gu et al ldquoInvolvement of p53 in oroxylinA-induced apoptosis in cancer cellsrdquoMolecular Carcinogenesisvol 48 no 12 pp 1159ndash1169 2009

[23] H Ohshima H Tazawa B S Sylla and T Sawa ldquoPreventionof human cancer by modulation of chronic inflammatoryprocessesrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 591 no 1-2 pp 110ndash122 2005

[24] P P Tak and G S Firestein ldquoNF-kappaB a key role ininflammatory diseasesrdquoe Journal of Clinical Investigation vol107 pp 7ndash11 2001

[25] E Heiss C Herhaus K Klimo H Bartsch and C GerhauserldquoNuclear factor 120581B is a molecular target for sulforaphane-mediated anti-inflammatory mechanismsrdquo e Journal of Bio-logical Chemistry vol 276 no 34 pp 32008ndash32015 2001

[26] A J Dannenberg N K Altorki J O Boyle et al ldquoCyclo-oxygenase 2 a pharmacological target for the prevention ofcancerrdquoe Lancet Oncology vol 2 no 9 pp 544ndash551 2001

[27] Y-C Chen L-L Yang and T J-F Lee ldquoOroxylin A inhi-bition of lipopolysaccharide-induced iNOS and COX-2 geneexpression via suppression of nuclear factor-120581B activationrdquoBiochemical Pharmacology vol 59 no 11 pp 1445ndash1457 2000

[28] D-F Lee andM-CHung ldquoAdvances in targeting IKK and IKK-related kinases for cancer therapyrdquoClinical Cancer Research vol14 no 18 pp 5656ndash5662 2008

[29] Z Han D L Boyle A M Manning and G S Firestein ldquoAP-1and NF-kappaB regulation in rheumatoid arthritis and murinecollagen-induced arthritisrdquo Autoimmunity vol 28 no 4 pp197ndash208 1998

[30] D Liu X Wang and Z Chen ldquoTumor Necrosis Factor-alphaa regulator and therapeutic agent on breast cancerrdquo CurrentPharmaceutical Biotechnology vol 17 no 6 pp 486ndash494 2016

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

[32] R van Horssen T L M ten Hagen and A M M EggermontldquoTNF-120572 in cancer treatment molecular insights antitumoreffects and clinical utilityrdquoeOncologist vol 11 no 4 pp 397ndash408 2006

[33] M M Ip S F Shoemaker and K M Darcy ldquoRegulation ofrat mammary epithelial cell proliferation and differentiation bytumor necrosis factor-alphardquo Endocrinology vol 130 no 5 pp2833ndash2844 1992

[34] J Zhang M A Warren S F Shoemaker and M MIp ldquoNF120581B1p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells impli-cations for NF120581B2p52 and RelBrdquo Endocrinology vol 148 no 1pp 268ndash278 2007

[35] M Liang P Zhang and J Fu ldquoUp-regulation of LOX-1expression by TNF-120572 promotes trans-endothelial migration ofMDA-MB-231 breast cancer cellsrdquo Cancer Letters vol 258 no1 pp 31ndash37 2007

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom