6-Shogaol, an Active Constituent of Dietary Ginger, Impairs Cancer

Jan 26, 2015 - CC-Chemokine Ligand 2 (CCL2) in Tumor-Associated Dendritic Cells. Ya-Ling Hsu,. †. Jen-Yu Hung,. ‡,#. Ying-Ming Tsai,. †,#. Eing-...
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6-Shogaol, an active constituent of dietary ginger, impairs cancer development and lung metastasis by inhibiting the secretion of CCL2 in tumor-associated dendritic cells Ya-Ling Hsu, Jen-Yu Hung, Ying-Ming Tsai, Eing-Mei Tsai, Ming-Shyan Huang, Ming-Feng Hou, and Po-Lin Kuo J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 Jan 2015 Downloaded from http://pubs.acs.org on January 28, 2015

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6-Shogaol, an active constituent of dietary ginger, impairs cancer

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development and lung metastasis by inhibiting the secretion of CCL2

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in tumor-associated dendritic cells

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Ya-Ling Hsu,‡ Jen-Yu Hung,∫,# Ying-Ming Tsai,‡,# Eing-Mei Tsai,‡ Ming-Shyan

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Huang,∫,# Ming-Feng Hou,† and Po-Lin Kuo†,§,¶,*

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Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University,

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Kaohsiung 807, Taiwan ∫

School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung

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807, Taiwan #

Division of Pulmonary and Critical Care Medicine, Kaohsiung Medical University

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Hospital, Kaohsiung 807, Taiwan †

Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University,

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Kaohsiung 807, Taiwan §

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Research Center for Environmental Medicine, Kaohsiung Medical University,

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Kaohsiung 807, Taiwan ¶

Institute of Medical Science and Technology, National Sun Yat-Sen University,

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Kaohsiung 804, Taiwan

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Corresponding Author

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* Po-Lin Kuo, PhD. Institute of Clinical Medicine, College of Medicine, Kaohsiung

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Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. E-mail

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address: [email protected]; Phone: +886-7-312-1101 ext.2512 #33. Fax:

25

+886-7-321-0701

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ABSTRACT

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This study has two novel findings: it is not only the first to demonstrate that

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tumor-associated dendritic cells (TADCs) facilitate lung and breast cancer metastasis

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in vitro and in vivo by secreting inflammatory mediator CC-chemokine ligand 2

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(CCL2), but it is also the first to reveal that 6-shogaol can decrease cancer

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development and progression by inhibiting the production of TADC-derived CCL2.

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Human lung cancer A549 and breast cancer MDA-MB-231 cells increase TADCs to

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express high levels of CCL2, which increase cancer stem cell features, migration and

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invasion, as well as immuno-suppressive tumor-associated macrophage infiltration.

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6-Shogaol decreases cancer-induced up-regulation of CCL2 in TADCs, preventing the

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enhancing effects of TADCs on tumorigenesis and metastatic properties in A549 and

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MDA-MB-231 cells. A549 and MDA-MB-231 cells enhance CCL2 expression by

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increasing the phosphorylation of signal transducer and activator of transcription 3

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(STAT3), and the activation of STAT3 induced by A549 and MDA-MB-231 is

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completed inhibited by 6-shogaol. 6-Shogaol also decreases the metastasis of lung and

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breast cancers in mice. 6-Shogaol exerts significant anti-cancer effects on lung and

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breast cells in vitro and in vivo by targeting the CCL2 secreted by TADCs. Thus,

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6-shogaol may have the potential of being an efficacious immuno-therapeutic agent

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for cancers. 2

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KEYWORDS: 6-Shogaol; CCL2; lung cancer; breast cancer; metastasis

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INTRODUCTION

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The immune system plays the primary role of protecting the host against tumor

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development

by

eliminating

pathogens/viral

infections,

timely

control

of

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inflammation response, and removal of cancer cells expressing tumor-specific

50

antigens.1 However, the immune system may not always be sufficiently potent to

51

suppress the development of cancer. The dysfunction of dendritic cells (DC) induced

52

by cancer playa a major role in escaping immuno-surveillance.2,3 Cancer

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cells produce soluble factors that impair immune cell development or function.4,5 In

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turn, DCs co-exist in the tumor micro-environment and produce a multitude of soluble

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factors and nutrients that promote tumor progression and growth.6,7 Understanding the

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factors that contribute to this vicious cycle between tumor and tumor-associated DCs

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(TADC) may help in the development of agents that will target these mediators as

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new therapies for cancer treatment.

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Chronic inflammation is an important tumor mediator and accelerator of many

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malignancies. Elevated cytokine or chemokine levels in patients have been

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increasingly correlated with poor prognosis.8,9 CC-chemokine ligand 2 (CCL2;

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MCP-1) is a well-recognized inflammatory factor that can induce the migration of

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monocytes, DCs, and T cells to sites of inflammation via the activation of CCR2.10-12 3

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CCL2 expression in cancers is strongly associated with higher histologic grades and

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acts as a biomarker for early relapse tumor-associated macrophages (TAM) and

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monocyte infiltration.12 Moreover, CCL2 has been demonstrated to directly stimulate

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cancer cell proliferation, survival, migration, and metastasis. Thus, it may play a role

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in mediating cancer development via DC-cancer cell interaction.

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Ginger (Zingiber officinale) is a well known plant used in cooking worldwide,

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and has long been reputed to have medicinal properties. It is an herbaceous,

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rhizomatous perennial plant widely distributed throughout tropical and subtropical

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regions,13 and is cultivated on a large scale in Nigeria, India, Bangladesh, Sri Lanka,

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Taiwan and other East Asian countries.13 Its biological constituents are

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phenylpropanoid-derived compounds such as gingerols and shogaols, which possess

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anti-cancer and anti-inflammatory activities, as has been reported in numerous

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studies.14-19 The present study reveals that 6-shogaol inhibits TADCs’ role in the

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switching of cancer stem cell-like phenotype, and enhancing cancer migration and

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invasion by decreasing CCL2 secretion. These results suggest that 6-shogaol acts as

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an effective therapeutic agent for cancer treatment by impeding the vicious cycle

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between tumors and TADCs.

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MATERIALS AND METHODS

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Reagents and Antibodies. 6-Shogaol (6-SH) and 6-gingerol (6-GL) were purchased 4

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from Sigma Chemicals Co (St. Louis, MO). Recombinant Human (rh) CCL2 protein

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was obtained from R&D Systems (Minneapolis, MN).

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Cell culture. Human breast cancer MDA-MB-231, lung cancer A549, mouse

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mammary tumor 4T1, and Lewis lung carcinoma (LLC) cells were purchased from

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American Type Culture Collection (ATCC) (Manassas, VA, USA).

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MDA-MB-231 cells were cultured in Leibovitz's L-15 Medium supplemented with

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10% fetal bovine serum (FBS), penicillin, and streptomycin in a CO2-free incubator.

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The A549, 4T1 and LLC cells were cultured in F-12K Medium (Kaighn's

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Modification of Ham's F-12 Medium) or RPMI1640 and DMEM containing 10% FBS

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in a 5% CO2 incubator. For the conditioned medium (CM) collection, the

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MDA-MB-231 and A549 cells (1Χ106) were seeded in a 10 cm dish and the

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supernatants collected, filtered (0.22 mm), and defined as MDA-CM and A549-CM.

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Isolation of CD14+ monocytes and differentiation of monocyte-derived DCs

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(mdDCs). Monocytes were purified from peripheral blood mononuclear cells

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(PBMCs) obtained from healthy consenting donors. Mononuclear cells were isolated

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from the blood by the Ficoll-Hypaque gradient (GE Healthcare Bio-Sciences, Little

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Chalfont, UK). CD14+ monocytes were purified using CD14+ monoclonal

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antibody-conjugated magnetic beads (MACS MicroBeads, Miltenyi Biotec),

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according to the manufacturer’s protocol. Monocyte-derived DCs (mdDCs) were 5

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generated by culturing CD14+ monocytes in RPMI 1640 medium containing 10% FBS

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(Invitrogen, Carlsbad, CA) and 20 ng/mL GM-CSF and 10 ng/mL IL-4 (R&D

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Systems, Minneapolis, MN) with or without 6-SH (1 or 10 µM) for 5 days. The

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medium was replaced with fresh medium containing GM-CSF and IL-4 on day 3. The

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hospital’s Institutional Review Board approved the study and all of the patients

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provided informed consent in accordance with the Declaration of Helsinki. A549 and

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MDA-MB-231 tumor-associated mdDCs (A549-TADCs) and MDA-MB-231-TADCs

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were generated by culturing CD14+ monocytes in RPMI 1640 medium containing

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FBS, GM-CSF, and IL-4 presenting in A549-CM (20%) and MDA-MB-231-CM

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(20%) with or without 6-SH (1 or 10 µM) for 5 days. After washing, the supernatants

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were collected and frozen and stored at -80°C, and thawed singly for the study.

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Measurement of Secreted Factors. Supernatants from mdDCs, A549-TADC,

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MDA-TADCs, 6-shogaol-treated mdDC and TADC were collected. CD11c+F4/80-

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cells were isolated from mice and cultured in RPMI1640 medium. After 24 h, the

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supernatants were harvested. CXCL1, CCL2, and CCL4 levels were quantified using

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human and mouse MILLIPLEX MAP kits.

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Quantitative real-time PCR (qRT-PCR). Isolation of RNA was performed using the

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TRIzol reagent (Invitrogen). cDNA was prepared using an oligo (dT) primer and

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reverse transcriptase (Takara, Shiga, Japan) following standard protocols. Real-time 6

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PCR was performed using SYBR Green on the ABI StepOnePlus™ real-time PCR

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instrument (Applied Biosystems, Foster City, CA, USA). Each PCR reaction mixture

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contained 100 nM of each primer, 5 µl of 2x SYBR Green PCR Master Mix (Applied

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Biosystems, Foster City, CA), 2.5 µl of cDNA and RNase-free water for a total

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volume of 10 µl. The PCR reaction was conducted with a denaturation step at 95°C

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for 10 min, and then for 40 cycles at 95°C for 15 s, and 60°C for 1 min. All of the

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PCRs

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glyceraldehyde-3-phosphate dehydrogenase

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expression was presented using the 2–△△CT method.

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Tumor Spheroid Formation. A549 and MDA-MB-231 cells were labeled with

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PKH67 (Sigma-Aldrich) following the manufacturer’s procedures. The cells were

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seeded in ultra-low attachment wells with or without 6-shogaol pre-set conditioned

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media (20%) of mdDCs or TADCs for tumor sphere formation. On day 10, the tumor

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spheres were assessed by fluorescence microscopy, or dissociated into single cells for

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ALDHFLOR analysis.

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Flow Cytometry Analysis. Aldehyde dehydrogenases (ALDH) activity was assessed

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using an ALDHFLOR kit (Stem Cell Technologies, Vancouver, BC, Canada). Briefly,

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cells were incubated in Aldefluor assay buffer containing ALDH substrate

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(1 µmol l−1 per 1 × 106cells), with or without diethylaminobenzaldehyde (DEAB), a

were

performed

in triplicate

and

normalized (GAPDH)

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internal

mRNA.

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control relative

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specific ALDH inhibitor. After incubation at 37°C for 30 min, the cells were

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centrifuged and re-suspended in cold Aldefluor buffer. ALDH activity was determined

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by an Acuri C6 flow cytometer (Becton Dickinson Immuno-cytometry Systems). The

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cells isolated from the lungs and mammary tumors of mice were stained by antibodies

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against mouse CD11b labeled by FITC (BD Biosciences, San Jose, CA), F4/80

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labeled by allophycocyanin (APC) (Biolegend). The expression of each molecule was

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analyzed using an Acuri C6 flow cytometer (BD Biosciences).

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Cell Migration and Invasion Analysis. Quantitative migration and invasion assay

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was conducted using the QCM™ 24-well Cell Migration Assay and Invasion System

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(Millipore Corp., Billerica, MA, USA). Briefly, 3 × 104 cells A549 or MDA-MB-231

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cells were seeded into the top chamber, and CMs of mdDC, TADC, 6-shogaol-treated

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mdDC and TADC were added to the bottom wells as chemo-attractant for 24 h (for

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migration) or 48 h (for invasion). At the end of treatment, the cells were post-stained

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with CyQuant GR dye in cell lysis buffer for 15 min at room temperature.

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Fluorescence of the migratory or invaded cells was read using a fluorescence plate

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reader at excitation and emission wavelengths of 485 and 540 nm, respectively.

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Immunoblot Analysis. CD14+ monocytes (4×106) were pre-treated with 6-shogaol

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for 1 h and the A549- or MDA-MB-231-CMs were added for 30 min. Total cell

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extracts were prepared in RIPA lysis buffer (Millipore). Equivalent amounts of protein 8

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were resolved by the SDS-PAGE and transferred to PVDF membranes. After the

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membranes were blocked in Tris-buffer saline containing 0.05% Tween 20 (TBST)

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and 5% non-fat powdered milk, they were incubated with primary antibodies against

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p-STAT3, STAT3 and GAPDH (Cell Signaling Technology) at 4°C overnight. After

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washing three times with TBST, the membranes were incubated with horseradish

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peroxidase-labeled secondary antibody for 1 h and then re-washed. Detection was

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performed using an enhanced chemiluminescence blotting detection system

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(Millipore).

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Chromatin Immuno-precipitation (ChIP). Chromatin immuno-precipitation (ChIP)

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was conducted according to the protocol of the Pierce Agarose ChIP kit (Pierce,

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Thermo Scientific, Rockford, IL). Immuno-precipitation conducted using anti-STAT3

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transcription factors (Cell Signaling Technology) or by normal rabbit IgG (Cell

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Signaling Technology) as the negative control. Input DNA and immuno-precipitated

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DNA were analyzed by quantitative PCR. Primer sequences were obtained from

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Qiagen (GPH1005667(-)01A).

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Lung Metastasis of Lung and Breast Cancer In Vivo. Male C57BL and Female

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BALB/c mice (5-6 wk old) from the National Science Council Animal Center (Taipei,

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Taiwan) were maintained in pathogen-free conditions. 4T1 cells (2×106) were

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implanted into mammary fat pads of BALB/c mice and LLC cells were implanted into 9

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C57BL mice via tail vein injection. The mice were then randomly divided into two

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groups: the 6-shogaol-treated group was given intra-peritoneal injection (ip) daily

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with 0.2 ml of 6-shogaol (dose: 30 mg/kg of body weight) while the control group

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was given an equal volume of normal saline. Tumor-bearing mice were sacrificed

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14-21 days after transplantation. Lung (LLC bearing mice) and mammary tumors

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(4T1 bearing mice) were collected and minced. Single cell suspensions were obtained

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after enzymatic digestion (1 mg/ml collagenase A; Roche Diagnostics) and 100 IU/ml

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type I DNase (Sigma-Aldrich) for 2 h at 37°C and 5% CO2 in RPMI 1640 medium.

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Single cell suspension was filtered through a 70 µm nylon mesh (BD Biosciences)

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and cells were washed twice by PBS. CD11c+ and CD11b+ cells were purified using

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anti-CD11c or CD11b monoclonal antibody-conjugated magnetic beads (MACS

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MicroBeads, Miltenyi Biotec). F4/80+ cells were depleted from CD11c+ cells by

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using F4/80+ antibody-biotin beads (Miltenyi Biotec). Alternatively, the lungs were

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removed and the metastatic tumor nodules counted. All animal experiments were

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performed according to a protocol approved by the Institutional Animal Care and Use

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Committee of Kaohsiung Medical University Hospital.

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Statistical Analysis. Data were expressed as means ± SD. Statistical analyses

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between control and experimental groups were analyzed by an unpaired

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Student's t-test. Multiple comparisons were evaluated by a one-way ANOVA, and 10

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differences in the mean values among groups were conducted by a Fischer post

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hoc analysis. p values < 0.05 were considered to be statistically significant.

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RESULTS

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Lung cancer and breast cancer-derived TADCs secrete high levels of CCL2,

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which are decreased by 6-shogaol and 6-gingerol. Previous studies and data show

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that lung cancer A549 and breast cancer MDA-MB-231 cells increase the expression

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of some inflammatory chemokines in TADCs.6 This study therefore assessed whether

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the active components of ginger, 6-shogaol (6-SH) and 6-gingerol (6-GL), could

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decrease the expression of these cytokines/chemokines in TADCs. The qRT-PCR data

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demonstrated that A549- and MDA-MB-231-CMs (MDA-CM) increased levels of

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CXCL1, CCL2, and CCL4 mRNA transcripts in TADC, when compared to mdDCs

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(Figs. 1A and 1B). As determined by ELISA, the CMs of lung and breast cancers

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increased the expression of CXCL1, CCL2, and CCL4 in protein levels in TADCs

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(Fig. 1C to 1H). 6-Shogaol decreased the production of CCL2 in TADCs in protein

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levels (Figs. 1E and 1F). The inhibitory effect of 6-shogaol on CCL2 secretion in

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TADCs was greater than that of 6-gingerol. In addition, 6-shogaol decreased the

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expression of CCL2 in TADCs in a dose-dependent manner ranging from 0.1-10 µM

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(Figs 1I and 1J). Neither 6-shogaol nor 6-gingerol prevented the simulative effect of

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cancers on the expression of CXCL1 and CCL4 in TADCs (Figs. 1C, 1D, 1F and 1G). 11

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To eliminate the possibility of cytokines being reduced due to altered cell viability,

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we assessed the effect of 6-shogaol and 6-gingerol (0.1-10 µM) on the cell viability of

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TADCs. The data shows that 6-shogaol and 6-gingerol do not affect the viability of

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TADCs (data not shown).

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6-Shogaol decreases the effect of TADC-derived CCL2 on the formation of tumor

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spheroids, migration and invasion. Because CCL2 has been reported to increase

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cancer-stem cell like property and enhance cancer progression,20 this study assessed

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the effect of 6-shogaol on the bio-activities of TADC-derived CCL2. The stem

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cell-like phenotype is determined by tumor spheroid formation and aldehyde

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dehydrogenases (ALDH) activity. A549- and MDA-MB-231-TADC-CM increased

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tumor spheroid formation in A549 and MDA-MB-231 cell lines, respectively (Figs.

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2A and 2B). However, TADCs lost their inductive effect on tumor spheroid formation

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if they were generated in the presence of 6-shogaol (Figs. 2A and 2B). The

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PKHhigh cells undergo fewer divisions during sphere formation, which implies they

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possess a stem cell phenotype, in contrast to high proliferation non-stem PKHlow cells.

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The ratio of PKHhigh cells increased in tumor spheroids cultured in TADC-CMs when

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compared to those cultured in mdDC-CMs. 6-Shogaol treatment decreased the ratio of

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PKHhigh cell population in tumor spheroids, revealing 6-shogaol reduces the stem cell

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characteristics in both lung and breast cancers (Fig. 2A and 2B). Moreover, the 12

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activity of ALDH, a stem cell marker, was enhanced in tumor spheroids cultured in

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TADC-CMs when compared to those cultured in mdDC-CMs. However, the ALDH

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activity of tumor spheroids was reduced when the TADCs were generated in the

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presence of 6-shogaol (Fig. 2C and 2D). We next assessed the effect of 6-shogaol on

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TADC-mediated cancer progression. A549- and MDA-MB-231-TADC-CM increased

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A549 and MDA-MB-231 cell migration, respectively. However, TADCs lost their

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inductive effect on cancer migration if they were generated in the presence of

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6-shogaol (Figs. 3A and 3B). Similarly, A549- and MDA-MB-231-TADC-CM

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increased cell invasion in A549 and MDA-MB-231 cells, but not if the TADC-CMs

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were derived in the presence of 6-shogaol (Figs. 3C and 3D). In contrast, the addition

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of rhCCL2 reduced the inhibitory effect of 6-shogaol on TADC-mediated A549 and

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MDA-MB-231 cell migration (Fig. 3E and 3F).

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6-Shogaol improves CCL2-mediated immuno-surveillance in lung and breast

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cancers in vivo. Because CCL2 increases immuno-suppressive response by

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decreasing macrophage infiltration in mice with cancer,20 this study assessed if

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6-shogaol could alter immuno-surveillance in mice. LLC were transplanted into mice

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by tail vein injection. The mice were sacrificed after 14 days, and TADC

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(CD11c+F4/80-) cells from metastatic tumor nodules in the lungs were isolated and

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examined. CCL2 expression in TADCs (CD11c+/F4/80-) of the lungs of mice with 13

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LLC transplantation was higher than the dendritic cells isolated from the lungs of

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normal mice. Administration of 6-shogaol decreased CCL2 levels in TADCs of the

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lungs of mice with LLC transplantation (Fig. 4A). 6-Shogaol treatment also decreased

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tumor associated macrophage (TAM) (CD11b+F4/80+) infiltration when compared to

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vehicle-treated mice with LLC transplantation (Fig. 4B). To assess the effect of

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6-shogaol on immuno-surveillance in breast cancer in vivo, 4T1 was implanted in the

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mammary fat pads of mice. On day 14 post-tumor implantation, TAM and TADCs

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were isolated from both mammary tissue and lungs with metastatic 4T1 tumors. 4T1

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breast cancer cells also had increased CCL2 expression in TADCs when compared to

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dendritic cells isolated from normal mammaries. This CCL2 up-regulation was

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inhibited by 6-shogaol administration (Fig. 4C). Similarly, TAM infiltration also

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decreased in 6-shogaol-treated mice (Fig. 4D).

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6-Shogaol decreases CCL2 expression by inhibiting STAT3 activation in TADCs.

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Since STAT3 was reported to bind to and activate the promoter of CCL2,20 this study

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assessed if 6-shoagol could decrease the activation of STAT3. The CMs of lung

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cancer A549 and breast cancer MDA-MB-231 cells increased the phosphorylation of

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STAT3 in TADCs more than in mdDCs (Fig. 5A). The CMs of lung and breast cancers

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also enhanced the nuclear translocation of activated STAT3 (Fig. 5B). These effects,

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including STAT3 activation and nuclear translocation, were inhibited by 6-shogaol in 14

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TADCs. ChIP analysis further revealed that CMs of A549 and MDA-MB-231 cancer

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increased the binding of STAT3 on the promoter of CCL2 gene, but this was inhibited

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by 6-shogaol treatment (Figs. 5C and 5D).

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6-Shaogol decreases metastasis in lung and breast cancers in vivo. To assess the

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activity of 6-shogaol on the metastasis of lung and breast cancers, LLC (by tail vein

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injection) and 4T1 (by mammary fad pad implantation) were transplanted into mice,

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which were sacrificed after 14 and 21 days, respectively. The tumors in the lungs of

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mice were collected and counted. 6-Shogaol treatment decreased the number of LLC

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tumor nodules in mice (Fig. 6A). Similarly, 6-shogaol also reduced the lung

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metastasis of breast cancer 4T1 in mice (Fig. 6B).

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DISCUSSION

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Tumor micro-environment is characterized by a stroma of hematopoietic

285

precursors, inflammatory immune cells, dysregulated vessel endothelial cells, and

286

proteolytic enzymes.21,22 TADCs have been shown to lose their antigen-presenting

287

function, and also to promote cancer progression by modulating multiple components

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in the cancer niche, thereby creating a permissive and supportive microenvironment

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for tumor survival, proliferation, and spread.7,23,24 This study has two novel findings.

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It is the first study to demonstrate that TADCs facilitate lung and breast cancer

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metastasis in vitro and in vivo by secreting inflammatory mediator CCL2. It is also the 15

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first study to demonstrate that 6-shogaol decreases cancer development and

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progression by inhibiting the production of TADC-derived CCL2 (Fig. 7).

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CCL2 has been implicated in cancer progression and metastasis. Serum CCL2

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levels are significantly correlated to the cancer stage and have been shown to have

296

significant prognostic value for relapse-free survival.25-27 CCL2 appears to play a dual

297

role in regulating tumor immuno-surveillance and in sustaining the development and

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advancement of established malignancy. CCL2 has also been implicated in directly

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affecting cancers by switching the cancer stem cell-like phenotype and by enhancing

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cancer cell survival, growth, angiogenesis, migration, and metastasis.28,29 It has also

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been reported to indirectly facilitate carcinogenesis and metastasis by altering the

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host’s immune response, including the accumulation of suppressive immune cells

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TAM, MDSCs, and regulatory DC and T cells.20,30-32 CCL2 expression is modulated

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in the interaction between tumor and niche cells, including fibroblasts and

305

adipocytes.20,30

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This study demonstrates that cancer cells stimulate TADCs to express high levels

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of CCL2, which in turn increase tumorigenesis by increasing cancer stem cell-like

308

properties. TADC-derived CCL2 also increases cancer migration and invasion; key

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characteristics of metastatic cancer. However, these enhancing effects of TADCs on

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cancer development are prevented by 6-shogaol treatment in vitro and in vivo. 16

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6-shogaol administration decreases immuno-suppressive TAM infiltration in cancer,

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suggesting that 6-shogaol may restore the host’s immune system.

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Accumulating evidence supports a role for the STAT3 pathway in the

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establishment of the inflammatory microenvironment. Constitutively activated STAT3

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can drive various pro-tumorigenic and inflammatory mediator expressions in cancer

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cells per se, as well as in niche cells.20,33,34 Breast cancer stimulates fibroblasts to

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express high levels of CCL2 via a STAT3-dependent mechanism, resulting in

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increased tumorigenesis. In contrast, inhibition of STAT3 in DCs enhances its ability

319

to prime T cells that have elevated IFNγ production,35 suggesting that STAT3 is a

320

potential target for developing a strategy that restores the host’s immunity against

321

tumors.

322

The results of this study reveal that cancer increases the activation and DNA

323

binding activity of STAT3, which in turn enhances the expression of CCL2 in TADCs.

324

6-Shogaol decreases the cancer-induced activation of STAT3, subsequently reducing

325

CCL2 levels in TADCs. According to the studies presented here, 6-shogaol may have

326

a novel mechanism for inhibiting STAT3 activation in TADCs, resulting in decreased

327

pro-tumorigenesis and inflammatory mediators in cancer niches.

328

In summary, this study provides novel insights for understanding the paracrine

329

interaction initiated by cancer cells to induce CCL2 production by TADCs. 17

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TADC-derived CCL2 promotes cancer progression by regulating cancer stem cell-like

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properties, migration/invasion, and immuno-suppressive immune cell infiltration.

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6-Shogaol has also been demonstrated to inhibit the effect of TADCs on cancer

333

tumorigenesis and progression by decreasing CCL2 production. 6-Shoagol also

334

reduces the accumulation of immuno-suppressive cells, suggesting that 6-shogaol may

335

serve as a future therapeutic agent for efficiently blocking the cancer-DC interaction

336

to overcome immune dysregulation and cancer progression.

337

Funding

338

This study was supported by grants from the National Science Council of Taiwan

339

(NSC

340

102-2628-B-037-002-MY3;

341

102-2314-B-037-035-MY3), the Ministry of Science and Technology (MOST

342

103-2320-B-037-006-MY3 and MOST 103-2314-B-037-052), the Excellence for

343

Cancer Research Center Grant, the Ministry of Health and Welfare, Executive Yuan,

344

Taipei, Taiwan (MOHW 103-TD-B-111-05), the Kaohsiung Medical University “Aim

345

for the Top 500 Universities Grant, Grant No. KMU-DT103008”, and the Kaohsiung

346

Medical

347

KMU-TP103A19 and KMU-TP103A20”.

348

Notes

101-2628-B-037-001-MY3;

University

“Aim

NSC

NSC

for

101-2320-B-037-043-MY3;

102-2632-B-037-001-MY3;

the

Top

Universities

18

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Grant,

and

Grant

NSC NSC

Nos.

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The authors declare that there are no competing financial interests.

350

Acknowledgment

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The authors also thank the Center for Resources, Research, and Development of

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Kaohsiung Medical University for its support with the instrumentation.

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FIGURE LEGENDS

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Figure 1. 6-Shogaol and 6-gingerol decreased the expression of CCL2 in TADC.

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The up-regulation of inflammatory chemokines in (A) A549- and (B)

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MDA-MB-231-derived TADC, as determined by qRT-PCR. The active

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components of ginger failed to inhibit the up-regulation of CXCL1 in both (C)

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A549- and (D) MDA-MB-231-derived TADCs. 6-Shogaol (10 µM) and

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6-gingerol (10 µM) decreased the expression of CCL2 in (E) A549- and (F)

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MDA-MB-231-derived TADCs. 6-Shogaol (10 µM) and 6-gingerol (10 µM) did

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not affect the stimulatory effect of (G) A549 and (H) MDA-MB-231 cells on

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CCL4 expression of TADCs. 6-Shogaol decreased the expression of CCL2 in (I)

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A549 and (J) MDA-MB-231-derived TADCs in a dose-dependent manner. The

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expression of mRNA and protein was assessed by qRT-PCR and MILLIPLEX

495

MAP kits. All results are representative of at least three independent experiments,

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and each value is the mean ± SD of three determinations. The results were

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reported as mean ± SD; *p