Effect of Kumatakenin Isolated From Cloves on the Apoptosis of

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Kumatakenin isolated from cloves induces cancer cell apoptosis and inhibits the alternative activation of tumour-associated macrophages Jeong-Hwa Woo, Ji-Hye Ahn, Dae Sik Jang, Kyung-Tae Lee, and Jung-Hye Choi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01543 • Publication Date (Web): 01 Aug 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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Kumatakenin isolated from cloves induces cancer cell apoptosis and inhibits the alternative activation of tumour-associated macrophages

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Jeong-Hwa Woo,†, § Ji-Hye Ahn, § Dae Sik Jang, †, § Kyung-Tae Lee, †, §, and Jung-Hye Choi†, §,*

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†

College of Pharmacy, Kyung Hee University, Seoul 02447, South Korea

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§

Department of Life & Nanopharamceutical Sciences, Kyung Hee University, Seoul 02447, South

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Korea

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Running title : Anti-cancer activities of kumatakenin

13 14 15 16 17

*Correspondence

18 19 20 21 22 23


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ABSTRACT

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The flower bud of Syzygium aromaticum (clove) has been used for a centuries as a spice and

26

herbal medicine. The biological activities of kumatakenin, a flavonoid that has recently been

27

isolated from cloves, are poorly characterized. In the present study, the anti-cancer effects of

28

kumatakenin in human ovarian cancer cells and tumour-associated macrophages (TAM) were

29

investigated. We found that kumatakenin exhibited significant cytotoxic activity in human

30

ovarian cancer cells, SKOV3 and A2780. A propidium iodide and Annexin V-FITC staining

31

assay showed that kumatakenin induces apoptosis in ovarian cancer cells. Kumatakenin

32

treatment increased the activity of caspase-3, -8, and -9, and caspase inhibitors attenuated

33

kumatakenin-induced SKOV3 cell death. In addition, kumatakenin was found to reduce the

34

expressions of MCP-1 and RANTES, which are major determinants of macrophage

35

recruitment at tumour sites, in ovarian cancer cells. Moreover, kumatakenin inhibited the

36

expression of M2 markers and cancer-promoting factors, including IL-10, MMP-2/-9, and

37

VEGF, in macrophages stimulated by the ovarian cancer cells. In conclusion, these results

38

suggest that kumatakenin shows anti-cancer activities by inducing apoptosis of ovarian

39

cancer cells and inhibiting the alternative activation of TAM.

40 41 42

KEYWORDS:

43

(TAM)

kumatakenin, clove, ovarian cancer, apoptosis, tumour-associated macrophage

44 45 46 47 48



49

INTRODUCTION

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The flower buds of Syzygium aromaticum, commonly known as cloves, are a useful spice

51

that has long been used for many medicinal purposes.1 Cloves have been shown to possess a

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variety of biological activities, including anti-fungal, anti-bacterial, anti-oxidant, and anti-

53

inflammatory activities.2 Additionally, its potential anti-cancer activities have also been

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suggested.3-7 However, the bioactive components responsible for the anti-tumour activities

55

and their mechanisms of action were poorly understood.

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Development of cancer is mainly due to the uncontrolled growth of malignant cells as a

58

result of the imbalance between cell growth and apoptosis. Apoptosis is the best-studied

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modality of programed cell death,8 and triggering apoptotic cell death in cancer cells has been

60

widely used as a strategy to control cancer cell growth.9 In fact, many conventional cancer

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drugs are known to promote tumour cell death through the apoptotic pathways. Apoptosis is

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characterized by chromatin condensation, formation of apoptotic bodies, nucleosomal DNA

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fragmentation, and activation of caspases.

64 65

The tumour microenvironment, composed of various non-cancer cells, such as fibroblasts,

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endothelial cells, T cells, and macrophages, has become recognized as a major factor

67

influencing cancer development and progression. For example, most tumours contain a large

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number of macrophages as a major component of host leukocyte infiltration. It has been

69

suggested that circulating macrophages and monocytes are actively mobilized into tumours

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by locally produced factors such as RANTES and MCP-1.10 Macrophage infiltration has been

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implicated in poor clinical prognosis in various type of tumours.11-14 It has been found that

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the macrophages are converted into tumour-associated macrophages (TAMs) with an M2

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phenotype, which can change the tumour microenvironment to accelerate tumour progression.


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In fact, TAMs have been demonstrated to promote tumour growth, metastasis, angiogenesis,

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and immune suppression by producing a wide diversity of protease enzymes, cytokines, and

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growth factors.15 In this regard, TAM has been suggested as a potential therapeutic target for

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cancer treatment.16

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In the developed countries, the most lethal gynaecological cancer is ovarian cancer. Due

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to no or vague symptoms, most patients are diagnosed in stage III or IV. Despite achieve

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improvements in the standard management of taxane/platinum-based chemotherapy, the long-

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term-survival rates remains poor.17 Thus, it is important to discover the new remedial agents

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that increase patient survival rates and provide high-quality life for ovarian cancer patients. In

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this study, we evaluated the cell growth inhibitory effect of kumatakenin, a flavonoid isolated

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from cloves, in SKOV3 and A2780 cells, which are the most commonly used cellular models

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of ovarian cancer. In addition, the effects of kumatakenin on M2 polarization and pro-tumour

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activation of macrophages stimulated by ovarian cancer cells were investigated.

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MATERIALS AND METHODS

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Sample preparation

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Compounds and extracts that were used for the present study were prepared in our previous

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study.18 Briefly, cloves, flower buds of Syzygium aromaticum, were obtained from Kyung

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Dong Traditional Market in Seoul, South Korea, in June 2013. A voucher herbarium

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specimen (SYAR1-2013) was stored at College of Pharmacy, Kyung Hee University. The

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origin of cloves was confirmed by Dr. Dae Sik Jang (Kyung Hee University). The 70% EtOH

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extract was prepared by extraction of dried and powdered cloves (2.1 kg) with 20 L of EtOH

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(70 %) at 60 °C in a water bath (2 h). After extraction, the solvent was removed in vacuo at



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40 °C. The 70% EtOH extract (625.0 g) was successively partitioned with n-hexane, EtOAc,

100

and BuOH to give n-hexane- (303.1 g), EtOAc- (132.0 g), BuOH- (93.0 g), and water-soluble

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(95.7 g) extracts, respectively. Kumatakenin (9.0 mg), pachypodol (5.5 mg), quercetin (35.1

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mg), luteolin (14.0 mg), rhamnazin-3-O-β-D-glucoside (20.3 mg), and rhamnazin-3-O-β-D-

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glucuronide-6"–methylester (8.0 mg) were purified from EtOAc fraction of the 70% EiOH

104

extract as described previously in detail.18

105 106

Materials

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Fetal bovine serum (FBS), streptomycin, penicillin, and RPMI 1640 media were procured

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from Life Technologies Inc. (Grand Island, NY, USA). MTT was obtained from Molecular

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Probes Inc. (Eugene, OR, USA). Annexin V-fluorescein isothiocyanate (FITC), BD

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OptEIA™ set for MCP-1, and phenylmethylsulfonylfluoride (PMSF) were procured from BD

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Biosciences (San Diego, CA, USA). 2-Mercaptoethanol, phorbol myristate acetate (PMA),

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and propidium iodide (PI), were bought from Sigma Chemical (St. Louis, MO, USA). All

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inhibitors for caspases were from Calbiochem (Bad Soden, Germany). Enhanced

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chemiluminescence (ECL) reagent was from EMD Millipore (Billerica, MA, USA). Tris-

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buffered saline was purchased from Boster Biological Technology Ltd. (Wuhan, China).

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Caspase-3, caspase-9, and MMP-2 antibodies were procured from Cell Signaling Technology

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(Beverly, MA, USA). Caspase-8, MMP-9, CD206, and Trem-2 antibodies were procured rom

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Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibody was purchased from

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The Jackson Laboratory (West Grove, PA, USA).

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Oligonucleotide primers for real-time PT-PCR experiments were procured from Bioneer

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(Seoul, South Korea). Easy Blue® kit and protein lysis buffer were obtained from Intron

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Biotechnology (Seoul, South Korea). First-Strand cDNA synthesis kit was obtained from

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Amersham Pharmacia Biotech. (Oakville, ON, Canada). SYBR ® Premix Ex Taq was


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procured from (Takara, Tokyo, Japan). ELISA kits for RANTES, IL-10, and VEGF were

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purchased from Koma Biotech Inc. (Seoul, South Korea).

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Cell culture

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Human ovarian cancer cell lines (A2780 and SKOV3) and human monocytic cell line (THP-1)

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were originally from American Type Culture Collection (ATCC). Ovarian cancer cells were

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maintained in RPMI 1640 containing with streptomycin sulfate (100 µg/mL), penicillin

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(100 U/mL), and FBS (5%). THP-1 cells were maintained in RPMI 1640 containing with

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streptomycin sulfate (100 µg/mL), penicillin (100 U/mL), 2-mercaptoethanol (0.05 mM), and

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FBS (10%). To differentiate the THP-1 cells into macrophages, the cells were treated with

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PMA (100 nM) for 24 h. Tumour associated macrophages (TAMs) were prepared by

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stimulating THP-1 macrophages with conditioned medium from SKOV3 ovarian cancer cells

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for 24 h. To obtain the conditioned medium from ovarian cancer cells, SKOV3 cells (1 x 106)

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were seeded in 60 mm culture dish in 3 ml complete medium for 48 h, and the medium was

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collected from these cultures. After centrifugation of the medium for 3 min at 2500 rpm, the

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supernatants (i.e. conditioned medium) were harvested and stored at - 80 °C until use.

140 141

Cell viability assay

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In order to evaluate cell viability, MTT assay was carried out. After trypsinization of cells, the

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cells (5 × 104) were seeded per well of a 96-well plate. After overnight, various

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concentrations of extracts and compounds were added to each well containing the cells. After

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48 h incubation, 25 µL of MTT solution (5 mg/mL) was added into each well. After

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incubation for 4 h, media in each well was removed and 50 µL of DMSO was added to

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solubilize the formazan blue. Spectra Max (Molecular Devices, Sunnyvale, CA, USA) was

148

used to evaluate the absorbance (at 540 nm) of the samples.



149 150

Flowcytometric analysis for cell cycle and apoptosis

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After treatment with kumatakenin, the cells were harvested in ice cold phosphate-buffered

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saline (PBS). Following wash twice with cold PBS, cells were fixed with EtOH (70%), and

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stored at 4℃ for 1 h. Fixed cells were suspended in a staining solution supplemented with

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RNase (250 µg/mL) and PI (50 µg/mL). The suspended cells were incubated in the dark at the

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room temperature for 20 min. To analysis the cell distribution in the cell cycle, fluorescent

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intensity of the cells was measure using a Guava easyCyte flow cytometry system (EMD

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Milipore, Bilerica, MA, USA). As for apoptosis analysis, Annexin V-FITC and PI double-

158

staining were carried out in this study. After treatment of ovarian cancer cells with

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kumatakenin, the cells were suspended in ice cold PBS. After wash twice with ice cold PBS,

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cells were re-suspended with a binding buffer (140 mM NaCl, 2.5 mM CaCl2, 10 mM

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HEPES/NaOH, pH 7.4) containing PI (50 mg/mL) and Annexin V- FITC, and incubated in

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the dark at the room temperature for 20 min. The cells were centrifuged and apoptosis was

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measured with Guava easyCyte flow cytometry system.

164 165

Western blot analysis

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After treatment with kumatakenin, the cells were rinsed twice with ice cold PBS and total

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cellular protein were extracted using a protein lysis buffer (Intron Biotechnology) containing

168

protease inhibitors (0.5 mM PMSF and 5 µg/mL aprotinin). Bradford assay was used to

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determine the concentrations of the cellular protein. Total proteins (30 µg) were separated on

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10 – 15 % SDS-PAGE gel. After electrophoresis, the proteins in the gel were transferred onto

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polyvinylidene fluoride (PVDF) membranes by electroblotting for 2 - 4 h. The membranes

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were incubated with blocking solution, Tris-buffered saline supplemented with 5 % skimmed

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milk, at room temperature for 1 h. After rinse with Tris-buffered saline, the membranes were


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incubated with primary antibodies using Tris-buffered saline supplemented 5% skimmed milk

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and Tween20 (0.1%) overnight at 4°C or at room temperature for 4h. After discard of primary

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antibody solution, the membranes were washed four times to get rid of the primary antibodies.

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It was further incubated with various concentration of a horseradish peroxidase-conjugated

178

secondary antibody (1:1000 – 1:3000) at room temperature for 1 - 2 h. After incubation with

179

ECL solution for 10 min, the signals for the immunoreactivity bands were visualized and

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analyzed by Image Quant LAS-4000 (Fujifilm Life science, Tokyo, Japan).

181 182

Real-time RT-PCR analysis

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Reverse transcription of total RNA (1 µg) was performed using First-Strand cDNA synthesis

184

kit based on the manufacturer’s instruction. Total RNA was extracted using Easy Blue® kit.

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The cDNA was amplified using Thermal Cycler Dice Real Time PCR System and SYBR ®

186

Premix Ex Taq (Takara, Tokyo, Japan). A dissociation curve analysis revealed a single peak.

187

Expression of the gene of interest was analyzed using comparative Ct method, in which the

188

mean Ct of target cDNA is normalized to that of a reference gene, GAPDH. The sequences of

189

the primers used for real-time RT-PCR are listed in Suppl. Table S1.

190 191

Measurement of cytokine production

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The secretion of cytokines RANTES, IL-10, VEGF, and MCP-1 were quantified using a

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commercial ELISA kits, based on the manufacturer’s instruction. Plates were read using a

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microplate spectrophotometer (Spectra Max). All cytokines were measured separately in

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conditioned medium collected after treatment of the cells with kumatakenin for 24 h.

196

197

Statistical analysis



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One-way ANOVA or Student’s t-test were performed to determine statistically significant

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differences. P-values less than 0.05 were regarded as statistically significant.

200 201 202 203 204

RESULTS Kumatakenin from the ethyl acetate fraction of cloves inhibited the growth of human ovarian cancer cells

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The effect of solvent fractions of cloves on the viability of human ovarian cancer cells

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(A2780 and SKOV3) was investigated using MTT assay. Among the four fractions, only the

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ethyl acetate fraction (EtOAc) exhibited a significant cell growth inhibition with IC50 values

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below 100 µM in both A2780 and SKOV3 cells (Table 1). Among the 19 flavonoids isolated

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from the EtOAc fraction, six flavonoids (kumatakenin, pachypodol, quercetin, luteolin,

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rhamnazin-3-O-β-D-glucuronide-6"-methylester, and rhamnazin-3-O-β-D-glucoside), which

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showed some cytotoxicity in a previous study,18 were selected for further confirmation of the

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growth inhibitory activity in two ovarian cancer cells, A2780 and SKOV3. As shown in Fig. 1,

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rhamnazin-3-O-β-D-glucuronide-6"-methylester and rhamnazin-3-O-β-D-glucoside, each

214

with a sugar moiety, presented only mild growth inhibitory activity in both A2780 and

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SKOV3 cells. Luteolin and pachypodol markedly inhibited the viability in A2780 cells, but

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not in SKOV3 cells. Kumatakenin and quercetin had a potent growth inhibitory effect on

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both A2780 and SKOV3 cells. Quercetin, commonly found in many fruits, vegetable, leaves,

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and grains, has been intensively studied for its anticancer activities.19 Kumatakenin has been

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reported to be isolated from several plants including Buddlej and Alpinia species.20-21

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However, little has been reported about the biological activities of kumatakenin. Therefore,

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we further explored the molecular mechanism of the inhibitory effect of kumatakenin on

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ovarian cancer cell viability.


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Kumatakenin-induced apoptotic death in human ovarian cancer cells

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To investigate whether apoptotic cell death or cell cycle arrest involve in the inhibitory

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effect of kumatakenin (Fig. 2A) on the viability in SKOV3 human ovarian cancer cells, cell

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cycle distribution was investigated using flow cytometry analysis following PI staining.

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Kumatakenin induced an increase in sub G1 population of SKOV3 cells (Fig. 2B). No

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significant cell cycle arrest following kumatakenin treatment was observed. To assess

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whether kumatakenin-induced cell death is related to the induction of apoptotic cell death, an

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Annexin V-FITC and PI double-staining assay was carried out. As shown in Fig. 3,

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kumatakenin induced a marked increase in the population of apoptotic cells (Annexin V-

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FITC-positive cells). The data suggested that the growth inhibitory effect of kumatakenin in

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human ovarian cancer cells is associated with apoptosis, but not a cell cycle arrest.

235 236

Kumatakenin induced caspase-dependent apoptotic cell death in human ovarian cancer

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cells

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Apoptosis has been suggested to require caspase activation in many cases.22 Therefore,

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the effect of kumatakenin on the activation of caspase-3, -8, and -9 was evaluated.

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Kumatakenin markedly increased the cleaved forms of caspase-3, -8, and -9 in SKOV3 cells.

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To further confirm the involvement of caspase in kumatakenin-induced apoptotic cell death,

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caspase inhibitors were used (Fig. 4B). z-DEVD-fmk (caspase-3-specific inhibitor), z-IEVD-

243

fmk (caspase-8-specific inhibitor), and z-LEHD-fmk (caspase-9-specific inhibitor), and z-

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VAD-fmk (a broad caspase inhibitor) significantly suppressed the inhibitory effect of

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kumatakenin on cell viability. In addition, a specific inhibitor of caspase-3 (z-DEVD-fmk)

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significantly reversed the kumatakenin-induced apoptosis (Fig. 4C). These findings show that



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the apoptosis induced by kumatakenin is mediated by the caspase-dependent pathway in

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human ovarian cancer cells.

249 250

Kumatakenin inhibited chemokine MCP-1 and RANTES expression in human ovarian

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cancer cells

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It has been demonstrated that MCP-1 and RANTES are highly expressed in many cancer

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cells and play a critical role in macrophage recruitment to the tumour site.10 We investigated

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the effect of kumatakenin on the expression of MCP-1 and RANTES in human ovarian

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cancer cells. As shown in Fig. 5A, kumatakenin significantly reduced the mRNA levels of

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both MCP-1 and RANTES in SKOV3 cells. In addition, kumatakenin inhibited the secretion

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of MCP-1 and RANTES (Fig. 5B). These data indicate that kumatakenin may suppress TAM

258

recruitment to the tumour region by suppressing the secretion of chemokine MCP-1 and

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RANTES from cancer cells by regulating their expression in ovarian cancer cells.

260 261

Kumatakenin inhibited the expression of M2 phenotype markers and cancer-promoting

262

factors in macrophages stimulated by ovarian cancer cells

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It has been demonstrated that macrophages recruited to tumour sites can be induced by

264

cancer cells to be converted into TAMs with an M2 phenotype and tumour-promoting

265

activities.23-25 We examined the effect of kumatakenin on M2 polarization and pro-tumour

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activation of macrophages. As shown in Fig. 6, TAMs, macrophages stimulated by

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conditioned medium (CM) of SKOV3 cells, showed enhanced expression of M2 phenotype

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markers CD206 and Trem-2,26 compared to control macrophages, and kumatakenin

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significantly suppressed CD206 mRNA and protein expression in TAMs. In addition,

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kumatakenin significantly inhibited the expression and production of known tumour-

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promoting factors IL-10, VEGF, MMP-2, and MMP-9 in TAMs (Fig. 7). These data suggest


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that kumatakenin inhibits the alternative activation of macrophages, which contribute to

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ovarian cancer progression.

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DISCUSSION

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Cloves, commonly used as a food flavouring spice, have been suggested to possess anti-

278

cancer activities.2-7 For example, clove infusion has been shown to inhibit the incidence of

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hyperplasia and dysplasia in a mice model of benzo[a]pyrene (BP)-induced lung cancer.7

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Clove extract stimulates apoptotic cell death and cell cycle arrest in colon cancer cell HT-29

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and tumour growth was inhibited in a colon cancer xenograft model.5 Dwivedi et al have

282

suggested that clove oil extract has cell growth inhibition activity in various cancer cell lines,

283

including breast cancer (MCF7 and MDA-MB231), cervical cancer (HeLa), and oesophageal

284

cancer (TE13) cells.3 However, most studies used crude extracts to elucidate the anti-cancer

285

activity of cloves. the flower buds of Syzygium aromaticum. Key bioactive components

286

responsible for its anti-cancer activities, and their mechanisms of action, remain poorly

287

understood.

288

Until now, only a few compounds derived from cloves have been reported to possess anti-

289

cancer inhibitory effects. For example, eugenol, a phenylpropanoid present in essential oils of

290

cloves, increased apoptotic cell death through the activation of caspase-3 and p53 in colon

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cancer cells.27 In addition, it induced apoptosis via the ERα pathway in MCF7 breast cancer

292

cells.28 Oleanolic acid, a pentacyclic triterpenoid compound isolated from cloves, inhibits the

293

growth in various cancer cells5 and this inhibition was shown to be related to the PI3K/Akt

294

and MAPK pathway.29-30 In a recent study, we isolated 19 flavonoids from an EtOAc-soluble

295

fraction from the 70% EtOH extract of cloves and found that six flavonoids (kumatakenin,

296

pachypodol,

quercetin,

luteolin,

rhamnazin-3-O-β-D-glucuronide-6"-methylester,


and


297

rhamnazin-3-O-β-D-glucoside) are cytotoxic to human cancer cells. Among the six

298

flavonoids, we chose kumatakenin for further study because it demonstrated potent

299

cytotoxicity in two tested ovarian cancer cells and its biological activities remain poorly

300

characterized. It has been isolated from Buddlej and Alpinia species.20-21 However, there are

301

only two published works concerning its biological activities, to the best of our knowledge.

302

Zong et al. isolated kumatakenin along with other compounds from Buddlej aalbiflora, and

303

suggested that it possesses insecticidal activity against third-instar Plutella xylostella.31

304

Kumatakenin isolated from a dichrolomethane fraction of Pogostemon cablin extract has

305

anti-mutagenic activities against 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1).32

306

In this study, we have shown, for the first time, a potential anti-cancer activity by inducing

307

apoptosis in cancer cells and inhibiting alternative activation of macrophages.

308 309

It is generally accepted that caspase family are key mediator of apoptotic cell death. The

310

caspase-dependent apoptotic cell death is mainly activated by two signalling pathways: the

311

intrinsic or extrinsic pathway. Caspase-9 is an initiator caspase for the mitochondrial-

312

mediated intrinsic pathway while the activation of caspase-8 is associated with the extrinsic

313

(death receptor-mediated) pathway. Active initiator caspases caspase-9 and caspase-8 can

314

stimulate the effector caspases including caspase-3.33 Here, kumatakenin has shown to

315

stimulate the activation of both caspase-8 and -9. Additionally, their specific inhibitors (z-

316

IEVD-fmk and z-LEHD-fmk) markedly attenuated the kumatakenin-induced apoptosis in

317

SKOV3 cells. The results suggested that kumatakenin induces caspase-dependent apoptotic

318

cell death, and the two main apoptotic pathways are associated with kumatakenin-stimulated

319

apoptotic cell death in human ovarian cancer cells. It is of note that SKOV3 cells were more

320

sensitive to kumatakenin than A2780 cells. SKOV3 cells have been demonstrated to express

321

high levels of Neu/HER-2/ErbB-2, a member of the human epidermal growth factor receptor


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family, which have been regarded as a tumour promoting factor in various cancers, especially

323

breast cancer

324

human ovarian cancer. Thus, it is possible that the levels of HER-2 are associated with

325

kumatakenin-induced apoptotic cell death. Further studies should explore the effect of

326

kumatakenin on the HER-2 pathway and its detailed mechanism of action in human ovarian

327

cancer cells.

34-35

. Interestingly, HER-2 overexpression has been reported previously in

328 329

TAMs play an essential role in cancer progression, and targeting the M2 polarization and

330

pro-tumour activation of TAMs, as well as the aggressiveness of the cancer cell itself, is

331

considered a potentially effective therapeutic strategy for cancer patients.15,

332

classically activated macrophages (M1 macrophages) with high cytotoxicity against tumour

333

cells, M2 macrophages have been suggested to promote angiogenesis and tissue remodelling,

334

as well as to elicit an immunosuppressive phenotype by secreting various soluble factors such

335

as IL-10, VEGF, and MMPs.37-39 It is noteworthy that many natural products have been

336

demonstrated to exert anti-cancer activities by inhibiting TAM activation. For example,

337

epigallocatechin gallate, an antioxidant catechin commonly found in black and green tea, can

338

inhibit tumour growth through the suppression of M2 polarization as well as TAM infiltration

339

in an ex vivo and in vivo breast cancer model.40 Dietary flavonoid isoliquiritigenin effectively

340

suppresses colitis-associated tumourigenesis by interfering with the polarization of M2

341

macrophage.41 In addition, onionin A, isolated from onions, showed anti-cancer activities by

342

inhibiting the pro-tumour functions of TAMs as well as the cell proliferation in in vitro and in

343

vivo ovarian cancer model.16 Similarly, in this study, kumatakenin was found to inhibit the

344

production of MCP-1 and RANTES, major determinants of macrophage recruitment at

345

tumour sites, resulting in the inhibition of the alternative activation of macrophages. In

346

addition, kumatakenin inhibited the expression of M2 marker and the production of cancer-


36

Unlike


347

promoting cytokines (IL-10 and VEGF) and proteinase enzymes (MMP-2 and MMP-9) in

348

TAMs.

349

In conclusion, our findings show that kumatakenin promotes apoptosis in human ovarian

350

cancer cells and inhibits the activation of TAMs, indicating that the anti-cancer properties of

351

kumatakenin are related to its inhibitory effect on TAM, as well as on cancer cells. In the

352

future, detail investigations into the in vivo effect of kumatakenin and its mechanism of

353

action should be carried out.

354 355 356 357

ABBREBIATIONS

358

PI, propidium iodide; MTT, 3[4-dimethylthiazol-2-71]-2-5-diphenyl tetrazolium bromide;

359

PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen activated protein kinase; MMP-2/9,

360

matrix

361

interleukin-10; VEGF, vascular endothelial growth factor; RANTES, regulated on activation,

362

normal T cell expressed and secreted;

metalloproteinase-2/9; MCP-1,

monocyte

chemoattractant protein-1; IL-10,

363 364 365

AUTHOR INFORMATION

366 367

Corresponding Author

368

* (J.-H. Choi) Phone: +82-2-961-2172. Fax: +82-2-962-3885. E-mail: [email protected]

369 370

Funding

371

Basic Science Research grant (to J.-H. Choi) funded by the National Research Foundation of


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Korea (NRF) (NRF-2013R1A2A2A01067888 and NRF-2016R1A2B4008476).

373 374

Notes

375

The authors have nothing to declare.

376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396



397

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510


FIGURE LEGENDS

511 512

Figure 1. Effect of flavonoids from the EtOAc fraction of cloves on cell viability in

513

human ovarian cancer cells

514

A2780 and SKOV3 cells were treated with the indicated concentration (1.563, 3.125, 6.25,

515

12.5, 25, 50, 100 µM) of compounds for 48 h. The effect of kumatakenin (A), pachypodol (B),

516

quercetin (C), luteolin (D), rhamnazin-3-O-β-D-glucuronide-6"-methylester (E), and

517

rhamnazin-3-O-β-D-glucoside (F) on cell viability was determined by MTT assays.

518 519

Figure 2. Effects of kumatakenin on cell cycle regulation in human ovarian cancer cells.

520

(A) Chemical structure of kumatakenin isolated from cloves (B) SKOV3 Cells were treated

521

with the indicated concentration of kumatakenin (5, 15, 30 µM) for 48 h and then stained

522

with propidium iodide (PI). The cell cycle distribution profiles of the cells were determined

523

by flow cytometry. The graph indicates the percentages of cells in the sub G1, G0/G1, S, and

524

G2/M phases of cell cycle. The data are representative of three independent experiments.

525 526

Figure 3. PI / Annexin V-FITC staining assay for analysis of the apoptotic cell death in

527


528

SKOV3 cells were treated with 30 µM kumatakenin for the indicated times (0, 12, 24, 48 h)

529

and then co-stained with PI and Annexin V-FITC. The translocation of phosphatidylserine

530

was detected by flow cytometry. The data are representative of three independent experiments.

531 532

Figure 4. Involvement of caspase in kumatakenin-induced cell death in human ovarian

533

cancer cells



534

(A) SKOV3 cells were treated with kumatakenin (5, 15, 30 µM) for 48 h. Caspase-3, -8, -9

535

levels were determined by Western blot assay. β-Actin was used as a control. A representative

536

protein immunoblot of three independent experiments is shown. Data are shown as mean

537

band density normalized relative to β-actin. Data are presented as the means ± S.D. of three

538

independent experiments. * p< 0.05 (B) SKOV3 cells were pretreated with broad caspase

539

inhibitor z-VAD-fmk (50 µM), caspase-3 inhibitor z-DEVD-fmk (50 µM), caspase-8 inhibitor

540

z-IETD-fmk (50 µM), and caspase-9 inhibitor z-LEHD-fmk (50 µM) for 30 min, and then

541

treated with kumatakenin (15 µM) for 48 h. MTT assay was performed to determine the cell

542

viability after kumatakenin treatment. # p < 0.05 as compared with the control group and * p

543

< 0.05 as compared with the kumatakenin-treated group. (C) SKOV3 cells were pretreated

544

with caspase-3 inhibitor z-DEVD-fmk (50 µM) for 30 min, and then treated with

545

kumatakenin (15 µM) for 48 h. The cells were co-stained with PI and Annexin V-FITC. The

546

translocation of phosphatidylserine was detected by flow cytometry.

547 548

Figure 5. The effect of kumatakenin on the expression of MCP-1 and RANTES in

549


550

(A) SKOV3 cells were treated with kumatakenin (5, 15, 30 µM) for 24 h. Real-time RT-PCR

551

was performed to measure the mRNA levels of MCP-1 and RANTES in SKOV3 cells.

552

GAPDH was used as an internal control. (B) SKOV3 cells were treated with kumatakenin (30

553

µM) for 24 h. Levels of MCP-1 and RANTES in the culture media were quantified using

554

ELISA kits. Data are presented as the means ± S.D. of three independent experiments. * p
100

82.3 ± 8.1

Water fraction

> 100

> 100

588 589 590

a) IC50 is defined as the concentration that results in a 50% decreased in the number of cells compared to that of

591

the control. The values represent the means of results from three independent experiments with similar patterns.

592 593 594 595 596 597 598 599 600 601


Page 25 of 34


602 603 604

Supplementary Table S1

Primer sequences for real-time PCR analysis

605

Gene

sense primer

anti-sense primer

CD206

ACCTCACAAGTATCCACACCATC

CTTTCATCACCACACAATCCTC

Trem-2

TTGCCCCTATGACTCCATGA

CGCAGCGTAATGGTGAGAGT

MMP-2

ACCGCGACAAGAAGTATGGC

CCACTTGCGGTCATCATCGT

MMP-9

CGATGACGAGTTGTGGTCCC

TCGTAGTTGGCCGTGGTACT

VEGF

ATGGCAGAAGGAGGAGGGCA

ATCGCATCAGGGGCACACAG

IL-10

GACCAGCTGGACAACATACTGCTAA

GATAAGGCTTGGCAACCCAAGTAA

MCP-1

GCTCATAGCAGCCACCTTCA

GGACACTTGCTGCTGGTGAT

RANTES

CCTCATTGCTAGGCCCTCT

GGTGTGGTGTCCCGAGGAAT

GAPDH

GAGTCAACGGATTTGGTCGT

TTGATTTTGGAGGGATCTCG

606 607 608



fig 1 88x58mm (300 x 300 DPI)


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fig 2 87x60mm (300 x 300 DPI)



fig 3 81x42mm (300 x 300 DPI)


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fig 4A 86x56mm (300 x 300 DPI)



fig 4B, 4C 68x62mm (300 x 300 DPI)


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fig 5 82x60mm (300 x 300 DPI)



fig 6 90x63mm (300 x 300 DPI)


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fig 7A 82x59mm (300 x 300 DPI)



fig 7B, 7C 73x50mm (300 x 300 DPI)


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Effect of Kumatakenin Isolated From Cloves on the Apoptosis of

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