Chrysin, Abundant in Morinda citrifolia Fruit Water–EtOAc Extracts

May 3, 2016 - HPLC data revealed the predominance of chrysin in water−EtOAc extracts ... chrysin combined with apigenin also suppressed tumor growth...
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Chrysin, abundant in Morinda Citrifolia fruit water-EtOAc extracts, combined with apigenin synergistically induced apoptosis and inhibited migration in human breast and liver cancer cells Hsiu-Chen Huang, Cheng Huang, Yu Xuan Wei, Man Ching Shen, Yu-Hsuan Tu, and chia chi Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00766 • Publication Date (Web): 03 May 2016 Downloaded from http://pubs.acs.org on May 5, 2016

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Chrysin, abundant in Morinda Citrifolia fruit water-EtOAc extracts, combined

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with apigenin synergistically induced apoptosis and inhibited migration in

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human breast and liver cancer cells Cheng Huang†, Yu Xuan Wei# and Man Ching Shen#, Yu-Hsuan Tu #, Chia-Chi Wang #, Hsiu Chen Huang#* † National Research Institute of Chinese Medicine, Taipei 11221, Taiwan, ROC # Department of Applied Science, National Hsinchu University of Education, Hsinchu

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30014, Taiwan, ROC

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Running title: Chrysin combined with apigenin synergistically induced apoptosis and inhibited migration

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*

Corresponding authors: Hsiu-Chen Huang Dr. Hsiu-Chen Huang Department of Applied Science National Hsinchu University of Education Address: No.521, Nanda Rd., Hsinchu City 30014, Taiwan Tel: +886-3-5213132 ext.2756 Fax: +886-3- 5257178 E-mail: [email protected]

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Abstract

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The composition of Morinda citrifolia were determined using High-Performance

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Liquid Chromatography (HPLC), and evaluated the anticancer effects of Morinda

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citrifolia extract in HepG2, Huh7, and MDA-MB-231 cancer cells. Morinda citrifolia

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fruit extracts were obtained by using five different organic solvents, including hexane

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(Hex), methanol (MeOH), ethyl acetate (EtOAc), chloroform (CHCl3), and ethanol

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(EtOH). The water-ethyl acetate extract from Morinda citrifolia fruits was found to

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have the highest anticancer activity. HPLC data revealed the predominance of chrysin

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in Water-EtOAc extracts of Morinda Citrifolia fruit. Furthermore, the combined

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effects of co-treatment with apigenin and chrysin on liver and breast cancer were

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investigated. Treatment with apigenin plus chrysin for 72-96 h reduced HepG2 and

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MDA-MB-231 cell viability, and induced apoptosis through downregulation of

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S-phase

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receptor-related protein 6 (LRP6) expression. However, the combination treatment for

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36 h synergistically decreased MDA-MB-231 cell motility but not cell viability

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through downregulation of MMP2, MMP9, fibronectin, and snail in MDA-MB-231

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cells. Additionally, chrysin combined with apigenin also suppressed tumor growth in

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human MDA-MB-231 breast cancer cells xenograft through downregulation of ki-67

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and Skp2 protein. The experimental results showed that chrysin combined with

kinase-associated

protein-2

(Skp2)

and

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low-density

lipoprotein

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apigenin can reduce HepG2 and MDA-MB-231 proliferation, cell motility and induce

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apoptosis. It also offers opportunities for exploring new drug targets, and further

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investigations are underway in this regard.

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Keywords : Chrysin; apigenin; epithelial-mesenchymal transition; apoptosis;

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xenografts

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Introduction

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Herbal preparations from various medicinal plants have often been used for

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treatment and symptom relief. Despite the lack of solid evidence regarding their

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therapeutic benefits, around 41% of breast cancer patients are utilizing

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complementary and alternative medicines (CAM) according to data of the World

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Health Organization.1 Flavonoids as complementary medicine have been reported to

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possess various pharmacological properties including antioxidation, anti-inflammation,

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hepatoprotective, anti-atherosclerosis, anticancer, hypolipidemia, and hypoglycemia

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effects, antiviral, antibacterial, antiallergy, and so on. Flavonoids exist in plant and

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contains mamy polyphenolic compounds, such as o flavonols, flavones, flavanones

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and isoflavonoids.2 Over 4,000 varieties of biologically active flavonoids have been

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isolated and played an important role in human health. Chrysin and apigenin are

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flavones that differ in number of hydroxyl groups on their B ring. Apigenin has one

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hydroxyl group on its B ring on position 4' while chrysin did not have any hydroxyl

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group on its B ring. Previous studies have shown that hydroxyl group numbers on the

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B ring affected cytochrome P450 (CYP) 1 A enzyme activity.3 Although chrysin and

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apigenin have similar chemical structures and both compounds exert anticancer

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effects, they affect cancer cell growth through different mechanisms. Previous

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findings revealed enhancement in biological activities of chrysin when combined with

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apigenin.

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Both chrysin and apigenin are abundantly present in several varieties of fruits,

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vegetables, and herbs. Chrysin has prominent effects in preventing and treating cancer,

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including breast cancer and liver cancer.4 In addition, chrysin has also been reported

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to decrease chronic diseases such as diabetes5, cardiovascular diseases6, and

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inflammation. Apigenin exhibited a wide variety of anticarcinogenic effects in skin,

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prostate, colon, cervical, and breast cancer cells.7 The detailed mechanisms of chrysin

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and apigenin in breast and liver cancer prevention is still a lot unknown, and the

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synergistic anticancer effects between the two compounds also need to examine.

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Morinda Citrifolia (also called Noni) have been used to treat many diseases, including

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bacterial infections, parasitic infection, inflammation, cancer and many others.8 All

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parts of Morinda Citrifolia have been used as food and medicine including its fruit.

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Previous studies have shown that Methanol (MeOH) extracts of Morinda Citrifolia

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leaves contain many flavonoids, such as kaempferol, apigenin and luteolin. Ethyl

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acetate (EtOAc) extracts contain different flavonoids including quercetin.9 Morinda

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Citrifolia is a rich source of phytochemical constituents, namely flavonoids,

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triterpenoids, iridoids, and anthraquinones which have potent anticancer activity. Thus,

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Morinda Citrifolia has been used as complementary medicine. However, the chemical

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components of Morinda Citrifolia have not been extensively examined and their

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bioactivities merit further studies.

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This study focused on evaluating the composition and biological potential of

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Morinda Citrifolia and determining whether Morinda Citrifolia fruits contain chrysin

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and

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apigenin –induced cell growth inhibition in cancer cell and nude mice were also

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examined.

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Materials and methods

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Preparation of Morinda Citrifolia fruit extracts

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Morinda Citrifolia fruits were collected from Taiwan. For preparation of five different

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organic solvents extracts, 100 g of dried fruit of Morinda Citrifolia were immersed in

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1000 mL of Hex, CHCl3, EtOAc, EtOH or MeOH for 24 h at 4°C and then filtered

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with a filter paper. The eluates were dried after evaporation under vacuum at 55°C.

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For preparation of water-ethyl acetate (Water-EtOAc) extracts, 100 g of dried fruit of

apigenin.

The

mechanisms

responsible

for

chrysin

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combined

with

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Morinda Citrifolia were immersed in 1000 mL of deionized water and boiled for more

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than thirty minutes. The cooling water solvent pass through a filter paper. Water

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extracts of Morinda Citrifolia fruit were evaporated to dryness using vacuum rotary

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evaporator (80°C), followed by reconstitution with distilled water and sequential

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partitioning with equal volumes of EtOAc. Similarly, the eluates were dried by

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evaporation under vacuum at 55°C. The concentration used in the experiment was

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determined according to the dry weight of the extract.

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Cell viability test by

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tetrazolium bromide] assay

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Cells were treated with Morinda Citrifolia extracts, chrysin, apigenin, or chrysin plus

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apigenin for 72-96 h. Cell viability was then examined using MTT assay. The MTT

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formazan dye crystals were formed by metabolically viable cells and were dissolved

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in DMSO. The DMSO solution was then collected and detected using ELISA

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microplate readers at wavelength of 550 nm.

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Flow Cytometry

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The cells were washed with cold 1X phosphate-buffered saline (PBS). Next, the cells

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were fixed in ice-cold 80% ethanol overnight at -20℃. After fixation, cells were

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removed ethanol and washed with cold PBS. The fixed cells were incubated in 0.5%

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Ttriton X-100/PBS/ 1mg/ml RNase for 30min at 37℃. In order to stain DNA, the

MTT [3-(4,

5-dimethylthiazol-2-yl) -2,5-diphenyl

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fixed and permeabilized cells were added 1mg/ml propidium iodide (PI) for 30min.

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Finally, fluorescence dye emitted by the PI-DNA complex was quantified by

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FACScan flow cytometry.

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High performance liquid chromatography (HPLC) analysis of Morinda Citrifolia

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extracts

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The concentrations of polyphenolic compound in Morinda Citrifolia extracts were

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determined by HPLC with C18-MS packed column. In gradient elution, the mobile

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phase consists mobile phase A (15% Acetonitrile, 4% Ethyl acetate, 0.1% Formic

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Acid and 80.9% ddH2O) and mobile phase B (45% Acetonitrile, 4% Ethyl acetate,

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0.1% Formic Acid and 50.9% ddH2O). The polyphenols were detected by UV at 280

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nm.

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Morphological assay

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MDA-MB-231 and HepG2 cells were treated with DMSO, 10μM chrysin, 10μM

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apigenin, or 10μM chrysin plus 10μM apigenin for 96h. Cells morphology were

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examined using light microscopy.

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Trypan blue dye exclusion assay

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MDA-MB-231 and HepG2 cells were seeded into 6-well plates at 5 x 104 cell/well in

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Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum

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(FBS). After 24 hours of incubation, the medium was then changed and cells were

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treated with DMSO, 10µM chrysin, 10µM apigenin, or 10µM chrysin plus 10µM

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apigenin for 72-96 h in DMEM containing 2% FBS. After treatment with drugs for

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72-96 h, cells were harvested using treatment with 1X trypsin/EDTA solution and then

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stained with trypan blue. The dead cells stained with trypan blue in a hemocytometer

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were counted.

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Analysis of nuclear morphology

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MDA-MB-231 and HepG2 cells were plated on the coverslips in 6-well plates at 5 x

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104 cell/well and were treated with DMSO 10µM chrysin, 10µM apigenin, or 10µM

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chrysin plus apigenin for 96 h. After treatment, cells were fixed with 4%

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formaldehyde in PBS for 30 min at room temperature, and then stained the coverslips

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with cells using 4 mg/mL Hoechst 33258 for 30 min. The coverslips with Hoechst

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33258 staining were inverted onto glass slides and mounted with Vectashield. Finally,

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nuclear morphological changes was viewed under a microscope.

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Western blot analysis

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The assay were performed as we previously method10. After drugs treatment, cells

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were lysed with Gold lysis buffer and then centrifuged for 30 min at 12,000 rpm at 4

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℃. The supernatants were collected and were quantified by the Bio-Rad assay dye.

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Proteins

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polyvinylidene difluoride (PVDF) membrane. The membrane was then incubated with

were

separated

using

SDS-PAGE

and electrotransferred

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a different primary antibody, followed with horseradish peroxidase -conjugated

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secondary antibody. The blots were enhanced by chemiluminescence reagent.

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Immunofluorescence assay

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MDA-MB-231 cells were plated on coverslips in 6-well plates at 5 x 104 cell/well and

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were treated with DMSO 10µM chrysin, 10µM apigenin, or 10µM chrysin plus

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apigenin for 96 h. After treatment, the coverslips with cells were fixed with 4%

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formaldehyde in PBS for 30 min at room temperature, and blocked at 4℃ for 1hr.

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Then, the coverslips were stained with apoptosis-inducing factor (AIF) primary

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antibodies at 4℃ overnight, follow with the fluorescein isothiocyanate-conjugated

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secondary antibodies and were viewed under a microscope.

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Soft agar assay

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MDA-MB-231 cells were treated with DMSO, 10µM chrysin, 10µM apigenin, or

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10µM chrysin plus 10µM apigenin in 20% FBS/2XDMEM medium and mixed with

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0.7% agarose. Then, the mixture was plated in 0.35% agarose. Colonies were

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observed and counted under a microscope.

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Wound-healing assay

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MDA-MB-231 cells were grown to full confluency in 6-well plates in 10%

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FBS/DMEM medium. After incubated overnight, the cells were washed with 1XPBS

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and added with drugs in 2% FBS/DMEM medium for the indicated time. Wound gap

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in MDA-MB-231 cells were produced by scratching using yellow plastic tip. The

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migration of individual cells across the wound gap was observed.

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Animals assay

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2 x 106 cancer cells were implanted subcutaneously into female BALB/c nude mice.

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which purchased from the National Laboratory Animal Breeding and Research Center

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(Taipei, Taiwan). After tumors size about 200 mm3, the mice were then randomly

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divided into 4 groups (4 mice/group) for drugs treatment by intraperitoneal (IP)

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administration. The tumor tissues were fixed in 4% paraformaldehyde and embedded

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in paraffin for hematoxylin and eosin (H&E) and immunohistochemical (IHC)

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stainings.

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Statistical Analysis

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All values were expressed as Standard Error of the Mean (SEM). Student’s t-test was

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used for statistical comparison. Symbol (*,#) indicates that the values are significantly

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different from the control (*,p < 0.05; **, p < 0.01; ***, p < 0.001; #,p < 0.05;

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< 0.01;

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multiple groups. Symbol (§) indicates that the values are significantly more effective

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than either agent alone (§, p < 0.05; §§, p < 0.01; §§§, p < 0.001).

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Results

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Anticancer activities of various extracts against liver and breast cancer cells

##

,p

###

, p < 0.001). The analysis of variance (ANOVA) is used to compare

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Plant extracts rich in polyphenolic compounds have been safely employed as

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traditional Chinese medicine. The cell viability effect of Hex, CHCl3, EtOH, MeOH,

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EtOAc or Water-EtOAc extracts of Morinda citrifolia fruit on liver (HepG2 and Huh7)

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and breast (MDA-MB-231) cancer cells were evaluated by MTT assay. Figure 1A

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shows the growth response of three cell lines to six different extracts of Morinda

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Citrifolia fruit at various concentrations. As can be seen, the growth of HepG2 and

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Huh7 cell lines was inhibited by EtOH, EtOAc or Water-EtOAc extracts of Morinda

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Citrifolia fruit. Water-EtOAc extracts also showed the highest inhibition followed by

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EtOAc and EtOH extracts in MDA-MB-231 cells. These data suggested that

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Water-EtOAc extracts have the highest anticancer activity. Thus, Water-EtOAc

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extracts were selected for further analysis to evaluate the impact of anticancer activity

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in different cancer types. To identify the mechanisms behind the antiproliferative

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effect of Water-EtOAc extracts, MDA-MB-231, HepG2 and Huh7 cells were treated

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50-200 µg/ml Water-EtOAc extracts for 72 h and 96 h and the cell cycle distributions

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were measured using flow cytometric analysis. After Water-EtOAc extracts treatment,

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the percentage of HepG2 and MDA-MB-231 cells in the sub-G1 fraction increased

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(Figure 1B).

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Chemical composition of Water-EtOAc extracts of Morinda citrifolia fruit

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To establish the chemical composition of Water-EtOAc

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concentrations of polyphenols were determined using HPLC. In HPLC analysis, we

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used 21 different polyphenols standard including apigenin, chrysin, catechin, b

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epigallocatechin gallate (EGCG), picatechin gallate (ECG), kaempferol, luteolin,

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narigenin,

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theaflavin-3-gallate (TF2), caffeic acid, chlorogenic acid, p-coumaric acid, corilagin,

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ellagic acid, gallic acid, and syringic acid to analyze the contents of these polyphenols

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in Water-EtOAc extracts of Morinda citrifolia fruit. The contents of these polyphenols

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in WaterEtOAc extracts of Morinda citrifolia fruit were summarized in Table 1 and

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Figure 1C. The retention times of gallic acid, corilagin, and chrysin were 4.55, 10.76,

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and 65.9 min, respectively. Three compounds in Water-EtOAc extracts have the same

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retention time. These results indicated that Water-EtOAc extracts contained gallic acid,

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corilagin, and chrysin at concentrations of 36.54, 28.62, and 16.35 µg/g, respectively.

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Furthermore, combinations of Water-EtOAc extracts and chrysin were injected into

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the HPLC. As seen in Figure 1C, injection of Water-EtOAc extracts combined with

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chrysin resulted in overlapping peaks with identical HPLC retention times of 65.9 min.

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These results suggested that chrysin were predominantly present in Water-EtOAc

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extracts of Morinda citrifolia fruit.

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Anticancer activity of chrysin combined with apigenin against breast and liver

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cancer cell lines

myricetin,

quercetin,

rutin,

resveratrol,

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theaflavin

(TF1),

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To determine the most active component of Water-EtOAc extracts of Morinda

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citrifolia fruit, the cytotoxicity of chrysin pure compounds was examined. The cell

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viability effects of co-treatment with apigenin and chrysin on AU565, MDAMB231,

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and HepG2cells were evaluated using the MTT test. As shown in Figure 2A and 2B,

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the combination of 10 µM chrysin plus 10 µM apigenin in HepG2, MDA-MB-231,

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and AU565 cells synergistically dose- and time-dependently inhibited cancer cells

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growth, compared with cells treated with either agent alone. The combination index

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(CI) for chrysin in combination with apigenin consistently less than 1 represented

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synergism (data not shown). Furthermore, the cell cycle distributions were measured

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using flow cytometric analysis. The percentages of apoptotic HepG2 cancer cells were

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0.07, 1.57, 3.18, and 20.05% and 0.09, 3.17, 8.28, and 61.66% after treatment with

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DMSO, 10 µM chrysin, 10 µM apigenin, and 10µM chrysin plus 10µM apigenin for

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72 h (Figure 3A) for 96 h (Figure 3B) respectively. Similar results were obtained for

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MDA -MB-231, and AU565 cancer cells. Our results demonstrated that apigenin in

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combination with chrysin synergistically induced HepG2, MDA-MB-231, and AU565

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cancer cells apoptosis. After 10 µM chrysin and 10 µM apigenin cotreatment for 96 h,

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the morphology of HepG2 and MDA-MB-231 cells had more dramatic changes than

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either agent alone (Figure 4A). The above data were assessed using the trypan blue

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dye exclusion method and Hochest assay. Compared with either agent alone treatment,

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co-treatment with 10 µM chrysin plus 10 µM apigenin in HepG2 and MDA-MB-231

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cells induced cell death (Figure 4B), DNA fragmentation and chromatin condensation

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(Figure 4C). To identify the molecular mechanisms underlying synergistic, the

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apoptosis-related proteins such as Wnt signaling, Skp2, cyclin dependent kinase

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(CDK), Cdc25c, and Survivin were determined by western blotting analysis. Figure

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4D shows that exposure to chrysin plus apigenin for 96 h synergistically decreased

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p-LRP6, LRP6, β-catenin, Skp2, CDK1, CDK4, Cdc25c, and Survivin in HepG2 and

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MDA-MB-231 cells. Apoptosis-inducing factor (AIF) is a critical mediator of

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caspase-independent cell death. To determine whether the combined treatment of

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chrysin and apigenin induced caspase-independent cell death, the localization of AIF

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in MDA-MB-231 cells was observed using immunofluorescence staining experiments.

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As shown in Figure 4E, exposure to chrysin plus apigenin for 96 h activates nuclear

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translocation of AIF.

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Antimotility activity of chrysin combined with apigenin in MDA-MB-231 cells

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Anchorage independence growth played an important role in cancer cell motility,

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invasion, and metastasis.11-13 The effects of chrysin combined with apigenin on

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anchorage independence in MDA-MB-231 cells were examined using soft agar assay.

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The colony sizes exceeding 50 µm were scored. As shown in Figure 5A, 10 µM

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chrysin combined with 10 µM apigenin synergistically reduced the colony-forming 14

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capacity. The effects of chrysin combined with apigenin on cell motility were

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examined using wound-healing assay. The results revealed significant cytotoxic

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activity, of 10 µM chrysin combined with 10 µM apigenin in MDA-MB-231 cells for

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72h. In order to decrease the interference in antimotility evaluation, the

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wound-healing assay was performed after 24-36 h exposure to 10 µM chrysin plus 10

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µM apigenin. Figure 5B shows synergistically inhibited motility in MDA-MB-231

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cells after exposure to 10 µM chrysin combined with 10 µM apigenin for 24-36 h.

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However, there was no inhibition in cell growth (data not shown). Thus, these data

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suggest that 10 µM chrysin combined with 10 µM apigenin synergistically reduced

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MDA-MB-231 cell motility but not cell viability. Cell motility was affected by

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change in expression of specific proteins involved in cell adhesion such as MMP2,

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MMP9, and those related to epithelial-mesenchymal transition (EMT). To further

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clarify whether chrysin combined with apigenin resulted from dysregulation of

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metastasis- and EMT-related proteins such as MMP2, MMP9, fibronectin, snail, twist,

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slug, and vimentin were examined by western blotting analysis. Exposure to chrysin

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plus apigenin for 36 h synergistically decreased MMP2, MMP9, fibronectin, and snail

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in MDA-MB-231 cells (Figure 5C).

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Antitumor growth of apigenin in combination with chrysin in BALB/c nude mice

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xenograft tumor model

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To establish xenograft tumors for human breast cancer, MDA-MB-231 cells were

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implanted subcutaneously into female BALB/c nude mice, and the tumor growth

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inhibitory effects were observed after the intraperitoneal injection (IP) of DMSO,

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chrysin (10 mg/kg), apigenin (10 mg/kg), or chrysin (10 mg/kg) plus apigenin (10

306

mg/kg). As shown in Figure 6A and 6B, the chrysin plus apigenin group on day 28

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decreased tumor size, tumor weights, and the nuclear/cytoplasmic (N/C) ratio more

308

than either single agents alone treatment group. Furthermore, Cyclin A, Cyclin B,

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Cyclin D, Cdk1/CDC2, Cdk4, Skp2, cleaved caspase 3, cleaved caspase 8 and cleaved

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PARP expression levels in drug-treated and DMSO-treated mice were examined. The

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chrysin plus apigenin group decreased the expression levels of Cyclin A, Skp2, and

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Cdk1/CDC2 more than either single agents alone treatment group (Figure 6C). On the

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other hand, the cleaved caspase 3, caspase 8 and PARP protein levels were increased

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in the chrysin plus apigenin group. IHC data showed that, Ki-67 and Skp2 were

315

decreased in chrysin plus apigenin group more than either single agents alone

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treatment group (Figure 6D). Our findings suggested that chrysin plus apigenin

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co-treatment inhibited tumor growth in vitro and in vivo.

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Discussion

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Morinda citrifolia products have been gaining popularity because of their health

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benefits and are sold as dietary supplements around the world. The fruit of Morinda

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citrifolia is widely used as traditional medicine to treat many health disorders such as

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tumor, diabetes, arthritis, inflammation and chronic diseases.14-15 In the present study,

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we examined the cytotoxic activity of Morinda citrifolia fruit extract with solvents of

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different polarities. Water-EtOAc extracts of Morinda citrifolia fruit exhibited the

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highest decrease in Huh-7, Hep-G2, and MDA-MB-231 cell viability by increasing

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cell apoptosis. The HPLC data reported here for the first time indicated that chrysin

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was a predominantly bioactive compound present in Water-EtOAc extracts of

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Morinda citrifolia fruit. Combinations of phytochemicals are naturally found in plant

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foods, and many clinical studies have also used the different phytochemicals mixtures

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in cancer prevention. Thus, synergistic interactions between phytochemicals would

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promote the anti-carcinogenic activity.

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regarding synergistic interactions between phytochemicals. Thus, it's very important

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to identify their combined effects in patients with cancer. Apigenin is also found in

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Morinda citrifolia with no apparent toxicity reported.9 Chrysin and apigenin are both

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abundantly present in Morinda citrifolia, but differ in their anticancer mechanism. To

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our knowledge, this is the first report on chrysin combined with apigenin

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synergistically inducing apoptosis in cancer cells and in nude mice xenograft tumor

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cells through caspase-dependent and -independent pathways.

339

16

However, there was limited information

Skp2 and LRP6 have oncogenic potential and are overexpressed in breast and

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liver cancer. The LRP6/Wnt/β-catenin signaling is significantly upregulated in

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20-36% of human breast tissues 17 Skp2 is highly expressed in estrogen receptor (ER)

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-negative tumors.18-19 Scientists recently discovered that Skp2 is a downstream target

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gene of LRP6/Wnt/β-catenin signaling.20 Prior research has shown that the

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combination of chrysin with 1,2,3,4,6-penta-O-galloyl-β-D-glucose (5GG) decreased

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LRP6 and Skp2 and resulted in a synergistic inhibition of tumor cell growth.21 In this

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current study, we found that the combination of chrysin and apigenin also inhibited

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LRP6 and Skp2 protein expression and resulted in a synergistic inhibition of cell

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mobility and tumor cell growth in vivo and in vitro. The overall data suggest that the

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combination of chrysin and apigenin or 5GG could be a promising new treatments for

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breast cancer patients.

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EMT is the first step in the metastatic cascade. Numerous studies have indicated

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an important role of EMT in cancer drug resistance, cancer stem cell transformation,

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and cancer metastasis.22-26 Therefore, EMT is an important targeted therapeutic

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strategy against invasive breast cancer. The present results demonstrated that the

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combination of apigenin and chrysin for 36 h synergistically inhibited cancer cell

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migration and decreased EMT-related proteins including MMP2, MMP9, fibronectin,

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and snail in MDA-MB-231. Yang et al. (2014) reported that chrysin under serum-free

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condition exerts anti-metastatic activities in triple-negative breast cancer (TNBC)

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cells through downregulating EMT protein expression and suppressing Akt

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activation.27 In view of possible drug disturbance by serum, many studies preferred

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not to use serum media in order to enhance the drug effect. However, serum is

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required to ensure that cells are healthy and the absence of serum will cause stress to

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the cells. Previous experience showed an optimal amount of 2% serum used during

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drug treatment; and also indicated that chrysin combined with apigenin, in a low dose,

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synergistically decreased EMT-related protein expression. The present findings are

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consistent with previous results. However, whether chrysin combined with apigenin

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could decrease EMT protein by downregulation of Akt phosphorylation needs further

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study.

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In summary, the abilities of Water-EtOAc extracts of Morinda citrifolia fruit to

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induce apoptosis of breast and liver cancer cells were identified. Chrysin, abundant in

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Morinda Citrifolia fruit water-EtOAc extracts, combined with apigenin synergistically

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induced apoptosis in MDA-MB-231, AU565, and HepG2 cells and inhibited

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migration in MDA-MB-231 cells and BALB/c nude mice xenografts of

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MDA-MB-231 cells. According to the present findings, the combination of apigenin

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and chrysin could be considered for improving effectiveness in breast or liver cancer

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therapy.

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Conflict of Interest The authors declare no conflict of interest. Acknowledgements This work was supported by grants from the National Science Council, Taiwan.

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(NSC 100-2313-B-134 -001 -MY3, MOST 103-2313-B-134 -001 -MY3).

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Figure Legends

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Figure 1. (A) Cell viability comparison of HepG2, Huh7 and MDA-MB-231 cells

promotes

insights

into

triple-negative

breast

TNFalpha-induced

cancer

metastasis

epithelial-mesenchymal

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by

activating

transition

in

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treated with Hex, CHCl3, EtOH, MeOH, EtOAc or Water-EtOAc extracts of Morinda

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Citrifolia fruit. MTT assay showed different responses of HepG2, Huh7 and

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MDA-MB-231 cancer cells treated with different extracts of Morinda Citrifolia fruit

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for 72-96 h. (B) Effect of different extracts of Morinda Citrifolia fruit on cell cycle

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distribution of HepG2 and MDA-MB-231 cancer cells. Cell cycle distribution was

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assessed by flow cytometry. (C) Analysis of polyphenol content in Morinda Citrifolia

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fruit. Polyphenols in Water-EtOAc extracts of Morinda Citrifolia fruit were analyzed

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by HPLC.

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280 nm. Peaks: 1, ellagic acid; 2, ellagic acid + gallic acid; 3, chlorogenic acid; 4,

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catechin; 5, syringic acid + epigallocatechin gallate (EGCG) + rutin + caffeic acid; 6,

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corilagin; 7, narigenin + p-coumaric acid; 8, picatechin gallate (ECG); 9, myricetin;

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10, theaflavin (TF1); 11, resveratrol; 12, theaflavin-3-gallate (TF2); 13, luteolin; 14,

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quercetin; 15, apigenin; 16, kaempferol; and 17, chrysin.

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Figure 2. Effect of chrysin plus apigenin on cell viability of AU565, HepG2, and

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MDA-MB-231 cancer cells. Cells were cultured in DMEM supplemented with 10%

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fetal calf serum for 24 h, and then treated with DMSO, chrysin, apigenin, or chrysin

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plus apigenin for 72 h (A) or 96 h (B). Cell growth inhibition was determined using

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MTT assay. The number of viable cells after treatment is expressed as a percentage of

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the vehicle-only control. Data are presented as mean ± SE of three independent

HPLC chromatograms of the polyphenol standard mixture recorded at

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experiments. The combination of chrysin with apigenin was more effective than either

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agent alone: *, P < 0.05; **, p