Inhibitory Effects of Metabolites of 5-Demethylnobiletin on Human

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Inhibitory effects of metabolites of 5-demethylnobiletin on human non-small cell lung cancer cells Mingyue Song, Noppawat Charoensinphon, Xian Wu, Jinkai Zheng, Zili Gao, Fei Xu, Minqi Wang, and Hang Xiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01367 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 24, 2016

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Journal of Agricultural and Food Chemistry

Inhibitory effects of metabolites of 5-demethylnobiletin on human non-small cell lung cancer cells

Mingyue Song1#, Noppawat Charoensinphon1#, Xian Wu1, Jinkai Zheng1,3, Zili Gao1, Fei Xu1, Minqi Wang1, Hang Xiao*1,2 1

Department of Food Science, University of Massachusetts, Amherst, MA, USA College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan, P. R. China 3 Institute of Agro-Products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing, P.R. China 2

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Mingyue Song and Noppawat Charoensinpon contributed equally to this work.

* Corresponding Author: Hang Xiao (Tel: (413) 545-2281; Fax: (413) 545-1262; Email: [email protected])

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ACS Paragon Plus Environment


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Abstract

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5-Demethylnobiletin is a unique flavonoid found in citrus fruits with potential

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chemopreventive effects against human cancers. We previously identified three metabolites

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of 5DN, namely 5,4´ -didemethylnobiletin (M1), 5,3´,4´- tridemethylnobiletin (M2), and 5,3´-

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didemethylnobiletin (M3) in mice fed 5DN. Herein, we investigated the inhibitory effects of

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these three metabolites on NSCLC cells. Our results demonstrated that M1, M2, and

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especially M3 showed stronger inhibition on the growth and colony formation of H460 and

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H1299 cells compared to 5DN. Three metabolites significantly inhibited the tumorsphere

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formation of A549 cells. Flow cytometry analysis showed that all metabolites induced cell

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cycle arrest and cellular apoptosis, and these effects were also stronger than that of 5DN. The

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inhibitory effects of these metabolites were associated with their ability in modulating the key

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signaling proteins related with cell proliferation and apoptosis. Overall, our results provided

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basis for utilizing 5DN and its metabolites for chemoprevention of lung cancer.

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Keywords: 5-demethylnobiletin / metabolites / nobiletin / lung cancer cells /

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chemoprevention

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Introduction Extensive research has suggested that many bioactive compounds derived from fruits

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and vegetables may have various health-promoting activities 1. Polymethoxyflavones (PMFs)

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from citrus fruits have been documented to exhibit a broad spectrum of beneficial properties

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including anti-carcinogenic activities 2. Nobiletin (NBT) is one of the most abundant

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polymethoxyflavones found in orange and has also been the most studied PMF for its

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potential health benefits 2. Recently, a unique sub-class of PMFs, 5-demethylated PMFs have

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been isolated from citrus fruits and they have attracted increasing attention due to their potent

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biological activities. For example, 5-demethylnobiletin (5-hydroxy-6,7,8,3´,4´-

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pentamethoxyflavone, 5DN) have shown inhibitory effects against multiple type of human

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cancer cells3–5.

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Biotransformation can greatly influence the biological activities of dietary compounds6.

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Once absorbed in gastrointestinal tract, these compounds may undergo various

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biotransformation, leading to the production of metabolites with the different chemical

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structures that may influence their biological actions. Therefore, it is important to investigate

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the biological activities of the metabolites of dietary compounds to better understand their in

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vivo efficacy. Recently, the metabolites of NBT have been identified and studied for their

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health promotion actions. For example, 3´-demethylnobiletin7, 4´-demethylnobiletin8, and

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3´,4´-didemethynobiletin9 were reported as major metabolites of NBT in rodents. 4´-

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hydroxy-5, 6,7,8-tetramethoxyflavone was also reported as one of the metabolites of

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tangeretin10.

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Several studies have demonstrated that metabolites of NBT such as 3´,4´-

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didemethynobiletin had stronger anti-cancer11, anti-inflammatory12,and antioxidant 13 effects

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than its parental compound, nobiletin. We recently isolated and identified three metabolites of

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5DN from mice fed with 5DN, namely 5,4´ -didemethylnobiletin (5,4´dihydroxy-6,7,8,3´-

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tetramethoxyflavone, M1), 5, 3´,4´- tridemethylnobiletin (5, 3´,4´- trihydroxy-6,7,8-

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trimethoxyflavone, M2) ,and 5, 3´-didemethylnobiletin (5, 3´-dihydroxy-6,7,8,4´-

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tetramethoxyflavone, M3) 14. However, the biological activities of 5DN metabolites remained

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unknown.

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Lung cancer is the leading cause of cancer-related mortality in both men and women

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globally, and non-small cell lung cancer (NSCLC) is responsible for more than 80% of all

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lung carcinoma cases15. Therefore, the identification of potential agents for combating this

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disease is critically needed. 5DN has been reported to promote the polymerization of

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microtubules, induce G2/M phase arrest and trigger JNK-induced autophagy and apoptosis in

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NSCLC cells5, but the effects of the 5DN metabolites on NSCLC cells are unknown. Herein,

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we investigated the growth inhibitory effects of three 5DN metabolites (M1, M2 and M3) in 3

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human NSCLC cell lines H460, H1299 and A549.

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Materials and methods

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Cell cultures and treatments

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Human NSCLC cell lines H460, H1299, and A549 were purchased from American Type

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Cell Collection (ATCC, Manassas, VA) All cell lines were cultured in RPMI 1640 media

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(Mediatech, Herndon, VA) supplemented with 5% heat-inactivated FBS, 0.1 mg/mL of

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streptomycin and 100 units/mL of penicillin at 37 oC in humidified atmosphere with 5% CO2.

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Cells were kept sub-confluent and sub-cultured every 3-4 days. Cells between 4 and 25

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passages were used in all experiments. NBT, 5DN, M1, M2, and M3 (Figure 1) were

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chemically synthesized and identified as previously described14. DMSO was used as a vehicle

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to deliver all treatments to the cells, and the final concentration of DMSO in cell culture

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media was 0.1% v/v in all experiments.

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Cell viability assay

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H460 or H1299 cells were seeded at a density of 2,000 cells/well in 96-well plates. After

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24 hrs of incubation for cell attachment, cells were treated with different treatments in serum

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complete media. The cell viability was then determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (MTT) assay as we previously reported16,17.

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Colony formation assay

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H460 cells were seeded at a density of 750 cells/well in 6-well plates. At 24 hrs of

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incubation, cells were treated with NBT, 5DN and its metabolites in serum complete media.

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The media was refreshed every 4 days. After 12 days of treatment, the colonies formed were

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washed with PBS, then were stained with 0.2% crystal violet for 10 minutes. The residual dye

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was then washed with double distilled water. H460 colonies formed were quantified by

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scanning with a high-resolution scanner (HP Inc.).

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Flow cytometric analysis of cell cycle and apoptosis

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H460 or H1299 were seeded at a density of 5x104 cells/well in 6-well plates. After 24

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hrs of incubation for cell attachment, cells were treated with NBT, 5DN and its metabolites in

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serum complete media. After 24 hrs or 48 hrs of treatment, floating cells in media and

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adherent cells that were detached by brief trypsinization (0.25% trypsin-EDTA; Mediatech)

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were harvested. Cell pellets were washed with 1 mL of ice-cold PBS then were subjected to

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cell cycle and apoptosis analysis as we described previously3,17.

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Immunoblotting

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Whole Cell lysate were prepared as previously described3. Cells were seeded in 10 cm

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culture dishes. After 24 hrs of incubation for cell attachment, cells were treated with 5DN,

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and its metabolites. After another 24 or 48 hrs of treatment, cells were harvested, combined

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with floating cells, if any, and extracted with RIPA lysis buffer (Tris-Hcl pH 7.2, 25 mM;

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SDS 0.1%; Triton X-100 1%; sodium deoxycholate 1%; NaCl 0.15 M;

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ethylenediaminetetraacetic acid (EDTA) 1 mM) (Boston Bioproducts, Ashland, MA, USA)

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containing 1% protease inhibitor cocktail. Then the lysates were subject to western blotting

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analysis as we previously described3. Antibodies for p21Cip1/Waf1, p53, Cdc-2 (CDK-1), CDK-

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4, CDK-6, cyclin D1, cyclin B1, cleaved caspase-3 (Asp175), poly ADP ribose polymerase

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(PARP) and Bax were purchased from Cell Signaling Technology (Beverly, MA, USA). β-

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Actin (Sigma-Aldrich) was used as a loading control.

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Lung cancer cell tumorsphere formation assay

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A549 cells were cultured in serum-free Dulbecco modified Eagle medium: Nutrient

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mixture F-12 (DMEM-F12) (Gibco, Grand Island, NY) supplemented with B27 (Invitrogen,

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Waltham, MA), 20 ng/mL epidermal growth factor (EGF) (Sigma), 20ng/mL basic fibroblast

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growth factor (bFGF) (Sigma, St. Louis, MO), 100U /mL of penicillin and 0.1 mg/mL of

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streptomycin (Cellgro, Manassas, VA). Cells were then passed through sterile cell strainer

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(40 µm) in order to acquire single cell suspension that was seeded in 24-well ultra-low

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attachment plate (5000 cell/well) (Corning, Corning, NY) in same medium. After 24h,

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treatments were directly added into the wells, and cells were incubated for 14 days to allow

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the formation of tumorspheres. Then tumorspheres were gently collected with cell strainers,

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washed with PBS, and then transferred into 6-well plates that contained RPMI 1640 media

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supplemented with 5% heat-inactivated FBS, 100U /mL of penicillin and 0.1 mg/mL of

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streptomycin to allow cell attachment. After 20 hours incubation, tumorspheres were counted

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under microscope as previously described18,19.

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Statistical analysis

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Data were presented as mean ± SD for the indicated number of independently performed experiments. Analyses of variance (ANOVA) model was used to compare the differences 6


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among more than two groups followed by a post-hoc test for multiple comparisons (The

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Tukey’s Range Honesty Significant Difference test as described in figure legends). A p-

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value < 0.01 was considered statistically significant.

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Results

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Metabolites of 5DN showed stronger inhibitory effects on the growth of NSCLC cells

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than 5DN.

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H460 and H1299 NSCLC cells were treated with serial concentrations of NBT (10 – 50

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µM), 5DN (3 – 15 µM), M1 (2– 10 µM in H460, and 1 – 5 µM in H1299), M2 (2– 10 µM in

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H460, and 1 – 5 µM in H1299), and M3 (0.2– 1 µM in H460, and 0.1 –0. 5 µM in H1299)

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(Figure 2). After 72 hours of incubation, all 5 compounds inhibited the growth of two types

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of NSCLC cells in dose-dependent manner. 5DN showed stronger inhibitory effects than

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NBT. Moreover, all three metabolites of 5DN showed more potent inhibition than 5DN. In

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H460 cells, M2 showed stronger inhibition than M1, i.e., at 10 µM, M1 and M2 inhibited cell

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growth by 68% and 90%, respectively. In H1299 cells, M1 and M2 showed similar inhibition,

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i.e., at 10 µM, M1 and M2 inhibited cell growth by 70% and 69%, respectively. Markedly,

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M3 demonstrated much stronger inhibitory effects than both M1 and M2, i.e., M3 at only 1

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µM and 0.5 µM inhibited cell growth by 85% and 84% in H460 and H1299 cells,

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respectively. The estimated IC50 values (at 72 h) of NBT, 5DN, M1, M2, and M3 were 76.3

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µM, 12.2 µM, 7.0 µM, 3.8 µM, and 0.46 µM in H460 cells, respectively (Figure 3). The

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estimated IC50 values (at 72 h) of NBT, 5DN, M1, M2, and M3 were 54.8 µM, 12.2 µM, 3.3

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µM, 2.7 µM, and 0.25 µM in H1299 cells, respectively. It is noteworthy that the IC50 values

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of M3 were about 27-fold and 49-fold lower than those of 5DN in H460 and H1299 cells,

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respectively.

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Metabolites of 5DN showed stronger inhibitory effects on the colony formation of

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NSCLC cells than 5DN.

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We determined the inhibitory effects of NBT, 5DN and 5DN metabolites on the colony

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formation capacity of NSCLC cells. H460 cell line was selected in this assay as a model

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because they could form well defined and distinct colonies compared to the other two cell

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lines. H460 cells were subjected to the treatments with NBT (50 µM), 5DN (10 µM), M1 (5

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µM), M2 (5 µM), and M3 (0.5 µM) for 12 days. NBT and 5DN at the test concentrations

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showed trend of inhibition on the colony formation of H460 cells, but the results were not

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statistically significant (Figure 4). However, M1, M2, and M3 at lower concentrations exerted

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stronger inhibitory effects than NBT and 5HN. For instance, M1 and M2 at 5 µM inhibited

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the colony formation by 34% and 77%, respectively. Strikingly, treatment of M3 at only 0.5

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µM completely abolished all colonies formed. Overall, our results indicated the stronger

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inhibitory effects of 5DN metabolites than 5DN and NBT on colony formation of H460 cells.

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Metabolites of 5DN induced cell cycle arrest.

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To elucidate the mechanism of inhibition on cancer cells, the effects of NBT, 5DN, and

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5DN metabolites on cell cycle progression were determined in NSCLC cells. As shown in

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figure 5, NBT (50 µM) did not cause significant change in the cell cycle distribution of H460

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cells, but led to an accumulation of H1299 cells in G0/G1 phase. 5DN (10 µM in H460 cells,

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and 5 µM in H1299 cells) did not cause significant changes in the cell cycle distribution of

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H460 or H1299 cells. In contrast, M1 (10 µM in H460 cells, and 5 µM in H1299 cells)

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increased the cell population in G0/G1 phase by 40% and 50% in H460 and H1299 cells

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compared to the control, respectively. M2 (10 µM in H460 cells, and 5 µM in H1299 cells)

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increased the cell population in G2/M phase by 130% and 330% in H460 and H1299 cells

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compared to the control, respectively. Furthermore, M3 (1 µM in H460 cells, and 0.5 µM in

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H1299 cells) increased the cell population in G2/M phase by 440% and 340% in H460 and

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H1299 cells compared to the control, respectively.

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Metabolites of 5DN induced cellular apoptosis

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Triggering apoptosis in cancer cells is one of the effective strategies in cancer

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chemoprevention. Herein, we examined the effects of 5DN metabolites on cellular apoptosis

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of H460 and H1299 cells, and compared them with those produced by their parent compound

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5DN and NBT. Early and late apoptotic cells were quantified by Annexin V/Propidium

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Iodine (PI) double staining assay using flow cytometry. As shown in figure 6, NBT (50 µM)

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or 5DN (10 µM in H460 cells, and 5 µM in H1299 cells) did not significantly induce cellular

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apoptosis in H460 or H1299 cells. In contrast, all three 5DN metabolites significantly

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increased cellular apoptosis in both H460 and H1299 NSCLC cells. In H460 cells, M1 at 10

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µM, M2 at 10 µM, and M3 at only 1 µM increased early apoptotic cells by 2.8-fold (from

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3.0% to 8.5%), 6.1-fold (from 3.0% to 18.2%), and 12-fold (from 3.0% to 35.9%) compared

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to the control, respectively. In H1299 cells, M1 at 5 µM, M2 at 5 µM, and M3 at only 0.5 µM

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increased early apoptotic cells by 5.2-fold (from 1.7% to 8.8%), 15.1-fold (from 1.7% to

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25.7%), and 18.7-fold (from 1.7% to 31.8%) compared to the cells treated with vehicle

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control, respectively. Furthermore, treatment of M2 at 5 µM, M3 at 0.5 µM also increased the

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late apoptotic cells in H1299 cells by 5.7-fold (from 2.7% to 15.3%) and 6.4-fold (from 2.7%

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to 17.4%) compared to the cells treated with vehicle control, respectively.

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Metabolites of 5DN modulated expression of key signaling proteins related with cell

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cycle and apoptosis regulation

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In order to obtain more information on the molecular mechanisms underlying the

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inhibitory activities of 5DN metabolites on human NSCLC cells, we examined their effects

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on expression of key proteins related with cell cycle and apoptosis regulation in H460 cells. 9



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Our results showed that treatments with 5DN metabolites extensively altered the expression

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of these crucial signaling proteins (Figure 7). M3 at 1 µM (10-fold lower than the

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concentrations of 5DN, M1 and M2 used in this section) significantly elevated the levels of

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p21Cip1/Waf1 and p53, while decreased the levels of Cdc-2 (CDK-1) and cyclin B1. M3 was

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also found to elevate the levels of Bax, cleaved caspase-3 and cleaved PARP. Similar results

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were also observed for M2 (10 µM) but with less extent of those produced by M3. M1 (10

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µM) decreased the levels of cyclin D1, CDK-6 and CDK-4. M1 also elevated the levels of

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Bax, cleaved caspase-3 and cleaved PARP but with less extent of what produced by M2 and

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M3. The effects of 5DN on these signaling proteins were either insignificant or much less

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than those produced by M1, M2, or M3.

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NBT, 5DN, and 5DN metabolites inhibited the tumorsphere formation of human lung

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cancer cells.

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Accumulating evidence has supported that cancer stem cells might be initiators of the

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occurrence, progression, and recurrence of malignant tumors 20. The ability to form tumor

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spheroid bodies under serum-free condition is one of the cancer stem cells (CSCs)

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properties21. Lung CSCs were reported to have the capacity to survive under serum-free

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condition containing epidermal growth factor (EGF) and basic fibroblast growth factor

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(bFGF) and proliferate as cellular clusters known as “tumorspheres”22,23. We determined the

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inhibitory effects of NBT, 5DN, and 5DN metabolites on tumorsphere formation of human

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NSCLC A549 cells that formed well defined and distinct tumorsphere (H460 or H1299 cells

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did not form well defined tumorsphere). We found that all 5 compounds significantly

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suppressed tumorsphere formation of A549 cells compared to the control group (Figure 8).

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NBT at 50 µM inhibited tumorsphere formation by 50%, while 5DN at 10 µM reduced

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tumorsphere formation by 63%. M1 and M2 both at 10 µM reduced tumorsphere formation

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by 66% and 48%, respectively. M3 at only 2.5 and 5 µM resulted 73% and 81% inhibition on 10


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the tumorsphere formation. It is noteworthy that 5DN metabolites, especially M3 decreased

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the size of the tumorsphere formed.

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Discussion

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Accumulating evidence has revealed that PMFs had a broad spectrum of biological

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activities including anti-inflammatory, anti-carcinogenesis, and anti-atherogenic effects.

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These biological effects were closely associated with their chemical structures. In this study,

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5DN showed stronger inhibitory activity on human NSCLC cells than its permethylated

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counterpart NBT despite a small difference in their structure: demethylation at 5 position

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(figure 1). For example, the IC50 concentrations of 5DN were approximately in the range of 4

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to 6-fold lower than those of NBT in H460 and H1299 cells (figure 2 and 3), and 5DN was

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about 5-fold more effective in suppressing tumorsphere formation of A549 cells than NBT

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(figure 8). These results were consistent with our previous reports showing that 5-

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demethylated PMFs namely 5DN and 5-demethyltangeretin (5DT) had stronger effects in

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inhibiting both human lung3,16 and colon4 cancer cell growth than their permethylated PMF

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counterparts namely NBT and tangeretin. Overall, our results reinforced the notion that

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demethylation of PMFs at 5 position may lead to enhanced biological effects.

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Biotransformation dictates the overall biological activities of many dietary compounds

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in vivo because it may transform these compounds to a wide range of metabolites with

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different biological effects. Recently, we have identified M1, M2 and M3 as metabolites of

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5DN in mice fed with 5DN, and we also developed methods to chemically synthesize these

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metabolites that can be used for further investigation on their biological effects 14. We

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demonstrated that all three metabolites had stronger inhibitory effects on NSCLC cells. M1,

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M2, and M3 all showed lower IC50 concentrations than 5DN in inhibiting the growth of H460

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and H1299 cells, and they also had stronger inhibition on colony formation of H460 cells

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than 5DN. It is striking that M3 showed much more potent inhibition on cancer cells than

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5DN. For example, the IC50 concentration of M3 was approximately in the range of 27 to 49-

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fold lower than that of 5DN (figure 3). M3 at 0.5 µM abolished the colony formation of H460

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cells, while 5DN at 10 µM (20 fold higher than that of M3) showed marginal effects (figure

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4). Moreover, M3 at 2.5 µM led to stronger inhibition on tumorsphere formation of A549

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cells than 5DN at 10 µM (4 fold higher than that of M3) (figure 8). M1, M2 and M3 are

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demethylated derivatives of 5DN with demethylation at 4´-, at both 3´- and 4´-, and at 3´-

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positions, respectively (figure 1). Our results suggested that further demethylation of 5DN at

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4´-position (M1) or 3´-position (M3) significantly enhanced their inhibitory activities on

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cancer cells growth. Especially, demethylation at 3´-position (M3) alone led to a drastic

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enhancement of the inhibitory effects. However, demethylation at both 3´- and 4´-positions

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(M2) decreased the inhibitory activities on cancer cells growth compared to M3. These

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results demonstrated that biotransformation of 5DN generates metabolites (M1, M2, and M3)

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with enhanced inhibitory activities against human NSCLC cells in comparison with 5DN

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itself. These findings are important to provide insights for the action of orally administered

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5DN. For example, M1, M2 and M3 may contribute to the anticancer effects of 5DN in vivo

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after dietary administration of 5DN.

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Induction of cell cycle arrest and cellular apoptosis in cancer cells is one of the effective

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strategies to control cancer progression. Several PMFs have been reported to induce

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apoptosis in difference cancer cells. Demethylation of PMFs, especially at the 5-position, was

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found to be critical for their pro-apoptotic activities in lung3,16, colon24, and breast cancer

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cells25. Our flow cytometry analysis clearly indicated that 5DN metabolites (M1, M2, and

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M3) not only significantly induced cell cycle arrest, but also resulted in extensive cellular

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apoptosis in both H460 and H1299 human NSCLC cells. These effects were consistently

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stronger than those produced by 5DN. Especially, M3 at the concentrations 10-fold lower

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than that of 5DN induced much stronger cell cycle arrest and cellular apoptosis than 5DN.

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Our results at least partially explained the more potent inhibitory activities of M1, M2 and

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M3 on human NSCLC cells compared to those of 5DN. Our results also indicated that

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different 5DN metabolites induced cell cycle arrest at different phases. M1 caused cell cycle

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arrest at G0/G1 phase, while M2 and M3 caused cell cycle arrest at G2/M phase. These

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findings suggested that M1, M2 and M3 inhibited cancer cell growth through different

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mechanism of actions. The difference in chemical structures of M1, M2 and M3 might lead to

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their interaction with different cellular proteins and/or interaction with the same cellular

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proteins but with different affinity levels. These different interactions may ultimately result in

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different biological outcomes such as cell cycle arrest at different phases.

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Cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors play important roles in

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regulating cell cycle progression. The formation of Cyclin/CDK complexes drives the cell

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cycle transition while CDK inhibitors induce cell cycle arrest26,27. We found that M3

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decreased the levels of both cyclin B1 and Cdc2 (cdk-1), which at least in part, led to cell

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accumulation in G2/M phase (figure 7). The complex of cyclin B1/Cdc2 is mandatory for a

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cell to enter into mitosis at G2/M transition28. Furthermore, M3 caused an increase in the

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expression level of p21Cip1/Waf1 in the human NSCLC cells, which also contributed to the cell

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cycle arrest observed. p21Cip1/Waf1 is a negative CDK regulator that can directly bound to the

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cyclin B1/Cdc2 complex and inhibit its activity, which blocks cells in G2/M phase29,30. In

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addition, p21Cip1/Waf1 can diminish Cdc2 protein levels by decreasing Cdc2 mRNA

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transcriptions and its promoter activity31. H460 cell contains wild-type p53 that has a diverse

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spectrum of cellular functions and plays crucial roles in inhibiting cancer progression by

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impeding cell cycle progression, inducing cellular apoptosis, and DNA repair32. Our results

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showed that M2 and M3 treatments elevated the expression of p53, which further lead to

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G2/M cell cycle arrest by downregulating transcription of both cyclin B1 and Cdc229,31,33,34.

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Moreover, p21Cip1/Waf1 is one of the transcriptional targets of p53, and increased levels of p53

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can result in increased levels of p21Cip1/Waf1, which in turn contributes to induce cell cycle

284

arrest at G2/M phase29,35. M1 decreased the expression levels of CDK-4, CDK-6, and Cyclin

285

D1 (Figure 7). The complex of cyclin D1/CDK-4 or a complex of cyclin D1/CDK-6 is

286

required for entry into S phase26. Therefore, decrease in these complexes may contribute to

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the effect of M1 in inducing cell cycle arrest at G0/G1 phase in H460 cells. In addition, we

288

also found that M1, M2, and M3 activated caspase 3 and PARP, which are the hallmarks of

289

apoptosis. Moreover, we also observed that M1, M2, and M3 increased the expression levels

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of Bax which is one of the pro-apoptotic Bcl-2 family proteins36. Our results demonstrated

291

that M1, M2, and especially M3 profoundly modulated signaling proteins regulating cell

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cycle progression and cellular apoptosis.

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Overall, we demonstrated, for the first time, the potent inhibitory activities of 5DN

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metabolites M1, M2 and M3 against human NSCLC cells. The inhibition on the lung cancer

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cells by 5DN metabolites were associated with induction of cell cycle arrest and apoptosis as

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the result of alteration of related signaling proteins. Most importantly, we demonstrated for

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the first time that metabolites of 5DN showed more potent inhibitory effects on human

298

NSCLC cells than their parent compound 5DN. Future animal and clinical studies are

299

warranted to establish the tissue distribution of M1, M2 and M3 after dietary administration

300

of 5DN. This information will provide guidance for the rationale design of chemoprevention

301

strategies to utilize 5DN and other PMFs.

302 303 304

Abbreviations:

305

5DN, 5-Demethylnobiletin; M1, 5,4´ -didemethylnobiletin; M2, 5,3´,4´- tridemethylnobiletin;

306

M3, 5,3´-didemethylnobiletin; NBT, nobiletin; NSCLC, non-small cell lung cancer; PARP,

307

poly ADP ribose polymerase; EGF, epidermal growth factor; PI, Propidium Iodine; CSCs,

14


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cancer stem cells; bFGF, basic fibroblast growth factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-

309

2,5-diphenyl-tetrazolium bromide.

15



310

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This work was partially supported by a NIH grant (CA139174), an AICR grant, a USDA

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Special Grant on bioactive food componets, and a grant from National Natural Science

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Foundation of China (NSFC31428017). The authors have declared no confilct of interests.

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FIGURE CAPTIONS:

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Figure 1. Chemical structures of nobiletin (NBT), 5-demethylnobiletin (5DN), and metabolites of 5DN, namely M1, M2, and M3.

421 422 423 424 425 426 427 428

Figure 2. The growth inhibitory effects of NBT, 5DN, and 5DN’s metabolites (M1, M2, and M3) on H460 (A) and H1299 (B) human NSCLC cells. Cells were treated with serial concentrations of test compounds for 72 hrs. The growth inhibition was measured by MTT assay as described in materials and methods. Data represent mean ± SD (n=6), “a” indicated significant difference between the effects of 5DN metabolites at different concentrations and the effect of NBT at 50µM (p

Inhibitory Effects of Metabolites of 5-Demethylnobiletin on Human

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