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May 19, 2015
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Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated
apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2
activation
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Yu-Jen Jou1,2
Chih-Ho Lai6
Jung-Yie Kao2,¶
Chao-Jung Chen3,4
Chun-Hung Hua7
Cheng-Wen Lin1,9*
Yu-Ching Liu4
Tzong-Der Way5
Ching-Ying Wang8
Su-Hua Huang9
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Department of Medical Laboratory Science and Biotechnology, China Medical
University, Taichung, Taiwan
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Department of biochemistry, College of life sciences, National Chung Hsing
University, Taichung, Taiwan
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Graduate Institute of Integrated Medicine, China Medical University, Taichung,
Taiwan
Proteomics Core Laboratory, Department of Medical Research, China Medical
University Hospital, Taichung, Taiwan
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Department of Biological Science and Technology, China Medical University,
Taichung, Taiwan
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Department of Microbiology, School of Medicine, China Medical University,
Taichung, Taiwan
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Department of Otolaryngology, China Medical University Hospital, Taichung,
Taiwan
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School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China
Medical University, Taichung, Taiwan
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Department of Biotechnology, College of Health Science, Asia University, Wufeng,
Taichung, Taiwan
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Co-corresponding author
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*Corresponding author: Cheng-Wen Lin, PhD, Professor. Department of Medical
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Laboratory Science and Biotechnology, China Medical University, No. 91,
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Hsueh-Shih Road, Taichung 404, Taiwan
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Fax: 886-4-22057414
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Abstract
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γ-Bisabolene, one of main components in cardamom, showed potent in vitro and
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in vivo antiproliferative activities against human oral squamous cell carcinoma
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(OSCC).
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memebrane potential, leading to apoptosis of OSCC cell lines (Ca9-22 and SAS), but
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not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated
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with γ-bisabolene was identified using TiO2-PDMS plate and LC-MS/MS, then
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confirmed using Western blotting and real-time RT-PCR assays. Phosphoproteome
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profiling revealed that γ-bisabolene increased the phosphorylation of ERK1/2, protein
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phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone
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deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein-protein
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interaction network analysis proposed the involvement of PP1-HDAC2-p53 and
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ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis. Subsequent assays
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indicated γ-bisabolene eliciting p53 acetylation that enhanced the expression of
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p53-regulated apoptotic genes. PP1 inhibitor-2 restored the status of HDAC2
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phosphorylation, reducing p53 acetylation and PUMA mRNA expression in
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γ-bisabolene-treated Ca9-22 and SAS cells. Meanwhile, MEK and ERK inhibitors
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significantly decreased γ-bisabolene-induced PUMA expression in both cancer cell
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lines. Notably, the results ascertained the involvement of PP1-HDAC2-p53 and
γ-Bisabolene
activated
caspases-3/9
and
decreased
mitochondrial
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ERK1/2-p53 pathways in mitochondria-mediated apoptosis of γ-bisabolene-treated
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cells. This study demonstrated γ-bisabolene displaying potent antiproliferative and
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apoptosis-inducing activities against OSCC in vitro and in vivo, elucidating molecular
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mechanisms of γ-bisabolene-induced apoptosis. The novel insight could be useful for
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developing anti-cancer drugs.
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Keywords: γ-Bisabolene, oral squamous cell carcinoma, phosphoproteomics, histone
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deacetylase, p53 acetylation
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1. Introduction
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Oral squamous cell carcinoma (OSCC), accounting for over 90% of head and
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neck cancers, is highly invasive and metastatic, with high mortality [1, 2]. OSCC
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prevalence rises noticeably in Asia [3, 4]: e.g., the fourth most common fatal cancer in
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Taiwanese males. Tobacco, alcohol and betel quid are the most common risk factors,
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synergistically initiating mucosal changes: e.g., leukoplakia, erythroplakia, and
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carcinogenesis in the oral cavity [5]. Far less than half of OSCC cases survive five
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years in recent decades, even those receiving surgery, radiotherapy or chemotherapy
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[6]. Consequently, developing effective therapeutic agents against OSCC appears
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paramount for reducing mortality and morbidity.
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Many potential agents of apoptotic induction become anti-cancer therapeutics in
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pre-clinical or clinical trials [7]. Apoptosis, a mechanism of programmed cell death,
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initially triggers Fas receptor-mediated (caspase 8) or mitochondria-dependent
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(caspase
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cytomorphological
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degradation, and apoptotic body formation [8]. B-cell lymphoma (Bcl)-related family
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proteins, such as Bcl-2, BclXL, Bax, Bak, BAD, BIM, and BclXS, exhibit both anti-
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and pro-apoptotic manners in regulating mitochondria-dependent apoptotic pathways
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[8]. Tumor suppressor protein p53 has the proapoptotic activity via binding with
9)
pathways,
subsequently
changes:
e.g.,
activates
DNA
caspase-3,
fragmentation,
and
results
cytoskeletal
in
protein
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mitochondrial Bcl-2 that causes the release of cytochrome C from mitochondria [8, 9].
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Meanwhile, p53 induces transcriptional up-regulation of pro-apoptotic genes: e.g.,
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PUMA, Noxa, and Death receptor 5 (DR5) [8, 10]. Histone deacetylase 2 (HDAC2)
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overexpresses in human cancer cells of hematological, colon and colorectal,
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esophageal squamous cell carcinoma, renal, hepatic, lung, and skin [11]. HDAC2, an
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oncogenic factor, modulates p53 transcriptional activity via deacetylation of p53 at
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lysine320 [12]. Meanwhile, knockdown or inactivation of HDAC2 raises
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p53-dependent trans-activation of genes for cell cycle control and apoptosis,
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suppressing the proliferation of cancer cells [13]. Inhibitors of protein phosphatases
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(PP1 and PP2A) indicate HDAC2 phosphorylation relating with deacetylase-catalyzed
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transcriptional repression [14].
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Cardamom fruits (Elettaria cardamomum L.) are renowned ancient and aromatic
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spices in India and Sri Lanka, as Chinese herbal medicine for treatment of infectious
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diseases, inflammation, cardiovascular, neuronal and digestive disorders [15].
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Cardamom exhibits cholinergic and Ca2+ antagonistic effects on modulating gut
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excitatory, blood pressure, diuretic, and sedative activities [16]; it also demonstrates
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chemopreventive, anti-proliferative, anti-oxidative, and pro-apoptotic activities
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against non-melanoma skin and colon cancer [17, 18]. Phytochemical constituents of
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cardamom include α-terpineol, myrcene, heptane, subinene, limonene, cineole,
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menthone, α-pinene, β-pinene, linalool, nerolidol, β-sitostenone, phytol, eugenyl
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acetate, bisabolene, borneol, carvone, citronellol, geraniol, geranyl acetate,
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stigmasterol, and terpinene [19]. Some of these bioactive compounds show
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anti-oxidant, anti-microbe, anti-inflammatory, and anticancer effects [15, 20-21].
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Cineole manifests in vitro and in vivo inhibitory effects on human colorectal cancer
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and leukemia cells through inactivation of survivin and Akt as well as activation of
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p38 and apoptosis signaling [22]. Limonene, a potential chemotherapeutic agent,
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induces mitochondria-dependent apoptosis of human colon cancer cells by
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suppressing the PI3K/Akt pathway [23]. Geraniol has in vitro and in vivo
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antiproliferative and apoptosis-inducing actions on pancreatic, hepatic, breast, prostate,
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skin, and oral cancer via inhibiting mevalonate synthesis and cell cycle regulation
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[24-27]. Nerolidol suppresses azoxymethane-induced neoplasia of rat intestine [28],
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exhibiting an antioxidant activity with a significant rise of superoxide dismutase and
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catalase in mice against oxidative stress [29]. Derivatives of carvone and limonene
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also have antiproliferative ability against human prostate cancer via ERK activation
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and p21(waf1) induction [30].
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Cardamom and its derived compounds, exhibiting therapeutic potential against
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cancers, are further investigated in vitro and in vivo antiproliferative and apoptotic
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activities against human OSCC. Among cardamom-derived compounds, γ-bisabolene
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(4-(1,5-Dimethyl-4-hexenylidene)-1-methylcyclohexene)
definitely inhibited
the
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growth of oral cancer cell lines (Ca9-22 and SAS cells) with 50% cytotoxic
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concentration (CC50) of less than 30 µM. γ-Bisabolene concentration-dependently
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induced apoptosis via activation of caspases 3 and 9. Liquid chromatography-tandem
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mass spectrometry (LC-MS/MS), Western blot, and quantitative PCR analysis
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asserted the involvement of PP1-HDAC2-p53 and ERK1/2-p53 signaling pathways in
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γ-bisabolene-induced apoptosis of Ca9-22 and SAS cells.
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2. Materials and Methods
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2.1 Cell cultures
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Human gingival Ca9-22 and oral squamous cell carcinoma SAS cells, as well as
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oral fibroblast (OF) normal cells, were used in this study. Cells were cultured in
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DMEM medium (HyClone Laboratories, US) supplemented with 10% fetal bovine
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serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 1 mM
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sodium pyruvate, incubating at 37℃ in a humidified atmosphere of 5% CO2.
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2.2 Cardamom extraction and its marker compounds
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Cardamom fruits purchased from Chinese Herbal Medicine Department at
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China Medical University Hospital were identified by Professor Chao-Lin Kuo at the
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School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources of
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China Medical University, Taiwan. Cardamom fruits were grinded to powder; crude
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extract powder was dissolved in water via sonication for 90 min at room temperature.
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Water extract was collected following centrifugation at 12,000 rpm for 20 min,
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filtration with a Whatman No. 1 filter paper, and then lyophilization in a freeze dryer
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(IWAKI FDR-50P). Lyophilized extract powder was kept in sterile bottles at -20°C.
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Stock water extract (10 mg/ml) was dissolved in phosphate buffered saline.
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γ-Bisabolene
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(S)-(-)-Limonene
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diene),
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(S)-(+)-p-Mentha-6,8-dien-2-one) were purchased from Alfa Aesar-A Johnson
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Matthey Company. Sabinene hydrate, (−)-Borneol (endo-(1S)-1,7,7-Trimethylbicyclo
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[2.2.1]heptan-2-ol)
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3-Hydroxy-3,7,11-trimethyl- 1,6,10-dodecatriene) were obtained from Sigma-Aldrich
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Chemical Co. (St. Louis, MO).
and
(4-(1,5-Dimethyl-4-hexenylidene)-1-methylcyclohexene),
((S)-(-)-4-Isopropenyl-1-methylcyclohexene
(S)-(+)-Carvone
and
(-)-p-Mentha-1,8-
((S)-(+)-5-Isopropenyl-2-methyl-2-cyclohexenone
Nerolidol
(3,7,11-Trimethyl-1,6,10-dodecatrien-3-ol
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2.3 In vitro cytotoxicity Test
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Cytotoxic activity of cardamom extract and its marker compounds was assessed
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by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
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OSCC (Ca9-22 and SAS) or normal (OF) cells (4 × 104 cells/mL) plated in 96-well
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plates overnight and then treated with indicated concentrations of cardamom extract
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(0.1, 1, 10, and 100 μg/mL) and marker compounds (0.1, 1, and 10 μM) for 48 h at 37
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℃ in humidified atmosphere of 5% CO2. Cells in each well reacted with 10 μL of a
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MTT solution at 5 mg/mL for other 4 h incubation; formazan crystals in cells were
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dissolved in 100 μL of stop solution (29.9 ml HCl and 100 μL isopropanol). Finally,
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optical density (OD, 570-630 nm) in each well was measured with micro-ELISA
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reader; survival rate was calculated as the ratio of OD570-630 nm of treated cells to
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OD570-630 nm of mock cells, used to indicate 50% cytotoxic concentration (CC50) of
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γ-bisabolene on Ca9-22 and SAS cells.
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2.4 Apoptosis and cell cycle assay by flow cytometry
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Cells were treated with or without 0.1, 1, 5 and 10 μM of γ-bisabolene for 24
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and 48 h, harvested and washed in cold phosphate-buffered saline (PBS) for cell cycle
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analysis by propidium iodide (PI) staining, and apoptosis assays by annexin V-FITC
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and PI staining. In cell cycle assays, cells were fixed by 70% ethanol at -20℃
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overnight, washed in cold PBS, then re-suspended in PI solution (BioLegend) plus
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with 0.1mg/mL RNase. After 30-min incubation at room temperature in darkroom,
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cells underwent flow cytometry with excitation wavelength of 488nm and emission
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wavelength of >575 nm; sub-G1 (apoptotic), G1, S, and G2 phase cells were rated by
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BD FACSCanto™ system. In apoptosis assays, cells were resuspended in annexin V
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binding buffer and stained with the reagents of Annexin V-FITC and PI in Annexin
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V-FITC apoptosis Detection Kit (BioVision, CA). After incubation at room
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temperature for 15 min in the dark, fractions of early or late phase of apoptotic cells
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were analyzed using flow cytometry with excitation wavelength of 488nm and
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emission wavelength of 530 nm for FITC signal and >575 nm for PI signal
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(Becton-Dickinson, USA).
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2.5 Western blot
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Cells were treated with or without 0.1, 1, 5 and 10 μM of γ-bisabolene for 24
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and 48 h, collected, washed in cold phosphate-buffered saline (PBS), and lysed in 100
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µl of RIPA lysis buffer containing phosphatase and protease inhibitors (Roche
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Diagnostics). Lysates (100 µg) were pre-incubated in sample buffer (0.5mM Tris-HCl
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[pH 6.8], 10% SDS, 10% glycerol, 0.5% brilliant blue R) at 100℃ for 8 min, then
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separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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(SDS-PAGE). Proteins in gels were transferred onto nitrocellulose membranes
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(Millipore, Billerica, MA). After blocking with 5% skim milk in TBST (Tris-Buffered
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Saline, 0.1% Tween-20) buffer at 4℃ for 2 h, the membranes were incubated
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overnight with specific primary antibodies against caspase 3 (Calbiocem), caspase 9
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(upstate), p53, phospho-p53(S15), phospho-Akt(S473), acetylated-Lysine (Cell
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signaling), phospho-HDAC2(S394) (Bioss), 3-phosphoinositide dependent protein
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kinase-1 (PDK1), casein kinase 2α (CK2α), and β-actin (Cell signaling). Subsequently,
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the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG
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antibodies (Invitrogen, Carlsbad, CA) after TBST washing. Immunoreactive bands for
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the proteins of interest were developed with ECLTM Western Blotting Detection
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Reagents (GE Healthcare), and then visualized by autoradiography (X-ray film from
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Kodak, Rochester, NY).
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2.6 Mitochondrial membrane potential (MMP) detection assays
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MMP changes were detected by JC-1 (BioVision) and DiOC6(3) staining
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(CALBIOCHEM). For imaging, cells treated with or without γ-bisabolene were
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stained with JC-1 (10 µg/ml) at room temperature in the dark for 1 h, then visualized
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by fluorescent microscope (Olympus) post-washing with cold PBS. Green
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fluorescence of monomer JC-1 indicated MMP loss, as photographed with excitation
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wavelength of 488nm and emission wavelength of 530 nm; red fluorescence of JC-1
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specified a higher MMP in stained cells, and then was pictured with the emission
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wavelength of 590 nm. For quantitating MMP changes, cells were incubated with
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DiOC6(3) (20 nM) at 37℃ in the dark for 1 h, then immediately analyzed with a flow
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cytometer with an excitation wavelength of 488nm and an emission wavelength of
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530 nm.
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2.7 Cell lysis and gel-assisted digestion
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Cells treated with γ-bisabolene for 24 h were collected, washed, and then lysed in
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100 µl of RIPA (radioimmunoprecipitation assay) lysis buffer, as described above in
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Western blot assays. Total lysates (400 µg/50 μl) were dissolved in solutions of 18.5
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μl of acrylamide (40%, 29:1), 2.5 μl of 10% ammonium persulfate, and 1 μl of
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TEMED (tetramethylethylenediamine). Gel was cut into small pieces and washed
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repeatedly with 500 μl of ABC (ammonium bicarbonate) containing 50% (v/v) ACN
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(acetonitrile), samples dehydrated with 100% ACN and thoroughly dried by
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SpeedVac. Proteolytic digestion was performed with trypsin (1:25 (w/w)
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trypsin-to-protein ratio) in 25 mM ABC overnight at 37°C. Peptides were derived
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from gel via sequential extraction with 200 μl of 25 mM ABC, 200 μl of 0.1% (v/v)
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TFA in water, 200 μl of 0.1% (v/v) TFA (trifluoroacetic acid) in ACN, and 200 μl of
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100% ACN. All extracted solutions were combined and concentrated in SpeedVac,
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then applied for purification of phosphopeptides and nanoLC-MS/MS analysis.
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2.8 Phosphopeptide enrichment by TiO2-PDMS coated plate
On-plate
phosphopeptide
enrichment
method
using
the
TiO2-
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polydimethylsiloxane (PDMS) coated plate was reported by Chen et al. (2014) [31].
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In brief, PDMS-coated plate was first fabricated by coating PDMS prepolymer on a
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glass plate. A roller flattened the PDMS prepolymer to make a thin layer on the plate
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that was incubated in an oven for polymerization at 80°C for 1 h. Once PDMS film
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formed, the plate was washed with 0.1% FA solution to remove incompletely
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polymerized monomers [32]. Aliquots (5 µL) of TiO2 particle solution (1mg in 500 μl,
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70% ACN) were deposited on PDMS-coated plate to make TiO2 spot arrays; the plate
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was subsequently incubated in an oven at 80°C for 10 min, flushed with water, then
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washed with 80% ACN/2% TFA solution to remove contaminants and nonspecifically
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adsorbed compounds. Protein digest samples dissolved in loading buffer (80% ACN,
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2% TFA, and 20-200 mg/mL of DHB) were loaded onto TiO2 spots, incubated for 1
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min, then washed in 80% ACN/2% TFA solution to remove non-phosphorylated
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peptides. Phosphopeptides were eluted with 3-5 µL of 0.05% NH4OH, dried by
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centrifuge, and followed by resuspension in 0.1% FA solution and nanoLC-MS/MS
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analysis.
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2.9 NanoLC-MS/MS
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Identification of phosphopeptides was performed with a nanoflow UPLC system
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(UltiMate 300 RSLCnano system, Dionex, Ameterdam) coupled with a captive spray
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ion source and a Q-TOF mass spectrometer (maXis impact, Bruker). Samples were
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injected into a home-made tunnel-frit trap column (C18, 5 μm, 180 μm x 20 mm) with
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a flow rate of 10 μL/min for a duration of 4 min [33, 34]. The trapped peptides were
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separated by a commercial analytical column (Acclaim PepMap C18, 2 μm, 100 Å, 75
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μm x 250 mm, Thermo Scientific) with the acetonitrile/water gradient of 1-40% at a
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flow rate of 300 nL/min. For MS detection, peptides with a charge of 2+, 3+, or 4+
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and intensity above 50 counts were chosen for data-dependent acquisition, set to 1 full
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MS scan (400-2000 m/z) with 1 Hz, and switched to 10 product ion scans (100-2000
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m/z) with 5 Hz.
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2.10 Protein Identification
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NanoLC-MS/MS spectra were deisotoped, centroided, and converted to .xml files
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by DataAnalysis software (version 4.1, Bruker Daltonics). To identify proteins, mass
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spectra obtained were compared to SwissPort database (release 51.0) via MASCOT
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algorithm (version 2.2.07) with search parameters of peptide and MS/MS mass
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tolerance
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modification-cabamidomethyl (Cys), variable modification-oxidation (Met), and
set
at
0.05
Da,
taxonomy-human,
enzyme-trypsin,
fixed
18
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phosphorylation (Ser, Thr, Tyr). Peptides were identified if MASCOT individual ion
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scores exceeded 30.
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2.11 Label-Free Quantification
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LC-MS/MS spectra were converted to xml files using DataAnalysis (Version 4.1,
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Bruker), then the files were searched against Swissport database with the MASCOT
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algorithm (version 2.2.07). Label-free quantitative proteomics was accomplished by
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LC-MS replicated runs of different groups (n=4); the results were processed to exhibit
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the molecular feature using DataAnalysis 4.1 and ProfileAnalysis software 2.0
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(Bruker Daltonics). For inter-group comparison, results were transferred to
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ProteinScape 3.0 (Bruker Daltonics) using t-tests, together with protein identification
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and quantified peptide information of every protein from each group.
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2.12 Pathway analysis with META core software
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Biological pathways of regulated proteins in responses to γ-bisabolene were
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analyzed by GeneGO Meta-Core software (GeneGo Inc, Encinitas, CA). Direct
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networks, molecular pathways, and biological diseases among identified proteins were
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considered, and then derived from statistical significance of association between
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treatment with or without γ-bisabolene (p-value cutoff ±10).
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2.13 Real-time RT-PCR
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Total RNA (1000 ng) was extracted from cells treated with or without
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γ-bisabolene using the RNA purification kit (Invitrogen), reverse-transcribed into
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cDNA with oligo dT primer and SuperScript III reverse transcriptase (Invitrogen). To
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quantify gene expression in response to γ-bisaboene, two-step RT-PCR using SYBR
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Green I was performed as in our prior report [35]. Oligonucleotide primer pairs in our
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study: (1) forward primer 5’-CAGTGGAGGCCGACTTCTTG-3’ and reverse
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primer5’-TGGCACAAAGCGACTGGAT-3’ for human caspase 3, (2) forward primer
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5’-TGTCCTACTCTACTTTCCCAGGTTTT-3’
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5’-GTGAGCCCACTGCTCAAAGAT-3’ for human caspase 9, (3) forward primer
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5’-AGTGGGTATTTCTCTTTTGACACAG-3’
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5’-GTCTCCAATACGCCGCAACT-3’ for human Bim, (4) forward primer
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5’-GACGACCTCAACGCACAGTA-3’
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5’-CACCTAATTGGGCTCCATCT-3’ for human PUMA, (5) forward primer
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5’-GAGATGCCTGGGAAGAAGG-3’ and reverse primer 5’-TTCTGCCGGAAGTT
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CAGTTT-3’
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5’-CACCTCACACCGGTAATCC-3’
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5’-AGAGAGGCACAGGAGGCATA-3’ for human
for
human
and
and
and
Noxa,
reverse
reverse
reverse
(6)
and
forward
reverse
cAMP-dependent
primer
primer
primer
primer
primer
PKAreg
20
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(PKA-R1), (7) forward primer 5’-GATGTGAATGGGCAGTTAGTC-3’ and reverse
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primer 5’-ATGGTGCCTATACTCCA-3’ for human 3-phosphoinositide-dependent
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protein kinase 1 (PDK1), (8) forward primer 5’-GAACGCTTTGTCCACAGTGA-3’
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and reverse primer 5’-TATCGCAGCAGTTTGTCCAG-3’ for human protein kinase
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CK2α, (9) forward primer 5’-AATGGAGAGTATGCTCATCAGTG-3’ and reverse
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primer 5’-ACTCTTCTAACTGCCATAGCACC -3’ for human T-complex protein 1
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(TCP1), and (10) forward primer 5’-CCACCCATGGCAAATTCC-3’ and reverse
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primer 5’-TGGGATTTCCATTGATGACAAG-3’ for human GAPDH. Real-time
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PCR reaction mixture contained 2 μl of cDNA (reverse transcription mixture), 200
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nM of each primer pair in SYBR Green I master mix (LightCycler TaqMAn Master,
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Roche Diagnostics). PCR was performed by amplification protocol consisting of 1
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cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 sec, and
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60°C for 1 min. Specific products were amplified and detected in ABI PRISM 7300
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sequence detection system (PE Applied Biosystems). Relative changes in mRNA
327
levels of indicated were gene normalized by housekeeping gene GAPDH.
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2.14 Inhibitory effect of protein phosphatase 1 inhibitor-2 on p53 acetylation
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Protein phosphatase 1 inhibitor-2 (I-2) was purchased from New England
331
Biolabs. To examine the direct translocation of I-2 across cell plasma membranes,
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Ca9-22 cells were pre-treated with I-2 at 4 °C or 37 °C for 1 h, washed three times
333
with PBS for 5 min, treated with or without γ-bisabolene for 24 h, then followed by
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cell cycle analysis with flow cytometry. For evaluating the acetylation status of p53
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using immunoprecipitation, lysate of cells treated with single or both of γ-bisabolene
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and I-2 was incubated with anti-p53 antibodies for 4 h at 4 °C, followed by addition
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of protein A-Sepharose beads and additional 2 h of incubation. After centrifugation,
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pellets were washed with NET buffer (150 mM NaCl, 0.1 mM EDTA, 30 mM
339
Tris-HCl, pH 7.4), and then dissolved in 2X SDS-PAGE sample buffer for Western
340
blot assays, as described above. Resulting blots were probed with primary antibodies
341
against p53 and acetylated-Lysine (Cell signaling), interacted with HRP-conjugated
342
anti-mouse IgG antibodies (Invitrogen, Carlsbad, CA), then followed by enhanced
343
chemiluminescence detection after TBST washing. The ratio of acetylated p53 to p53
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was calculated according to the relative density of indicated protein band. In addition,
345
relative mRNA levels of p53 target gene PUMA in each cell group treated with or
346
without I-2 were performed using real-time PCR, as described above.
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2.15 In vivo studies
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Animal experiment was conducted following institutional guidelines and
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approved by Institutional Animal Care and Use Committee (IACUC) at China
22
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Medical University. Balb/c-nu/nu nude mice (4-6 weeks age) were purchased from
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BioLASCO Taiwan Co., Ltd (Taiwan), and maintained in Laboratory Animal Center
353
at China Medical University. The mouse xenograft model and in vivo tumor volumes
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were modified as previously described [36]. In brief, oral cancer SAS cells
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(1 × 107/0.1 ml DMEM) were subcutaneously injected into the dorsal site of the nude
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mice. After 1 week, tumor volumes approximately reached 100 mm3, and then the
357
mice initially received the intraperitoneal treatment with or without 30 μL of 50
358
mg/kg γ-bisabolene every 2 days. After 10 times treatment, mice were sacrificed and
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tumors were collected, weighted, and measured the volumes.
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2.16 Statistical analysis
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Three independent experiments were performed for measuring mean ± standard
363
error (mean ± S.E.). Group means were compared using Student's t-test; P < 0.05 was
364
considered statistically significant.
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366
3. Results
367
3.1 Growth inhibition and apoptosis induction of oral cancer cells by cardamom
368
extract and γ-bisabolene in a dose-dependent manner
369
To examine the growth inhibitory ability of cardamom extract and its
23
370
derived compounds, the survival rates of human oral cancer cell lines (Ca9-22 and
371
SAS) and normal oral fibroblast cells were measured using MTT assays 2 days post
372
treatment (Supp. Fig. 1, Fig. 1A). Cardamom extract concentration-dependently
373
inhibited the growth of oral cancer Ca9-22 cells, exhibiting a CC50 value of 81.2
374
μg/ml (Supp. Fig. 1A). Among cardamom derived compounds, only γ-bisabolene had
375
a significant reduction of human oral cancer cell growth in a concentration-dependent
376
manner (Supp. Fig. 1B). The CC50 value of γ-bisabolene to human oral cancer cells
377
ranged from 5.15 μM (Ca9-22 cells) to 29.5μM (SAS cells). Meanwhile, cell cycle
378
analysis using PI staining showed γ-bisabolene increasing sub-G1 fractions of Ca9-22
379
and SAS oral cancer cells in a time-dependent manner (Figs. 1B and 1C). However,
380
γ-bisabolene was less to cytotoxic to normal human oral fibroblast cells (Fig. 1A); no
381
sub-G1 fraction was observed in γ-bisabolene-treated oral fibroblast cells (Fig. 1D).
382
The results indicated γ-bisabolene, a cardamom derived compound, exhibiting the
383
anti-proliferative activity on human oral cancer cell lines, but not normal oral
384
fibroblast cells.
385
386
3.2 Activation of caspase 9-dependent and mitochondria-mediated apoptosis induced
387
by γ-bisabolene
388
To test whether γ-bisabolene induces apoptosis of human oral cancer cells
24
389
and normal fibroblast cells, γ-bisabolene-treated cells were analyzed using annexin
390
V-FITC and PI staining. The fractions of early (annexin-V positive/PI negative) and
391
late (annexin-V positive/PI positive) apoptosis were determined by flow cytometer
392
(Figs. 2A-2B). γ-Bisabolene elicited the dose-dependent increase of early apoptosis
393
on human oral cancer cells, but no apoptotic effect on normal oral fibroblast cells. To
394
further examine the influence of γ-bisabolene on caspase expression, oral cancer cell
395
lines Ca9-22 and SAS were treated with or without γ-bisabolene, then harvested 24 h
396
post treatment for analyzing the mRNA levels of caspases 3 and 9 using real-time
397
PCR (Figs. 2C and 2D). Quantitative RT-PCR revealed γ-bisabolene significantly
398
up-regulating the mRNA expression of caspase 9 in both oral cancer cell lines.
399
Subsequently, Western blot was used to analyze active forms of caspases in
400
γ-bisabolene-treated Ca9-22 and SAS cells 24 h post treatment (Figs. 2E and 2F).
401
Active forms of caspases 3 and 9 obviously elevated in Ca9-22 and SAS cells in a
402
concentration-dependent manner. The results demonstrated the apoptosis-inducing
403
ability of γ-bisabolene to human oral cancer cells.
404
Since caspase 9 is an initiator caspase linked with mitochondria death
405
damage [37], the change of mitochondrial membrane potential (MMP) in
406
γ-bisabolene-treated cancer cells was subsequently explored using JC-1 and DiOC6(3)
407
staining (Fig. 3). The images of oral cancer cells stained with JC-1 indicated
25
408
γ-bisabolene
409
concentration-dependent manners, appearing that γ-bisabolene triggered the loss of
410
MMP in Ca9-22 cells (Fig. 3A). Quantitative analysis of MMP changes using
411
DiOC6(3) staining demonstrated γ-bisabolene strongly declining the MMP of human
412
oral cancer Ca9-22 and SAS cells (Figs. 3B-3D); a 92.3% decrease in MMP was
413
found in Ca9-22 cells treated with 1 μM of γ-bisabolene (Fig. 3C). Therefore, the
414
results
415
mitochondria-mediated apoptosis of human oral cancer cells.
decreasing
revealed
that
in
the
red/green
γ-bisabolene
fluorescence
induced
caspase
intensity
ratio
9-dependent
by
and
416
417
3.3 Proteomic profiling of human oral cancer cells induced by γ-bisabolene
418
To differentiate specific phosohoproteomic profiling induced by γ-bisabolene,
419
phosphopeptides of Ca9-22 oral cancer cells were enriched by the TiO2-PDMS plate
420
(TP plate) and identified using quantitative LC-MS/MS (Supp. Tables 1-4, Figs.
421
Supp. 2A-2B). Supplemental Figures 2A and 2B showed MS/MS spectra for
422
mapping phosphopeptides of p53 and HDAC2. A total of 358 phosphopeptides and
423
142 nonphosphorylated peptides in mock-control and treated oral cancer cells with 5
424
µM γ-bisabolene were identified and quantified using LC-MS/MS with Bruker
425
DataAnalysis and ProfileAnalysis software (Supp. Tables 1-4). Analyzing cell lysates
426
by Western blot confirms protein profiling of Ca9-22 and SAS cells induced by
26
427
γ-bisabolene (Figs. 4A and 4C): i.e., a significant rise of phospho-p53 and PDK1 as
428
well as an obvious decline of phospho-HDAC2 and CK2α in γ-bisabolene-treated
429
cells compared to mock-control cells, as consistent with LC-MS/MS data (Supp.
430
Tables 1-4). Moreover, quantitative real-time PCR indicated greater 6-fold increases
431
of PKA-R1, PDK1 and TCP1, as well as 2-fold decrease of CK2-α in
432
γ-bisabolene-treated Ca9-22 and SAS cells than mock cells (Figs. 4B and 4D), as in
433
agreement with LC-MS/MS and Western blot data (Supp. Tables 1-4, Figs. 4A and
434
4C). In addition, GeneGO Meta-Core analysis of phosohoproteomic profiling
435
proposed γ-bisabolene-induced apoptotic pathways of human oral cancer cells, in
436
which HDAC2 inhibition and ERK1/2 activation would be responsible for
437
p53-mediated transcriptional activities for apoptosis induction by γ-bisabolene (Fig.
438
4E). Therefore, HDAC2- and ERK1/2-mediated pathways were consequently
439
examined to confirm the proposed mechanisms of γ-bisabolene-induced apoptosis.
440
441
3.4 Increase of p53 acetylation via PP1-mediated dephosphorylation of HDAC2 in
442
γ-bisabolene-induced apoptotic cells
443
HDAC2 was phosphorylated by CK2, and dephosphorylated by protein
444
phosphatase 1 (PP1) [14, 38]. Meanwhile, HDACs phosphorylation promoted their
27
445
enzymatic activity to suppressed p53 transcriptional activities via the loss of p53
446
acetylation [39-42]. Therefore, the PP1-HDAC2-p53 acetylation pathway was
447
proposed to be involved in apoptosis induction by γ-bisabolene. Firstly, p53
448
transcriptional activities for gene expression of PUMA, NOXA, and Bim were
449
quantified in both cancer cell lines using real-time RT-PCR (Figs. 4F-4G).
450
γ-bisabolene significantly induced a concentration-dependent upregulation of PUMA
451
mRNA expression in Ca9-22 and SAS cells. To confirm whether γ-bisabolene
452
triggered the PP1-HDAC2-p53 acetylation pathway involved triggering apoptosis,
453
PP1 inhibitor-2 (I-2) was used to ascertain the relation between HDAC2
454
phosphorylation and p53 acetylation (Figs. 5A-5D). Initially, the translocation of I-2
455
across cell membrane was evaluated by the inhibitory ability of I-2 pre-treatment at 4
456
°C versus 37 °C (Fig. 5A). I-2 pre-treatment at 37 °C reduced a higher decrease of
457
γ-bisabolene-induced apoptosis (sub-G1 phase) compared to I-2 pre-treatment at 4 °C,
458
because the permeability of cell membrane was low at 4 °C. The result revealed the
459
direct translocation of I-2 across cell membrane. Subsequently, I-2 was used to assess
460
the PP1-HDAC2-p53 acetylation pathway in γ-bisabolene-induced apoptosis.
461
Immunofluorescence assay demonstrated I-2 concentration-dependently elevating the
462
phosphorylation of HDAC2 in γ-bisabolene-treated cancer cells (Fig. 5B). Meanwhile,
463
I-2 reduced γ-bisabolene-induced p53 acetylation and p53 transcriptional activities in
28
464
oral cancer cells in concentration-dependent manners (Figs.5C and 5D). The results
465
demonstrated the inhibition of PP1 activity by I-2 caused the increase of HDAC2
466
activity, resulting in the decrease of p53 acetylation. The results revealed the
467
involvement of PP1-HDAC2-p53 acetylation pathway in γ-bisabolene-induced
468
apoptosis of human oral cancer cells.
469
470
3.5 Activation of ERK1/2-mediated apoptosis of human oral cancer cells by
471
γ-bisabolene
472
GeneGO Meta-Core pathway analysis also indicated ERK1/2-p53 and
473
ERK1/2-Akt pathways as responsible for γ-bisabolene-induced apoptosis (Fig. 4E).
474
ERK1/2 regulated apoptosis through increasing p53-mediated gene expression and
475
decreasing Akt activation [54, 55], thus we investigated whether ERK1/2 activation
476
links
477
γ-bisabolene-induced apoptosis (Figs. 5E and 5F). Western blotting showed
478
γ-bisabolene lessened the phosphorylation of Akt in human oral cancer Ca9-22 and
479
SAS cells, linking with activation of ERK1/2-mediated apoptosis. Likewise,
480
ERK1/2- and MEK-specific inhibitors (PD98059 and U0126) significantly
481
suppressed the up-regulation of p53-mediated gene PUMA induced by γ-bisabolene
482
(Fig. 5F). Results revealed ERK1/2 activation was notably responsible for
with
the
Akt
activation
and
p53-mediated
gene
expression
in
29
483
p53-medaited apoptosis of γ-bisabolene-treated Ca9-22 and SAS cells.
484
485
3.6 Inhibition of oral cancer cell xenograft growth by γ-bisabolene
486
To examine in vivo antitumor efficacy of γ-bisabolene, Balb/c-nu/nu nude
487
mice were implanted with human oral cancer cell xenografts in the dorsal
488
subcutaneous site for one week, then intraperitoneally injected with(out) 50 mg/kg
489
γ-bisabolene every 2 days (Fig. 6). Treatment with γ-bisabolene had a significantly
490
inhibitory effect on the tumor volume of oral cancer cell xenografts (Fig. 6B), but
491
only slightly reduced body weight (Fig. 6A). Tumor volume growth curves indicated
492
exponential growth of oral cancer cell xenografts in the untreated control (113.8,
493
700.2, 1761.8, and 3175.9 mm3 at Day 0, 7, 14 and 21), but not in
494
γ-bisabolene-treated group (95.4, 338.2, 824.3, and 793.8 mm3 at Day 0, 7, 14 and 21)
495
(Fig. 6B). In addition, γ-bisabolene treatment significantly reduced the tumor weight
496
(1.2 ± 0.6 g) of oral cancer cell xenografts, as markedly lower than that in untreated
497
group (4.7 ± 0.8 g) at Day 21 (Fig. 6C). The results indicated γ-bisabolene
498
significantly constrained the xenograft growth of human oral cancer cells in nude
499
mice.
500
30
501
4. Discussion
502
Cardamom and its derived compound γ-bisabolene inhibited the proliferative
503
activities of human oral cancer cells (Supp.Fig. 1, Fig. 1). Antiproliferative ability of
504
cardamonin, limonene and carvone on human oral cancer cells was less that of
505
γ-bisabolene with CC50 of 5.15 μM for CA9-22, and 29.5 μM for SAS oral cancer
506
cells (Supp. Fig.1B, Fig 1A). Importantly, γ-bisabolene significantly suppressed the
507
growth of human oral cancer cells in mouse xenograft model (Fig. 6). Cardamom and
508
its derived compounds cardamonin, limonene, and carvone have demonstrated the
509
growth inhibition of colon cancer cells, breast, skin, and prostate cancer cells, as well
510
as chemopreventive and antioxidant actions on skin cancer [18, 21, 27, 30, 45].
511
Besides cardamom, γ-bisabolene was also detected in essential oil of Croton flavens L.
512
leaf, Zingiber officinale, Magnolia grandiflora and Magnolia virginiana flower
513
[46-48]. These essential oils containing γ-bisabolene exhibited anti-inflammatory
514
properties and anti-proliferative activities on human prostate cancer, glioblastoma,
515
lung carcinoma, breast carcinoma, and colon adenocarcinoma cell lines [46-48].
516
However, γ-bisabolene alone seldom demonstrated the anticancer action in vitro and
517
in vivo. The study firstly reported potent antiproliferative activities of γ-bisabolene on
518
human oral squamous cell carcinoma cell lines in vitro and in vivo.
519
γ-Bisabolene exerts apoptosis-inducing activities against human oral cancer
31
520
cells, triggering apoptosis in time- and concentration-dependent manners (Figs. 1B,
521
1C, 2A, and 2B). γ-Bisabolene up-regulated the mRNA expression of caspase 9 in
522
Ca9-22 and SAS cells, then triggered the activation of caspases 3 and 9 in oral cancer
523
cells (Figs. 2C-2F), in which linked with the loss of mitochondria membrane potential
524
in γ-bisabolene-treated cancer cells (Fig. 3). Results demonstrated the involvement of
525
the mitochondrial apoptotic pathway in γ-bisabolene-induced apoptosis of human oral
526
squamous cell carcinoma.
For elucidating the mechanism of γ-bisabolene-induced apoptosis in oral cancer
527
528
cells,
comprehensive
529
γ-bisabolene-treated Ca9-22 cells was examined using LC-MS/MS and confirmed
530
using Western blotting and real-time RT-PCR assays (Supp. Tables 1-4, Fig. 4, Supp.
531
Fig.
532
phosphopeptides identified indicated γ-bisabolene activating the proteins of apoptotic
533
process, induction of apoptosis, and regulation of cell cycle, such as
534
melanoma-associated antigen 4 (MAGE-A4), tensin-3, insulin-like growth factor 2
535
mRNA-binding protein 2, and tumor suppressor p53. For example, the C-terminal
536
fragment of MAGE-A4 with proapoptotic activity induces apoptosis in human cells
537
after genotoxic stress [50]. Tumor suppressor p53 shows the proapoptotic activity on
538
the mitochondrial apoptotic pathway, exerting transcriptional activities on
2).
PANTHER
and
quantitative
classification
system
phosphoproteome
[49]
analysis
profiling
of
of
up-regulated
32
539
up-regulation of pro-apoptotic genes: e.g., PUMA, Bim, and NOXA [8-10]. On the
540
other
541
phosphopeptides shows γ-bisabolene constraining the proteins of negative regulation
542
of apoptotic process or anti-apoptotic process, including BAG family molecular
543
chaperone regulator 3 (BAG-3), HDAC2, and double-stranded RNA-specific
544
adenosine deaminase (ADAR). Anti-apoptotic protein BAG-3 interacts with Bcl-2 in
545
blocking Fas/FasL-mediated apoptosis [51]. ADAR, catalyzing the conversion of
546
adenosine to inosine, exhibits a protection against stress-induced apoptosis and
547
measles virus-induced apoptosis [63, 64]. HDAC2, a negative regulator of apoptotic
548
process, overexpresses in many types of human cancers: e.g., hematological, colon
549
and colorectal, esophageal squamous cell carcinoma, renal, hepatic, lung, skin tumors,
550
pancreatic cancer [11]. Clinical studies indicated HDAC inhibitors displaying
551
anticancer activities against ovarian cancer, T-cell lymphoma, Hodgkin lymphoma,
552
and myeloid malignancies [54, 55]. Therefore, phosphoproteome profiling presents
553
γ-bisabolene modulating the biological processes of apoptotic, anti-apoptotic,
554
induction of apoptosis, and negative regulation of apoptosis.
hand,
PANTHER
classification
system
analysis
of
down-regulated
555
GeneGO Meta-Core pathway analysis of proteomic profiling implied the
556
involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in γ-bisabolene-induced
557
apoptosis (Fig. 4E). γ-Bisabolene provoked the increases of p53 phosphorylation and
33
558
p53 acetylation, in which were linked with up-regulation of p53 transcriptional
559
activities on apoptotic genes in concentration-dependent manners (Figs. 4F and 4G).
560
PP1 inhibitor-2 (I-2) treatment reduced γ-bisabolene-induced apoptosis, and restored
561
the phosphorylated level of HDAC2 in γ-bisabolene-treated oral cancer cells (Figs.
562
5A and 5B). I-2 treatment decreased p53 acetylation, and then reduced p53
563
transcriptional activity in a concentration-dependent manner (Figs. 5C-5D). Notably,
564
the results were in agreement with reports on the inhibitory effect of PP1 on the
565
deacetylase activity of HDAC2 via dephosphorylation [14]; inactivation of HDAC2
566
augmented p53 acetylation and transcriptional activity [12, 13]. For examining the
567
ERK1/2-p53 pathway, Western blotting indicated that γ-bisabolene decreased Akt
568
phosphorylation (Fig. 5E); ERK1/2- and MEK-specific inhibitors significantly
569
reduced the expression of p53-dependent apoptotic genes (Fig. 5F). The finding was
570
consistent with prior studies on the mechanism of ERK-mediated apoptosis [43, 44].
571
Therefore, the results ascertained the essential role of PP1-HDAC2-p53 and
572
ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis.
573
This study demonstrated γ-bisabolene exerting potent antiproliferative and
574
apoptosis-inducing activities on human oral squamous cell carcinoma. γ-Bisabolene
575
activated PP1-HDAC2-p53 and ERK1/2-p53 pathways, eliciting
576
dependent and mitochondria-mediated apoptosis of human oral cancer cells. The
caspase 9-
34
577
results suggested γ-bisabolene as a potential anti-cancer drug for treatment of human
578
oral squamous cell carcinoma.
579
580
581
Acknowledgments
This work was supported by the National Science Council of Taiwan
582
(NSC102-2320-B-039-044-MY3)
583
(CMU102-ASIA-15).
and
China
Medical
University
584
585
Conflict of Interest
586
YJ Jou and CW Lin have a patent pending on γ-bisabolene against OSCC.
587
588
References
589
[1] Parkin, D. M., Bray, F., Ferlay, J., Pisani, P., Global cancer statistics, 2002. CA
590
591
592
Cancer J Clin. 2005, 55, 74–108.
[2] Neville, B. W., Day, T. A., Oral cancer and precancerous lesions. CA Cancer J
Clin. 2002, 52, 195–215.
593
[3] Ko, Y. C., Huang, Y. L., Lee, C. H., Chen, M. J. et al., Betel quid chewing,
594
cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J
35
595
596
597
Oral Pathol Med. 1995, 24, 450–453.
[4] Sturgis, E. M., A review of social and behavioral efforts at oral cancer
preventions in India. Head Neck. 2004, 26, 937–944.
598
[5] Zhang, C., Li, K., Wei, L., Li, Z., et al., p300 expression repression by
599
hypermethylation associated with tumour invasion and metastasis in oesophageal
600
squamous cell carcinoma. J Clin Pathol. 2007, 60, 1249-1253.
601
602
603
604
605
606
607
608
[6] Vokes, E. E., Weichselbaum, R. R., Lippman, S. M., Hong, W. K., Head and
neck cancer. N Engl J Med. 1993, 328, 184–194.
[7] Liu, Q., Wang, H. G., Anti-cancer drug discovery and development: Bcl-2 family
small molecule inhibitors. Commun Integr Biol. 2012, 5, 557-565.
[8] Elmore, S., Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007,
35, 495-516.
[9] Mancini, F., Moretti, F., Mitochondrial MDM4 (MDMX): an unpredicted role in
the p53-mediated intrinsic apoptotic pathway. Cell Cycle. 2009, 8, 3854-3859.
609
[10] Fujita, T., Ishikawa, Y., Apoptosis in heart failure. The role of the β-adrenergic
610
receptor-mediated signaling pathway and p53-mediated signaling pathway in the
611
apoptosis of cardiomyocytes. Circ J. 2011, 75, 1811-1818.
36
612
[11] Wagner, T., Brand, P., Heinzel, T., Krämer, O. H., Histone deacetylase 2
613
controls p53 and is a critical factor in tumorigenesis. Biochim Biophys
614
Acta. 2014, PMID:25072962.
615
[12] Brandl, A., Wagner, T., Uhlig, K. M., Knauer, S. K., et al., Dynamically
616
regulated sumoylation of HDAC2 controls p53 deacetylation and restricts
617
apoptosis following genotoxic stress. J Mol Cell Biol. 2012, 4, 284-293.
618
[13] Jung, K. H., Noh, J. H., Kim, J. K., Eun, J. W., et al., HDAC2 overexpression
619
confers oncogenic potential to human lung cancer cells by deregulating
620
expression of apoptosis and cell cycle proteins. J Cell Biochem. 2012, 113,
621
2167-2177.
622
[14] Galasinski, S. C., Resing, K. A., Goodrich, J. A., Ahn, N. G., Phosphatase
623
inhibition leads to histone deacetylases 1 and 2 phosphorylation and disruption of
624
corepressor interactions. J Biol Chem. 2002, 277, 19618-19626.
625
[15] Hatziieremia, S., Gray, A. I., Ferro, V. A., Paul, A., Plevin, R., The effects of
626
cardamonin on lipopolysaccharideinduced inflammatory protein production and
627
MAP kinase and NFkB signalling pathways in monocytes/macrophages. Br J
628
Pharmacol. 2006, 149, 188-198.
629
[16] Gilani, A. H., Jabeen, Q., Khan, A. U., Shah, A. J., Gut modulatory, blood
37
630
pressure
lowering,
diuretic
and
631
Ethnopharmacol. 2008, 115, 463-472.
sedative
activities
of
cardamom.
J
632
[17] Das, I., Acharya, A., Berry, D. L., Sen, S., et al., Antioxidative effects of the
633
spice cardamom against non-melanoma skin cancer by modulating nuclear factor
634
erythroid-2-related factor 2 and NF-κB signalling pathways. Br J Nutr. 2012, 108,
635
984-997.
636
[18] Bhattacharjee, S., Rana, T., Sengupta, A., Inhibition of lipid peroxidation and
637
enhancement of GST activity by cardamom and cinnamon during chemically
638
induced colon carcinogenesis in Swiss albino mice. Asian Pac J Cancer Prev.
639
2007, 8, 578-582.
640
641
[19] Gopalakrishnan, M., Narayanan, G. S., Grenz, M., Nonsaponifiable lipid
constituents of Cardamom. J Agric Food Chem. 1990, 38, 2133–2136.
642
[20] Held, S., Schieberle, P., Somoza, V., Characterization of alpha-terpineol as an
643
anti-inflammatory component of orange juice by in vitro studies using oral
644
buccal cells. J Agric Food Chem. 2007, 55, 8040-8046.
645
[21] Qiblawi,
S., Al-Hazimi,
A., Al-Mogbel,
M., Hossain,
A., Bagchi,
D.,
646
Chemopreventive Effects of Cardamom (Elettaria cardamomum L.) on
647
Chemically Induced Skin Carcinogenesis in Swiss Albino Mice. J Med Food.
38
648
649
650
2012, 15, 576–580.
[22] Murata, S., Shiragami, R., Kosugi, C., Tezuka, T., et al., Antitumor effect of 1,
8-cineole against colon cancer. Oncol Rep. 2013, 30, 2647-2652.
651
[23] Jia, S. S., Xi, G. P., Zhang, M., Chen, Y. B., et al., Induction of apoptosis by
652
D-limonene is mediated by inactivation of Akt in LS174T human colon cancer
653
cells. Oncol Rep. 2013, 29, 349-354.
654
[24] Jin, X., Sun, J., Miao, X., Liu, G., Zhong, D., Inhibitory effect of geraniol in
655
combination with gemcitabine on proliferation of BXPC-3 human pancreatic
656
cancer cells. J Int Med Res. 2013, 41, 993-1001.
657
[25] Duncan, R. E., Lau, D., El-Sohemy, A., Archer, M. C., Geraniol and beta-ionone
658
inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2
659
activity in MCF-7 breast cancer cells independent of effects on HMG-CoA
660
reductase activity. Biochem Pharmacol. 2004, 68, 1739-1747.
661
[26] Madankumar, A., Jayakumar, S., Gokuladhas, K., Rajan, B., et al., Geraniol
662
modulates tongue and hepatic phase I and phase II conjugation activities and
663
may contribute directly to the chemopreventive activity against experimental oral
664
carcinogenesis. Eur J Pharmacol. 2013, 705, 148-155.
39
665
[27] Chaudhary, S. C., Siddiqui, M. S., Athar, M., Alam, M. S., Geraniol inhibits
666
murine skin tumorigenesis by modulating COX-2 expression, Ras-ERK1/2
667
signaling pathway and apoptosis. J Appl Toxicol. 2013, 33, 828-837.
668
[28] Wattenberg, L. W., Inhibition of azoxymethane-induced neoplasia of the large
669
bowel
by
3-hydroxy-3,7,11-trimethyl-1,6,10-dodecatriene
670
Carcinogenesis. 1991, 12, 151-152.
(nerolidol).
671
[29] Nogueira Neto, J. D., de Almeida, A. A., da Silva Oliveira, J., Dos Santos, P. S.,
672
de Sousa, D. P., de Freitas, R. M., Antioxidant effects of nerolidol in mice
673
hippocampus after open field test. Neurochem Res. 2013, 38, 1861-1870.
674
[30] Chen, J., Lu, M., Jing, Y., Dong, J., The synthesis of L-carvone and limonene
675
derivatives with increased antiproliferative effect and activation of ERK pathway
676
in prostate cancer cells. Bioorg Med Chem. 2006, 14, 6539-6547.
677
[31] Chen, C. J., Lai, C. C., Tseng, M. C., Liu, Y. C., et al., A novel titanium
678
dioxide-polydimethylsiloxane plate for phosphopeptide enrichment and mass
679
spectrometry analysis. Anal Chim Acta. 2014, 812, 105-113.
680
[32] Chen, C. J., Lai, C. C., Tseng, M. C., Liu, Y. C., et al., Simple fabrication of
681
hydrophobic surface target for increased sensitivity and homogeneity in
682
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
40
683
analysis of peptides, phosphopeptides, carbohydrates and proteins. Anal Chim
684
Acta. 2013, 783, 31-38
685
[33] Chen, C. J., Chen, W. Y., Tseng, M. C., Chen, Y. R., Tunnel frit: a nonmetallic
686
in-capillary
frit
for
nanoflow
ultra
high-performance
liquid
687
chromatography-mass spectrometryapplications. Anal Chem. 2012, 84, 297-303.
688
[34] Hsieh, J. Y., Chang, C. T., Huang, M. T., Chang, C. M., et al., Biochemical and
689
functional characterization of charge-defined subfractions of high-density
690
lipoprotein from normal adults. Anal Chem. 2013, 85, 11440-11448.
691
[35] Wang,
C.
Y., Huang,
S.
C., Zhang,
Y., Lai,
Z.
R., et
al.,
692
Antiviral Ability of Kalanchoe gracilis Leaf Extract against Enterovirus 71 andC
693
oxsackievirus A16.
694
PMCID:PMC3361180.
Evid
Based
Complement
Alternat
Med. 2012,
695
[36] Lu, K. W., Chen, J. C., Lai, T. Y., Yang, J. S., et al., Gypenosides suppress
696
growth of human oral cancer SAS cells in vitro and in a murine xenograft model:
697
the role of apoptosis mediated by caspase-dependent and caspase-independent
698
pathways. Integr Cancer Ther. 2012, 11, 129-140.
699
700
[37] Riedl, S. J., Salvesen, G. S., The apoptosome: signalling platform of cell death.
Nat Rev Mol Cell Biol. 2007, 8, 405-413.
41
701
[38] Rush, J., Moritz, A., Lee, K.A., Guo, A., et al., Immunoaffinity profiling of
702
tyrosine phosphorylation in cancer cells, Nat Biotechnol. 2005, 23, 94-101.
703
[39] Pluemsampant, S., Safronova, O. S., Nakahama K, Morita I. Protein kinase CK2
704
is a key activator of histone deacetylase in hypoxia-associated tumors. Int J
705
Cancer. 2008, 122, 333-341.
706
[40] Juan, L. J., Shia, W. J, Chen, M. H., Yang, W. M., et al., Histone deacetylases
707
specifically down-regulate p53-dependent gene activation. J Biol Chem. 2000,
708
275, 20436-20443.
709
[41] Sun, G., Yu, R. T., Evans, R. M., Shi, Y., Orphan nuclear receptor TLX recruits
710
histone deacetylases to repress transcription and regulate neural stem cell
711
proliferation. Proc Natl Acad Sci U S A. 2007, 104, 15282-15287.
712
713
714
715
[42] Tang, Y., Zhao, W., Chen, Y., Zhao, Y., Gu, W., Acetylation is indispensable for
p53 activation. Cell. 2008, 133, 612-626.
[43] Zhuang, S., Schnellmann, R. G., A death-promoting role for extracellular
signal-regulated kinase. J Pharmacol Exp Ther. 2006, 319, 991-997.
716
[44] Sinha, D., Bannergee, S., Schwartz, J. H., Lieberthal, W., Levine, J. S., Inhibition
717
of ligand-independent ERK1/2 activity in kidney proximal tubular cells deprived
42
718
of soluble survival factors up-regulates Akt and prevents apoptosis. J Biol
719
Chem. 2004, 279, 10962-10972.
720
[45] Miller, J.A., Thompson, P.A., Hakim, I.A., Lopez, A.M., et al., Safety and
721
Feasibility of Topical Application of Limonene as a Massage Oil to the Breast,
722
J Cancer Ther. 2012, PMCID:PMC3824622.
723
[46] Sylvestre, M., Pichette, A., Longtin, A., Nagau, F., Legault, J., Essential oil
724
analysis and anticancer activity of leaf essential oil of Croton flavens L. from
725
Guadeloupe. J Ethnopharmacol. 2006, 103, 99-102.
726
[47] Bayala, B., Bassole, I. H., Gnoula, C., Nebie, R., et al., Chemical composition,
727
antioxidant, anti-inflammatory and anti-proliferative activities of essential oils of
728
plants from burkina faso. PLoS One. 2014, 9, e92122.
729
[48] Farag, M. A., Al-Mahdy, D. A., Comparative study of the chemical composition
730
and biological activities of Magnolia grandiflora and Magnolia virginiana flower
731
essential oils. Nat Prod Res. 2013, 27, 1091-1097.
732
[49] Mi, H., Muruganujan, A., Casagrande, J. T., Thomas, P. D., Large-scale gene
733
function analysis with the PANTHER classification system. Nat Protoc. 2013, 8,
734
1551-1566.
43
735
[50] Sakurai, T., Itoh, K., Higashitsuji, H., Nagao, T., et al., A cleaved form of
736
MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. Chem. 2004,
737
279, 15505-15514.
738
[51] Lee, J. H., Takahashi, T., Yasuhara, N., Inazawa, J., et al., Bis, a Bcl-2-binding
739
protein that synergizes with Bcl-2 in preventing cell death. Oncogene. 1999, 18,
740
6183-6190.
741
[52] Wang, Q., Miyakoda, M., Yang, W., Khillan, J., et al., Stress-induced apoptosis
742
associated with null mutation of ADAR1 RNA editing deaminase gene. J Biol
743
Chem. 2004, 279, 4952-4961.
744
[53] Toth, A. M., Li, Z., Cattaneo, R., Samuel, C. E., RNA-specific adenosine
745
deaminase ADAR1 suppresses measles virus-induced apoptosis and activation of
746
protein kinase PKR. J Biol Chem. 2009, 284, 29350-29356.
747
748
749
750
[54] Khabele, D., The therapeutic potential of class I selective histone deacetylase
inhibitors in ovarian cancer. Front Oncol. 2014, 4, 111.
[55] Prince, H. M., Bishton, M. J., Harrison, S. J., Clinical studies of histone
deacetylase inhibitors. Clin Cancer Res. 2009, 15, 3958-3969.
751
44
752
Figure captions
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Fig. 1. Survival rates and cell cycle analysis of human oral cancer and normal
oral fibroblast cells in response to γ-bisabolene. For antiproliferative assay
(A), Ca9-22 and SAS oral cancer cells as well as normal oral fibroblast (OF)
cells were incubated with various concentrations of γ-bisabolene for 48 h.
Survival rate was calculated as MTT data. For cell cycle analysis, Ca9-22 (B),
SAS (C) and OF (D) cells were fixed by 70% ethanol, incubated with PI
solution, then examined using flow cytometry. *, p value < 0.05; **, p value <
0.01 compared with untreated cells.
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Fig. 2. Apoptosis analysis of human oral cancer and normal oral fibroblast cells
in responses to γ-bisabolene. Cells were harvested after 24- or 48-h post
treatment, stained by Annexin V-FITC/PI dye, and then analyzed using flow
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cytometry. Annexin V positive/PI negative was early phase of apoptosis (A);
Annexin V positive/PI positive was late apoptosis(B). Relative mRNA levels of
caspases-3, and 9 were normalized to GAPDH in real-time PCR assays (C, D).
Active forms of caspases 3/9 in Ca9-22 and SAS cells were characterized using
Western blotting (E, F). *, p value < 0.05; **, p value < 0.01 compared with
untreated cells.
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Fig. 3. Loss of mitochondrial membrane potential (ΔΨM) in OSCC cells by
γ-bisabolene. For imagine analysis of ΔΨM change, treated or mock cells were
stained with JC-1 solution, and visualized under a fluorescence microscope (A).
For quantifying ΔΨM change, cells were stained using DiOC 6(3), and then
measured by flow cytometry (B). Relative changes in low MMP of Ca9-22 and
SAS cells treated with γ-bisabolene were shown (C, D). *, p value < 0.05; **, p
value < 0.01 compared with untreated cells.
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Fig. 4. Validation of γ-bisabolene-induced phosphoproteome profiling by Western
blotting and real-time PCR. Western blot analysis of lysates from treated and
mock cells was performed; the blots were probed with primary and secondary
antibodies (A, C). Relative fold levels in treated and mock cells appear as ratio
of indicated mRNA/GAPDH mRNA after performing real time PCR assays (B,
D, F, and G). GeneGO Meta-Core pathway analysis of proteomic profiling
predicts protein-protein interaction networks in treated cells (E).
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Fig. 5. Influence of protein phosphatase inhibitor-2 (I-2) and MEK-specific
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inhibitors on γ-bisabolene-induced apoptosis. Cells were pre-treated with I-2
at 4 °C or 37 °C for 1 h, treated with γ-bisabolene for 24 h, then harvested for
analyzing sub-G1 phase using PI staining (A). HDAC2 phophorylation and p53
acetylation in treated cells were detected using immunofluorescence and
immunoprecipitation assays (B, C). Relative mRNA levels of PUMA in treated
cells, normalized by GAPDH, were performed using real-time PCR (D, F).
Western blot analysis of lysates was performed with primary and secondary
antibodies (E).
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Fig. 6. Growth inhibition of human oral cancer cells in a mouse xenograft model
by γ-bisabolene. SAS cells were implanted intramuscularly to nude mice.
When tumor volumes approximately reached 100 mm3, mice were i.p. injected
with PBS control or 50 mg/kg γ-bisabolene very other day after three weeks.
After 10 times treatment, body weight of each mouse was measured (A), then
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mice were sacrificed. Furthermore, tumor volume (B) and tumor weight (C)
were calculated.
46