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Chemical constituents of Rhododendron formosanum show pronounced growth inhibitory effect
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on non-small cell lung carcinoma cells
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Tzong-Der Way1,2, Shang-Jie Tsai3, Chao-Min Wang3, Chi-Tang Ho4, Chang-Hung Chou1,3,5*
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University, Taichung 40402, Taiwan
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2Department
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Taichung 41354, Taiwan
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3Research
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4Department
of Food Science, Rutgers University, New Brunswick, New Jersey, USA
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5Department
of Biological Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
Department of Biological Science and Technology, College of Life Sciences, China Medical
of Health and Nutrition Biotechnology, College of Health Science, Asia University,
Center for Biodiversity, China Medical University, Taichung 40402, Taiwan
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*Author for correspondence:
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Chang-Hung Chou, Ph.D
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Academician, Academia Sinica
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Chair Professor and Director
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Research Center for Biodiversity and Graduate Institute of Ecology and Evolutionary Biology
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China Medical University, Taichung, Taiwan
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Address: Room 720, 7F, Lifu Hall, 91, Hsueh-Shih Road, Taichung, 40402, Taiwan.
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E-mail: choumasa@mail.cmu.edu.tw
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Tel: +886-4-2205-3366 ext. 1633
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Fax: +886-4-2207-1500
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ABBREVIATION
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ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; BuOH, n-butanol; DAPI, 4’,6-
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diamidino-2-phenylindole; DCM, dichloromethane; DR, death receptor; EA, ethyl acetate; FASN, fatty
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acid synthase; FBS, fetal bovine serum; LC, liquid chromatography; MeOH, methanol; mTOR,
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mammalian target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;
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NSCLC, non-small cell lung carcinoma; PI, propidium-iodide; PS, phosphatidylserine
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ABSTRACT
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The aim of the present study was to investigate whether Rhododendron formosanum Hemsl.
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(Ericaceae), an endemic species in Taiwan, exhibit anti-neoplastic potential against non-small cell lung
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carcinoma (NSCLC). We successively extracted the R. formosanum with methanol, and then separated
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into dichloromethane (RFL-DCM), ethyl acetate (RFL-EA), n-butanol (RFL-BuOH), and water (RFL-
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H2O) fractions. Among these extracts, RFL-EA exhibited the most effective anti-neoplastic effect. Our
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study also demonstrated that fractions 2 and 3 from the RFL-EA extract (RFL-EA-2, RFL-EA-3)
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possessed the strongest anti-neoplastic potential against NSCLC cells. The major phytochemical
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constituents of RFL-EA-2 and RFL-EA-3 were ursolic acid, oleanolic acid, and betulinic acid. Our
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studied indicated that ursolic acid demonstrated the most efficient anti-neoplastic effects on NSCLC
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cells. Ursolic acid inhibited growth of NSCLC cells in a dose- and time-dependent manner, and
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stimulated apoptosis. Apoptosis was substantiated by activation of caspase-3 and -9, and decrease in
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Bcl-2 and elevation of the Bax was also observed following ursolic acid treatment. Ursolic acid
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activated AMP-activated protein kinase (AMPK) and then inhibited the mammalian target of rapamycin
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(mTOR) which controls protein synthesis and cell growth. Moreover, ursolic acid decreased the
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expression and/or activity of lipogenic enzymes, such as acetyl-CoA carboxylase (ACC) and fatty acid
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synthase (FASN) via AMPK activation. Collectively, these data provide insight into the chemical
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constituents and anticancer activity of R. formosanum against NSCLC cells and worthy of continued
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study.
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KEYWORDS: Rhododendron formosanum; Non-small cell lung carcinoma; Ursolic acid, AMP-
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activated protein kinase
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INTRODUCTION
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Lung cancer is the second frequent types of cancer, being considered one of the most common
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causes of death by cancer worldwide.1 Despite the improvements in surgical techniques and other
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therapies, most patients may present with advanced disease and with an estimated 5-year relative
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survival rate of 17%.2 Non-small cell lung cancer (NSCLC) accounts for ~80% of primary lung cancer.
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Surgery, radiation, and chemotherapy are useful treatments for patients with NSCLC. However, patients
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considered favorable for therapeutic treatment will still keep a high rate of recurrence. Recent studies
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showed that numerous alterations in oncogenic pathways possess a critical role in NSCLC tumorigenesis
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and progression. Develop selective drugs to specifically target the NSCLC oncogenic pathways is very
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critical.
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Cell death plays an important role in the efficacy of cancer chemotherapy. The process of apoptosis,
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a major form of cell death, is regulated by programmed cellular signaling pathways. The main
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mechanism by which anti-cancer drugs kill cells is to induce apoptosis in cancer cells. Apoptosis is
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associated with characteristic morphological changes including the formation of apoptotic bodies,
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chromatin and nuclear condensation, and DNA fragmentation. The caspases, a family of cysteine
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proteases, play a critical role during apoptosis. In the intrinsic pathway, pro-apoptotic factors are
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released from the mitochondria, leading to caspase 9 and caspase 3 cleavages and then induce
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apoptosis.3,4
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The tumor suppressor LKB1 is mutated in at least 15%–30% of NSCLC and play a critical role in
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NSCLC metastasis.5 The canonical target of LKB1 is AMP-activated protein kinase (AMPK), a crucial
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cellular energy sensor, that is activated during metabolic stress.6 Phosphorylation of Thr172 in the T-loop
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of AMPK catalytic α subunit by LKB1 is necessary for AMPK catalytic activity. Extensive evidences
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have demonstrated that AMPK inhibits anabolic pathways that promote cell growth, such as synthesis of
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cholesterol, fatty acid, glycogen, triglyceride, protein, and ribosomal RNA synthesis. Cancer cells
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possess mutation or deletion of LKB1 that inactivate the AMPK pathway are highly malignant form of
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cancer.7,8 Since AMPK activation inhibits anabolic pathways, such as cell growth and proliferation–
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thereby antagonizing carcinogenesis. Many studies have verified the anti-cancer effects of AMPK in
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vitro and in vivo, including breast, lung, colorectum, skin, and hematological malignancies.9
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The mammalian target of rapamycin (mTOR) is one of the major growth regulatory pathways
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controlled by AMPK. The mTOR pathway plays a major role in proliferation, angiogenesis, and
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metastasis of NSCLC and other cancers. The target on mTOR signaling pathways is extensively
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investigated for cancer chemotherapy including NSCLC.10 Moreover, AMPK controlled lipid
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metabolism at transcriptional and post-translational levels. AMPK phosphorylates and inactivates
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metabolic enzyme acetyl-CoA carboxylase (ACC) that involves in regulating de novo biosynthesis of
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fatty acid and cholesterol. Phosphorylated ACC leaded to the decrease of malonyl-CoA levels, thus
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stimulating mitochondrial carnitine palmitoyltransferase 1 (CPT1) and promoting fatty acid oxidation.
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Moreover, AMPK inhibits the transcription factor SREBP1c, which controls the entire fatty acid
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synthetic pathway, or by directly inhibiting the expression of fatty acid synthase (FASN).11
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Rhododendron formosanum Hemsl. (Ericaceae), is an evergreen, broad leafed tree native to Taiwan
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and ubiquitously distributed from 800 m to 2,000 m. The vegetation exhibits a unique pattern and forms
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pure dominant vegetation.12,13 We studied whether R. formosanum exhibited pharmacological activities
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for NSCLC and investigated its bioactive phytochemical constituents. Our results indicated that
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treatment of NSCLC cells with R. formosanum had a very potent inhibitory effect on cellular growth.
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MATERIALS AND METHODS
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Materials. The leaves of R. formosanum were collected after flowering in April 2010 and July
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2010 at the Dasyueshan site (24°14'6.49"N, 120°57'7.29"E at 1911 m asl.). Ursolic acid, oleanolic acid,
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betulinic
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diphenyltetrazolium (MTT), and propidium iodide (PI) were purchased from Sigma Chemical Co. (St.
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Louis, MO, USA). The antibody for LKB1 was purchased from Santa Cruz Biotechnology (Santa Cruz,
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CA, USA). Primary antibody against phospho-AMPK (Thr 172), AMPK, Bcl-2, Bax, caspase 9, caspase
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3, FASN, phospho-ACC (Ser 79), mTOR, phospho-mTOR (Ser 2448) and phospho-p70S6k (Thr 389)
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were purchased from Cell Signaling Technology (Beverly, MA, USA). β-actin antibody was purchased
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from sigma Chemical Co. (St. Louis, MO, USA). HRP-conjugated Goat anti-Rabbit IgG and Goat anti-
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Mouse IgG were obtained from Millipore (Billerica, MA, USA). Methanol (MeOH), n-hexane,
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dichloromethane (DCM), n-butanol (BuOH), and ethyl acetate (EA) were purchased form Seedchem Co.
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(Melbourne, Australia). Silica gel 60 was purchased from Merck KGaA (Darmstadt, Germany).
acid,
4’,6-diamidino-2-phenylindole
(DAPI),
3-[4,5-dimethylthylthiazol-2-yl]-2,5-
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Plant collection, chemical extraction and isolation. The leaves of R. formosanum were air-dried
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for chemical analysis. The air-dried and powdered leaves of R. formosanum (5.5 kg) were extracted with
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MeOH for three days at room temperature (three times), and the combined extracts were concentrated in
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vacuo (under 35 °C) to obtain a dark green gum (1,540 g), which was suspended in H2O and partitioned
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sequentially with DCM, EA, and BuOH. The EA extract (3.5 g) was subjected to column
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chromatography on silica gel using n-hexane, n-hexane-EA and EA-MeOH mixtures of increasing
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polarity for elution to furnish 10 fractions.
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The quantification of ursolic acid, oleanolic acid and betulinic acid in RFL-EA-2 and RFL-
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EA-3 fractions. The EA fractions of R. formosanum were prepared at concentration of 1 mg/mL in
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MeOH. The samples were passed through 0.22-µm filters (Millipore) and placed in sample vials for
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liquid chromatography (LC) analysis. LC mass analysis for the quantification of betulinic acid, oleanolic
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acid and ursolic acid was carried out with an Atlantis T3 RP-18 column (150 × 2.1 mm; 3 µm; Waters,
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Milford, MA, USA). In each case the injection volume was 5 mL. The column was eluted with buffer A
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(distilled water/acetonitrile/formic acid; 95/5/0.1, v/v/v) and buffer B (acetonitrile/formic acid; 100/0.1,
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v/v) at a flow rate of 0.25 mL/min at 25 °C. The column was eluted initially with 100% buffer A,
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followed by a linear increase in buffer B to 30% from 0 to 5 min, and maintained in 30% buffer B for
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another 5 min. From 10 to 20 min, a linear increase in buffer B to 80% was carried out and the column
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was maintained in 80% buffer B for another 10 min. The column was further eluted with a linear
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increase in buffer B to 95% from 30 to 40 min. The column was finally equilibrated with buffer A for 10
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min. Quantification of the triterpenoids in EA fraction was performed in the ion monitoring mode
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selected. Both positive and negative ionization mode MS analyses were undertaken. The molecular ion
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peaks and mass spectra recorded were compared to those of reference substances. Analysis was carried
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out using data-dependent MS/MS scanning from m/z 100 to 1000. The temperature of the ion source
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was maintained at 100 °C, the dry temperature was 365 °C and the desolvation gas, N2, had a flow rate
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of 12 L/min. Product ion scans for mass were performed by low-energy collision (20 eV) using argon as
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the collision gas. All liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-
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ESI-MS/MS) data were processed by Bruker Daltonics Data analysis software (version 4.0). All
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chemicals were prepared at a concentration range of 0.01–1000 µg/mL. Quantification of triterpenoids
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was performed with the selected ion monitoring (SIM) mode. The separated [M-H]– ion chromatogram
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was selected at m/z 455 for the specific parent ion of triterpenoids. The linearity of the calibration
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curves was demonstrated by the good determination of coefficients (r2) obtained for the regression line.
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Good linearity was achieved over the calibration range, with all coefficients of correlation greater than
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0.995. All samples were freshly prepared. The mean values for the regression equation were y=6E+07x
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+ 1E+07 (r2=0.9997) for betulinic acid, y=552577x+ 5E+07 (r2=0.9983) for oleanolic acid and
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y=353895x + 3E+07 (r2=0.9985) for ursolic acid. The limits of quantification (LOQ) and determination
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(LOD), defined as signal to noise ratios of 3:1 and 10:1, were in the range 0.01–0.1 mg/mL and 0.1
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mg/mL, receptively.
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Cell lines and cell culture. Both A549 cells (human lung adenocarcinoma cell line) and H460 cells
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(human non-small cell lung cancer cell line) were used to study and acquired from American Type
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Culture Collection. A549 cells were cultured in DMEM (Invitrogen Carlsbad, CA, USA) and H460 cells
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were cultured in RPMI-1640 (Invitrogen Carlsbad, CA, USA) supplemented with 10% fetal bovine
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serum (FBS, 10%) (Invitrogen Carlsbad, CA, USA), streptomycin (100 g/mL), and penicillin (100
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IU/mL) (Invitrogen Carlsbad, CA, USA) in a humidified incubator at 37 oC with 5% CO2.
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Cell viability assay. To measure the cell viability, A549 cells or H460 cells were incubated on the
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96-well cell culture cluster (1 × 104 cells/well). MTT stock solution was prepared at con. 5 mg/mL in
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PBS, and working solution (500 μg/mL) was diluted form stock solution. Remove the medium from
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each well, add 100 μL MTT working solution for 1 h at 37 °C. When the crystals were formed, remove
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the solution and add 80 μL DMSO to dissolve the crystals. Finally, use OD 570 nm to detect the
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absorbance by the ELISA reader. MTT assay was done as described previously.14
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Western blot analysis. Seed cells (2 × 106) onto the 10 cm cell culture dish overnight, and treat
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with ursolic acid (30 μM) for 0, 12, 24, and 48 h. After treatment, cells were collected in 1.5 mL
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eppendorf, and lysed in the lysis buffer (0.1% SDS, 1% NP-40, 10 g/mL leupeptin, 1% sodium
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deoxycholate, 1 mM PMSF, 10 mM Tris-HCl pH7.5, 150 mM NaCl and 10 g/mL aprotinin). Extract
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each protein sample and quantify by Bio-Rad protein assay kit, each group was took 50 μg total proteins
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to run SDS-PAGE, followed transfer to PVDF membrane. Western Blot was done as described
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previously.15 The results were analyzed and quantified by using Image J software.
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Cell cycle and apoptosis analysis. A549 cells (5×105) were cultured in 6 cm cell culture dish for
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24 h, and then treated with ursolic acid (10-30 μM) for 24 h and 48 h. After treatment, cells were
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harvested in a 15 mL tube, washed with PBS, resuspended in PBS, and fixed in 2 mL of 70 % ethanol at
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20 oC overnight. The cell cycle state and apoptosis was determined by using PI staining as reported
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previously.15
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Fluorescence microscopy. After treatment with ursolic acid (30 μM) for 48 h, A549 cells fixed by
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70 % ethanol at -4 °C for 6 h or overnight. Cells stained with DAPI (1 μg/mL DAPI, 0.1% Triton X-
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100) at room temperature for 15 min. The morphology of cell nuclei was observed by Nikon TE2000-U
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fluorescence microscope at 400× magnification. Fluorescence microscopy was done as described
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previously.16
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RNA interference suppression of LKB1. A549 cells were transfected with LKB1 small
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interfering RNA (siRNA) using siRNA transfection reagent (Santa Cruz Biotechnology; Santa Cruz, CA,
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USA) and incubated for 6 h. The LKB1 siRNA was obtained from Santa Cruz Biotechnology (sc-
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25816).
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Statistical analysis. Data were presented as the mean ± SD of at least three independent
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experiments. For statistical analysis, the independent Student’s t-test was used to compare the
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continuous variables between two groups, and the chi-squared test was applied for compare the
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dichotomous variables. Asterisk indicates that the values were significantly different from the control (*,
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P < 0.05).
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RESULTS
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R. formosanum induced NSCLC A549 cells growth inhibition. To evaluate the bioactive
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phytochemical constituents of R. formosanum, we extracted with methanol and separated the methanol
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extract into dichloromethane (RFL-DCM), ethyl acetate (RFL-EA), n-butanol (RFL-BuOH), and water
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(RFL-H2O) fractions (Figure 1A). We next examined anti-proliferative activity of R. formosanum
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extracts on NSCLC A549 cells. Among these R. formosanum extracts, RFL-EA was the most effective
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one in our assay (Figure 1B). Further fractionation of the RFL-EA by column chromatography was used
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to analyze the detailed bioactive phytochemical constituents (Figure 2A). Our study demonstrated that
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fractions 2 and 3 from the RFL-EA extract (RFL-EA-2, RFL-EA-3) possessed the strongest anti-
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proliferative activity against A549 cells (Figure 2B). The morphology variations of R. formosanum-
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treated A549 cells were investigated by microscopic inspection. After treatment with different
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concentrations of RFL-EA-2 or RFL-EA-3 for 48 h, apoptotic bodies were observed in A549 cells
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(Figure 2C). These results may provide a rationale for the potential use of R. formosanum against
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NSCLC.
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Comparative study of the phytochemical constituents of of RFL-EA-2 and RFL-EA-3. The
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HPLC fingerprint chromatogram was established to analyze the detailed phytochemical constituents of
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RFL-EA-2 and RFL-EA-3. Figure 3A showed the HPLC fingerprint chromatogram for the mixture of
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ursolic acid, oleanolic acid, and betulinic acid. As shown in Figure 3B, RFL-EA-3 contains ursolic acid,
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oleanolic acid, and betulinic acid. Interestingly, our results revealed that ursolic acid was the most
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abundant constituents in RFL-EA-2 and RFL-EA-3 (Table 1).
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Effect of oleanolic acid, ursolic acid, and betulinic acid on NSCLC cells proliferation. The
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potential anti-proliferative activities of oleanolic acid, ursolic acid, and betulinic acid were evaluated
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using MTT assay. A549 and H460 cells were treated with various concentrations of ursolic acid (Figure
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4A), oleanolic acid (Figure 4B), and betulinic acid (Figure 4C) for 24 and 48 h. Among these
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constituents, ursolic acid and betulinic acid exhibited the potent cytotoxic effects on NSCLC cells
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(Figure 4). Because ursolic acid was the most abundant constituents and exhibited the potent cytotoxic
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effects, we chose ursolic acid for the future experiments.
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Ursolic acid induces NSCLC cells apoptosis. To test whether ursolic acid could induce apoptosis,
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apoptosis and morphology variations were investigated by microscopic inspection. Treatment with
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different concentrations of ursolic acid for 48 h, apoptotic bodies were observed in A549 cells (Figure
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5A). We next elucidated whether ursolic acid induce chromatin condensation in A549 cells. After 30
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M ursolic acid treatment, chromatin condensation was seen in A549 cells, as evidenced by staining
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with DAPI. (Figure 5B). To further confirm ursolic acid-induced A549 cells apoptosis, flow cytometric
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analysis was performed. After treatment with various concentrations of ursolic acid for 24 and 48 h,
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A549 cells underwent apoptosis (Figure 5C). Apoptosis-related modulators were studied by western
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blot analysis. Treatment with 30 μM ursolic acid increased the cleavages of Caspase-3 and Caspase-9
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(Figure 5D). Moreover, there was a marked increase of proapoptotic protein Bax and marked decrease
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of antiapoptotic protein Bcl-2 in ursolic acid-treated A549 cells (Figure 5E). These results indicated
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that ursolic acid induced apoptosis in NSCLC cells.
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Ursolic acid decreases protein synthesis via the up-regulation of AMPK activity in NSCLC
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cells. Abnormalities in the AMPK function has emerged as an important pathway implicated in cancer
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development.9 We examined whether ursolic acid was involved in the regulation of AMPK. Figure 6A
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indicated that ursolic acid stimulated AMPK phosphorylation in a time-dependent manner. While
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AMPK activation resulted in marked inhibition of mammalian target of rapamycin (mTOR) and
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p70S6K in a time-dependent manner (Figure 6B). Our studies suggest that the phosphorylation of
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AMPK is required for ursolic acid to decrease protein synthesis in NSCLC cells.
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Ursolic acid inhibits lipogenesis through activation of AMPK in NSCLC cells. AMPK
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negatively regulated the activities of lipogenic enzymes FASN and ACC. We next examined whether
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reduction of FASN expression and ACC activity in ursolic acid-treated A549 cells. Our result indicated
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that ursolic acid decreased the expression of FASN and increased ACC phosphorylation in a time-
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dependent manner (Figure 6C). Our results suggested that ursolic acid suppressed lipogenesis through
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modulation of AMPK activity.
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Ursolic acid induces AMPK phosphorylation via a LKB1-dependent pathway. The canonical
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target of LKB1 is AMPK.6 To further investigate whether ursolic acid induced AMPK phosphorylation
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via a LKB1-dependent pathway, LKB1 siRNA was used to inhibit LKB1 expression in A549 cells.
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Treatment with ursolic acid, an increasing level of phosphorylated AMPK was observed, however,
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under LKB1 siRNA transfection, despite the addition of ursolic acid, AMPK phosphorylation was
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reduced (Figure 6D). Our results showed that the activation of AMPK by ursolic acid treatment might
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occur via a LKB1-dependent pathway.
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DISCUSSION
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The present study has clearly evaluated the bioactive constituents of R. formosanum and
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investigated the anti-proliferative activity on NSCLC. We extracted the R. formosanum with fractions of
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dichloromethane (RFL-DCM), ethyl acetate (RFL-EA), n-butanol (RFL-BuOH), and water (RFL-H2O).
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RFL-EA possesses the most effective bioactivity in our cell viability assay. Moreover, our study also
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demonstrated that fractions 2 and 3 from the RFL-EA extract (RFL-EA-2, RFL-EA-3) showed the most
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anti-proliferative effect on NSCLC cells. In our previous study, eighteen known compounds were
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isolated from the six fractions obtained by column chromatography from the methanolic extract of R.
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formosanum leaves.13 In this study, the major compositions of RFL-EA-2 and RFL-EA-3 were ursolic
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acid, oleanolic acid, and betulinic acid. Among the three constituents, ursolic acid was the most
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abundant compound in RFL-EA-2 and RFL-EA-3. Interestingly, our results also revealed that ursolic
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acid possessed the most efficient anti-proliferative effect on NSCLC cells.
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Ursolic acid is a natural pentacyclic triterpene acid, which exists in elder flower, apples, lavender,
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peppermint rosemary, basil, bilberries, cranberries, thyme, oregano, hawthorn, prunes and medicinal
13
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herbs. Since ursolic acid is relatively non-toxic, there is a growing interest in the elucidation of the
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pharmacological activities. Several reports described that ursolic acid has many health benefits including
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hepatoprotective, anti-inflammatory, and anti-cancer effects.17-19 Ursolic acid possesses strong
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anticancer activity against several cancers of prostate,20 breast,21 lung,22 pancreas,23 ovary,24 colon,25 and
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bladder26. In the present study, we focused on the anti-neoplastic effect of ursolic acid on NSCLC cells.
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These findings suggested that ursolic acid is a potent anti-cancer agent.
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Induction of cell apoptosis is thought to be the principal mechanism by which anti-cancer drugs kill
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cells. Ursolic acid has been reported to induce apoptosis through calcium-dependent and activation of
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sphingomyelinase.27,28 To confirm whether the cytotoxic effect of ursolic acid was due to apoptosis in
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NSCLC cells, phenotypic characteristics, cell cycle analysis, and Western blotting were used in the
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present study. Our results indicated that ursolic acid induced cleavages of caspase-9 and caspase-3 and
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also attenuated the expression of Bcl-2, indicating that ursolic acid induced apoptosis via caspase
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dependent pathway. The Bcl-2 family proteins play a crucial role in the mitochondrial pathway of
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apoptosis. The ratio between Bcl-2 and Bax proteins has been suggested as a primary event in
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determining the sensitivity to the mitochondrial pathway of apoptosis. Bax translocation to the
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mitochondria and insertion into the membrane and induces cytochrome c and other apoptogenic factors
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release that subsequently activates caspase-3 leading to downstream apoptotic responses.29 From the
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above results, we suggest that the enhancement of Bax and the attenuation of Bcl-2 a might be the
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important molecular pathway that is involved in ursolic acid-induced apoptosis in A549 cells.
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Recent studies have focused on the potential of targeting cellular metabolic pathways that may be
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altered during NSCLC tumorigenesis and progression. AMPK has recently been considered as a critical
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energy-sensing serine/threonine kinase in the regulation of cellular metabolism. The activation of
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AMPK switches on catabolic pathways that generate ATP, while switching off ATP-consuming
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processes. Recently, our and other groups have reported that sustained AMPK activation inhibits cancer
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cell growth and proliferation, and induces cancer cell apoptosis.15,30 These findings are given that cancer
14
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cell DNA replication, mitosis, cell growth, and proliferation are all ATP-consuming processes. In this
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study, ursolic acid induced growth inhibition and apoptosis in NSCLC A549 cells, and activation of
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AMPK may contribute to the process. Similarly, Zheng et al. suggested that activation of AMPK by
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ursolic acid contributes to growth inhibition and apoptosis in human bladder cancer T24 cells.31 Son et
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al. reported that ursolic acid induced apoptosis in HepG2 cells via AMPK activation and GSK3β
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phosphorylation.32 Lee et al. found that ursolic acid potentiated cell cycle arrest and UVR-induced
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apoptosis in skin melanoma cells via AMPK activation.33 These studies are consistent with our findings
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here that AMPK activation by ursolic acid inducing A549 cells apoptosis. However, how AMPK
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activation induces A549 cells apoptosis needs further study. Our study provides evidence that ursolic
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acid may as a potential cancer chemopreventive agent for NSCLC and AMPK might be the key
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mechanism for its action.
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Targeting mTOR pathway may lead to the development of novel drug for cancers. Many laboratories
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and pharmaceutical companies have focused intensely on developing approaches to block the mTOR
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pathway. Particularly due to the fact that mTOR pathway plays such a crucial role in cancer biology. The
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intervention of some target proteins will lower mTOR activity, for example, activation of AMPK results
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in decreased mTOR signaling and in turn inhibition of protein synthesis and cellular growth.14,15,34 The
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present data shows that ursolic acid inhibits protein synthesis via AMPK-mTOR pathway.
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In conclusion, R. formosanum is an endemic species distributed widely in Taiwan, the
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pharmacological activities of R. formosanum have not yet been fully explained. To the best of our
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knowledge, the anti-neoplastic activity of R. formosanum has no been explored in the previous studies.
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In this study, we have investigated the chemical constituents and anti-neoplastic activity of R.
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formosanum against NSCLC cells. Based on these studies, it is tempting to propose that R. formosanum
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could be developed as an anti-neoplastic agent for NSCLC.
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ACKNOWLEDGMENT
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This study was financially supported by grants from the National Science Council of Taiwan
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(NSC101-2811-B-039-013, NSC101-2621-B-039-001, NSC102-2313-B-039-001 and NSC102-2811-B-
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039-005) to C. H. Chou. Additionally, technical assistance with chemical data analyses from Instrument
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Analysis Centers at the China Medical University and National Chung Hsing University is greatly
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appreciated.
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Figure legends
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Figure 1. The anti-proliferation activities of different fractions from R. formosanum. (A) Leaf
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powder of R. formosanum was extracted by methanol and partitioned into four fractions including
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dichloromethane, ethyl acetate, n-butanol and water to get RFL-DCM, RFL-EA, RFL-BuOH, and RFL-
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H2O fractions. (B) A549 cells were treated with four fractions (40-160 μg/mL) for 24 h. The cell
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viability was then determined using MTT assay. This experiment was repeated three times. The data
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represented the mean ± S.D. Values significantly were different from the control group. *, P < 0.05.
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Figure 2. The anti-proliferation activities of different RFL-EA fractions against NSCLC A549
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cells. (A) RFL-EA was subjected into silica column and eluted with n-hexane, ethyl actate and methanol
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at different combination rates to get ten fractions. (B) A549 cells were treated with ten fractions (10-80
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μg/mL) for 24 h. The cell viability was then determined using MTT assay. This experiment was repeated
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three times. The data represented the mean ± S.D. Values significantly were different from the control
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group. *, P < 0.05. (C) A549 cells were treated with RFL-EA-2 (40-80 μg/mL) and RFL-EA-3 (40-80
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μg/mL) for 24 h and the cell morphology was observed by photomicroscope.
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Figure 3. Quantification of triterpenoids by LC-ESI-MS/MS analysis. (A) Individual standard
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triterpenoids including betulinic acid (B), oleanolic acid (O) and ursolic acid (U) were subjected to LC-
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ESI-MS/MS analysis for chemical identification and quantification. (B) Quantification of the betulinic
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acid, oleanolic acid and ursolic acid from RFL-EA-2 and RFL-EA-3 fractions.
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Figure 4. The anti-proliferation effect of ursolic acid, oleanolic acid and betulinic acid against
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NSCLC cells. A549 cells and H460 cells were treated with (A) ursolic acid, (B) oleanolic acid and (C)
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betulinic acid at concentration of 10-160 μM for 24 h or 48 h. The cell viability was then determined
499
using MTT assay. This experiment was repeated three times. The data represented the mean ± S.D.
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Values significantly were different from the control group. *, P < 0.05.
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Figure 5. Ursolic acid induced A549 cells apoptosis. (A) A549 cells were incubated with ursolic acid
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(10-40 μM) for 24 h, and morphology was observed by photomicroscope, and (B) the morphology of
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cell nuclei was observed by fluorescence microscope. (C) To observe cell cycle statements and apoptosis
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levels, cell were stained with PI and measured by flow cytometry. (D) A549 cells were treated with
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ursolic acid (30 μM) for the indicated time. Cells were then harvested and lysed for the detection of
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caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9 and β-actin. (E) A549 cells were treated with
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ursolic acid (30 μM) for the indicated time. Cells were then harvested and lysed for the detection of Bcl-
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2, Bax and β-actin. Western blot data presented are representative of those obtained in at least three
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separate experiments. The values below the figures represent change in protein expression of the bands
510
normalized to β-actin.
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Figure 6. Ursolic acid decreases general mRNA translation and the activity of fatty acid synthesis
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via activating of AMPK. (A) A549 cells were treated with ursolic acid (30 μM) for the indicated time.
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Cells were then harvested and lysed for the detection of phosphorylated AMPK (Thr 172), AMPK and
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β-actin. (B) A549 cells were treated with ursolic acid (30 μM) for the indicated time. Cells were then
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harvested and lysed for the detection of phosphorylated mTOR (Ser 2448), mTOR, phosphorylated
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p70S6K and β-actin. (C) A549 cells were treated with ursolic acid (30 μM) for the indicated time. Cells
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were then harvested and lysed for the detection of FASN, phospho-ACC (Ser79) and β-actin. (D) A549
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cells were transfected with 50 nmol/L LKB1-siRNA using Oligofectamine. A total of 24 h after
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transfection, cells were treated with ursolic acid (30 μM) for 24 h. After harvesting, cells were lysed and
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prepared for Western blotting analysis using antibodies against LKB-1, phosphorylated AMPK (Thr
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172) and β-actin. Western blot data presented are representative of those obtained in at least three
522
separate experiments. The values below the figures represent change in protein expression of the bands
523
normalized to β-actin.
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