Article pubs.acs.org/JAFC
Chemical Constituents of Rhododendron formosanum Show Pronounced Growth Inhibitory Effect on Non-Small-Cell Lung Carcinoma Cells Tzong-Der Way,†,§ Shang-Jie Tsai,‡ Chao-Min Wang,‡ Chi-Tang Ho,⊗ and Chang-Hung Chou*,†,‡,⊥ †
Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung 40402, Taiwan Department of Health and Nutrition Biotechnology, College of Health Science, Asia University, Taichung 41354, Taiwan ‡ Research Center for Biodiversity, China Medical University, Taichung 40402, Taiwan ⊗ Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States ⊥ Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan §
ABSTRACT: The aim of the present study was to investigate whether Rhododendron formosanum Hemsl. (Ericaceae), an endemic species in Taiwan, exhibits antineoplastic potential against non-small-cell lung carcinoma (NSCLC). R. formosanum was successively extracted with methanol and then separated into dichloromethane (RFL-DCM), ethyl acetate (RFL-EA), n-butanol (RFL-BuOH), and water (RFL-H2O) fractions. Among these extracts, RFL-EA exhibited the most effective antineoplastic effect. This study also demonstrated that fractions 2 and 3 from the RFL-EA extract (RFL-EA-2, RFL-EA-3) possessed the strongest antineoplastic potential against NSCLC cells. The major phytochemical constituents of RFL-EA-2 and RFL-EA-3 were ursolic acid, oleanolic acid, and betulinic acid. This study indicated that ursolic acid demonstrated the most efficient antineoplastic effects on NSCLC cells. Ursolic acid inhibited growth of NSCLC cells in a dose- and time-dependent manner and stimulated apoptosis. Apoptosis was substantiated by activation of caspase-3 and -9, and a decrease in Bcl-2 and an elevation of the Bax were also observed following ursolic acid treatment. Ursolic acid activated AMP-activated protein kinase (AMPK) and then inhibited the mammalian target of rapamycin (mTOR), which controls protein synthesis and cell growth. Moreover, ursolic acid decreased the expression and/or activity of lipogenic enzymes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) via AMPK activation. Collectively, these data provide insight into the chemical constituents and anticancer activity of R. formosanum against NSCLC cells, which are worthy of continued study. KEYWORDS: Rhododendron formosanum, non-small-cell lung carcinoma, ursolic acid, AMP-activated protein kinase
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INTRODUCTION Lung cancer is the second most frequent type of cancer, being considered one of the most common causes of death by cancer worldwide.1 Despite the improvements in surgical techniques and other therapies, most patients present with advanced disease with an estimated 5 year relative survival rate of 17%.2 Nonsmall-cell lung cancer (NSCLC) accounts for ∼80% of primary lung cancers. Surgery, radiation, and chemotherapy are useful treatments for patients with NSCLC. However, patients considered favorable for therapeutic treatment will still have a high rate of recurrence. Recent studies showed that numerous alterations in oncogenic pathways play a critical role in NSCLC tumorigenesis and progression. Development of selective drugs to specifically target the NSCLC oncogenic pathways is very critical. Cell death plays an important role in the efficacy of cancer chemotherapy. The process of apoptosis, a major form of cell death, is regulated by programmed cellular signaling pathways. The main mechanism by which anticancer drugs kill cells is by inducing apoptosis in cancer cells. Apoptosis is associated with characteristic morphological changes including the formation of apoptotic bodies, chromatin, and nuclear condensation and DNA fragmentation. The caspases, a family of cysteine proteases, play a critical role during apoptosis. In the intrinsic pathway, pro© 2014 American Chemical Society
apoptotic factors are released from the mitochondria, leading to caspase-9 and caspase-3 cleavages, and then induce apoptosis.3,4 The tumor suppressor LKB1 is mutated in at least 15−30% of NSCLC and plays a critical role in NSCLC metastasis.5 The canonical target of LKB1 is AMP-activated protein kinase (AMPK), a crucial cellular energy sensor, that is activated during metabolic stress.6 Phosphorylation of Thr172 in the T-loop of the AMPK catalytic α subunit by LKB1 is necessary for AMPK catalytic activity. Extensive evidence has demonstrated that AMPK inhibits anabolic pathways that promote cell growth, such as synthesis of cholesterol, fatty acid, glycogen, triglyceride, protein, and rRNA synthesis. Cancer cells possessing mutation or deletion of LKB1 that inactivates the AMPK pathway are a highly malignant form of cancer7,8 because AMPK activation inhibits anabolic pathways, such as cell growth and proliferation, thereby antagonizing carcinogenesis. Many studies have verified the anticancer effects of AMPK in vitro and in vivo, including breast, lung, colorectal, skin, and hematological malignancies.9 Received: Revised: Accepted: Published: 875
September 22, 2013 January 13, 2014 January 14, 2014 January 14, 2014 dx.doi.org/10.1021/jf404243p | J. Agric. Food Chem. 2014, 62, 875−884
Journal of Agricultural and Food Chemistry
Article
min. From 10 to 20 min, a linear increase in buffer B to 80% was carried out, and the column was maintained in 80% buffer B for another 10 min. The column was further eluted with a linear increase in buffer B to 95% from 30 to 40 min. The column was finally equilibrated with buffer A for 10 min. Quantification of the triterpenoids in EA fraction was performed in the ion monitoring mode selected. Both positive and negative ionization mode MS analyses were undertaken. The molecular ion peaks and mass spectra recorded were compared to those of reference substances. Analysis was carried out using data-dependent MS/MS scanning from m/z 100 to 1000. The temperature of the ion source was maintained at 100 °C, the dry temperature was 365 °C, and the desolvation gas, N2, had a flow rate of 12 L/min. Product ion scans for mass were performed by low-energy collision (20 eV) using argon as the collision gas. All liquid chromatography−electrospray ionization− tandem mass spectrometry (LC-ESI-MS/MS) data were processed by Bruker Daltonics data analysis software (version 4.0). All chemicals were prepared at a concentration range of 0.01−1000 μg/mL. Quantification of triterpenoids was performed with the selected ion monitoring (SIM) mode. The separated [M − H]− ion chromatogram was selected at m/z 455 for the specific parent ion of triterpenoids. The linearity of the calibration curves was demonstrated by the good determination of coefficients (r2) obtained for the regression line. Good linearity was achieved over the calibration range, with all coefficients of correlation >0.995. All samples were freshly prepared. The mean values for the regression equation were y = 6 × 107x + 1 × 107 (r2 = 0.9997) for betulinic acid, y = 552577x + 5 × 107 (r2 = 0.9983) for oleanolic acid, and y = 353895x + 3 × 107 (r2 = 0.9985) for ursolic acid. The limits of quantification (LOQ) and determination (LOD), defined as signal-tonoise ratios of 3:1 and 10:1, were in the ranges 0.01−0.1 and 0.1 mg/mL, respectively. Cell Lines and Cell Culture. Both A549 cells (human lung adenocarcinoma cell line) and H460 cells (human non-small-cell lung cancer cell line) were used in the study and were acquired from the American Type Culture Collection. A549 cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA), and H460 cells were cultured in RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS, 10%) (Invitrogen), streptomycin (100 μg/mL), and penicillin (100 IU/mL) (Invitrogen) in a humidified incubator at 37 °C with 5% CO2. Cell Viability Assay. To measure the cell viability, A549 cells or H460 cells were incubated on the 96-well cell culture cluster (1 × 104 cells/well). MTT stock solution was prepared at a conentration of 5 mg/ mL in PBS, and working solution (500 μg/mL) was diluted from stock solution. The medium was removed from each well, and 100 μL of MTT working solution was added for 1 h at 37 °C. When the crystals had formed, the solution was removed and 80 μL of DMSO was aded to dissolve the crystals. Finally, OD 570 nm was used to detect the absorbance by the ELISA reader. MTT assay was done as described previously.14 Western Blot Analysis. Cells (2 × 106) were seeded onto the 10 cm cell culture dish overnight and treated with ursolic acid (30 μM) for 0, 12, 24, and 48 h. After treatment, cells were collected in a 1.5 mL eppendorf and lysed in the lysis buffer (0.1% SDS, 1% NP-40, 10 g/mL leupeptin, 1% sodium deoxycholate, 1 mM PMSF, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 10 g/mL aprotinin). Each protein sample was extracted and quantified by using a Bio-Rad protein assay kit, and each group took 50 μg of total proteins to run SDS-PAGE, following transfer to the PVDF membrane. Western Blot was done as described previously.15 The results were analyzed and quantified by using Image J software. Cell Cycle and Apoptosis Analysis. A549 cells (5 × 105) were cultured in a 6 cm cell culture dish for 24 h and then treated with ursolic acid (10−30 μM) for 24 and 48 h. After treatment, cells were harvested in a 15 mL tube, washed with PBS, resuspended in PBS, and fixed in 2 mL of 70% ethanol at 20 °C overnight. The cell cycle state and apoptosis was determined by using PI staining as reported previously.15 Fluorescence Microscopy. After treatment with ursolic acid (30 μM) for 48 h, A549 cells were fixed by 70% ethanol at −4 °C for 6 h or overnight. Cells were stained with DAPI (1 μg/mL DAPI, 0.1% Triton X-100) at room temperature for 15 min. The morphology of cell nuclei
The mammalian target of rapamycin (mTOR) is one of the major growth regulatory pathways controlled by AMPK. The mTOR pathway plays a major role in proliferation, angiogenesis, and metastasis of NSCLC and other cancers. The target on mTOR signaling pathways is extensively investigated for cancer chemotherapy including NSCLC.10 Moreover, AMPK controlled lipid metabolism at transcriptional and post-translational levels. AMPK phosphorylates and inactivates metabolic enzyme acetyl-CoA carboxylase (ACC) that is involved in regulating de novo biosynthesis of fatty acid and cholesterol. Phosphorylated ACC led to the decrease of malonyl-CoA levels, thus stimulating mitochondrial carnitine palmitoyltransferase 1 (CPT1) and promoting fatty acid oxidation. Moreover, AMPK inhibits transcription factor SREBP1c, which controls the entire fatty acid synthetic pathway or directly inhibits the expression of fatty acid synthase (FASN).11 Rhododendron formosanum Hemsl. (Ericaceae) is an evergreen broad-leafed tree native to Taiwan and ubiquitously distributed from 800 to 2000 m. The vegetation exhibits a unique pattern and forms pure dominant vegetation.12,13 We studied whether R. formosanum exhibited pharmacological activities for NSCLC and investigated its bioactive phytochemical constituents. Our results indicated that treatment of NSCLC cells with R. formosanum had a very potent inhibitory effect on cellular growth.
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MATERIALS AND METHODS
Materials. The leaves of R. formosanum were collected after flowering in April 2010 and July 2010 at the Dasyueshan site (24°14′6.49″ N, 120°57′7.29″ E at 1911 m asl). Ursolic acid, oleanolic acid, betulinic acid, 4′,6-diamidino-2-phenylindole (DAPI), 3-[4,5dimethylthylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT), and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The antibody for LKB1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibody against phospho-AMPK (Thr 172), AMPK, Bcl-2, Bax, caspase-9, caspase-3, FASN, phospho-ACC (Ser 79), mTOR, phospho-mTOR (Ser 2448), and phospho-p70S6k (Thr 389) were purchased from Cell Signaling Technology (Beverly, MA, USA). β-Actin antibody was purchased from Sigma Chemical Co. HRP-conjugated goat anti-rabbit IgG and goat antimouse IgG were obtained from Millipore (Billerica, MA, USA). Methanol (MeOH), n-hexane, dichloromethane (DCM), n-butanol (BuOH), and ethyl acetate (EA) were purchased form Seedchem Co. (Melbourne, Australia). Silica gel 60 was purchased from Merck KGaA (Darmstadt, Germany). Plant Collection, Chemical Extraction, and Isolation. The leaves of R. formosanum were air-dried for chemical analysis. The airdried and powdered leaves of R. formosanum (5.5 kg) were extracted with MeOH for 3 days at room temperature (three times), and the combined extracts were concentrated in vacuo (under 35 °C) to obtain a dark green gum (1540 g), which was suspended in H2O and partitioned sequentially with DCM, EA, and BuOH. The EA extract (3.5 g) was subjected to column chromatography on silica gel using n-hexane, nhexane−EA, and EA−MeOH mixtures of increasing polarity for elution to furnish 10 fractions. Quantification of Ursolic Acid, Oleanolic Acid, and Betulinic Acid in RFL-EA-2 and RFL-EA-3 Fractions. The EA fractions of R. formosanum were prepared at concentration of 1 mg/mL in MeOH. The samples were passed through 0.22 μm filters (Millipore) and placed in sample vials for liquid chromatography (LC) analysis. LC mass analysis for the quantification of betulinic acid, oleanolic acid, and ursolic acid was carried out with an Atlantis T3 RP-18 column (150 × 2.1 mm; 3 μm; Waters, Milford, MA, USA). In each case the injection volume was 5 mL. The column was eluted with buffer A (distilled water/acetonitrile/ formic acid; 95/5/0.1, v/v/v) and buffer B (acetonitrile/formic acid; 100/0.1, v/v) at a flow rate of 0.25 mL/min at 25 °C. The column was eluted initially with 100% buffer A, followed by a linear increase in buffer B to 30% from 0 to 5 min, and maintained in 30% buffer B for another 5 876
dx.doi.org/10.1021/jf404243p | J. Agric. Food Chem. 2014, 62, 875−884
Journal of Agricultural and Food Chemistry
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Figure 1. Antiproliferation activities of different fractions from R. formosanum. (A) Leaf powder of R. formosanum was extracted by methanol and partitioned into four fractions including dichloromethane, ethyl acetate, n-butanol, and water to get RFL-DCM, RFL-EA, RFL-BuOH, and RFL-H2O fractions. (B) A549 cells were treated with the four fractions (40−160 μg/mL) for 24 h. Cell viability was then determined using the MTT assay. This experiment was repeated three times. The data represent the mean ± SD. Values were significantly different from the control group: ∗, P < 0.05. was observed by Nikon TE2000-U fluorescence microscope at 400× magnification. Fluorescence microscopy was done as described previously.16 RNA Interference Suppression of LKB1. A549 cells were transfected with LKB1 small interfering RNA (siRNA) using siRNA transfection reagent (Santa Cruz Biotechnology) and incubated for 6 h. The LKB1 siRNA was obtained from Santa Cruz Biotechnology (sc25816). Statistical Analysis. Data were presented as the mean ± SD of at least three independent experiments. For statistical analysis, the independent Student’s t test was used to compare the continuous variables between two groups, and the chi-squared test was applied for comparison of the dichotomous variables. An asterisk indicates that the values were significantly different from the control (∗, P < 0.05).
Among these R. formosanum extracts, RFL-EA was the most effective in our assay (Figure 1B). Further fractionation of the RFL-EA by column chromatography was used to analyze the detailed bioactive phytochemical constituents (Figure 2A). Our study demonstrated that fractions 2 and 3 from the RFL-EA extract (RFL-EA-2, RFL-EA-3) possessed the strongest antiproliferative activity against A549 cells (Figure 2B). The morphology variations of R. formosanum-treated A549 cells were investigated by microscopic inspection. After treatment with different concentrations of RFL-EA-2 or RFL-EA-3 for 48 h, apoptotic bodies were observed in A549 cells (Figure 2C). These results may provide a rationale for the potential use of R. formosanum against NSCLC. Comparative Study of the Phytochemical Constituents of RFL-EA-2 and RFL-EA-3. The HPLC fingerprint chromatogram was established to analyze the detailed phytochemical constituents of RFL-EA-2 and RFL-EA-3. Figure 3A shows the HPLC fingerprint chromatogram for the mixture of ursolic acid, oleanolic acid, and betulinic acid. As shown in Figure 3B, RFLEA-3 contains ursolic acid, oleanolic acid, and betulinic acid. Interestingly, our results revealed that ursolic acid was the most abundant constituent in RFL-EA-2 and RFL-EA-3 (Table 1).
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RESULTS R. formosanum Induced NSCLC A549 Cell Growth Inhibition. To evaluate the bioactive phytochemical constituents of R. formosanum, we extracted with methanol and separated the methanol extract into dichloromethane (RFL-DCM), ethyl acetate (RFL-EA), n-butanol (RFL-BuOH), and water (RFLH2O) fractions (Figure 1A). We next examined the antiproliferative activity of R. formosanum extracts on NSCLC A549 cells. 877
dx.doi.org/10.1021/jf404243p | J. Agric. Food Chem. 2014, 62, 875−884
Journal of Agricultural and Food Chemistry
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Figure 2. Antiproliferation activities of different RFL-EA fractions against NSCLC A549 cells. (A) RFL-EA was injected into a silica column and eluted with n-hexane, ethyl actate, and methanol at different combination rates to get 10 fractions. (B) A549 cells were treated with the 10 fractions (10−80 μg/ mL) for 24 h. Cell viability was then determined using the MTT assay. This experiment was repeated three times. The data represent the mean ± SD. Values were significantly different from the control group: ∗, P < 0.05. (C) A549 cells were treated with RFL-EA-2 (40−80 μg/mL) and RFL-EA-3 (40− 80 μg/mL) for 24 h, and cell morphology was observed by photomicroscope.
confirm ursolic acid-induced A549 cell apoptosis, flow cytometric analysis was performed. After treatment with various concentrations of ursolic acid for 24 and 48 h, A549 cells underwent apoptosis (Figure 5C). Apoptosis-related modulators were studied by Western blot analysis. Treatment with 30 μM ursolic acid increased the cleavages of caspase-3 and caspase-9 (Figure 5D). Moreover, there was a marked increase of proapoptotic protein Bax and a marked decrease of anti-apoptotic protein Bcl-2 in ursolic acid-treated A549 cells (Figure 5E). These results indicated that ursolic acid induced apoptosis in NSCLC cells. Ursolic Acid Decreases Protein Synthesis via the Upregulation of AMPK Activity in NSCLC Cells. Abnormalities in the AMPK function have emerged as an important pathway implicated in cancer development.9 We examined whether ursolic acid was involved in the regulation of AMPK. Figure 6A indicates that ursolic acid stimulated AMPK phosphorylation in a time-dependent manner, whereas AMPK activation resulted in marked inhibition of mTOR and p70S6K in a time-dependent manner (Figure 6B). Our studies suggest that the phosphor-
Effect of Oleanolic Acid, Ursolic Acid, and Betulinic Acid on NSCLC Cell Proliferation. The potential antiproliferative activities of oleanolic acid, ursolic acid, and betulinic acid were evaluated using the MTT assay. A549 and H460 cells were treated with various concentrations of ursolic acid (Figure 4A), oleanolic acid (Figure 4B), and betulinic acid (Figure 4C) for 24 and 48 h. Among these constituents, ursolic acid and betulinic acid exhibited potent cytotoxic effects on NSCLC cells (Figure 4). Because ursolic acid was the most abundant constituent and exhibited potent cytotoxic effects, we chose ursolic acid for the subsequent experiments. Ursolic Acid Induces NSCLC Cell Apoptosis. To test whether ursolic acid could induce apoptosis, apoptosis and morphology variations were investigated by microscopic inspection. After treatment with different concentrations of ursolic acid for 48 h, apoptotic bodies were observed in A549 cells (Figure 5A). We next elucidated whether ursolic acid induced chromatin condensation in A549 cells. After 30 μM ursolic acid treatment, chromatin condensation was seen in A549 cells, as evidenced by staining with DAPI (Figure 5B). To further 878
dx.doi.org/10.1021/jf404243p | J. Agric. Food Chem. 2014, 62, 875−884
Journal of Agricultural and Food Chemistry
Article
Figure 3. Quantification of triterpenoids by LC-ESI-MS/MS analysis. (A) Individual standard triterpenoids including betulinic acid (B), oleanolic acid (O), and ursolic acid (U) were subjected to LC-ESI-MS/MS analysis for chemical identification and quantification. Panel B shows quantification of the betulinic acid, oleanolic acid, and ursolic acid from RFL-EA-2 and RFL-EA-3 fractions.
Ursolic Acid Inhibits Lipogenesis through Activation of AMPK in NSCLC Cells. AMPK negatively regulated the activities of lipogenic enzymes FASN and ACC. We next examined reduction of FASN expression and ACC activity in ursolic acid-treated A549 cells. Our result indicated that ursolic acid decreased the expression of FASN and increased ACC phosphorylation in a time-dependent manner (Figure 6C). Our results suggested that ursolic acid suppressed lipogenesis through modulation of AMPK activity. Ursolic Acid Induces AMPK Phosphorylation via an LKB1-Dependent Pathway. The canonical target of LKB1 is AMPK.6 To further investigate whether ursolic acid induced
Table 1. Concentration of Betulinic, Oleanolic, and Ursolic Acid in the Fractions RF-EA-2 and RF-EA-3 concentration (μg/mg) betulinic acid oleanolic acid ursolic acid
RF-EA-2
RF-EA-3
0.37 2.55 550.2
