Article pubs.acs.org/JAFC
Cinnamtannin D1 from Rhododendron formosanum Induces Autophagy via the Inhibition of Akt/mTOR and Activation of ERK1/2 in Non-Small-Cell Lung Carcinoma Cells Tzong-Der Way,†,§ Shang-Jie Tsai,‡ Chao-Min Wang,‡ Yun-Lian Jhan,‡ Chi-Tang Ho,∥ and Chang-Hung Chou*,†,‡,⊥ †
Department of Biological Science and Technology, College of Biopharmaceutical and Food Sciences, and ‡Research Center for Biodiversity, China Medical University, Taichung 40402, Taiwan § Department of Health and Nutrition Biotechnology, College of Health Science, Asia University, Taichung 41354, Taiwan ∥ Department of Food Science, Rutgers University, New Brunswick, New Jersey United States ⊥ Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan S Supporting Information *
ABSTRACT: In our previous study, ursolic acid present in the leaves of Rhododendron formosanum was found to possess antineoplastic activity. We further isolated and unveiled a natural product, cinnamtannin D1 (CNT D1), an A-type procyanidin trimer in R. formosanum also exhibiting anticancer efficacy that induced G1 arrest (83.26 ± 3.11% for 175 μM CNT D1 vs 69.28 ± 1.15% for control, p < 0.01) and autophagy in non-small-cell lung carcinoma (NSCLC) cells. We found that CNT D1mediated autophagy was via the noncanonical pathway, being beclin-1-independent but Atg5 (autophagy-related genes 5)dependent. Inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in CNT D1-treated NSCLC cells. Moreover, CNT D1 inhibited the Akt/mammalian target of the rapamycin (mTOR) pathway and activated the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, resulting in induction of autophagy. KEYWORDS: Rhododendron formosanum, non-small-cell lung carcinoma, cinnamtannin D1, Akt/mTOR, ERK1/2
■
INTRODUCTION Non-small-cell lung cancer (NSCLC) has a relatively poor prognosis and accounts for 80−85% of all lung cancer cases.1 The treatments for NSCLC patients are surgical resection, adjuvant chemotherapy, radiation therapy, and targeted therapy. However, despite the use of these approaches, the management of this disease has not been improved in the past 30 years, with an overall 5-year survival rate of only 15%.2 The recent challenge is to identify new therapeutic targets and novel strategies for the treatment of NSCLC and to incorporate them into existing treatment regimens with the goal of improving therapeutic gain. Although most chemotherapeutic agents induce cancer cell death by activation of apoptosis, autophagy has received considerable attention in recent years for its role in the response to anticancer therapies. Autophagy, or “self-eating”, is an evolutionarily conserved catabolic process by which longlived proteins, damaged organelles, and intracellular pathogens are eliminated. Autophagy has a fundamental role in normal organisms, originally characterized as a survival response to nutrient deficiency.3 It is frequently induced in response to variety of physiological and pathophysiological roles,4 such as adaptation, starvation, antiaging, development, elimination of microorganisms,5 cell death, tumor suppression, and antigen presentation. There is a potential link between autophagy and various biological fields, including cancer, neurodegenerative disorders, © 2015 American Chemical Society
cardiomyopathy, amyotrophic lateral sclerosis, and prion diseases.6 Interestingly, cancer cells show less autophagy than normal cells, indicating that autophagy induction is an attractive modality of anticancer therapy. Even if autophagy was first used to describe the process of cell death, it has also been shown to play a pro-survival function in many stressful conditions, mainly via negative regulation of apoptosis. Under some situations, apoptosis and autophagy can exert synergetic effects, whereas in other conditions autophagy can be triggered only when apoptosis is suppressed.7 LC3, the mammalian autophagy-related genes 8 (Atg8) known as homologue microtubule-associated protein light chain 3, plays a critical role in autophagy formation and is considered a suitable marker for this process. LC3 exists in the cytosolic form (pro-form) and hAtg4B cleaves a C-terminal 22 amino acid fragment, thus converting pro-LC3 to LC3-I. When autophagy is induced, the cytosolic form LC3-I is converted to LC3-II, a 16 kDa protein that localizes to autophagosomal membranes.8 LC3-II formation is now regarded as a reliable marker protein for autophagy. The molecular machinery of autophagy is under the control of diverse signaling pathways. Several signaling pathways Received: Revised: Accepted: Published: 10407
May 1, 2015 November 12, 2015 November 15, 2015 November 15, 2015 DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry
NMR (Varian Inova 600) and mass spectrometry (Bruker Daltonics Esquire HCT). The isolated compound was identified by comparison of spectral data with literature reported previously.2,14,15 The compound was further purified by high-performance liquid chromatography (HPLC) (column, Gemini C6-Phenyl, 5 μm, 4.6 mm × 250 mm; solvent system, acetonitrile−0.2% formic acid with gradient elution; flow rate, 1 mL/min; UV detection, 280 nm). The purity of CNT D1 was over 95%. A flowchart of the extraction for CNT D1 is shown in Figure 1A.
control autophagy in mammalian cells. The Akt/mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) and the extracellular signal-regulated kinases 1/2 (ERK1/2) pathways are two major pathways that regulate autophagy induced by nutrient starvation.9 These pathways are also frequently associated with oncogenesis. It is wellestablished that the Akt/mTOR and ERK1/2 pathways are involved in regulating autophagy; however, their roles in autophagy in cancer are not yet fully understood. Rhododendron formosanum, an endemic species, is distributed widely in the central mountains from 800 to 2000 m in Taiwan.10−12 Our previous study found that a major phytochemical constituent, ursolic acid, in the leaves of R. formosanum exhibited antineoplastic potential against NSCLC cells and stimulated apoptosis.13 In the present study, we isolated cinnamtannin D1 (CNT D1) from R. formosanum extracts and found that the compound exhibited efficient cytotoxic effects on NSCLC cells.
■
MATERIALS AND METHODS
Chemicals. Acridine orange (AO), propidium iodide (PI), 4′,6diamidino-2-phenylindole (DAPI), 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium (MTT), and XAD-2 gel were purchased from Sigma Chemical Co. (St. Louis, MO). DMEM medium, RPMI-1640, and fatal bovine serum (FBS) were purchased from Invitrogen Co. (Carlsbad, CA). Methanol (MeOH) was purchased from Avantor (Center Valley, PA). Primary antibodies against cyclin D1 and CDK6 were purchased from St John’s Laboratory Ltd. (London, UK). Silica gel 60 and 3-methyladenine (3MA) were purchased from Merck KGaA (Darmstadt, Germany). Toyoperal HW-40F gel was purchased from Tosoh Co. (Tokyo, Japan). Primary antibodies against caspase 3, caspase 9, LC3, beclin-1, phosphor-mTOR (Ser2448), phosphor-Akt (Ser473), JNK, phosphor-JNK (Thr183/Tyr185), ERK1/2, phosphorERK1/2 (Thr202/Tyr204), p53, p27, and poly(ADP-ribose)-polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA). Primary antibody against Atg5 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PE conjugate goat anti-rabbit IgG, horseradish peroxidase (HRP) conjugate goat anti-rabbit IgG, and goat anti-mouse IgG were purchased from Life Technologies Co., (Carlsbad, CA). Plant Materials. The leaves of R. formosanum were collected in April and July of 2010 from the Yuanzui Mountain (24°14′6.49″ N, 120°57′7.29″ E at 1911 m asl) in Hopin township of Taichung County, Taiwan. The plant species was identified by Dr. Tsai-Wen Hsu, Key Laboratory of High Altitude Experimental Station, Taiwan Endemic Species Research Institute. The voucher specimens (20100708-Wang) were deposited in the Chemical Ecology Laboratory, Research Center for Biodiversity, China Medical University, Taichung, Taiwan. Isolation and Identification of CNT D1. Five kilograms of airdried leaves of R. formosanum was extracted with methanol three times following the standard extraction procedures.1 The methanolic extract was concentrated to yield 1540 g of dry residue that was then partitioned by dichloromethane (DCM), ethyl acetate (EtOAc), and butanol (BuOH) with H2O to obtained portion of DCM (262 g), EtOAc (220 g), BuOH (423 g), and an aqueous layer (420 g). The EtOAc portion was subjected to a silica gel 60 column using gradient elution with a mixed solvent composed of hexane−ethyl acetate− methanol and led to 31 fractions (EA-1−EA-31). By gradient elution with MeOH−H2O (60−100%), fraction EA-17 (7.9 g) was separated via an Amberlite XAD-2 (Sigma-Aldrich) gel column to obtain 6 subfractions (EA-17-1−EA-17-6). Fraction EA-17-2 (970.7 mg) was further fractionated by Toyopearl HW-40F chromatography by gradient elution with MeOH−H 2 O (40−100%) to give 14 subfractions (EA-17-2-1−EA-17-2-14). Finally, CNT D1 (214.5 mg) was isolated from the subfraction EA-17-2-13. Purified compound was subjected to spectroscopic identification by using 1H NMR and 13C
Figure 1. (A) Flowchart of extraction for cinnamtannin D1 (CNT D1) from R. formosanum. (B) The chemical structure of CNT D1.
Cell Lines and Cell Culture. In this study, A459 cells and H460 cells obtained from American Type Culture Collection (ATCC) were used. The A549 cell line, human alveolar basal epithelial cells, was first developed in 1972 by Giard et al. through explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male. The H460 cell line was developed by Gazdar and associates in 1982 from the pleural fluid of a patient with large-cell cancer of the lung. A549 cells were cultured in DMEM medium, and H460 cells were cultured in RPMI-1640 medium. DMEM medium and RPMI-1640 medium contained 10% FBS, penicillin (100 IU/mL), and streptomycin (100 μg/mL). Both cells were incubated at 37 °C with 5% CO2. The morphology of cells was observed by photomicroscope. MTT Assay. To determine the cell viability, MTT reagent was used. Cells (1 × 104 per each well) were incubated in 96-well plate and treated with CNT D1 (0−200 μM) or 3-MA (1 mM) for 24, 48, and 72 h. After treatment, the medium was removed and MTT reagent (500 μg/mL) was added at 37 °C for 1 h, then the solution was removed from each well and 80 μL of DMSO was added to dissolve the crystal. Finally, the absorbance at OD 570 nm was detected by an 10408
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry
ice-cold 100% methanol for 15 min at −20 °C. After removing methanol and washing with PBS, cells were blocked with blocking buffer (PBS contained 5% FBS and 0.3% Triton X-100). Then blocking buffer was removed from the cells by PBS and hybridized with primary antibody (LC3 antibody; 1:200) (antibody dilution buffer; PBS contained 1% BSA and 0.3% Triton X-100) overnight at 4 °C. The cells were washed with PBS and hybridized with PE conjugated secondary antibody (1:200) for 2 h at 37 °C in the dark. After removing the secondary antibody and washing with PBS, the cell nucleuses were stained with DAPI (1 μg/mL DAPI, and 0.1% Triton X-100) for 15 min at RT in the dark. Finally, cell morphology was observed by confocal microscope. Statistical Analysis. Each treatment was repeated at least three times, and results are expressed as means ± SD. The control and different experiment groups were analyzed by independent Student’s t test, and the P value statistical significance is presented with asterisks (*, P < 0.05; **, P < 0.005; ***, P < 0.001).
ELISA reader. The suppressant effect was presented as a percentage relative to the control. Bromodeoxyuridine (BrdU) Cell Proliferation Assay. A BrdU cell proliferation assay kit was purchased from Cell Signaling Technology (Beverly, MA), and method followed was the protocol of the BrdU cell proliferation assay kit #6813. After cells (5 × 105) were cotreated with BrdU and CNT D1 (150 μM) for 72 h, they were fixed with fixing/denaturing solution for 30 min at room temperature (RT). Detection antibody solution was added and the mixture was incubated for 1 h at RT and washed three times with wash buffer. HRP conjugate solution was added and the mixture was incubated for 30 min at RT and washed three times with wash buffer. Then samples were reacted with TMB substrate for 30 min at RT, and stop solution was added. Finally, the absorbance at OD 440 nm was detected by an ELISA reader. The suppressant effect was presented as a percentage relative to the control. Cell Cycle Analysis. Cell cycle distribution and apoptosis proportion were determined by PI staining. A549 cells (5 × 105) were seeded in a 6 cm culture dish and treated with CNT D1 (125− 175 μM) for 72 h. Cells were collected in a 15 mL tube, washed with PBS, and subsequently fixed with 70% ethanol in −20 °C overnight. Then the 70% ethanol was removed from the cells with PBS, and the cells were stained with PI (20 μg/mL PI, 0.1% Triton X-100, and 0.1 mg/mL RNase A) for 15 min at RT. Finally, cell cycle distribution was analyzed by cytometry (FAC-Scan, BD Biosciences, San Jose, CA). Protein Extraction and Quantification. The total proteins were isolated to analyze target protein expression. A549 cells (1 × 106) were collected after treatment, and to them was added HEPES lysis buffer (20 mM HEPES, 2 mM MgCl2, 10 mM KCl, 1% NP-40, 10 μM Na3VO4, and 10 μg/mL protease inhibitor, pH 7.4) to lysis cells for 15 min on ice. Unlysed parts was removed by centrifugation at 12 000 rpm for 30 min. A Bio-Rad protein assay kit was used to quantify the total proteins concentration, and protocol followed was that included in the Bio-Rad protein assay kit. Western Blot Analysis. Protein expressions were analyzed by Western blot according to a previous publication.13 A 50 μg portion of total proteins sample was separated by SDS−PAGE and transferred to PVDF membrane. After blocking with blocking buffer [TBST (Trisbuffered saline and Tween 20) which contained 5% nonfat milk and 0.1% NaN3] the samples were hybridized separately with primary antibodies, namely, p53, p27, CDK6, cyclin D1, caspase 3, caspase 9, PARP, LC3, Atg5, beclin-1, phosphor-mTOR, phosphor-Akt, phosphor-ERK1/2, ERK, phosphor-JNK, JNK, and β-actin. Primary antibodies were diluted by antibody buffer (TBST which contained 5% BSA and 0.1% NaN3) with the ratios of 1:1000. Finally, membrane was hybridized with secondary antibody (goat anti-mouse IgG or goat antirabbit IgG; 1:5000) and protein bands were detected using ECL Western blotting detection reagents (GE Healthcare UK Ltd.). Apoptosis Detection. A 488 annexin V/dead cell apoptosis kit was purchased from Life Technologies (Eugene, OR), and the procedure for detection followed the instruction from the manufacturer. Cells were collected in the 15 mL tube and diluted to ∼1 × 106 cells/mL with 1× annexin-binding buffer. To a 100 μL portion of cell suspension from each sample were added 5 μL of Alexa Fluor 488−annexin V and 1 μL of PI solution (100 μg/mL). Cells were stained for 15 min at RT, and 400 μL of 1× annexin-binding buffer was added. Finally, cell apoptosis analysis was measured by cytometry (FAC-Scan, BD Biosciences, San Jose, CA). AO Staining and Flow Cytometry Analysis. AO staining was used to observe and measure cell autophagy. A549 cells were treated with CNT D1 in dose-dependent and time-dependent manners. After treatment, cells were collected and washed with PBS and then stained with AO staining (1 μg/mL) for 15 min at 37 °C in the dark. The autophagy of cell morphology was observed by fluorescence microscopy (Nikon TE2000-U), and cell autophagy distribution analysis was measured by cytometry (FAC-Scan, BD Biosciences). Confocal Microscopy. LC3 protein accumulation was observed by immunostainning following the protocol accompanying the LC3 A/ B antibody #4108 (Cell Signaling Technology). A549 cells (5 × 105) were treated with CNT D1 (150 and 175 μM) for 72 h and fixed with
■
RESULTS Identification of CNT D1. The ESI-MS of CNT D1 recorded in both positive- and negative-ion modes [Figure S1, Supporting Information (SI)] exhibited a sodiated ion [M + Na]+ at m/z 887.1 and deprotonated ion [M − H]− at m/z 863.1, indicating a molecular formula of C45H36O18, suggested a triflavonoid moiety (trimeric A-type procyanidin) having only one C−O−C interflavanoid linkage in the structure. All 1H and 13 C NMR resonances of CNT D1 were assigned by analysis of the 2D NMR (HSQC, HMBC, COSY, NOESY) data (Figure S2−S5, SI). In the 1H NMR spectrum, the presence of the AB coupling system at δH 3.45 and 4.00 (each d, J = 3.5 Hz) also indicated an A-type unit in CNT D1. This doubly linked structure was also supported from the one ketal carbon signal at δC 100.0 in the 13C-resonance. The NMR data of the GHI moiety of CNT D1 (unit III in Table S1, SI), appearing at δH 3.94 (d, J = 9 Hz), 3.67 (m), 3.05 (dd, J = 16.2, 6.0), and 2.42 (dd, J = 16.2, 10.1) and δC 83.2, 70.0, and 30.6 consistent with the terminal unit, were consistent with a catechin moiety. The 1 H and 13C spectroscopic data of the DEF moiety of CNT D1 (unit II in Table S1, SI) at δH 5.51 (brs), 4.06 (d, J = 1.8), and δC 78.6, 72.4 suggested that unit II is an epicatechin. Oligomeric procyanidins are generally linked from C-4 of one flavan unit to C-6 or C-8 of another, and when doubly connected it is often from C-2 of the upper unit to the hydroxyl group of the next unit at the C-5 or C-7 position. The spectroscopic data indicated that the lineages between units were connected at position C-4 of unit I/II to C6 of unit II/III, which were confirmed by HMBC correlations between H-4 (Cring) and C-7, C-8, and C-9 (D-ring) and between H-4 (Fring) and C-7, C-8, and C-9 (G-ring), respectively (Table S1, Figure S6, SI). Comparison of the 1H and 13C NMR spectroscopic data (Table S1, SI) with the literature14,15 established it as CNT D1, previously isolated from Cinnamomum cassia,14 the leaves of Machilus philippinensis,15 and the bark of Parameria laevigata.2 Characterization data for CNT D1: pale red amorphous powder; [α]20 D +34.5° (c = 0.2, MeOH); ESI-MS [M − H]− 863.1 m/z, [M + Na]+ 887.1 m/z (calcd for C45H36O18 864.1); UV λMeOH nm (log ε) 230 (3.63), max 243 (3.74), 280 (2.84); The 1H and 13C NMR resonances of CNT D1 are listed in Table S1 (SI). Cytotoxic Effects of CNT D1 on NSCLC Cells. To evaluate the antiproliferative activities of CNT D1 (Figure 1B) on NSCLC cells, A549 and H460 cells were treated with various concentrations of CNT D1 for 24, 48, and 72 h. The cell viability was evaluated using the MTT assay. As shown in 10409
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry
Figure 2. Effect of CNT D1 on antiproliferation activity. (A) A459 and (B) H460 cells were treated with CNT D1 at different concentrations for 24, 48, and 72 h and measured by the MTT assay. After (C) A459 and (D) H460 cells were treated with CNT D1 for 72 h, the cell proliferation ability of A549 and H460 cells was measured by the BrdU cell proliferation assay. These experiments were repeated three times. The data represent the mean ± SD.
Changes in Caspase Activities and PARP Cleavage. To investigate whether the cell death induced by CNT D1 treatment was due to apoptosis, the activities of two types of caspase were examined. CNT D1 showed no noticeable effect on the activities of these caspases, although some weak elevations of caspase activities were observed (Figure 4A,B). We also examined the effects of CNT D1 on PARP cleavage. PARP is well-known as an endogenous substrate for caspase-3 and an early marker of apoptosis. After treatment with CNT D1, the content of the intact form of PARP did not decrease, and the cleaved form of PARP did not appear (Figure 4A,B). These data suggested that apoptosis was not a major cause of cell death in CNT D1-treated A549 cells. Our result also found that CNT D1 did not induce apoptosis in H460 cells (Figure 4C). We further used the annexin V/PI double staining assay to test whether CNT D1 induced cell death through apoptosis. As shown in Figure 4D, our results showed that CNT D1 did not induce significant apoptosis in A549 cells. Formation of Autophagic Vacuoles by Treatment with CNT D1. AO is a fluorescent molecule used to identify autophagy. It can accumulate in acidic organelles in which it becomes protonated, forming aggregates that emit bright red fluorescence. We next studied whether the accumulation of AO in cells was stimulated by CNT D1 treatment. A549 (Figure 5A) and H460 (Figure 6A) cells were treated with the indicated concentrations of CNT D1 for 72 h and then analyzed by fluorescence microscopy. Our data showed that after treatment with CNT D1, AO-labeled fluorescent dots were clearly
Figure 2, our results showed that the exposure of A549 (Figure 2A) and H460 (Figure 2B) cells to CNT D1 decreased cellular viability in a dose- and time-dependent manner. We next used the BrdU incorporation assay to examine the antiproliferative activities of CNT D1. Our results found that CNT D1 decreased the proliferation of A549 (Figure 2C) and H460 (Figure 2D) cells in a dose- and time-dependent manner. These results showed that CDT D1 exhibited efficient cytotoxic effects on NSCLC cells. CNT D1 Induces G1-Phase Cell Cycle Arrest in NSCLC Cells. The ability of an anticancer agent to affect cell cycle distribution can provide information regarding its cytotoxic mechanism of action. We next investigated the effect of CNT D1 on cell cycle distribution in NSCLC cells. A549 cells were treated with the indicated concentrations of CNT D1 for 72 h and subjected to cell cycle analysis. As shown in Figure 3A, CNT D1 caused an obvious G1 arrest in A549 cells. To determine whether CNT D1 induced cell cycle arrest specifically at G1 phase, we examined the expression of p53 and cyclin D1 in CNT D1-treated cells. CNT D1 increased the expression of p53 and decreased the expression of cyclin D1 in a dose- (Figure 3B) and time-dependent manner (Figure 3C). The level of CDK6 and CDK inhibitor p27 was also determined in CNT D1-treated cells. Our results showed that CNT D1 increased the expression of p27 and decreased the expression of CDK6 in a dose- (Figure 3B) and timedependent manner (Figure 3C). Taken together, our results showed that CNT D1 induced cell cycle arrest at G1 phase. 10410
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry
Figure 3. Effect of CNT D1 on cell cycle distribution and related protein expression. (A) To observe the state of the cell cycle, A549 cells were treated with CNT D1 at 125, 150, and 175 μM for 72 h. Cells were stained with PI and measured by flow cytometry. Results are described as means ± SD, and the statistical significance between the control group and different experimental groups is presented with asterisks (*, P < 0.05; **, P < 0.005; ***, P < 0.001). (B) A549 cells were treated with CNT D1 at 125, 150, and 175 μM for 72 h. (C) A549 cells were treated with CNT D1 (150 μM) for the indicated time. The protein expression of p53, p27, CDK6, and cyclin D1 was measured by Western blot. Western blot data presented are representative of those obtained in at least three separate experiments. The values below the figures represent the change in protein expression of the bands normalized to β-actin.
5C) and dose-dependent (Figure 5D) manner. To further verify that CNT D1 did induce autophagy, we evaluated the effect of CNT D1 on the intracellular localization of LC3-II. As shown in Figure 5E, representative fluorescence micrographs showed the punctated pattern of LC3-II. After 72 h treatment with CNT D1 (0, 150, and 175 μM), a marked elevation in the number of cells with visibly increased punctated fluorescence, particularly in the perinuclear region of the cytoplasm, was observed. This suggested that CNT D1 induced autophagosome formation in this cancer cell line. Inhibition of CNT D1-Induced Autophagy Enhanced Cell Death. The induction of autophagy in response to metabolic and therapeutic stresses can have a prodeath or a prosurvival role, which contributes to the anticancer efficacy.9 To evaluate whether CNT D1-induced autophagy promoted cell death or survival, we applied pharmacologic inhibition of autophagy. 3-MA is a known autophagy blocker at an early stage of autophagy. Figure 7A shows that pretreatment with 3-
detected, and the number of these dots increased during treatment in a dose-dependent manner. We next performed FACS analysis of AO-stained acidic vacuoles on CNT D1treated A549 cells. We used the red to green ratio as an indicator of acidic vacuolar organelle (AVO) accumulation and therefore autophagic progression. Quantification of AVOs showed a dose-dependent increase of AVOs in A549 cells (Figure 5B). To quantify the incidence of CNT D1-induced autophagy, we carried out immunoblot analysis and it revealed that CNT D1 caused a time- (Figure 5C) and dose-dependent (Figure 5D) increase in the levels of LC3-II proteins in A549 cells. Moreover, CNT D1 also increased the levels of LC3-II proteins in H460 cells (Figure 6B). Beclin-1 and Atg5 are important regulators of the autophagic pathway. We next measured the expression of beclin-1 and Atg5 using Western blot analysis. CNT D1 did not enhance the accumulation of beclin-1; however, CNT D1-induced autophagy was accompanied by enhanced accumulation of Atg5 in a time- (Figure 10411
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry
Figure 4. Effect of CNT D1 on cleavage of caspase 3, caspase 9, and PARP. (A) A549 cells treated with CNT D1 at 125, 150, and 175 μM for 72 h. (B) A549 and (C) H460 cells were treated with CNT D1 (150 μM) for the indicated time. The protein expression of caspase 3, cleaved caspase 3, caspase 9, cleaved caspase 9, PARP, and cleaved PARP were measured by Western blot. Western blot data presented are representative of those obtained in at least three separate experiments. The values below the figures represent the change in protein expression of the bands normalized to βactin. (D) After A549 cells were treated with CNT D1 for 72 h, cells were stained with annexin V-FITC and PI and measured by flow cytometry.
decrease of phospho-Akt in a dose- (Figure 8A) and timedependent (Figure 8B) manner. Effects of the ERK1/2 and JNK Signaling Pathways on CNT D1-Treated Cells. To determine whether ERK1/2 and JNK phosphorylation was closely related to CNT D1-induced autophagy, we treated A549 cells with CNT D1 and then detected the levels of p-ERK1/2 and p-JNK. CNT D1 treatment increased the expression levels of p-ERK1/2; however, the expression levels of p-JNK slightly decreased in a dose- (Figure 8C) and time-dependent (Figure 8D) manner.
MA potentiated CNT D1-induced cell death compared with CNT D1 or 3-MA treatment alone in A549 cells. Figure 7B demonstrated that pretreatment with 3-MA reduced the percentage of cell proliferation compared with CNT D1 or 3MA treatment alone in A549 cells. CNT D1 Suppresses the Akt/mTOR Signaling Pathway. Our next objective was to identify and characterize the molecular pathways involved in CNT D1-induced autophagy. The Akt/mTOR pathway is the main regulatory pathway that negatively regulates autophagy.9 Therefore, we examined the effect of CNT D1 on this pathway. To investigate whether mTOR was affected by CNT D1, we determined the status of phosphorylated mTOR at Ser2448 using immunoblot analysis. CNT D1 decreased the expression of phosphorylated mTOR in a dose- (Figure 8A) and time-dependent (Figure 8B) manner. Akt was constitutively phosphorylated at residue Ser473 in A549 cells, and treatment with CNT D1 induced a significant
■
DISCUSSION Proanthocyanidins, also known as condensed tannins, are a class of polyphenols found in a variety of plants. Proanthocyanidins with A-type linkages have been recognized as the bioactive compounds in blueberries, peanuts, cranberries, curry, and plums.16 A-Type proanthocyanidins have been reported to play a significant role in the health benefits. Recent studies 10412
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry
Figure 5. CNT D1-induced autophagy in A549 Cells. (A) A549 cells treated with CNT D1 at 125, 150, and 175 μM for 72 h and stained with AO. The autophagy of cell morphology was observed by fluorescence microscopy. Scale bar: 50 μm. (B) The AO-stained acidic vacuoles on CNT D1treated A549 cells were measured by flow cytometry. Scale bar: 50 μm. Autophagy data are described as means ± SD, and the statistical significance between the control group and different experimental groups is presented with asterisks (*, P < 0.05; **, P < 0.005; ***, P < 0.001). (C) A549 cells treated with CNT D1 at 125, 150, and 175 μM for 72 h. (D) A549 cells were treated with CNT D1 (150 μM) for the indicated time. The protein expression of LC3 II, Atg 5, and Beclin 1 were measured by Western blot. Western blot data presented are representative of those obtained in at least three separate experiments. The values below the figures represent the change in protein expression of the bands normalized to β-actin. (E) Detecting the LC3 accumulation by immunofluorscence. A549 cells were stained with rabbit monoclonal anti-LC3 and PE-conjugated secondary antibodies (red), and nuclei were stained with DAPI (blue). Results were observed by confocal microscope. Scale bar: 10 μm.
found that A-type proanthocyanidins reduce peroxide-induced pancreatic acinar cell injury, generate insulin-like biological activity, and improve insulin sensitivity in vivo.17 CNT D1, a major A-type proanthocyanidins in Cinnamomum tamala, could protect pancreatic β-cells from palmitic acid (PA)-induced apoptosis via attenuating oxidative stress in pancreatic β-cell line and primary cultured murine islets.18 However, the effects of CNT D1 have not yet been explored for antineoplastic potential. The present work focused on understanding the
mechanisms of CNT D1-exhibited efficient cytotoxic effects on NSCLC cells. An increasing number of anticancer agents has been shown to induce autophagy in different types of cancer. Autophagy can enhance the efficacy of anticancer agents through autophagymediated mechanisms of cell death in some apoptosis-resistant cancer cells.19 The induction of autophagic processes can be therapeutically useful to evade chemoresistance; therefore, to identify novel autophagic enhancers for chemoresistant cancer 10413
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry
Figure 6. CNT D1-induced autophagy in H460 cells. (A) H460 cells treated with CNT D1 at 125, 150, and 175 μM for 72 h and stained with AO. The autophagy of cell morphology was observed by fluorescence microscopy. Scale bar: 100 μm. (B) H460 cells were treated with CNT D1 (150 μM) for the indicated time. The protein expression of LC3 II was measured by Western blot. Western blot data presented are representative of those obtained in at least three separate experiments. The values below the figures represent change in protein expression of the bands normalized to βactin.
D1 from R. formosanum extracts and identified the cytotoxic effects of CNT D1 on NSCLC cells. AO staining and flow cytometry analysis showed that the number of autophagic vacuoles containing cellular materials increased in A549 cells treated with CNT D1. These findings indicated that CNT D1 treatment induced autophagy but not apoptosis in A549 cells. Recent studies found that autophagy is induced in various cancer cells in response to treatment by some natural compounds. Some important examples are resveratrol,21 genistein,22 curcumin,23 rottlerin,24 and quercetin,25 all of which have been shown to induce autophagy in various cancer cells. To the best of our knowledge, this study is the first to demonstrate that CNT D1 induces autophagy in NSCLC cells. The main pathway involved in autophagy is the formation of a double membrane around cytoplasmic substrates, resulting in the organelle known as an autophagosome. The autophagosome then fuses with a lysosome and its cargo is degraded and recycled. The formation of an autophagosome is controlled by Atgs and their protein products. Beclin-1, one of the major Atg proteins, is a 60 kDa protein that has been implicated as an important regulator of the autophagic pathway. Autophagy can be regulated by various canonical and noncanonical pathways, including beclin-1-dependent,26 beclin-1-dependent and Atg5/ Atg7-independent,27 beclin-1-independent and Vps34-independent,28 or beclin-1-independent and Vps34-dependent29 pathways. In this study, our findings constituted evidence that CNT D1-mediated autophagy was via the noncanonical pathway, being beclin-1-independent but Atg5-dependent. Autophagy plays two different roles in cells, depending on cell type. It may function as a protective mechanism against cellular stress, or it may induce autophagic cell death.30 To investigate whether autophagy induced by CNT D1 had a cytoprotective function or led to autophagic cell death, we treated A549 cells with CNT D1 in the presence or absence of 3-MA (Figure 6A). 3-MA, a specific inhibitor of the early stage of the autophagic process, is often used in autophagy studies. Treatment with 1 mM 3-MA enhanced both LC3-II expression and the cytotoxic effect. Our study found that the inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in CNT
Figure 7. Autophagy inhibition by 3-MA. After A549 cells were pretreated with 1 mM 3-MA for 1 h, they were cotreated with 150 μM CNT D1 and 1 mM 3-MA for 72 h. (A) Protein expression of LC3 I, LC3 II, PARP, and cleaved PARP was determined by Western blot. (B) Cell viability was measured by MTT assay. Results are described as means ± SD, and between the CNT D1 group and 3-MA + CNT D1 group, the statistical significance is presented with asterisks (*, P < 0.05; **, P < 0.005; ***, P < 0.001).
cells is highly important.20 Many novel anticancer agents have been isolated from natural products. Here, we aimed to detect natural anticancer enhancers of autophagy. We isolated CNT 10414
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry
Figure 8. CNT D1 downregulated Akt/mTOR phosphorylation and upregulated ERK1/2 phosphorylation in A549 cells. (A) A549 cells treated with CNT D1 at 125, 150, and 175 μM for 72 h. (B) A549 cells were treated with CNT D1 (150 μM) for the indicated time. The protein expression of Akt and mTOR phosphorylation was measured by Western blot. (C) A549 cells treated with CNT D1 at 125, 150, and 175 μM for 72 h. (D) A549 cells were treated with CNT D1 (150 μM) for the indicated time. The protein expression of ERK1/2 and JNK phosphorylation was measured by Western blot. Western blot data presented are representative of those obtained in at least three separate experiments. The values below the figures represent the change in protein expression of the bands normalized to β-actin.
be one of the common mechanisms of autophagy induction by anticancer agents. In conclusion, we have shown for the first time that CNT D1 induced autophagy in NSCLC cells. As this effect of CNT D1 was investigated only in NSCLC, its effect on other types of cancer should also be examined. Furthermore, additional investigation is needed to precisely clarify the autophagy induction pathway, as it is thought to be a candidate pathway that can be targeted by a chemotherapeutic agent in cancer treatment.
D1-treated cells. Therefore, to further improve the rate of CNT D1-induced NSCLC cell death, complete inhibition of autophagy may be required. Diverse signaling pathways have been reported in the regulation of autophagy in mammalian cells, such as PI3Ks, mTOR, AMPK, MAPKs (ERK1/2 and JNK), and PKC. These signaling pathways may be part of an energy-sensing mechanism and stress response. For example, the Akt/mTOR pathway is an important repressor of autophagy and controller of cell growth and proliferation. Moreover, an increasing number of studies have suggested that ERK1/2 plays a role in modulating autophagy.23 Recent studies also showed that the inhibition of the Akt/mTOR pathway and the activation of the ERK1/2 pathway induces autophagy. Ellington et al. showed that the natural products triterpenoid B-group soyasaponins induced autophagy by inhibiting Akt signaling and enhancing ERK1/2 activity.31 Curcumin induces autophagy in malignant glioma cells through the inhibition of the Akt/mTOR/p70S6K and activates the ERK1/2 pathway.32 In our study, CNT D1 inhibited the Akt/mTOR pathway and activated the ERK1/2 pathway, resulting in autophagy in NSCLC cells. The autophagy regulated by these two different pathways differently influenced the cytotoxicity of CNT D1. Therefore, the combination of Akt inhibition and ERK1/2 activation might
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04375. Table S1 and Figures S1−S6 (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +886-4-2205-3366, ext. 1633. Fax: +886-4-2207-1500. Funding
This study was financially supported by grants from the National Science Council of Taiwan (NSC 102-2313-B-03910415
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry
growth inhibitory effect on non-small-cell lung carcinoma cells. J. Agric. Food Chem. 2014, 62, 875−884. (14) Killday, K. B.; Davey, M. H.; Glinski, J. A.; Duan, P. G.; Veluri, R.; Proni, G.; Daugherty, F. J.; Tempesta, M. S. Bioactive A-type proanthocyanidins from Cinnamomum cassia. J. Nat. Prod. 2011, 74, 1833−1841. (15) Lin, H. C.; Lee, S. S. Proanthocyanidins from the leaves of Machilus philippinensis. J. Nat. Prod. 2010, 73, 1375−1380. (16) Nandakumar, V.; Singh, T.; Katiyar, S. K. Multi-targeted prevention and therapy of cancer by proanthocyanidins. Cancer Lett. 2008, 269, 378−387. (17) Middleton, E., Jr.; Kandaswami, C.; Theoharides, T. C. The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 2000, 52, 673−751. (18) Wang, T.; Sun, P.; Chen, L.; Huang, Q.; Chen, K. X.; Jia, Q.; Li, Y. M.; Wang, H. Y. Cinnamtannin D-1 protects pancreatic beta-cells from palmitic acid-induced apoptosis by attenuating oxidative stress. J. Agric. Food Chem. 2014, 62, 5038−5045. (19) Kondo, Y.; Kanzawa, T.; Sawaya, R.; Kondo, S. The role of autophagy in cancer development and response to therapy. Nat. Rev. Cancer 2005, 5, 726−734. (20) Law, B. Y. K.; Chan, W. K.; Xu, S. W.; Wang, J. R.; Bai, L. P.; Liu, L.; Wong, V. K. W. Natural small-molecule enhancers of autophagy induce autophagic cell death in apoptosis-defective cells. Sci. Rep. 2014, 4, 5510. (21) Opipari, A. W., Jr.; Tan, L. J.; Boitano, A. E.; Sorenson, D. R.; Aurora, A.; Liu, J. R. Resveratrol-induced autophagocytosis in ovarian cancer cells. Cancer Res. 2004, 64, 696−703. (22) Gordon, P. B.; Holen, I.; Seglen, P. O. Protection by naringin and some other flavonoids of hepatocytic autophagy and endocytosis against inhibition by okadaic acid. J. Biol. Chem. 1995, 270, 5830− 5838. (23) Shinojima, N.; Yokoyama, T.; Kondo, Y.; Kondo, S. Roles of the Akt/mTOR/p70S6K and ERK1/2 signaling pathways in curcumininduced autophagy. Autophagy 2007, 3, 635−637. (24) Akar, U.; Ozpolat, B.; Mehta, K.; Fok, J.; Kondo, Y.; LopezBerestein, G. Tissue transglutaminase inhibits autophagy in pancreatic cancer cells. Mol. Cancer Res. 2007, 5, 241−249. (25) Psahoulia, F. H.; Moumtzi, S.; Roberts, M. L.; Sasazuki, T.; Shirasawa, S.; Pintzas, A. Quercetin mediates preferential degradation of oncogenic Ras and causes autophagy in Ha-RAS-transformed human colon cells. Carcinogenesis 2007, 28, 1021−1031. (26) Aita, V. M.; Liang, X. H.; Murty, V. V. V. S.; Pincus, D. L.; Yu, W. P.; Cayanis, E.; Kalachikov, S.; Gilliam, T. C.; Levine, B. Cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21. Genomics 1999, 59, 59−65. (27) Nishida, Y.; Arakawa, S.; Fujitani, K.; Yamaguchi, H.; Mizuta, T.; Kanaseki, T.; Komatsu, M.; Otsu, K.; Tsujimoto, Y.; Shimizu, S. Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 2009, 461, 654−659. (28) Scarlatti, F.; Maffei, R.; Beau, I.; Ghidoni, R.; Codogno, P. Noncanonical autophagy: an exception or an underestimated form of autophagy? Autophagy 2008, 4, 1083−1085. (29) Gao, P.; Bauvy, C.; Souquere, S.; Tonelli, G.; Liu, L.; Zhu, Y. S.; Qiao, Z. Z.; Bakula, D.; Proikas-Cezanne, T.; Pierron, G.; Codogno, P.; Chen, Q. A.; Mehrpour, M. The Bcl-2 homology domain 3 mimetic gossypol induces both beclin 1-dependent and beclin 1-independent cytoprotective autophagy in cancer cells. J. Biol. Chem. 2010, 285, 25570−25581. (30) Dalby, K. N.; Tekedereli, I.; Lopez-Berestein, G.; Ozpolat, B. Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer. Autophagy 2010, 6, 322−329. (31) Ellington, A. A.; Berhow, M. A.; Singletary, K. W. Inhibition of Akt signaling and enhanced ERK1/2 activity are involved in induction of macroautophagy by triterpenoid B-group soyasaponins in colon cancer cells. Carcinogenesis 2006, 27, 298−306. (32) Aoki, H.; Takada, Y.; Kondo, S.; Sawaya, R.; Aggarwal, B. B.; Kondo, Y. Evidence that curcumin suppresses the growth of malignant
001, NSC 102-2313-B-039-001-MY3, and NSC 102-2811-B039-005) and the Ministry of Science and Technology (MOST 103-2811-B-039-020 and MOST 104-2811-B-039-019) to C.H.C. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS Technical assistance with chemical data analyses from Proteomics Research Core Laboratory, Office of Research & Development at China Medical University and Instrument Analysis Centers at the National Chung-Hsing University is greatly appreciated. We also appreciate the identification of plant species by Dr. Tsai-Wen Hsu (Key Laboratory of High Altitude Experimental Station, Taiwan Endemic Species Research Institute).
■
ABBREVIATIONS USED AO, acridine orange; ATGs, autophagy-related genes; AVO, acidic vacuolar organelle; BuOH, butanol; CNT D1, cinnamtannin D1; DAPI, 4′,6-diamidino-2-phenylindole; DCM, dichloromethane; ERK1/2, extracellular signal-regulated kinases 1/2; EtOAc, ethyl acetate; FBS, fatal bovine serum; HRP, horseradish peroxidase; MeOH, methanol; mTOR, mammalian target of rapamycin; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium; NSCLC, non-small-cell lung carcinoma; PA, palmitic acid; PI, propidium iodide
■
REFERENCES
(1) Wang, C. M.; Chen, H. T.; Li, T. C.; Weng, J. H.; Jhan, Y. L.; Lin, S. X.; Chou, C. H. The Role of pentacyclic triterpenoids in the allelopathic effects of Alstonia scholaris. J. Chem. Ecol. 2014, 40, 90−98. (2) Kamiya, K.; Ohno, A.; Horii, Y.; Endang, H.; Umar, M.; Satake, T. A-type proanthocyanidins from the bark of Parameria laevigata. Heterocycles 2003, 60, 1697−1708. (3) Mizushima, N.; Levine, B. Autophagy in mammalian development and differentiation. Nat. Cell Biol. 2010, 12, 823−830. (4) Marino, G.; Lopez-Otin, C. Autophagy: molecular mechanisms, physiological functions and relevance in human pathology. Cell. Mol. Life Sci. 2004, 61, 1439−1454. (5) Deretic, V. Autophagy as an immune defense mechanism. Curr. Opin. Immunol. 2006, 18, 375−382. (6) Bursch, W.; Ellinger, A. AutophagyA basic mechanism and a potential role for neurodegeneration. Folia Neuropathol. 2005, 43, 297−310. (7) Eisenberg-Lerner, A.; Bialik, S.; Simon, H. U.; Kimchi, A. Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ. 2009, 16, 966−975. (8) Mizushima, N.; Yoshimori, T. How to interpret LC3 immunoblotting. Autophagy 2007, 3, 542−545. (9) Hasima, N.; Ozpolat, B. Regulation of autophagy by polyphenolic compounds as a potential therapeutic strategy for cancer. Cell Death Dis. 2014, 5, e1509. (10) Chou, S. C.; Krishna, V.; Chou, C. H. Hydrophobic metabolites from Rhododendron formosanum and their allelopathic activities. Nat. Prod. Commun. 2009, 4, 1189−1192. (11) Krishna, V.; Chang, C. I.; Chou, C. H. Two isomeric epoxysitosterols from Rhododendron formosanum: H-1 and C-13 NMR chemical shift assignments. Magn. Reson. Chem. 2006, 44, 817−819. (12) Wang, C. M.; Li, T. C.; Jhan, Y. L.; Weng, J. H.; Chou, C. H. The impact of microbial biotransformation of catechin in enhancing the allelopathic effects of Rhododendron formosanum. PLoS One 2013, 8, e85162. (13) Way, T. D.; Tsai, S. J.; Wang, C. M.; Ho, C. T.; Chou, C. H. Chemical constituents of Rhododendron formosanum show pronounced 10416
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
Article
Journal of Agricultural and Food Chemistry gliomas in vitro and in vivo through induction of autophagy: Role of Akt and extracellular signal-regulated kinase signaling pathways. Mol. Pharmacol. 2007, 72, 29−39.
10417
DOI: 10.1021/acs.jafc.5b04375 J. Agric. Food Chem. 2015, 63, 10407−10417
