Growth-inhibiting and apoptosis-inducing activities of Myricanol from the bark of Myrica rubra in human lung adenocarcinoma A549 cells

Phytomedicine 21 (2014) 1490–1496

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Growth-inhibiting and apoptosis-inducing activities of Myricanol from the bark of Myrica rubra in human lung adenocarcinoma A549 cells G.H. Dai a,∗ , G.M. Meng b , Y.L. Tong a , X. Chen a , Z.M. Ren a , K. Wang c , F. Yang a,∗∗ a

Institute of Basic Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou 310007, China Key Laboratory of Tongde Hospital of Zhejiang Province, Hangzhou 310012, China c College of Animal Sciences, Zhejiang University, Hangzhou 310058, China b

a r t i c l e

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Article history: Received 13 December 2013 Received in revised form 13 March 2014 Accepted 20 April 2014 Keywords: Myricanol Anti-cancer activity Cell growth inhibition Apoptosis

a b s t r a c t Myrica rubra (Lour.) Sieb. Et Zucc. is a myricaceae Myrica plant. It is a subtropical fruit tree in China and other Asian countries. The bark of M. rubra is used in Chinese folk medicine because of its antibacterial, antioxidant, anti-inflammatory, and anticancer activities. However, the mechanisms underlying such activities remain unclear. This study investigated whether or not Myricanol extracted from M. rubra bark elicits anti-cancer effects on human lung adenocarcinoma A549 cells by inducing apoptosis in vivo. Myricanol was extracted from M. rubra bark through system solvent extraction and silica gel layer column separation. The results of tritiated thymidine assay, colony formation assay, and flow cytometry indicated that Myricanol inhibited the growth of A549 cells. The effects of Myricanol on the expression of key apoptosis-related genes in A549 cells were evaluated by quantitative PCR and Western blot analyses. Myricanol significantly inhibited the growth of A549 cells in a dose-dependent manner, with a half maximal inhibitory concentration of 4.85 ␮g/ml. Myricanol significantly decreased colony formation and induced A549 cell apoptosis. Myricanol upregulated the expression of Caspase-3, Caspase-9, Bax, and p21 and downregulated the expression of Bcl-2 at the mRNA and protein levels. These changes were associated with apoptosis. Based on these results, we propose that Myricanol elicits growth inhibitory and cytotoxic effects on lung cancer cells. Therefore, Myricanol may be a clinical candidate for the prevention and treatment of lung cancer. © 2014 Elsevier GmbH. All rights reserved.

Introduction Lung cancer, which is generally divided into small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). NSCLC accounts for approximately 75–85% of all lung cancers (Jemal et al., 2009) and is still one of the leading causes of neoplasia-related fatalities (Divisi et al., 2006). Despite the availability of new chemotherapy regimens and cytotoxic combinations investigated in previous clinical trials, no significant improvement in the prognosis of patients with lung cancer has been achieved. The five-year survival rate for all patients diagnosed with lung cancer is approximately 15%, which is only 5%

∗ Corresponding author. Tel.: +86 13989494472; fax: +86 571 8884 5196. ∗∗ Corresponding author. Fax: +86 571 8884 5196. E-mail addresses: [email protected] (G.H. Dai), [email protected] (F. Yang). http://dx.doi.org/10.1016/j.phymed.2014.04.025 0944-7113/© 2014 Elsevier GmbH. All rights reserved.

higher than the survival rate 40 years ago (Korpanty et al., 2011). In addition, many lung cancer cells are resistant to chemotherapeutic drugs. Therefore, the development of new therapeutic drugs for lung cancer is clinically important. Previously, scientists have focused on the potential of extracts from traditional Chinese medicinal herbs as alternative and complementary medications for cancer treatment (Han et al., 2003; Roy et al., 2007; Li et al., 2009). The bark of Myrica rubra contains flavonoids, tannins, triterpenes, and diarylheptanoids (Zhang et al., 2008). Diarylheptanoids are used not only as food flavoring agents but also as medicines because of their many beneficial properties, including antitumor (Ishida et al., 2000), antiviral (Konno et al., 2011), antioxidant (Tao et al., 2008), and anti-inflammatory (Aguilar et al., 2011). Medicinal chemists and pharmaceutical researchers have recently focused on natural plants. Myricanol exerts potent anticancer effects on many cancer cell lines, including HL-60 and HepG2 (data not shown). However, the detailed

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antitumor mechanisms of Myricanol remains unclear. The present study aims to investigate the mechanisms underlying the effects of Myricanol on A549 cells in vitro.

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Materials and methods Chemicals and reagents The dried bark of M. rubra was provided by the Yongjia County Pankeng Sensen Farm (Wenzhou, Zhejiang, China) and authenticated by our experts at the Zhejiang Academy of TCM, Hangzhou, China. The voucher specimen was maintained in Zhejiang Academy of TCM. The Myricanol standard (>98% pure) used in highperformance liquid chromatography (HPLC) was purchased from BioBioPha Co., Ltd. (Kunming, Yunnan, China). Tritiated thymidine (3 H-TdR) was purchased from SINAP of CAS (Shanghai, China). Fluorouracil (5-FU) injection was purchased from Tianjin Jin Yao Amino Acid Co., Ltd. (Tianjin, China). Silica gel for column chromatography (200–300) was purchased from Qingdao Haiyang Chemical Co., Ltd. (Qingdao, Shandong, China). Annexin V-FITC/PI Apoptosis Assay kit was purchased from Zoman Bio Co., Ltd. (Beijing, China). Culture reagents, such as phosphate-buffered saline (PBS), RPMI-1640 medium, 0.25% trypsin, and 0.02% ethylenediaminetetraacetic acid were purchased from Gino Biological Pharmaceutical Technology Co., Ltd. (Hangzhou, Zhejiang, China). Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, Zhejiang, China). Primary rabbit monoclonal antibodies against Caspase-3, Caspase-9, Cleaved Caspase-9, Bax, Bcl-2, p21, and ␤-tubulin were purchased from Epitomics (Burlingame, CA, USA). All other chemicals used were of analytical grade and were purchased from Sigma–Aldrich (St. Louis, MO, USA).

H3CO OH

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Fig. 1. Chemical structure of Myricanol.

Cell culture The human lung carcinoma A549 cell line was obtained from the Integrated Traditional Chinese and Western Medicine Cancer Research Laboratory (Zhejiang Cancer Hospital, China). A549 cells were cultured in RPMI-1640 medium containing 100 U/ml of penicillin, 100 ␮g/ml of streptomycin, and 10% heat-inactivated FBS at 37 ◦ C in a humidified atmosphere of 5% CO2 . Upon reaching 80–90% confluence, the cells were trypsinized, harvested, and seeded into a new cell culture dish. The cells were treated the following day with Myricanol (at a final concentration range of 1.56–50.0 ␮g/ml), vehicle (0.1% DMSO), and 5-FU in RPMI-1640 medium for 48 h. A549 cells were used to determine the growth inhibitory and apoptotic effects of Myricanol. Cytotoxicity assay

Preparation of Myricanol from M. rubra bark Dried M. rubra bark was ground into fine powder and sifted through a 20-mesh sieve. The powdered bark (200 g) was extracted with 2000 ml of 85% ethanol at a constant temperature of 80 ◦ C for 2 h. This extraction process was repeated thrice, and the resulting extracts were combined. The solutions were filtered and concentrated in a rotary evaporator (RE200A, Shanghai, China) under reduced pressure. The residue (60.2 g) was designated as the ethanol extract. The ethanol extracts were further extracted with 300 ml of chloroform at room temperature for 1 h. The extraction process was repeated thrice, and the resulting extracts were combined. After evaporating the solvent using a rotary evaporator, the residue (6.0 g) was designated as the chloroform extract. Approximately 0.5 g of the chloroform extract was fed to a column chromatography silica gel layer column separation with mobile phase elution (petroleum ether:ethyl acetate = 7:2). Each 30 ml bottle was combined with the same components for thin-layer chromatography. Myricanol content was detected by HPLC (Varian Prostar, Germany). Chromatographic separation was performed by using a YMC-pack ODS-A column (250 mm × 4.6 mm, 5 ␮m). The mobile phase comprised water/acetonitrile 55:45 (v/v), the flow rate was 1.0 ml/min, and the separation temperature was 25 ◦ C. The UV detector was set at 250 nm. The chemical structure of Myricanol is shown in Fig. 1. Myricanol at 100 mg/ml was 100% dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution, which was subsequently stored at −20 ◦ C and diluted with medium before use in experiments. The final DMSO concentration did not exceed 0.1% throughout the study. All control groups contained 0.1% DMSO.

The cytotoxic effect of Myricanol was measured using the assay. A549 cells (5 × 103 /well) were seeded in 96-well microtiter plates and incubated for 24 h to allow cell attachment prior to treatment with Myricanol. Myricanol was dissolved in DMSO and filled with the medium until the final concentration of the vehicle (DMSO) in the cell medium did not exceed 0.1% (v/v). After incubation for 48 h at 37 ◦ C in a humidified incubator, each well was added with 3 H-TdR (0.5 ␮ci/50 ␮l in RPMI-1640 medium) and then incubated for 16 h. The cells were collected, and the counts per minute for each sample were determined by liquid scintillation counting (Packard Model 2050). The growth inhibitory effect of Myricanol was assessed to obtain cell viability percentage. Assays were performed in triplicate using three independent experiments. 3 H-TdR

Colony formation assay The toxicity of Myricanol on A549 cells was assessed using colony formation assay as previously described with modifications (Zhao et al., 2011). The cells were subcultured into 24-well plates (200 cells/well) and incubated for 18 h in 5% CO2 at 37 ◦ C. Myricanol was dissolved in DMSO and added with the medium to obtain a final vehicle (DMSO) concentration of <0.1%. The cells were treated with Myricanol (final concentration range of 1.56–50.0 ␮g/ml), vehicle (0.1% DMSO), and 5-FU. Subsequently, cells were incubated for 10 d under standard conditions. The plates were viewed under a microscope every other day. At termination of culture, the medium was decanted, and the cells were rinsed with PBS. The cells were fixed and stained with crystal violet (0.5% in 95% ethanol) for 5 min and then gently washed to remove the dye. Colonies containing at least 50 cells were counted. All assays were performed in triplicate.

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Fig. 2. Myricanol content detection through HPLC. (A) Myricanol standard, 98.0%. (B) Extracts of Myricanol, 96.02%. (C) Chloroform fractions, 24.84%. (D) Extracts of ethanol, 2.47%.

Cell apoptosis analysis by flow cytometry (FCM) The early stages of apoptosis were characterized by perturbations in the cellular membrane, which resulted in the redistribution of phosphatidylserine to the external side of the cell membrane. This process provoked a flux of calcium, which was required by the Annexin V-labeled dye FITC to selectively bind to phosphatidylserine. Thus, cells undergoing apoptosis were identified. The cells were also stained with propidium iodide (PI) to distinguish early and late apoptotic cells from necrotic cells. The Annexin V-FITC/PI apoptosis assay kit was used following the protocol specified by the manufacturer. The cells (1.5 × 105 /well) were placed overnight in six-well plates and then incubated for 24 h to allow cell attachment. The cells were treated with Myricanol (5.0 ␮g/ml), 5-FU (5.0 ␮g/ml), or vehicle (0.1% DMSO) for 48 h. The cells (2 × 105 ) were resuspended in 500 ␮l PBS, incubated in the dark with both FITC-Annexin V and PI for 15 min, and then analyzed by FCM (Beckman Coulter, Model EPICS XL-4). For each measurement, at least 10,000 cells were counted. The experiments were repeated thrice. Total RNA isolation and quantitative real-time PCR (qPCR) analysis A549 cells were pretreated with assigned concentrations of Myricanol (2.5, 5.0, and 10.0 ␮g/ml) for 48 h. The vehicle (0.1% DMSO) served as the control. The total RNA of each sample was isolated using an E.Z.N.A Total RNA Kit (Omega Bio-tech Inc., USA) according to the manufacturer’s protocol. Total RNA samples were suspended in diethylpyrocarbonate-treated water. The purity and concentration of all RNA samples were measured using a Nano Drop spectrophotometer (ND-2000, NanoDrop Technologies, USA). For cDNA synthesis, 1 ␮g of total RNA was used in a 25 ␮l reaction volume using a First-Strand cDNA synthesis kit (Gene Copoeia, USA).

The reaction products of reverse transcription were maintained at −20 ◦ C until use. All oligonucleotide primers were designed using Perlprimer software and synthesized commercially (Sangon Biotechnology, Shanghai, China). The sequences of the primers are as follows: 5 -GAGAAACCTGCCAAGTATGATGAC3 (forward) and 5 -TAGCCGTATTC ATTGTCATACCAG-3  (reverse) for GAPDH, 5 -ATCACAGCAAAAG GAGCAGTTT-3 (forward) and 5 -ATCACAGCAAAAGGAGCAGTTT-3 (reverse) for Caspase-3, 5 -TCTGG AGGATTTGGTGATGTC-3 (forward) and 5 -CAT TTTCTTGGCATC AGGTC-3 (reverse) for Caspase-9, 5 AAGCTGAGCGAGTGTCTCAAG-3 (forward) and 5 -CAAAGTAGAA AAGGGCGACAAC-3 (reverse) for Bax, 5 -ATGTGTGTGGAGAGCG TCAAC-3 (forward) and 5 -AGAGACAGCCAGGAGAAATCAAAC-3 (reverse) for Bcl-2, 5 -TTAGCAGCGG AACAAGGAGT-3 (forward) and 5 -AGAAACGGGAACCAG GACA C-3 (reverse) for p21. qPCR was performed on the Mastercycler Ep Realplex (Eppendorf, Hamburg, Germany) using a SYBR premix EX Taq (TaKaRa, Dalian, China) following the manufacturer’s protocol. The reactions were conducted in duplicate in a 25 ␮l reaction volume in a 96-well plate, and reaction mixtures with no cDNA served as the negative control. The two-step PCR reaction condition was as follows: initial denaturation at 95 ◦ C for 30 s, followed by 40 cycles of denaturation at 95 ◦ C for 5 s; annealing and extension at 60 ◦ C for 30 s, followed by confirmation with melting curve analysis at 95 ◦ C for 15 s and at 50 ◦ C and 95 ◦ C for 15 s. The real-time qPCR products were confirmed by DNA sequencing, electrophoresed with 1.5% agarose gel after staining by GoldView (SBS Genetech, Beijing, China), and then visualized under UV light. GAPDH was used as a housekeeping gene to normalize the expression of the target genes (Caspase-3, Caspase-9, Bax, Bcl-2, and p21) using 2−Ct , where Ct is Ct (drug treatment) – Ct (background), Ct is Ct (target gene) – Ct (GAPDH), and Ct is the threshold cycle (Whyte et al., 2007).

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temperature. After extensive washing with TBST, the immunoreactive protein bands on the membrane were developed for 3 min in 10 ml of alkaline phosphatase for Western color development on buffer [100 mM Tris–Cl (pH 9.5), 5 mM MgCl2 , and 50 mM NaCl] mixed with 100 ␮l of a nitro blue tetrazolium chloride (NBT)/5bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP) solution (18.75 mg/ml of NBT and 9.4 mg/ml BCIP in 67% DMSO, v/v). The results of western blot analysis were evaluated using Quantity One software.

Results Fig. 3. Growth inhibitory effect of Myricanol on A549 cells after 72-h treatment (5-FU served as positive control).

Western blot analysis A549 cells were pretreated with assigned concentrations of Myricanol (2.5, 5.0, and 10.0 ␮g/ml) for 48 h. The vehicle (0.1% DMSO) and 5-FU (5.0 ␮g/ml) served as the negative and positive controls, respectively. The cells were harvested by lysing buffer [10% glycerol, 0.5% NP-40, 1 mM leupeptin, 50 mM Tris–Cl (pH 7.5), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol] on ice and by centrifugation at 12,000 g for 10 min at 4 ◦ C. Protein concentrations in the supernatants were determined using a Bicinchoninic Acid Protein Assay Kit (Beyotime Biotechnology, China). Subsequently, equal amounts of cellular protein (30 ␮g) were subjected to 12–15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The gels were transferred to polyvinylidene fluoride membranes, and the membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 [TBST; 20 mM Tris–Cl (pH 7.4), 150 mM NaCl, and 0.02% Tween 20] for 30 min at room temperature. The blots were incubated overnight with primary antibodies for 1 h at room temperature or at 4 ◦ C overnight. Thereafter, the membranes were washed thrice with TBST and incubated with a 1:10,000 dilution of alkaline phosphatase-conjugated secondary antibody for 1 h at room

Myricanol content HPLC (Varian Prostar, 330 DAD, 410 Auto Sampler) detection (Fig. 2) was performed in gradient mode using a YMC-pack ODSA C(18) column (250 mm × 4.6 mm, 5 ␮m) with mobile phases of acetonitrile and water (45:55) at 1 ml/min. The results obtained at a detection wavelength of 250 nm demonstrated that Myrica bark comprised a high level of Myricanol.

The growth-inhibitory effect of Myricanol on A549 cells The potential cytotoxic effects of Myricanol (Fig. 3) on A549 cells were investigated by 3 H-TdR assay. Myricanol showed significant growth inhibitory effects on A549 cells in a dose-dependent manner, with a half maximal inhibitory concentration (IC50 ) of 4.85 ␮g/ml. The inhibitory effect of Myricanol on cell growth was evaluated under a microscope. As shown in Fig. 4, the 0.1% DMSO-treated cells showed good adhesion with intact morphology (Fig. 4A). After Myricanol treatment for 48 h, the number of adherent cells significantly decreased, and a large number of cells were suspended in the culture medium (Fig. 4B–D). Myricanol-induced morphological injuries resulted in cell necrosis with debris.

Fig. 4. Representative photos of the morphology of the Myricanol-treated A549 after 48 h treatment using bright field microscopy (100×). (A) Vehicle (0.1% DMSO) control. (B) Myricanol, 6.25 ␮g/ml. (C) Myricanol, 12.5 ␮g/ml. (D) Myricanol, 25.0 ␮g/ml.

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Effects of Myricanol on mRNA expression of apoptotic pathway in A549 cells According to Affymetrix Gene Expression (data not shown), Myricanol inhibited cell growth via the apoptotic pathway. The relative expression levels of four apoptosis-associated genes were measured by quantitative real-time reverse transcriptasepolymerase chain reaction. As shown in Fig. 7, Myricanol treatment significantly upregulated the mRNA expression level of Caspase3, Caspase-9, Bax, and p21 and significantly downregulated the mRNA expression level of Bcl-2 in a dose-dependent manner. All gene expression changes contributed to cell apoptosis (Huang et al., 2012). Fig. 5. Inhibitory effect of Myricanol on formation of colonies for A549 cells after a 10-d treatment (5-FU served as positive control).

The results of colony formation assay showed that Myricanol significantly reduced the number of A549 cell colonies in a dosedependent manner (Fig. 5). Apoptotic effect of Myricanol on A549 cells A549 cells were treated with Myricanol (5.0 ␮g/ml) for 48 h. The apoptotic rate induced by Myricanol was quantified by FCM, after cell labeling with Annexin V-FITC and PI (Fig. 6). The apoptotic rate increased from 4.13% (untreated cells) to 34.3% (Myricanol-treated cells), indicating that Myricanol can induce the apoptosis of A549 cells.

Myricanol induced A549 cells apoptosis by regulating the expression level of associated proteins We measured the expression levels of proteins (Caspase-3, Caspase-9, Cleaved Caspase-9, Bax, Bcl-2, and p21) associated with the mitochondrial pathway of apoptosis to further elucidate the mechanism involved in the Myricanol-mediated apoptosis of A549 cells. As shown in Fig. 8, Myricanol treatment increased the expression of the pro-apoptotic proteins Bax, Cleaved Caspase-9, and p21 in a dose-dependent manner (2.5, 5.0, and 10.0 ␮g/ml). The expression of the anti-apoptotic protein Bcl-2 was reduced by Myricanol treatment in a dose-dependent manner. These result suggested that Myricanol can induce A549 cell apoptosis.

Fig. 6. Effects of Myricanol on quantitation of apoptosis in A549 cells. The cells were treated with Myricanol (5.0 ␮g/ml) for 48 h, and then labeled with Annexin V-FITC and PI. (A) Vehicle (0.1% DMSO) control. (B) 5-FU, 5.0 ␮g/ml. (C) Myricanol, 5.0 ␮g/ml. Annexin−/PI-(LL), viable cells; annexin+/PI-(LR), early apoptosis cells; annexin+/PI+ (UR), late apoptosis cells. LL: lower left; LR: lower right; UR: upper right.

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Fig. 7. Effects of Myricanol on Caspase-3, Caspase-9, Bax, Bcl-2, and p21 mRNA expression in A549 cells. As result shown, the mRNA expression of Caspase-3, Caspase-9, Bax, and p21 were raised dramatically in dose-dependent of Myricanol. And also significantly downregulated the expression of Bcl-2 mRNA. All these gene expression change are important in apoptosis pathway.

Discussion In Asia, Chinese medicinal herbs have been widely used and historically well documented for centuries. However, information on Chinese medicinal herbs is limited. M. rubra bark is an important medicinal plant in Asian countries because of its medicinal properties, including antibacterial (Matsuda et al., 2001), antioxidant (Akazawa et al., 2010), anti-inflammatory (Wang et al., 2010), and anticancer (Zhang et al., 2009). Previous pharmacological studies have isolated many bioactive agents from M. rubra bark (Mochida, 2008; Tong et al., 2009). Some of these chemicals exhibit anticancer activities (Kuo et al., 2004). Myricanol is a bioactive agent that can be extracted from Myrica bark (Liu et al., 2009; Tene et al., 2000). Myricanol belongs to a class of cyclic diarylheptanoids. Several compounds with such a chemical structure have been identified; these compounds include Myricanol, Myricanone, Alnusone,

Fig. 8. Western blot analysis of apoptosis related proteins. Total protein extracts were prepared after treatment of A549 cells with: 2.5, 5.0, and 10.0 ␮g/ml Myricanol for 48 h and then analyzed by Western blotting with antibodies to Caspase-3, Caspase-9, Cleaved Caspase-9, Bax, Bcl-2, p21, and ␤-tubulin. Western blots were representative of three independent experiments.

Alusdiol, and Asadanin (Zhang et al., 2008). Myricanol and Myricanone are both components of M. rubra bark, but Myricanol is more abundant (approximately 0.7%) than Myricanone. Myricanol has significant antitumor activity. Myricanol synthesis has not been reported as of this writing. We will perform a structural modification of Myricanol in future functional studies. Myricanol has many biological activities, including reversal of Alzheimer’s disease (Jones et al., 2011), inhibition of nitric oxide production (Tao et al., 2002), protection of liver injuries (Ohta et al., 1992), and anti-androgenic activity (Matsuda et al., 2001). However, information on the anticancer activity of Myricanol is lacking. The present study is the first to investigate and demonstrate Myricanol-induced inhibition of A549 cell proliferation. The effect of Myricanol on A549 cell growth was investigated by 3 HTdR assay. Myricanol demonstrated significant cytotoxic effects on A549 cells, with an IC50 value of 4.85 ␮g/ml. Moreover, a colony formation assay was performed to detect the growth inhibitory effect of the compound on the anchorage-independent growth of colonies (Li et al., 2009). In the present study, Myricanol effectively inhibited colony formation in A549 cells in a dose-dependent manner. This finding agreed with the results of 3 H-TdR assay and microscopic examination. Myricanol possibly induced the apoptosis of A549 cells by upregulating Caspase-3, Caspase-9, Bax, and p21 and downregulating Bcl-2 at the mRNA and protein levels. P21 is a wellstudied cyclin-dependent kinase inhibitor that indicates apoptosis signaling. Myricanol caused cell cycle arrest in A549 cells partly by promoting p21 accumulation. Caspases, as primary executors of apoptosis, are highly conserved (Olsson and Zhivotovsky, 2011). Therefore, Myricanol exhibits anticancer activity. Apoptosis is a normal physiological process that occurs during embryonic development and tissue homeostasis in adult animals. Any dysregulation of apoptosis can result in abnormality, disease, and death (Wilson et al., 1998). Cancer is a result of uncontrolled cell proliferation and apoptosis dysregulation (Han et al., 2007). Therefore, the induction of apoptosis is a highly desirable goal in developing preventive strategies for cancer control (Reed and Pellecchia, 2005). The present results showed that Myricanol effectively induced the apoptosis of A549 cells. Myricanol possibly exerts anticancer activities by inducing the apoptosis and inhibiting the growth of cancer cells. In conclusion, Myricanol extracted from M. rubra bark can effectively inhibit the growth and induce the apoptosis of A549 cells. Therefore, Myricanol may be a clinical candidate for the

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