Phytomedicine 21 (2014) 1490–1496
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
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
i n f o
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
G.H. Dai et al. / Phytomedicine 21 (2014) 1490–1496
1491
OH
antitumor mechanisms of Myricanol remains unclear. The present study aims to investigate the mechanisms underlying the effects of Myricanol on A549 cells in vitro.
H3CO
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
HO
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
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
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
i n f o
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
G.H. Dai et al. / Phytomedicine 21 (2014) 1490–1496
1491
OH
antitumor mechanisms of Myricanol remains unclear. The present study aims to investigate the mechanisms underlying the effects of Myricanol on A549 cells in vitro.
H3CO
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
HO
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
