J Huazhong Univ Sci Technol [Med Sci] 34(4):497-503,2014 DOI 10.1007/s11596-014-1305-1 J Huazhong Univ Sci Technol［Med Sci］ 34(4):2014
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Anticancer Effect of Icaritin on Human Lung Cancer Cells through Inducing S Phase Cell Cycle Arrest and Apoptosis* Qian ZHENG (郑 倩), Wei-wei LIU (刘伟伟), Bin LI (李 斌), Hui-jie CHEN (陈慧洁), Wen-shan ZHU (祝文山), Guang-xiao YANG (杨广笑), Ming-jie CHEN (陈明洁)#, Guang-yuan HE (何光源)# The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014 Summary: Icaritin, a prenylflavonoid derivative from Epimedium Genus, has been shown to exhibit many pharmacological and biological activities. However, the function and the underlying mechanisms of icaritin in human non-small cell lung cancer have not been fully elucidated. The purpose of this study was to investigate the anticancer effects of icaritin on A549 cells and explore the underlying molecular mechanism. The cell viability after icaritin treatment was tested by MTT assay. The cell cycle distribution, apoptosis and reactive oxygen species (ROS) levels were analyzed by flow cytometry. The mRNA and protein expression levels of the genes involved in proliferation and apoptosis were respectively detected by RT-PCR and Western blotting. The results demonstrated that icaritin induced cell cycle arrest at S phase, and down-regulated the expression levels of S regulatory proteins such as Cyclin A and CDK2. Icaritin also induced cell apoptosis characterized by positive Hoechst 33258 staining, accumulation of the Annexin V-positive cells, increased ROS level and alteration in Bcl-2 family proteins expression. Moreover, icaritin induced sustained phosphorylation of ERK and p38 MAPK. These findings suggested that icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells. Key words: icaritin; A549 cells; apoptosis; ERK; p38 MAPK
Lung cancer is the most prevalent cancer worldwide with the highest cancer-related mortality[1, 2]. According to the data published by World Health Organization (WHO), lung cancer is ranking the first death in the world and accounts for about 19% of all malignant tumors. The American Cancer Society estimates that there would be 159 480 deaths from lung cancer and 228 190 new cases diagnosed in 2013. Of these, non-small cell lung cancer (NSCLC) accounts for about 85%[1]. Nowadays, although the methods of early diagnosis have been improved, and the survival rate has increased to some extent, the therapeutic interventions have little influence on the prognosis of patients with NSCLC. The 5-year survival rate of NSCLC remains less than 15% and recurrence rate still increases after surgery[3]. Cancer cells have the ability of malignant proliferation and evading apoptosis. So that, anticancer agents that can trigger tumor cells undergoing apoptosis are viable for treatment of NSCLC. Icaritin has long been used as a Chinese traditional medicine. Previous studies showed that icaritin exerted a variety of biological and pharmacological effects including inducible differentiation of embryonic stem cells into cardiomyocytes[4], prevention fsteroid-associated osQian ZHENG, E-mail: [email protected] # Corresponding authors, Ming-jie CHEN, E-mail: [email protected]; Guang-yuan HE, E-mail: [email protected] * This project was supported by grants from Wuhan Municipal Science and Technology Research Project, China (No. 201260523185), and the Public Science and Technology Research Funds Projects of Ocean, China (No. 201005013).
teonecrosis[5], neuroprotective effects[6], enhancement of osteoblastic and suppression of osteoclastic differentiation and activity[7], and inhibition of the proliferation of acute myeloid leukemia, breast, prostate, endometrial – and liver cancer cell lines by inducing apoptosis[8 13]. In the present studies, we investigated the effects of icaritin on the growth of human lung carcinoma cell line and the molecular basis of its regulation in vitro. The results revealed that icaritin strongly suppressed the growth of lung cancer A549 cells with cell cycle arrest and cell apoptosis. 1 MATERIALS AND METHODS 1.1 Reagents Icaritin at 98% purity was purchased from Must Biotechnology Co., Ltd. (China). Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were acquired from HyClone Laboratories (USA). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma (USA). Propidium iodide (PI), Hoechst 33258, DCFH-DA and BCA Protein Assay Kit were obtained from Beyotime (China). An Annexin V-FITC/PI apoptosis detection kit was purchased from MultiSciences Biotech Co., Ltd (China). Primary antibodies specific to Cyclin A, CDK2, Bax, Bcl-2, p53, p21, and β-actin were obtained from Santa Cruz Biotechnology (USA). The secondary antibodies goat anti-mouse IgG HRP conjugate and goat anti-rabbit IgG HRP conjugate were obtained from Boster Biological Technology, Ltd (China).
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J Huazhong Univ Sci Technol［Med Sci］ 34(4):2014
1.2 Cell Culture A549 cell line was obtained from the China Center for Type Culture Collection (Wuhan University, China). The cells were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2. All experiments were performed one day after the cells were seeded. 1.3 MTT Assay The effects of icaritin on cell viability were determined by measuring the metabolic activity (MTT assay). A density of 2×104 cells/well with 200 µL culture medium per well was seeded in 96-well plates for 24 h. On the next day, the medium was replaced with fresh medium containing various concentrations of icaritin or vehicle (DMSO). After incubation for an additional 24 and 48 h, a total of 20 µL MTT solution was added to each well and incubated at 37°C for 4 h to metabolize MTT into formazan. Then the supernatant was discarded and 100 µL of DMSO was added to each well to terminate the reaction. The absorption (A) value at 490 nm was measured using a microplate reader (Sunrise, Switzerland). Three independent experiments were performed in triplicates. 1.4 Cell Cycle Analysis by PI Staining The cell cycle distributions were analyzed by flow cytometry after PI staining. The A549 cells were cultured with the indicated concentrations of icaritin for 48 h. Then the cells were harvested by centrifugation and washed with cold PBS. Finally, the cells were fixed with 70% ice-cold ethanol overnight at –20°C. After fixation, the cells were harvested and rinsed once with PBS and then incubated with 1 mL PI staining solution (50 µg/mL PI and 100 µg/mL RNase A) for 1 h at room temperature. The distributions of cells in the G1, S, and G2/M phases were measured. 1.5 Hoechst 33258 Staining Apoptotic cells could be detected by investigating condensed and fragmented nuclei stained with Hoechst 33258 under a fluorescent microscope. Cells were exposed to icaritin for 48 h, and then washed with PBS. Subsequently, the cells were stained with Hoechst 33258 Target genes Bax Forward Reverse Bcl-2 Forward Reverse Cyclin A Forward Reverse CDK2 Forward Reverse GAPDH Forward Reverse
for 10 min at room temperature and washed with PBS for 3 times. Finally the samples were photographed (×10 objective) with inverted epifluorescent microscope. 1.6 Apoptosis Detection with Annexin V-FITC/PI Staining Living cells and apoptotic cells could be detected using the Annexin V-FITC/PI apoptosis kit by flow cytometry. The A549 cells were treated with the indicated concentrations of icaritin for 48 h in 6-well plates. Then the cells were harvested and washed twice with ice-cold PBS. After discarding the supernatant, the cells were resuspended in 500 µL binding buffer and stained with 5 µL Annexin V-FITC and 10 µL PI for 5 min in the dark at room temperature. Then, the samples were detected by flow cytometry and the apoptotic population was analyzed using FlowJo Software (Treestar, USA). 1.7 Measurement of Intracellular Reactive Oxygen Species Intracellular reactive oxygen species (ROS) generation was assessed using the stable non-polar dye DCFH-DA. Cells were treated with or without icaritin for 48 h, then harvested and incubated with 10 µmol/L DCFH-DA for 20 min at 37°C. Finally, the cells were washed twice with serum-free medium and ROS levels were determined by flow cytometry using a flow cytometer. 1.8 Semi-quantitative Reverse Transcription-PCR Assay The A549 cells were exposed to the indicated concentrations of icaritin for 48 h. Then samples of (2–5)×105 cells were harvested and washed twice with PBS. Total RNA was extracted from A549 cells using the Animal Tissue/Cell total RNA extraction kit (Zoman, China) and the RNA quality was detected by gel electrophoresis using a 1.2% agarose gel. The cDNA synthesis was conducted with a reverse transcription kit (Promega, USA). The primers for cDNA fragment amplification were listed in table 1. The PCR products were analysed by agarose gel electrophoresis and Image Lab software was used to measure the relative light intensities.
Table 1 RT-PCR primer sequence list Primer sequences 5'-GGGCTGGACATTGGACTTCC-3' 5'-AGATGGTGAGTGAGGCGGTG-3' 5'-TCGCCCTGTGGATGACTGAG-3' 5'-CAGAGTCTTCAGAGACAGCCAGGA-3' 5'-GTCACCACATACTATGGACATG-3' 5'-AAGTTTTCCTCTCAGCACTGAC-3' 5'-TTTGGAGTCCCTGTTCGTAC-3' 5'-TGCGATAACAAGCTCCGTCC-3' 5'-CAAGGTCATCCATGACAACTTTG-3' 5'-GTCCACCACCCTGTTGCTGTAG-3'
1.9 Western Blotting The A549 cells were lysed in cell lysis buffer on ice, and then centrifuged at 12 000 g for 5 min at 4°C after treatment with the indicated concentrations of icaritin for 48 h. The protein concentration was determined using a BCA protein assay kit. The lysate containing 35 µg of protein was subjected to electrophoresis on sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked overnight with 1×TBST containing 0.1% Tween-20 and 5% skim milk at 4°C. Then the membrane
was washed three times and incubated with the appropriate primary antibodies for 2 h at room temperature. After that, the membrane was washed three times and incubated with the corresponding secondary antibody (1:3000) for 1.5 h at room temperature. Finally, the membrane was washed three times again and detected using an enhanced chemiluminescence (ECL) kit (Millipore, USA). 1.10 Statistical Analysis All values obtained were expressed as ±s. Significant differences between the groups were determined
J Huazhong Univ Sci Technol［Med Sci］ 34(4):2014
using Student’s t test and statistical significance was set at P
497
Anticancer Effect of Icaritin on Human Lung Cancer Cells through Inducing S Phase Cell Cycle Arrest and Apoptosis* Qian ZHENG (郑 倩), Wei-wei LIU (刘伟伟), Bin LI (李 斌), Hui-jie CHEN (陈慧洁), Wen-shan ZHU (祝文山), Guang-xiao YANG (杨广笑), Ming-jie CHEN (陈明洁)#, Guang-yuan HE (何光源)# The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014 Summary: Icaritin, a prenylflavonoid derivative from Epimedium Genus, has been shown to exhibit many pharmacological and biological activities. However, the function and the underlying mechanisms of icaritin in human non-small cell lung cancer have not been fully elucidated. The purpose of this study was to investigate the anticancer effects of icaritin on A549 cells and explore the underlying molecular mechanism. The cell viability after icaritin treatment was tested by MTT assay. The cell cycle distribution, apoptosis and reactive oxygen species (ROS) levels were analyzed by flow cytometry. The mRNA and protein expression levels of the genes involved in proliferation and apoptosis were respectively detected by RT-PCR and Western blotting. The results demonstrated that icaritin induced cell cycle arrest at S phase, and down-regulated the expression levels of S regulatory proteins such as Cyclin A and CDK2. Icaritin also induced cell apoptosis characterized by positive Hoechst 33258 staining, accumulation of the Annexin V-positive cells, increased ROS level and alteration in Bcl-2 family proteins expression. Moreover, icaritin induced sustained phosphorylation of ERK and p38 MAPK. These findings suggested that icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells. Key words: icaritin; A549 cells; apoptosis; ERK; p38 MAPK
Lung cancer is the most prevalent cancer worldwide with the highest cancer-related mortality[1, 2]. According to the data published by World Health Organization (WHO), lung cancer is ranking the first death in the world and accounts for about 19% of all malignant tumors. The American Cancer Society estimates that there would be 159 480 deaths from lung cancer and 228 190 new cases diagnosed in 2013. Of these, non-small cell lung cancer (NSCLC) accounts for about 85%[1]. Nowadays, although the methods of early diagnosis have been improved, and the survival rate has increased to some extent, the therapeutic interventions have little influence on the prognosis of patients with NSCLC. The 5-year survival rate of NSCLC remains less than 15% and recurrence rate still increases after surgery[3]. Cancer cells have the ability of malignant proliferation and evading apoptosis. So that, anticancer agents that can trigger tumor cells undergoing apoptosis are viable for treatment of NSCLC. Icaritin has long been used as a Chinese traditional medicine. Previous studies showed that icaritin exerted a variety of biological and pharmacological effects including inducible differentiation of embryonic stem cells into cardiomyocytes[4], prevention fsteroid-associated osQian ZHENG, E-mail: [email protected] # Corresponding authors, Ming-jie CHEN, E-mail: [email protected]; Guang-yuan HE, E-mail: [email protected] * This project was supported by grants from Wuhan Municipal Science and Technology Research Project, China (No. 201260523185), and the Public Science and Technology Research Funds Projects of Ocean, China (No. 201005013).
teonecrosis[5], neuroprotective effects[6], enhancement of osteoblastic and suppression of osteoclastic differentiation and activity[7], and inhibition of the proliferation of acute myeloid leukemia, breast, prostate, endometrial – and liver cancer cell lines by inducing apoptosis[8 13]. In the present studies, we investigated the effects of icaritin on the growth of human lung carcinoma cell line and the molecular basis of its regulation in vitro. The results revealed that icaritin strongly suppressed the growth of lung cancer A549 cells with cell cycle arrest and cell apoptosis. 1 MATERIALS AND METHODS 1.1 Reagents Icaritin at 98% purity was purchased from Must Biotechnology Co., Ltd. (China). Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were acquired from HyClone Laboratories (USA). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma (USA). Propidium iodide (PI), Hoechst 33258, DCFH-DA and BCA Protein Assay Kit were obtained from Beyotime (China). An Annexin V-FITC/PI apoptosis detection kit was purchased from MultiSciences Biotech Co., Ltd (China). Primary antibodies specific to Cyclin A, CDK2, Bax, Bcl-2, p53, p21, and β-actin were obtained from Santa Cruz Biotechnology (USA). The secondary antibodies goat anti-mouse IgG HRP conjugate and goat anti-rabbit IgG HRP conjugate were obtained from Boster Biological Technology, Ltd (China).
498
J Huazhong Univ Sci Technol［Med Sci］ 34(4):2014
1.2 Cell Culture A549 cell line was obtained from the China Center for Type Culture Collection (Wuhan University, China). The cells were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2. All experiments were performed one day after the cells were seeded. 1.3 MTT Assay The effects of icaritin on cell viability were determined by measuring the metabolic activity (MTT assay). A density of 2×104 cells/well with 200 µL culture medium per well was seeded in 96-well plates for 24 h. On the next day, the medium was replaced with fresh medium containing various concentrations of icaritin or vehicle (DMSO). After incubation for an additional 24 and 48 h, a total of 20 µL MTT solution was added to each well and incubated at 37°C for 4 h to metabolize MTT into formazan. Then the supernatant was discarded and 100 µL of DMSO was added to each well to terminate the reaction. The absorption (A) value at 490 nm was measured using a microplate reader (Sunrise, Switzerland). Three independent experiments were performed in triplicates. 1.4 Cell Cycle Analysis by PI Staining The cell cycle distributions were analyzed by flow cytometry after PI staining. The A549 cells were cultured with the indicated concentrations of icaritin for 48 h. Then the cells were harvested by centrifugation and washed with cold PBS. Finally, the cells were fixed with 70% ice-cold ethanol overnight at –20°C. After fixation, the cells were harvested and rinsed once with PBS and then incubated with 1 mL PI staining solution (50 µg/mL PI and 100 µg/mL RNase A) for 1 h at room temperature. The distributions of cells in the G1, S, and G2/M phases were measured. 1.5 Hoechst 33258 Staining Apoptotic cells could be detected by investigating condensed and fragmented nuclei stained with Hoechst 33258 under a fluorescent microscope. Cells were exposed to icaritin for 48 h, and then washed with PBS. Subsequently, the cells were stained with Hoechst 33258 Target genes Bax Forward Reverse Bcl-2 Forward Reverse Cyclin A Forward Reverse CDK2 Forward Reverse GAPDH Forward Reverse
for 10 min at room temperature and washed with PBS for 3 times. Finally the samples were photographed (×10 objective) with inverted epifluorescent microscope. 1.6 Apoptosis Detection with Annexin V-FITC/PI Staining Living cells and apoptotic cells could be detected using the Annexin V-FITC/PI apoptosis kit by flow cytometry. The A549 cells were treated with the indicated concentrations of icaritin for 48 h in 6-well plates. Then the cells were harvested and washed twice with ice-cold PBS. After discarding the supernatant, the cells were resuspended in 500 µL binding buffer and stained with 5 µL Annexin V-FITC and 10 µL PI for 5 min in the dark at room temperature. Then, the samples were detected by flow cytometry and the apoptotic population was analyzed using FlowJo Software (Treestar, USA). 1.7 Measurement of Intracellular Reactive Oxygen Species Intracellular reactive oxygen species (ROS) generation was assessed using the stable non-polar dye DCFH-DA. Cells were treated with or without icaritin for 48 h, then harvested and incubated with 10 µmol/L DCFH-DA for 20 min at 37°C. Finally, the cells were washed twice with serum-free medium and ROS levels were determined by flow cytometry using a flow cytometer. 1.8 Semi-quantitative Reverse Transcription-PCR Assay The A549 cells were exposed to the indicated concentrations of icaritin for 48 h. Then samples of (2–5)×105 cells were harvested and washed twice with PBS. Total RNA was extracted from A549 cells using the Animal Tissue/Cell total RNA extraction kit (Zoman, China) and the RNA quality was detected by gel electrophoresis using a 1.2% agarose gel. The cDNA synthesis was conducted with a reverse transcription kit (Promega, USA). The primers for cDNA fragment amplification were listed in table 1. The PCR products were analysed by agarose gel electrophoresis and Image Lab software was used to measure the relative light intensities.
Table 1 RT-PCR primer sequence list Primer sequences 5'-GGGCTGGACATTGGACTTCC-3' 5'-AGATGGTGAGTGAGGCGGTG-3' 5'-TCGCCCTGTGGATGACTGAG-3' 5'-CAGAGTCTTCAGAGACAGCCAGGA-3' 5'-GTCACCACATACTATGGACATG-3' 5'-AAGTTTTCCTCTCAGCACTGAC-3' 5'-TTTGGAGTCCCTGTTCGTAC-3' 5'-TGCGATAACAAGCTCCGTCC-3' 5'-CAAGGTCATCCATGACAACTTTG-3' 5'-GTCCACCACCCTGTTGCTGTAG-3'
1.9 Western Blotting The A549 cells were lysed in cell lysis buffer on ice, and then centrifuged at 12 000 g for 5 min at 4°C after treatment with the indicated concentrations of icaritin for 48 h. The protein concentration was determined using a BCA protein assay kit. The lysate containing 35 µg of protein was subjected to electrophoresis on sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked overnight with 1×TBST containing 0.1% Tween-20 and 5% skim milk at 4°C. Then the membrane
was washed three times and incubated with the appropriate primary antibodies for 2 h at room temperature. After that, the membrane was washed three times and incubated with the corresponding secondary antibody (1:3000) for 1.5 h at room temperature. Finally, the membrane was washed three times again and detected using an enhanced chemiluminescence (ECL) kit (Millipore, USA). 1.10 Statistical Analysis All values obtained were expressed as ±s. Significant differences between the groups were determined
J Huazhong Univ Sci Technol［Med Sci］ 34(4):2014
using Student’s t test and statistical significance was set at P
