MOLECULAR MEDICINE REPORTS 10: 1046-1050, 2014
1046
Germacrone inhibits the proliferation of glioma cells by promoting apoptosis and inducing cell cycle arrest BO LIU1, YUE-QIU GAO2, XIAO-MIN WANG1, YU-CHUN WANG1 and LI-QI FU1 1
Department of Neurosurgery, Daqing Oilfield General Hospital, Daqing, Heilongjiang 163411; 2 Department of Gastroenterology and Hepatology, The Fifth Affiliated Hospital of Harbin Medical University, Daqing, Heilongjiang 163316, P.R. China Received August 16, 2013; Accepted March 6, 2014 DOI: 10.3892/mmr.2014.2290
Abstract. Germacrone is one of the major bioactive components of the traditional Chinese Medicinal plant Curcuma aromatica Salisb. and has been shown to possess anti‑tumor properties. In the present study, the anti‑proliferative effect of germacrone on human glioma cells and the molecular mechanism underlying its cytotoxicity were investigated. Treatment of the U87 and U251 human glioma cell lines with germacrone inhibited the cell proliferation in a dose‑ and time‑dependent manner as assessed by MTT assay, while significantly lower effects were observed on normal human astrocytes. Flow cytometric analysis and DNA fragmentation revealed that germacrone promoted apoptosis of glioma cells, associated with an increased expression of p53 and bax and decreased expression of bcl‑2. Furthermore, flow cytometric cell cycle analysis revealed that germacrone induced G1 phase arrest, associated with an obvious decrease in the expression of cyclin D1 and CDK2 and an increased expression of p21. In conclusion, the present study suggested that germacrone may be a novel potent chemopreventive drug candidate for gliomas via regulating the expression of proteins associated with apoptosis and G1 cell cycle arrest. Introduction Gliomas are the most common tumor types of the central nervous system with a low 5‑year survival rate and high morbidity rate (1). The current clinical treatments for glioma include surgery, radiotherapy and chemotherapy (2). However, accumulating evidence shows that the resistance of glioma cells to conventional drugs is becoming a problem and it is imperative to find novel and effective drugs against glioma (3).
Correspondence to: Dr Bo Liu, Department of Neurosurgery, Daqing Oilfield General Hospital, No. 9 Zhong Kang Street, Daqing, Heilongjiang 163411, P.R. China E-mail: [email protected]
Key words: germacrone, glioma cells, apoptosis, cell cycle arrest
Traditional Chinese Medicinal plants are a significant source of drugs that serve as potential therapeutic compounds for the treatment of cancer (4). The rhizome of Curcuma aromatica Salisb. is widely used as a traditional herbal medicine for anti‑tumor therapy in China, Japan and other Asian countries (5). Findings of recent studies have demonstrated that germacrone, a major bioactive component of Curcuma aromatica Salisb., possesses anti‑tumor, anti‑inflammatory and neuroprotective properties. Claeson et al (6) found that germacrone had an obvious anti‑inflammatory activity against carrageenan‑induced hind paw edema in rats. Matsuda et al (7) and Morikawa et al (8) reported that germacrone exerted a potent protective effect on acute liver injury in mice induced by D‑galactosamine/lipopolysaccharide and tumor necrosis factor‑α. Another study showed that the treatment of HepG2 and Bel7402 hepatoma cell lines with germacrone promoted apoptosis associated with the upregulation of bax and the downregulation of bcl‑2. The upregulation of p53 and an increase in reactive oxygen species were observed, which suggested that germacrone is a novel potent drug candidate for liver cancer (9). Furthermore, germacrone was found to inhibit the proliferation of breast cancer cell lines MCF‑7 and MDA‑MB‑231 by inducing cell cycle arrest in the G0/G1 and G2/M phase as well as apoptosis through a mitochondria‑mediated caspase pathway (10). However, to the best of our knowledge, this is the first study to investigate the inhibitory effect of germacrone on human glioma cells. Therefore, in the present study, the anti‑proliferative effect of germacrone on glioma cells and normal human astrocytes as well as the mechanism of action of the anti‑tumor activity were investigated. Materials and methods Germacrone (>95%) was purchased from Sigma (St. Louis, MO, USA). RPMI‑1640 culture medium, Dulbecco's medium Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate‑buffered saline (PBS), penicillin‑streptomycin (PS) and 0.25% (w/v) trypsin/1 mM EDTA were purchased from Gibco (Grand Island, NY, USA). Cell lines and culture. The U87 and U251 human glioma cell lines were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China).
LIU et al: GERMACRONE INHIBITS THE PROLIFERATION OF GLIOMA CELLS
Cells were cultured in DMEM supplemented with 10% FBS, 2 mmol/l glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml), and maintained at 37˚C and 5% CO2 in a humid environment. Normal human astrocytes (NHA) were obtained as part of the human astrocytes kit (Gibco) and cultured in astrocyte medium. MTT assay. Cell proliferation was evaluated by MTT assay. Briefly, the cells were seeded into 96‑well plates at a density of 3x104 (cells/well) and left to adhere overnight. The cells were then incubated with or without 0‑250 µmol/l germacrone prior to addition of 10 ml of 5 mg/ml MTT and incubation in the dark at 37˚C for 2 h. Absorbance was determined at a wavelength of 492 nm. Flow cytometric analysis. Cells were incubated with germacrone at different concentrations (0‑250 µmol/l) for 24 h. The cells were washed with PBS, detached with trypsin and harvested. The cells were resuspended in 1 ml Hoechst 33258 for 5 min and washed with PBS three times. Apoptotic cells were detected by staining with annexin V‑FITC/PI according to the protocol of the annexin V‑FITC cell Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ USA). DNA fragmentation. For DNA laddering, following exposure to drug treatment, 2x106 cells were suspended in PBS and homogenized in buffer containing proteinase K and RNase. Subsequently, cell lysates were incubated at 50˚C for 30 min, and isopropranol was added. DNA was precipitated and washed by 70% ethanol. DNA was then analyzed using 1.2% agarose gel electrophoresis. Cell cycle. Cells were seeded at a density of 1.0x10 6 cells and incubated with germacrone at various concentrations (0‑250 µmol/l) for 24 h. Following two washes with PBS, the cells were harvested and collected by centrifugation, followed by fixation in ice‑cold 70% ethanol at ‑20˚C overnight. Cells were then collected and stained with 100 µl PI solution for 30 min in the dark followed by cell cycle analysis. Western blot analysis. Following incubation with germacrone at various concentrations (0‑250 µmol/l), total cell lysates were prepared and subjected to SDS‑PAGE. For western blot analysis, primary antibodies used included anti‑p53, B‑cell lymphoma 2 (bcl‑2), bcl‑2 associated X (bax), cyclin D1 and cyclin D kinase 2 (CDK2) (Cell Signaling, Beverly, MA, USA) as well as p21 and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Anti‑rabbit or anti‑mouse secondary antibody conjugated with horseradish peroxidase was also used (Pierce Chromatography Cartridges, Rockford, IL, USA). Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit for western blot analysis and the ChemiGenius bioimaging system (Syngene, Cambridge, UK). Statistical analysis. Data were presented as the mean ± standard deviation and processed for statistical analysis by the SPSS program (SPSS, Inc., Chicago, IL, USA). A comparison between groups was made using analysis of variance (ANOVA), and a statistically significant difference between values was defined as P
1046
Germacrone inhibits the proliferation of glioma cells by promoting apoptosis and inducing cell cycle arrest BO LIU1, YUE-QIU GAO2, XIAO-MIN WANG1, YU-CHUN WANG1 and LI-QI FU1 1
Department of Neurosurgery, Daqing Oilfield General Hospital, Daqing, Heilongjiang 163411; 2 Department of Gastroenterology and Hepatology, The Fifth Affiliated Hospital of Harbin Medical University, Daqing, Heilongjiang 163316, P.R. China Received August 16, 2013; Accepted March 6, 2014 DOI: 10.3892/mmr.2014.2290
Abstract. Germacrone is one of the major bioactive components of the traditional Chinese Medicinal plant Curcuma aromatica Salisb. and has been shown to possess anti‑tumor properties. In the present study, the anti‑proliferative effect of germacrone on human glioma cells and the molecular mechanism underlying its cytotoxicity were investigated. Treatment of the U87 and U251 human glioma cell lines with germacrone inhibited the cell proliferation in a dose‑ and time‑dependent manner as assessed by MTT assay, while significantly lower effects were observed on normal human astrocytes. Flow cytometric analysis and DNA fragmentation revealed that germacrone promoted apoptosis of glioma cells, associated with an increased expression of p53 and bax and decreased expression of bcl‑2. Furthermore, flow cytometric cell cycle analysis revealed that germacrone induced G1 phase arrest, associated with an obvious decrease in the expression of cyclin D1 and CDK2 and an increased expression of p21. In conclusion, the present study suggested that germacrone may be a novel potent chemopreventive drug candidate for gliomas via regulating the expression of proteins associated with apoptosis and G1 cell cycle arrest. Introduction Gliomas are the most common tumor types of the central nervous system with a low 5‑year survival rate and high morbidity rate (1). The current clinical treatments for glioma include surgery, radiotherapy and chemotherapy (2). However, accumulating evidence shows that the resistance of glioma cells to conventional drugs is becoming a problem and it is imperative to find novel and effective drugs against glioma (3).
Correspondence to: Dr Bo Liu, Department of Neurosurgery, Daqing Oilfield General Hospital, No. 9 Zhong Kang Street, Daqing, Heilongjiang 163411, P.R. China E-mail: [email protected]
Key words: germacrone, glioma cells, apoptosis, cell cycle arrest
Traditional Chinese Medicinal plants are a significant source of drugs that serve as potential therapeutic compounds for the treatment of cancer (4). The rhizome of Curcuma aromatica Salisb. is widely used as a traditional herbal medicine for anti‑tumor therapy in China, Japan and other Asian countries (5). Findings of recent studies have demonstrated that germacrone, a major bioactive component of Curcuma aromatica Salisb., possesses anti‑tumor, anti‑inflammatory and neuroprotective properties. Claeson et al (6) found that germacrone had an obvious anti‑inflammatory activity against carrageenan‑induced hind paw edema in rats. Matsuda et al (7) and Morikawa et al (8) reported that germacrone exerted a potent protective effect on acute liver injury in mice induced by D‑galactosamine/lipopolysaccharide and tumor necrosis factor‑α. Another study showed that the treatment of HepG2 and Bel7402 hepatoma cell lines with germacrone promoted apoptosis associated with the upregulation of bax and the downregulation of bcl‑2. The upregulation of p53 and an increase in reactive oxygen species were observed, which suggested that germacrone is a novel potent drug candidate for liver cancer (9). Furthermore, germacrone was found to inhibit the proliferation of breast cancer cell lines MCF‑7 and MDA‑MB‑231 by inducing cell cycle arrest in the G0/G1 and G2/M phase as well as apoptosis through a mitochondria‑mediated caspase pathway (10). However, to the best of our knowledge, this is the first study to investigate the inhibitory effect of germacrone on human glioma cells. Therefore, in the present study, the anti‑proliferative effect of germacrone on glioma cells and normal human astrocytes as well as the mechanism of action of the anti‑tumor activity were investigated. Materials and methods Germacrone (>95%) was purchased from Sigma (St. Louis, MO, USA). RPMI‑1640 culture medium, Dulbecco's medium Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate‑buffered saline (PBS), penicillin‑streptomycin (PS) and 0.25% (w/v) trypsin/1 mM EDTA were purchased from Gibco (Grand Island, NY, USA). Cell lines and culture. The U87 and U251 human glioma cell lines were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China).
LIU et al: GERMACRONE INHIBITS THE PROLIFERATION OF GLIOMA CELLS
Cells were cultured in DMEM supplemented with 10% FBS, 2 mmol/l glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml), and maintained at 37˚C and 5% CO2 in a humid environment. Normal human astrocytes (NHA) were obtained as part of the human astrocytes kit (Gibco) and cultured in astrocyte medium. MTT assay. Cell proliferation was evaluated by MTT assay. Briefly, the cells were seeded into 96‑well plates at a density of 3x104 (cells/well) and left to adhere overnight. The cells were then incubated with or without 0‑250 µmol/l germacrone prior to addition of 10 ml of 5 mg/ml MTT and incubation in the dark at 37˚C for 2 h. Absorbance was determined at a wavelength of 492 nm. Flow cytometric analysis. Cells were incubated with germacrone at different concentrations (0‑250 µmol/l) for 24 h. The cells were washed with PBS, detached with trypsin and harvested. The cells were resuspended in 1 ml Hoechst 33258 for 5 min and washed with PBS three times. Apoptotic cells were detected by staining with annexin V‑FITC/PI according to the protocol of the annexin V‑FITC cell Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ USA). DNA fragmentation. For DNA laddering, following exposure to drug treatment, 2x106 cells were suspended in PBS and homogenized in buffer containing proteinase K and RNase. Subsequently, cell lysates were incubated at 50˚C for 30 min, and isopropranol was added. DNA was precipitated and washed by 70% ethanol. DNA was then analyzed using 1.2% agarose gel electrophoresis. Cell cycle. Cells were seeded at a density of 1.0x10 6 cells and incubated with germacrone at various concentrations (0‑250 µmol/l) for 24 h. Following two washes with PBS, the cells were harvested and collected by centrifugation, followed by fixation in ice‑cold 70% ethanol at ‑20˚C overnight. Cells were then collected and stained with 100 µl PI solution for 30 min in the dark followed by cell cycle analysis. Western blot analysis. Following incubation with germacrone at various concentrations (0‑250 µmol/l), total cell lysates were prepared and subjected to SDS‑PAGE. For western blot analysis, primary antibodies used included anti‑p53, B‑cell lymphoma 2 (bcl‑2), bcl‑2 associated X (bax), cyclin D1 and cyclin D kinase 2 (CDK2) (Cell Signaling, Beverly, MA, USA) as well as p21 and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Anti‑rabbit or anti‑mouse secondary antibody conjugated with horseradish peroxidase was also used (Pierce Chromatography Cartridges, Rockford, IL, USA). Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit for western blot analysis and the ChemiGenius bioimaging system (Syngene, Cambridge, UK). Statistical analysis. Data were presented as the mean ± standard deviation and processed for statistical analysis by the SPSS program (SPSS, Inc., Chicago, IL, USA). A comparison between groups was made using analysis of variance (ANOVA), and a statistically significant difference between values was defined as P
