β-elemene, a plant-derived drug extracted from
Glioblastoma is the most common and aggressive subset of primary brain tumors, accounting for ~52% of all primary intracranial tumors. Glioblastomas are lethal tumors, characterized by high chemotherapy resistance and diffuse infiltration into the brain tissue. Despite the numerous advances in the development of cancer therapeutics in recent years, the prognosis for patients with glioblastoma remains discouraging. An effective antitumor medicine is the extract from
Glia maturation factor β (GMFβ, also known as GMF) is a 17 kDa intracellular regulator of stress-associated signal transduction and is predominantly expressed in astrocytes in the brain. GMFβ is necessary for the growth and maturation of brain glial cells and neurons (10.11). GMFβ is able to inhibit the growth of rat C6 and human HG-1 glioblastoma cells through G0/G1 cell cycle arrest
It was previously identified that β-elemene is able to arrest U87 and C6 glioblastoma cells in G0/G1 phase of the cell cycle and inhibit cell proliferation through the activation of GMFβ-MKK3/6-p38 and the downregulation of phosphorylated ERK1/2 (p-ERK1/2) (
In the present study, the regulatory action of GMFβ on the ERK1/2 pathway in the antiproliferative effect of β-elemene on glioblastoma was examined. The effect of β-elemene on the ERK1/2-B-cell lymphoma 2 (Bcl-2)/survivin pathway in association with GMFβ was examined. Furthermore, the effect of GMFβ silencing by transfecting small interfering (si)RNA into glioblastoma on the phosphorylation levels of ERK1/2 was assessed. In addition, the effect of β-elemene on the sensitivity of U87 glioblastoma cells to the chemotherapeutic temozolomide (TMZ) was tested. The results indicated that GMFβ-ERK1/2-Bcl-2/survivin pathway may be a putative target for novel molecular therapetic strategies against glioblastoma in the future.
β-Elemene (98% purity) was obtained from Jingang Pharmaceutical Co. (Dalian, China). TMZ was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against p-ERK1/2, ERK1/2, GMFβ, survivin, Bcl-2, Bcl-2-associated X protein (Bax) and GAPDH and PD98059 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). siRNAs for GMFβ and a negative control were obtained from Shanghai GenePharma Co., Ltd. (Pudongxinqu, Shanghai, China). A reverse transcription polymerase chain reaction (RT-PCR) kit was purchased from Takara Bio, Co., Ltd. (Dalian, China). The Lipofectamine 2000 transfection reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The Cell Counting kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). All of the other reagents were obtained from Sigma-Aldrich. The human U251 and U87 glioblastoma cell lines were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, maintained in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal calf serum (Invitrogen Life Technologies), 50 IU/ml penicillin (Invitrogen Life Technologies) and 50 mg/ml streptomycin (Invitrogen Life Technologies) and grown at 37°C in a humidified atmosphere with 5% CO2.
Cell viability was evaluated by a CCK-8 assay. The cells in exponential growth phase were cultured in a 96-well culture plates and treated according to the study design. Then, 10 μl of CCK-8 was added to each well and the mixture was incubated for 4 h at 37°C. The optical density (OD) of each well was measured at 450 nm using a spectrophotometric microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Five replicate wells were designed for each cell sample. Cells were examined using an inverted microscope (ECLIPSE TE2000-U; Nikon, Tokyo, Japan).
The cells were lysed with radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4), 1.0% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA, 150 mM NaCl, 1 mM aprotinin, 1 mg/ml phenylmethanesulfonyl fluoride, 1 μg/ml pepstatin and 1 μg/ml leupeptin]. The concentrations of total protein in the cellular extracts were measured using the bicinchoninic acid assay kit from Keygen Biotech. Co., Ltd. (Nanjing, China). Following separation by 12% SDS-PAGE, the proteins were transferred to nitrocellulose filter membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline with Tween-20 at 4°C overnight and probed with various primary antibodies at 4°C overnight. This included primary antibodies against p-ERK1/2 (goat polyclonal IgG), ERK1/2 (rabbit polyclonal IgG), GMFβ (mouse monoclonal IgG), survivin (mouse monoclonal IgG), Bcl-2 (mouse monoclonal IgG), Bax (rabbit polyclonal IgG) and GAPDH (mouse monoclonal IgG) purchased from Santa Cruz Biotechnology, Inc. (Santa Crux, CA, USA). Followed by incubation with horseradish peroxidase-conjugated secondary antibodies (donkey anti-goat IgG, goat anti-mouse IgG and mouse anti-rabbit IgG; Santa Cruz Biotechnology, Inc.) at 37°C for 3 h. The membranes were exposed to an enhanced chemiluminescence system (Amersham Biosciences, Uppsala, Sweden) and fluorescence was detected by exposing the membrane to X-ray film (Fujifilm Co., Ltd., Tokyo, Japan). The results were scanned with the Image Quant 5.2 software (Amersham Biosciences) and the gray bands were semi-quantitatively evaluated using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Rockville, MD, USA). The gray values were normalized to GAPDH.
The cells were plated at a density of 4×105 cells/well in six-well plates or 4×103/well in 96-well plates and cultured for 24 h. siRNA oligonucleotides were transfected into glioblastoma cells with Lipofectamine 2000 according to the manufacturer’s instructions. Post-transfection (24 h), the cells were treated with β-elemene for 24 h. siRNA oligonucleotides were obtained from Shanghai GenePharma Co., Ltd. and specific sequences identical to those used in a previous study by our group (
Values are expressed as the mean ± standard deviation of at least three independent experiments. Statistical analysis was performed using Student’s t-test. A level of P<0.05 was considered to indicate a statistically significant difference between groups and P<0.01 was considered to indicate a highly significant difference. Statistical analysis was performed with SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA).
To evaluate the antitumor effect of β-elemene on glioblastoma cells, human U87 and U251 glioblastoma cells were treated with β-elemene at variable doses or for different durations. Cell viability was measured with a CCK-8 assay. It was identified that the viability of human U87 and U251 glioblastoma cells treated with β-elemene evidently decreased as the drug dose (
To investigate the role of the ERK1/2 signalling pathway in the antiproliferative effect of β-elemene on glioblastoma cells, the expression levels of p-ERK1/2, ERK1/2, Bcl-2, Bax and survivin were examined by western blot analysis in β-elemene-treated U87 cells (
To further confirm the role of ERK1/2 inactivation in the anti-proliferative effect of β-elemene on glioblastoma cells, ERK1/2 was pretreated with 30 μM PD9805 for 1 h in U87 cells. Following treatment with 80 μg/ml β-elemene for 24 h, U87 cell viability was measured by the CCK-8 assay (
A previous study by our group reported that β-elemene inhibited the proliferation of U87 cells via activation of the GMFβ-MKK3/6-p38 signalling pathway (
As previously reported, the ERK1/2 signalling pathway is associated with drug resistance in numerous types of cancer, including glioblastoma and ovarian cancer (
Glioblastoma is the most common and lethal type of brain tumor. In spite of improvements in cancer therapeutics, the prognosis for patients with glioblastoma remains poor. The majority of chemotherapeutics are not effective in treating glioblastoma due to frequent drug resistance and the severity of the various associated side effects (
In the past ten years, numerous clinical and basic studies have been conducted on the antiproliferative effect of elemene on glioblastoma carcinoma. Elemene is able to significantly reduce the size of tumors and prolong the lifespan of patients, without serious side effects. Simultaneously, elemene has demonstrated strong antitumor activity on glioblastoma cell lines from humans, rats and glioblastoma-bearing nude mice by inducing tumor cell apoptosis, inhibiting cell proliferation and arresting cell cycle processes (
GMFβ is an intracellular protein primarily localized in the mammalian central nervous system and is important in regulating the growth and development of glial cells and neurons. GMFβ has been identified to mediate apoptosis in glioblastoma cells and the development of an inflammatory response (
In recent years, numerous basic studies on the anti-glioblastoma molecular mechanism of β-elemene have been performed. It has previously been reported that β-elemene is able to arrest cells at G0/G1 in the cell cycle and activate the GMFβ-MKK3/6-p38 pathway to inhibit the growth of human U87, U251 and rat C6 glioblastoma cells (
TMZ is a monofunctional alkylating agent widely used in the clinic as a first-line chemotherapeutic agent against newly diagnosed or recurrent glioblastoma (
In conclusion, the present study suggested that the antiproliferative effect of β-elemene on glioblastoma proceeds via the inactivation of the ERK1/2-Bcl-2/survivin pathway, which is dependent on GMFβ activation. The GMFβ-ERK1/2-Bcl-2/survivin pathway may be a putative target for molecular therapy against glioblastoma. In addition, β-elemene should be investigated further, to facilitate the development of a combined therapeutic regimen with TMZ for a more efficacious strategy in the treatment of glioblastoma primary brain tumors.
This study was supported by the National Natural Science Foundation of China (no. 30740027/30471778) and China Postdoctoral Science Foundation (2012M521921). The authors are also grateful to our colleagues in our research group for their generous support.
β-elemene inhibits the proliferation of human U87 and U251 glioblastoma cells dose- and time-dependently. (A) U87 and U251 cells were treated with β-elemene at different doses (0, 20, 40, 60 and 80 μg/ml) for 24 h, and then, an CCK-8 assay was performed to determine cell viability. (B) U87 and U251 cells were treated with β-elemene at a concentration of 60 μg/ml for various durations (0, 12, 24, 36 and 48 h), and cell viability was determined by a CCK-8 assay. The viability of the cells treated with β-elemene decreased with increasing drug dose and treatment time. (C) The cells of (A) were observed by an inverted microscope (magnification, ×100). All of the values are represented as the mean ± standard deviation (**P<0.01). CCK-8, Cell Counting kit-8.
β-elemene decreasesthe expression of p-ERK1/2, Bcl-2 and survivin in human glioblastoma cells. U87 cells were treated with β-elemene at various concentrations (0, 40 and 80 μg/ml) for 24 h, and the total protein was extracted for western blot analysis. (A) The expression levels of p-ERK1/2, ERK1/2, Bcl-2, Bax, survivin and GAPDH were detected with specific antibodies by western blot analysis. (B) Blots were semi-quantitatively evaluated. β-elemene decreased the phosphorylation levels of ERK1/2 and impaired the expression of Bcl-2 and survivin in human U87 cells (**P<0.01). However, the expression of total ERK1/2 and Bax was not affected by β-elemene. The results are representative of three independent experiments. Data are presented as the mean ± standard deviation. p-ERK1/2, phosphorylated-extracellular signal-regulated kinase 1/2; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2 associated X protein.
Inhibition of ERK1/2 by PD98059 enhances the antitumor effect of β-elemene and further decreases the expression of Bcl-2 and survivin in human U87 glioblastoma cells. U87 cells were pretreated with (or without) 30 μM PD9805 for 1 h, and then treated with 80 μg/ml β-elemene for 24 h. (A) Cell viability was measured by a CCK-8 assay (untreated U87 cells were used as the control) and (B) the expression levels of Bcl-2 and survivin were detected using western blot analysis. (C) The results of (B) were semi-quantitatively evaluated. The antiproliferative effect of β-elemene was increased by PD98059 and the inhibition of ERK1/2 further decreased the expression of Bcl-2 and survivin. Values are presented as the mean ± standard deviation (**P<0.01). ERK1/2, extracellular signal-regulated kinase 1/2; CCK-8, Cell Counting kit-8; Bcl-2, B-cell lymphoma 2.
Silencing the expression of GMFβ impairs the inactivating action of β-elemene on the ERK1/2 pathway. (A) RNA interference was performed with 53 nM GMFβ siRNA for 24 h to downregulate GMFβ expression in U87 cells. The interference efficiency was determined by western blot analysis. (B) GMFβ-silenced U87 cells were treated with 80 μg/ml β-elemene for 24 h and the expression levels of p-ERK1/2 were detected by western blot analysis. (C and D) The results of A and B, respectively, were semi-quantitatively evaluated. GMFβ siRNA significantly downregulated the expression of GMFβ and increased the p-ERK1/2 levels in U87 cells treated with β-elemene. The results are representative of three independent experiments. Values are presented as the mean ± standard deviation (**P<0.01). GMFβ, glia maturation factor β; p-ERK1/2, phosphorylated-extracellular signal-regulated kinase 1/2; siRNA, small interfering RNA.
Treatment with β-elemene increases the sensitivity of U87 cells to TMZ-induced cytotoxicity and this chemotherapy sensitising effect is promoted by PD98059. (A) Five groups were designed: a, untreated cells; b, treated with 80 μg/ml β-elemene for 24 h; c, treated with 300 μM TMZ for 24 h; d, treated with 80 μg/ml β-elemene combined with 300 μM TMZ for 24 h; and e, treated with 80 μg/ml β-elemene combined with 300 μM TMZ and 30 μM PD98059 for 24 h. (B) Cell viability was measured by a CCK-8 assay and quantified. The results demonstrated that the viability of the cells in group e was lower than that in groups a, b, c or d. Treatment with β-elemene increased the sensitivity of U87 cells to TMZ-induced cytotoxicity and this chemotherapy sensitising effect was enhanced by PD98059. Values are presented as the mean ± standard deviation (**P<0.01). TMZ, temozolomide; CCK-8, Cell Counting Kit-8.