Acta Biochim Biophys Sin, 2016, 48(6), 501–508 doi: 10.1093/abbs/gmw035 Advance Access Publication Date: 17 May 2016 Original Article
Original Article
Silence of bFGF enhances chemosensitivity of glioma cells to temozolomide through the MAPK signal pathway Qiong Wang1,†, Jixiang Du2,†, Bin Xu2,†, Lixia Xu1, Xiuyu Wang2, Jun Liu1, and Jinhuan Wang1,* 1
Tianjin Cerebral Vascular and Neural Degenerative Disease Key Laboratory, Tianjin Neurosurgery Institute, Tianjin Huanhu Hospital, Tianjin 300060, China, and 2The Graduate School, Tianjin Medical University, Tianjin 300070, China †
These authors contributed equally to this work. *Correspondence address. Tel: +86-22-60367668; Fax: +86-22-60367651; E-mail: [email protected]
Received 10 December 2015; Accepted 30 March 2016
Abstract Basic fibroblast growth factor (bFGF) is a multifunctional growth factor in glioma cells and has been proved to be associated with the grade malignancy of glioma and prognosis of patients. Although there is evidence showing that bFGF plays an important role in proliferation, differentiation, angiogenesis, and survival of glioma cells, the effect of bFGF on chemosensitivity of glioma has not been verified. In this study, we analyzed the relationship between bFGF and chemotherapy resistance, with the objective of offering new strategy for chemotherapy of glioma patients. Here, siRNA was used to silence the expression of bFGF in glioma cell lines including U87 and U251 followed by chemotherapy of temozolomide (TMZ). Then, the characters of glioma including proliferation, apoptosis, migration, and cell cycle were studied in U87 and U251 cell lines. Our results demonstrated that silencing bFGF enhanced the effect of TMZ by inhibiting proliferation and migration, blocking cell cycle in G0/G1, and promoting apoptosis. In addition, the phosphorylation level of MAPK was measured to explore the mechanism of chemosensitization. The results showed that bFGF could promote the activation of the MAPK signal pathway. Our data indicated that bFGF might be a potential target for chemotherapy through the MAPK signal pathway. Key words: gliomas, bFGF, temozolomide, combined chemotherapy, drug resistance, chemosensitivity
Introduction Glioma is one of the most common and malignant primary intracranial tumor in adults, which typically leads to progressive disability and eventually death. Up to now, the standard therapies for glioma include surgical resection followed by chemotherapy and radiotherapy. Unfortunately, even with this multimodality treatment, the median survival time of patients is still ~1 year [1]. Chemotherapy and radiotherapy after surgical resection are the necessary methods to prevent metastasis and recurrence [2]. But chemotherapy does not work well in some patients.
Basic fibroblast growth factor (bFGF), a heparin-binding multifunctional growth factor, can promote the proliferation and angiogenesis on glioma cells through autocrine way [3]. bFGF has been proved to be overexpressed in glioma cells [4], and the expression level of bFGF was associated with the grade and prognosis of patients [5]. In our previous study, the depolarization of mitochondria and apoptosis was induced by delivery of bFGF interfering RNA [6]. In addition, down-regulating the expression of bFGF has been proved to inhibit tumorigenicity and metastasis [7].
© The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: [email protected] 501
502 Temozolomide (TMZ), an alkylating agent, does not require hepatic metabolism for activation, because TMZ could be spontaneously converted to the active form of alkylating agent (5-(3-Nmethyltriazen-1-yl)-imidazole-4-carboxamide) (MTIC) [8]. TMZ can function as a broad-spectrum antitumor agent for several diseases such as melanoma, mesothelioma, lymphoma, sarcoma, leukemia, carcinoma of the ovary, carcinoma of the colon [9], and glioma [10]. Because of its extensive effects, chemotherapy inevitably has a wide range of side effects. Previous studies have shown the side effects including neutropenia, thrombocytopenia, lack of red blood cells, and hemoglobin when TMZ was administered in a dosedependent manner [11]. However, the role of bFGF in the resistance of chemotherapy is still unclear. In this study, we tried to explore the relationship between bFGF and chemosensitivity. bFGF expression was silenced with specific siRNA and combined with TMZ to investigate its biological impact on glioma cells, which may help to develop new treatment strategies for glioma patients.
Materials and Methods Cell culture and treatment Glioblastoma cell lines U87 and U251 were obtained from the Peking Union Medical College Cell Library (Beijing, China), and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Grand Island, USA) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in the appropriate atmosphere containing 5% CO2. The cells were divided into five groups: (1) siRNA negative control (NC) group (cells were transfected with NC); (2) TMZ group (cells were treated with TMZ alone); (3) TMZ + NC (cells were transfected with NC and then treated with TMZ); (4) bFGF-siRNA group (cells were transfected with bFGF-siRNA); and (5) union group (cells were transfected with bFGF-siRNA and then treated with TMZ). U87 and U251 cells were incubated in 6-well plates for 12 h, followed by cell transfections. At 24 h after transfection, cells were treated with or without TMZ (Sigma, St Louis, USA).
siRNA transfection siRNAs were purchased from GenePharma (Shanghai, China). The sequence of siRNA targeting bFGF (5’-CGAACTGGGCAGTAT AAAC-3’) was described as previously shown [9]. The sequence of NC was 5’-UUCUCCGAACGUGUCACGUTT-3’. U87 and U251 cells were seeded into 6- or 96-well plates (Corning Costar, Cambridge, USA) and cultured for 12 h. siRNAs were transfected into the cells with Lipofectamine™ 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instruction. The mixture of siRNA and Lipofectamine™ 2000 was added to culture medium for 6 h, and then the medium was changed with DMEM containing 10% FBS.
Western blot analysis U87 and U251 cells were lyzed with the lysis buffer (CW Biotech, Beijing, China) containing protease inhibitor cocktails (Roche, Basel, Switzerland) for 30 min, followed by centrifuging at 12,000 g for 15 min at 4°C. For each sample, 20 μg of total protein was subject to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinyl difluoride membranes (Bio-Rad, Hercules, USA). The membranes were blocked with 5% nonfat milk in tris buffered saline with tween (TBST) buffer for 1.5 h at room temperature, and incubated with primary antibodies overnight at 4°C.
bFGF-siRNA enhances chemosensitivity of glioma After being washed five times with TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody and developed with the electrochemiluminescence (ECL) kit (Millipore, Billerica, USA). The primary anti-Bcl-2, anti-Bax, antimatrix metalloproteinases-2 (MMP-2), and anti-MMP-9 antibodies were from Santa Cruz Biotechnology (Santa Cruz, USA), and the antip-ERK1/2, anti-ERK1/2, anti-p-p38, anti-p38, anti-p-JNK, and antiJNK primary antibodies were from Cell Signaling Technology (Beverly, USA).
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay U87 and U251 cells were seeded into 96-well plates and transfected with NC or bFGF-siRNA, and then treated with TMZ for 48 h. The cell proliferation rate was measured at 0, 24, 48, and 72 h after TMZ treatment. Then, 20 μl of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenylteyrazolium bromide (MTT) (5 mg/ml; Sigma, St Louis, USA) was added into the supernatant. After 4 h of incubation, the medium was exchanged with 150 μl of dimethyl sulfoxide (DMSO) (Sigma). The absorption at 490 nm was determined by a microplate reader.
Colony formation U87 and U251 cells were seeded into 6-well plates and transfected with NC or bFGF-siRNA, and then treated with TMZ for 48 h. The cells were re-seeded into 6-well plates at the density of 500 cells/well for colony formation assay. The medium was replaced every 4 days. After the cells were cultured for 2 weeks, the number of colonies was counted.
Migration assay The migration ability of U87 and U251 cells was detected using a transwell system with 8-μm pore size (Millipore). Cells were transfected with bFGF-siRNA, and then treated with TMZ for 48 h. The lower chamber was filled with 600 μl of culture medium containing 10% FBS, and the upper chamber was seeded with 104 cells from each group. After 24 h of incubation at 37°C in 5% CO2, the cells were fixed with 4% paraformaldehyde and then stained with 1% crystal violet for 20 min. The cells of upper chamber were scraped off using a cotton bud prior to taking photos. Five different fields were randomly chosen in each well and the photos were taken.
Cell apoptosis analysis U87 and U251 cells were seeded into 6-well plates and transfected with NC or bFGF-siRNA, and then treated with TMZ for 48 h. The cells (~1 × 105) were washed with phosphate buffered saline (PBS), and resuspended in 100 μl of binding buffer containing 5 μl of annexin V-FITC and 5 μl of propidium iodide (PI) (BD Pharmingen, San Diego, USA) in a tube. After 15 min of incubation at room temperature in the dark, another 400 μl of binding buffer was added into the tube. Samples were detected by fluorescence-activated cell sorter. The data were analyzed by FlowJo software (BD Pharmingen).
Flow cytometric analysis of cell cycle U87 and U251 cells were cultured in 25-cm2 cell culture bottles (Corning Co, Corning, USA). The cells were transfected with NC or bFGF-siRNA, and then treated with TMZ for 48 h. The cells were harvested and washed with cold PBS. Then, the cells were fixed in
503
bFGF-siRNA enhances chemosensitivity of glioma cold 70% ethanol at 4°C overnight. The cells were washed twice with PBS and incubated with stain working solution (containing 50 μg/ml PI, 1% Triton X-100, and 1 mg/ml RNaseA) for 20 min at room temperature. The percentage of cells in each phase was analyzed by flow cytometry (BD Pharmingen).
Statistical analysis The data were expressed as mean ± standard deviation (SD) of three independent experiments. Statistical analyses were performed using SPSS software 22.0, and compared with one-way analysis of variance. The P values
Original Article
Silence of bFGF enhances chemosensitivity of glioma cells to temozolomide through the MAPK signal pathway Qiong Wang1,†, Jixiang Du2,†, Bin Xu2,†, Lixia Xu1, Xiuyu Wang2, Jun Liu1, and Jinhuan Wang1,* 1
Tianjin Cerebral Vascular and Neural Degenerative Disease Key Laboratory, Tianjin Neurosurgery Institute, Tianjin Huanhu Hospital, Tianjin 300060, China, and 2The Graduate School, Tianjin Medical University, Tianjin 300070, China †
These authors contributed equally to this work. *Correspondence address. Tel: +86-22-60367668; Fax: +86-22-60367651; E-mail: [email protected]
Received 10 December 2015; Accepted 30 March 2016
Abstract Basic fibroblast growth factor (bFGF) is a multifunctional growth factor in glioma cells and has been proved to be associated with the grade malignancy of glioma and prognosis of patients. Although there is evidence showing that bFGF plays an important role in proliferation, differentiation, angiogenesis, and survival of glioma cells, the effect of bFGF on chemosensitivity of glioma has not been verified. In this study, we analyzed the relationship between bFGF and chemotherapy resistance, with the objective of offering new strategy for chemotherapy of glioma patients. Here, siRNA was used to silence the expression of bFGF in glioma cell lines including U87 and U251 followed by chemotherapy of temozolomide (TMZ). Then, the characters of glioma including proliferation, apoptosis, migration, and cell cycle were studied in U87 and U251 cell lines. Our results demonstrated that silencing bFGF enhanced the effect of TMZ by inhibiting proliferation and migration, blocking cell cycle in G0/G1, and promoting apoptosis. In addition, the phosphorylation level of MAPK was measured to explore the mechanism of chemosensitization. The results showed that bFGF could promote the activation of the MAPK signal pathway. Our data indicated that bFGF might be a potential target for chemotherapy through the MAPK signal pathway. Key words: gliomas, bFGF, temozolomide, combined chemotherapy, drug resistance, chemosensitivity
Introduction Glioma is one of the most common and malignant primary intracranial tumor in adults, which typically leads to progressive disability and eventually death. Up to now, the standard therapies for glioma include surgical resection followed by chemotherapy and radiotherapy. Unfortunately, even with this multimodality treatment, the median survival time of patients is still ~1 year [1]. Chemotherapy and radiotherapy after surgical resection are the necessary methods to prevent metastasis and recurrence [2]. But chemotherapy does not work well in some patients.
Basic fibroblast growth factor (bFGF), a heparin-binding multifunctional growth factor, can promote the proliferation and angiogenesis on glioma cells through autocrine way [3]. bFGF has been proved to be overexpressed in glioma cells [4], and the expression level of bFGF was associated with the grade and prognosis of patients [5]. In our previous study, the depolarization of mitochondria and apoptosis was induced by delivery of bFGF interfering RNA [6]. In addition, down-regulating the expression of bFGF has been proved to inhibit tumorigenicity and metastasis [7].
© The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: [email protected] 501
502 Temozolomide (TMZ), an alkylating agent, does not require hepatic metabolism for activation, because TMZ could be spontaneously converted to the active form of alkylating agent (5-(3-Nmethyltriazen-1-yl)-imidazole-4-carboxamide) (MTIC) [8]. TMZ can function as a broad-spectrum antitumor agent for several diseases such as melanoma, mesothelioma, lymphoma, sarcoma, leukemia, carcinoma of the ovary, carcinoma of the colon [9], and glioma [10]. Because of its extensive effects, chemotherapy inevitably has a wide range of side effects. Previous studies have shown the side effects including neutropenia, thrombocytopenia, lack of red blood cells, and hemoglobin when TMZ was administered in a dosedependent manner [11]. However, the role of bFGF in the resistance of chemotherapy is still unclear. In this study, we tried to explore the relationship between bFGF and chemosensitivity. bFGF expression was silenced with specific siRNA and combined with TMZ to investigate its biological impact on glioma cells, which may help to develop new treatment strategies for glioma patients.
Materials and Methods Cell culture and treatment Glioblastoma cell lines U87 and U251 were obtained from the Peking Union Medical College Cell Library (Beijing, China), and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Grand Island, USA) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in the appropriate atmosphere containing 5% CO2. The cells were divided into five groups: (1) siRNA negative control (NC) group (cells were transfected with NC); (2) TMZ group (cells were treated with TMZ alone); (3) TMZ + NC (cells were transfected with NC and then treated with TMZ); (4) bFGF-siRNA group (cells were transfected with bFGF-siRNA); and (5) union group (cells were transfected with bFGF-siRNA and then treated with TMZ). U87 and U251 cells were incubated in 6-well plates for 12 h, followed by cell transfections. At 24 h after transfection, cells were treated with or without TMZ (Sigma, St Louis, USA).
siRNA transfection siRNAs were purchased from GenePharma (Shanghai, China). The sequence of siRNA targeting bFGF (5’-CGAACTGGGCAGTAT AAAC-3’) was described as previously shown [9]. The sequence of NC was 5’-UUCUCCGAACGUGUCACGUTT-3’. U87 and U251 cells were seeded into 6- or 96-well plates (Corning Costar, Cambridge, USA) and cultured for 12 h. siRNAs were transfected into the cells with Lipofectamine™ 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instruction. The mixture of siRNA and Lipofectamine™ 2000 was added to culture medium for 6 h, and then the medium was changed with DMEM containing 10% FBS.
Western blot analysis U87 and U251 cells were lyzed with the lysis buffer (CW Biotech, Beijing, China) containing protease inhibitor cocktails (Roche, Basel, Switzerland) for 30 min, followed by centrifuging at 12,000 g for 15 min at 4°C. For each sample, 20 μg of total protein was subject to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinyl difluoride membranes (Bio-Rad, Hercules, USA). The membranes were blocked with 5% nonfat milk in tris buffered saline with tween (TBST) buffer for 1.5 h at room temperature, and incubated with primary antibodies overnight at 4°C.
bFGF-siRNA enhances chemosensitivity of glioma After being washed five times with TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody and developed with the electrochemiluminescence (ECL) kit (Millipore, Billerica, USA). The primary anti-Bcl-2, anti-Bax, antimatrix metalloproteinases-2 (MMP-2), and anti-MMP-9 antibodies were from Santa Cruz Biotechnology (Santa Cruz, USA), and the antip-ERK1/2, anti-ERK1/2, anti-p-p38, anti-p38, anti-p-JNK, and antiJNK primary antibodies were from Cell Signaling Technology (Beverly, USA).
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay U87 and U251 cells were seeded into 96-well plates and transfected with NC or bFGF-siRNA, and then treated with TMZ for 48 h. The cell proliferation rate was measured at 0, 24, 48, and 72 h after TMZ treatment. Then, 20 μl of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenylteyrazolium bromide (MTT) (5 mg/ml; Sigma, St Louis, USA) was added into the supernatant. After 4 h of incubation, the medium was exchanged with 150 μl of dimethyl sulfoxide (DMSO) (Sigma). The absorption at 490 nm was determined by a microplate reader.
Colony formation U87 and U251 cells were seeded into 6-well plates and transfected with NC or bFGF-siRNA, and then treated with TMZ for 48 h. The cells were re-seeded into 6-well plates at the density of 500 cells/well for colony formation assay. The medium was replaced every 4 days. After the cells were cultured for 2 weeks, the number of colonies was counted.
Migration assay The migration ability of U87 and U251 cells was detected using a transwell system with 8-μm pore size (Millipore). Cells were transfected with bFGF-siRNA, and then treated with TMZ for 48 h. The lower chamber was filled with 600 μl of culture medium containing 10% FBS, and the upper chamber was seeded with 104 cells from each group. After 24 h of incubation at 37°C in 5% CO2, the cells were fixed with 4% paraformaldehyde and then stained with 1% crystal violet for 20 min. The cells of upper chamber were scraped off using a cotton bud prior to taking photos. Five different fields were randomly chosen in each well and the photos were taken.
Cell apoptosis analysis U87 and U251 cells were seeded into 6-well plates and transfected with NC or bFGF-siRNA, and then treated with TMZ for 48 h. The cells (~1 × 105) were washed with phosphate buffered saline (PBS), and resuspended in 100 μl of binding buffer containing 5 μl of annexin V-FITC and 5 μl of propidium iodide (PI) (BD Pharmingen, San Diego, USA) in a tube. After 15 min of incubation at room temperature in the dark, another 400 μl of binding buffer was added into the tube. Samples were detected by fluorescence-activated cell sorter. The data were analyzed by FlowJo software (BD Pharmingen).
Flow cytometric analysis of cell cycle U87 and U251 cells were cultured in 25-cm2 cell culture bottles (Corning Co, Corning, USA). The cells were transfected with NC or bFGF-siRNA, and then treated with TMZ for 48 h. The cells were harvested and washed with cold PBS. Then, the cells were fixed in
503
bFGF-siRNA enhances chemosensitivity of glioma cold 70% ethanol at 4°C overnight. The cells were washed twice with PBS and incubated with stain working solution (containing 50 μg/ml PI, 1% Triton X-100, and 1 mg/ml RNaseA) for 20 min at room temperature. The percentage of cells in each phase was analyzed by flow cytometry (BD Pharmingen).
Statistical analysis The data were expressed as mean ± standard deviation (SD) of three independent experiments. Statistical analyses were performed using SPSS software 22.0, and compared with one-way analysis of variance. The P values
