Benzyl Isothiocyanate Alters the Gene Expression with Cell Cycle Regulation and Cell Death in Human Brain Glioblastoma GBM 8401 Cells
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ONCOLOGY REPORTS 35: 2089-2096, 2016 Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells NOU-YING TANG1, FU-SHIN CHUEH2, CHIEN-CHIH YU3, CHING-LUNG LIAO1, JEN-JYH LIN4, TE-CHUN HSIA5, KING-CHUEN WU6, HSIN-CHUNG LIU7, KUNG-WEN LU8* and JING-GUNG CHUNG7,9* 1School of Chinese Medicine, China Medical University, Taichung 404; 2Department of Health and Nutrition Biotechnology, Asia University, Taichung 413; 3School of Pharmacy, China Medical University, Taichung 404; 4Division of Cardiology and 5Department of Internal Medicine, China Medical University Hospital, Taichung 404; 6Department of Anesthesiology, E-Da Hospital/I-Shou University, Kaohsiung 824, Taiwan; 7Department of Biological Science and Technology, and 8School of Post-Baccalaureate Chinese Medicine, China Medical University, Taichung 404; 9Department of Biotechnology, Asia University, Taichung 413, Taiwan, R.O.C. Received November 3, 2015; Accepted December 12, 2015 DOI: 10.3892/or.2016.4577 Abstract. Glioblastoma multiforme (GBM) is a highly malig- downregulated and two genes were associated with receptor nant devastating brain tumor in adults. Benzyl isothiocyanate for cell responses to stimuli, the EGF containing fibulin-like (BITC) is one of the isothiocyanates that have been shown extracellular matrix protein 1 (EFEMP1) was inhibited 2.01- to induce human cancer cell apoptosis and cell cycle arrest. fold and the TNF receptor-associated protein 1 (TRAP1) Herein, the effect of BITC on cell viability and apoptotic cell was inhibited 2.08-fold. BITC inhibited seven mitochondria death and the genetic levels of human brain glioblastoma ribosomal genes, the mitochondrial ribosomal protein; tumor GBM 8401 cells in vitro were investigated. We found that protein D52 (MRPS28) was inhibited 2.06-fold, the mitochon- BITC induced cell morphological changes, decreased cell dria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the viability and the induction of cell apoptosis in GBM 8401 mitochondria ribosomal protein L23 (MRPL23) decreased cells was time-dependent. cDNA microarray was used to 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) examine the effects of BITC on GBM 8401 cells and we decreased 2.07-fold, the mitochondria ribosomal protein S12 found that numerous genes associated with cell death and (MRPS12) decreased 2.08-fold, the mitochondria ribosomal cell cycle regulation in GBM 8401 cells were altered after protein L12 (MRPL12) decreased 2.25-fold and the mito- BITC treatment. The results show that expression of 317 chondria ribosomal protein S34 (MRPS34) was decreased genes was upregulated, and two genes were associated 2.30-fold in GBM 8401 cells. These changes of gene expres- with DNA damage, the DNA-damage-inducible transcript sion can provide the effects of BITC on the genetic level and 3 (DDIT3) was increased 3.66-fold and the growth arrest are potential biomarkers for glioblastoma therapy. and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was Introduction Glioblastomas are the most frequent and aggressive primary brain cancers in adults (1) with a high recurrence and mortality rate (2). Glioblastoma prognosis is poor and there Correspondence to: Professor Jing-Gung Chung, Department are limited therapeutic options. In recent years, advances have of Biological Science and Technology, China Medical University, been made in multimodality including surgery, radiotherapy, 91 Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C. E-mail: [email protected] chemotherapy and biotherapy, but the overall 5-year survival rate is still <3% for patients with glioblastoma (3). Thus, we Professor Kung-Wen Lu, School of Post-Baccalaureate Chinese try to identify prognostic gene expression (upregulation or Medicine, China Medical University, 91 Hsueh-Shih Road, Taichung downregulation) that may contribute to evaluate a more effec- 404, Taiwan, R.O.C. tive treatment to improve patient survival and to address more E-mail: [email protected] precisely the use of comprehensive therapy. *Contributed equally Benzyl isothiocyanate (BITC), one of the isothio- cyanates, is present in cruciferous plants, it acts against Key words: benzyl isothiocyanate, gene, cell cycle arrest, regulation, carcinogenesis (4,5) and induces cell death through the glioblastoma induction of apoptosis and cell cycle arrest in various human cancer cells (6-10). In human prostate cancer cells, BITC promoted the phosphorylation of Bcl-xL with simultaneous 2090 tang et al: BITC changes gene EXpression and protein leVels in GBM 8401 cells cell cycle arrest and subsequent apoptosis (11). In our cDNA microarray assay for gene expression. GBM 8401 previous studies we have demonstrated that BITC inhibited cells (2.4x106 cells/dish) were maintained in a 10 cm dish migration and invasion in human colon (12) and gastric (13) for 24 h. Cells were treated with 6 µM BITC or DMSO for cancer cells in vitro. There is no available information to 48 h then cells were collected, and washed twice with PBS. show whether BITC affects human brain tumor cells, in All samples were further isolated in total RNA using Qiagen particular regarding the effects of BITC on gene expression RNeasy Mini kit (Qiagen, Valencia, CA, USA). The isolated in human glioblastoma cells. RNA was further conducted for cDNA synthesis, labeling In cell survival, to maintain the integrity of genomic and and microarray hybridization. The fluorescent-labeled cDNA mitochondrial DNA is critically important. It was reported hybridization (Affymetrix GeneChip Human Gene 1.0 ST that damage to nuclear and mitochondrial DNA can increase array; Affymetrix, Santa Clara, CA, USA) on the chip was the accumulation of defective cellular components leading conducted (18), and the fluorescence from each sample was to impact unfavorably on physiological functions, increasing measured by Asia BioInnovations Corporation (Taipei, entropy (14). If an agent induces DNA damage, the cell in order Taiwan). Expression Console software (Affymetrix) with to respond to the DNA damage, activates the cell cycle check- default RNA parameters (19,20) was used to analyze the data. points (G1, S and G2/M) to stop cell cycle progression in order BITC affecting gene expression with at least a 2-fold-change to allow time for repair, thereby preventing transmission of was considered significant and recorded. damaged or incompletely replicated chromosomes (15). Thus, the associated gene expression regarding cell cycle progres- Results sion, cell apoptosis and DNA damage in cells are important for cancer cell therapy. There is no previous study showing the Cytotoxic effects of BITC in GBM 8401 cells. To investigate anticancer properties of BITC at the genetic level of human the cytotoxic effects of BITC in GBM 8401 cells, after treat- glioblastoma. We investigated the effects of BITC on gene ment of cells with 6 µM BITC for 0, 12, 24 and 48 h, the expression in human brain cancer glioblastoma multiforme cell morphological changes and percentage of viable cells (GBM 8401) in vitro. were measured and results are presented in Fig. 1A and B, respectively. BITC induced cell morphological changes and Materials and methods decreased cell viability in GBM 8401 cells and these effects are time-dependent (Fig. 1A and B). Chemicals and reagents. BITC, dimethyl sulfoxide (DMSO), penicillin-streptomycin and trypsin-EDTA were obtained from Induction of cell apoptosis in GBM 8401 cells after exposure Sigma Chemical Co. (St. Louis, MO, USA). RPMI-1640 culture to BITC. In order to further examine whether cell death was medium and fetal bovine serum (FBS) were purchased from induced by BITC and through the induction of cell apoptosis, Gibco-BRL/Invitrogen (Carlsbad, CA, USA). Tissue culture the cells after treatment with 6 µM BITC were harvested and flasks and plates were obtained from Gibco-BRL/Invitrogen. apoptotic cells were measured by Annexin V/PI staining, and the results are presented in Fig. 2. Based on the data in Cell culture. Human brain glioblastoma GBM 8401 cells were Fig. 2, BITC-induced apoptotic cell death and these effects are purchased from the Food Industry Research and Development time-dependent. The treatment of cells with BITC increased Institute (Hsinchu, Taiwan) and cultured following the the total apoptotic cell death to 36.81% at 48 h (Table I). The supplier's instructions. Cells were grown in 75 cm2 culture result is consistent with the morphology and examination of flasks with RPMI-1640 medium supplemented with 10% FBS, total viable cells. 100 U/ml penicillin and 100 µg/ml streptomycin and main- tained in an atmosphere of 5% CO2 and 95% air at 37˚C. The BITC alters the regulations of gene expression in GBM 8401 medium was changed every 2 days (16). cells. GBM 8401 cells were treated with or without 6 µM BITC for 48 h and then harvested for total RNA extraction. Cell morphological changes and viability assays. GBM 8401 The expression of the top 10 up- and downregulated genes was cells (8x104 cells/ml) were seeded into a 12-well plate estimated by cDNA microarray analysis and the results are containing RPMI-1640 medium for 24 h. In addition, BITC presented in Tables II and III. BITC induced 317 upregulated was added to wells at the final concentration of 6 µM for 0, 12, genes and 182 downregulated genes of GBM 8401, respectively. 24 and 48 h. After treatment, cells were examined and photo- Fourty-six genes were upregulated in the range >3-<4-fold, graphed using contrast-phase microscopy at a magnification of and 198 genes were upregulated >2-<3-fold. One gene was x400 and then harvested for measuring the total percentage of downregulated >4-fold, and 11 genes were downregulated viable cells using flow cytometric assay (16). in the range >3-<4-fold, and 170 genes were downregulated >2-<3-fold (data not shown). Annexin V/PI staining for cell apoptosis.