| Summary: | 博士 === 慈濟大學 === 醫學科學研究所 === 103 === Glioblastoma is the most common and most aggressive primary brain malignancy. The multimodality treatments for this tumor including surgery, radiotherapy, and chemotherapy, are still not completely satisfying the current needs. Phenethyl isothiocyanate (PEITC), one member of the isothiocyanate family, has been shown to induce apoptosis, and inhibit metastasis in many human cancer cells. In our study, we investigated the effects of PEITC on apoptosis, gene expressions, migration and invasion in human brain glioblastoma multiforme GBM 8401 cells.
Our results revealed that PEITC induced the cell morphological changes and decreased the cell viability of GBM 8401 cells in a dose- and time-dependent manner. The analysis of cell cycle distribution showed that PEITC induced significantly sub-G1 phase (apoptotic population) in GBM 8401 cells. Moreover, PEITC promoted the production of reactive oxygen species (ROS) and Ca2+, but decreased the mitochondrial membrane potential (ΔΨm) in treated cells. PEITC also induced caspases activities in GBM 8401 cells. Data from Western blotting analysis indicated that PEITC promoted the expressions of Fas, FasL, FADD, TRAIL, caspase-8, -9, -3, increased the pro-apoptotic proteins (Bax, Bid and Bak), and inhibited the anti-apoptotic proteins levels (Bcl-2 and Bcl-xl). In addition, PEITC promoted the release of cytochrome c, AIF and Endo G. GADD153, GRP 78, XBP-1 and IRE-1α, Calpain I and II in treated cells. PEITC also promoted the protein expressions associated with endoplasmic reticulum (ER) stress. Taken together, PEITC induced apoptosis through the extrinsic (death receptor) pathway, dysfunction of mitochondria, ROS induced ER stress, intrinsic (mitochondrial) pathway in GBM 8401 cells.
Regarding to the gene expressions in GBM 8401 after PETIC treatment, many genes associated with the cell cycle regulation were changed: 504 genes were up-regulated, 361 were down-regulated. PEITC may induce G0/G1 cell cycle arrest through affecting proteins such as cdk2, cyclin E, p21 in GBM 8401 cells. The cell-division cycle protein 20 (CDC20), budding uninhibited by benzimidazole 1 homolog beta (BUB1B), cyclin A2, B1, B2 were down-regulated by 2.62, 2.29, 2.66, 2.34, 2.13-fold, respectively, and crystallin, alpha B (CRYAB), chromosome 4 open reading frame 34 (C4orf34) were up-regulated by 18.49, 5.29-fold, respectively in treated cells.
Our results also indicated the effects of PEITC on migration of GBM 8401 cells from scratch wound healing assay and Transwell migration assay were in a dose-dependent manner. The percentage of Transwell migration assay ranged from 46.89% to 15.75%, and invasion assay ranged from 27.80% to 7.31% after 48 h-treatment of PEITC. Results from Western blotting analysis indicated that PEITC decreased the levels of proteins associated with migration and invasion, Ras, uPA, Rho A, GRB2, p-p38, p-JNK, p-ERK, p65, SOS1, MMP-2, MM-9, MMP-13 in a dose-dependent manner. Results from real-time PCR analyses revealed that PEITC reduced the mRNA levels of MMP-2, MMP-7, MMP-9 and Rho A in a dose- and time-dependent manner. PEITC had potent anticancer activities through the inhibition of migration and invasion in GBM 8401 cells.
In our study, the possible molecular mechanisms and signaling pathways of anticancer properties of PEITC for human brain glioblastoma cells were postulated. Changes of gene expressions might provide insight into the effects of PEITC on the genetic levels and also the potential biomarkers for glioblastoma treatment. PEITC may be considered to be a therapeutic agent for human brain glioblastoma.
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