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Verotoxin-1 induction of apoptosis in Gb3-expressing human glioma cell lines
David Johansson1, Anders Johansson, Kjell Grankvist
1Department of Medical Biosciences, Clinical Chemistry, Umeå University, Umeå, Sweden.
Abstract:
The aim of this study was to examine the cytotoxicity and mechanism of apoptosis induction of verotoxin-1 (VT-1) in human glioma cell lines. VT-1 is a member of the shiga-toxin family expressed by some serotypes of Escherichia coli and Shigella dysenteriae. Shiga-toxins have been shown to induce apoptosis by binding to its membrane receptor Gb3. The human glioma cell lines SF-767, U-343 MG, and U-251 MG were studied together with BT4C, a rat glioma cell line. Cells were first screened for Gb3 expression by flow cytometry. Fluorescein diacetate was used to determine cell viability after VT-1 and irradiation exposure and apoptosis was studied by TUNEL staining, a mitochondrial membrane potential assay, and caspase activity assays. SF-767 and U-343 MG cells were found to express Gb3 and were also sensitive to VT-1-induced cytotoxicity, whereas nonGb3-expressing U-251 MG and BT4C glioma cells were not. VT-1 depolarized the mitochondrial membrane and activated caspase-9 and -3 of SF-767 and U-343 MG cells. VT-1 exposure for 72 h resulted in approx. 60 and 90% TUNEL-stained cells, respectively. D, L-Threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) an inhibitor of glucosylceramide synthesis was used to block Gb3 synthesis. Two mumol/L PPMP for 72 h abolished SF-767 and U-343 MG expression of Gb3 and made the cells completely resistant to VT-1 induced apoptosis. Key components of MAP kinase signalling pathways that control BAX and mitochondrial function were investigated. VT-1 induced JNK phosphorylation in both cell lines, suggesting that survival signal pathways were overruled by VT-1-induced JNK activation leading to mitochondrial depolarization, caspase-9 activation and apoptosis. Immunohistochemistry of cryostat section from glioma biopsies demonstrated expression of Gb3 was in the vascular endothelial cells as well as tumor cells, but not in astrocytes. The high specificity and apoptosis inducing properties of verotoxin-1 indicates that the toxin may be a potential anti-neoplastic agent for Gb3-expressing gliomas.
Insights
Verotoxin-1 (VT-1) induces apoptosis in human glioma cells expressing the Gb3 receptor, demonstrating its potential as an anti-cancer agent. Inhibition of Gb3 synthesis renders cells resistant to VT-1, highlighting the receptor's critical role.
Area of Science:
- Molecular Biology
- Oncology
- Toxicology
Background:
- Verotoxin-1 (VT-1), a Shiga-toxin, induces apoptosis by binding to the Gb3 receptor.
- Glioma is a primary brain tumor with limited treatment options.
- Understanding VT-1's mechanism in glioma cells is crucial for therapeutic development.
Purpose of the Study:
- To investigate the cytotoxicity and apoptosis-inducing mechanism of VT-1 in human glioma cell lines.
- To determine the role of Gb3 receptor expression in VT-1 sensitivity.
- To explore VT-1's potential as an anti-neoplastic agent for gliomas.
Main Methods:
- Screening of human (SF-767, U-343 MG, U-251 MG) and rat (BT4C) glioma cell lines for Gb3 expression using flow cytometry.
- Assessment of cell viability, apoptosis (TUNEL staining, mitochondrial membrane potential, caspase activity), and Gb3 synthesis inhibition (PPMP).
- Analysis of MAP kinase signaling pathways (JNK phosphorylation) and immunohistochemistry on glioma biopsy samples.
Main Results:
- Gb3-expressing glioma cells (SF-767, U-343 MG) exhibited sensitivity to VT-1-induced cytotoxicity and apoptosis.
- VT-1 triggered mitochondrial depolarization and activated caspase-9 and -3, leading to significant apoptosis.
- Inhibition of Gb3 synthesis using PPMP rendered cells resistant to VT-1-induced apoptosis.
- VT-1 induced JNK phosphorylation, suggesting activation of pro-apoptotic signaling pathways.
- Glioma biopsies showed Gb3 expression in tumor cells and vascular endothelial cells, but not astrocytes.
Conclusions:
- VT-1 induces apoptosis in Gb3-expressing human glioma cells via mitochondrial pathways and JNK activation.
- Gb3 receptor expression is essential for VT-1's cytotoxic effects on glioma cells.
- VT-1 demonstrates potential as a targeted anti-neoplastic agent for Gb3-positive gliomas.
