Mahmud Uzzaman, Gordon Keller and Isabelle M. Germano
Death receptor targeting is an attractive approach in experimental treatment for tumors such as malignant gliomas, which are resistant to radiation and chemotherapy. Among the family of cytokines referred to as death li gands, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has attracted clinical interest. The aim of this study was to assess whether TRAIL can be used as an adjuvant to temozolomide (TMZ) for apoptosis induction in malignant glioma cell lines.
Six human malignant glioma cell lines (A172, U87, U251, T98, U343, and U373) were exposed to human (h)TRAIL, TMZ, or an hTRAIL/TMZ combined treatment. Cell viability was assayed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and phase-contrast microscopy. Cell apoptosis was detected using the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling technique and quantified using flow cytometric analysis. The apoptosis signaling cascade was studied with Western blotting.
The additive effects of hTRAIL and TMZ resulted in a significant decrease in cell viability and an increased apoptotic rate. Expression of the death receptors DR5 and DR4 in two cell lines (A172 and U251) upregulated significantly when they were used in combination hTRAIL/TMZ treatment (p < 0.05 compared with baseline control), leading to activation of caspase-8 and caspase-3 (p < 0.05 compared with baseline control) and confirming an extrinsic apoptotic pathway. A cell intrinsic pathway through mitochondrial cytochrome c was not activated.
Based on this work, one may infer that hTRAIL should be considered as an adjuvant treatment for TMZ-resistant human malignant gliomas.
Mahmud Uzzaman, Ronald J. Benveniste, Gordon Keller and Isabelle M. Germano
For gene therapy strategies currently in clinical trials, viral vectors are used to deliver transgenes directly to normal and tumor cells within the central nervous system (CNS). The use of viral vectors is limited by several factors. The aim of this study was to assess whether embryonic stem cell (ESC)–derived astrocytes expressing a doxycycline-inducible transgene can be used as a vector for gene therapy.
The authors generated a pure population of ESC-derived astrocytes carrying a transgene, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), inserted in the chromosome under the control of a highly regulated doxycycline-inducible expression system. Fully differentiated ESC-derived astrocytes were stereotactically transplanted in the mouse brain, and then cell migration and transgene expression were studied.
The ESC-derived astrocytes started to migrate from the transplant site 48 hours after the procedure. They were found to have migrated throughout the brain tissue by 6 weeks. Transplanted ESC-derived astrocytes expressed the TRAIL transgene after doxycycline induction throughout the duration of the experiment. Teratoma formation was not observed in long-term experiments (12 weeks).
These data show that ESC-derived astrocytes can be used as delivery vectors for CNS tumors. This technique might have a major impact on the treatment of patients with malignant gliomas and a wide spectrum of other neurological diseases.
Isabelle M. Germano, Mahmud Uzzaman, Ronald J. Benveniste, Milana Zaurova and Gordon Keller
Embryonic stem (ES) cell–derived astrocytes have several theoretical and practical advantages as gene therapy vectors in the treatment of malignant gliomas. The aim of this study was to test the proapoptotic effects of ES cell–derived astrocytes expressing transgenic tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in human malignant glioma cells.
Mouse ES cells containing a doxycycline-inducible transgene were engineered with human TRAIL (hTRAIL) and then directed to differentiate into astrocytes. The ES cell-derived–TRAIL-expressing astrocytes were cocultured with human malignant glioma cells. Reverse transcriptase polymerase chain reaction, immunocytochemistry, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling, and flow cytometry were used to quantify results.
In vitro coculture of ES cell–derived astrocytes expressing hTRAIL with A172 human malignant glioma cells after doxycycline induction caused a significant decrease in cell viability from 85 ± 2% at baseline to 8 ± 2% posttreatment (p < 0.001). Labeling with apoptotic markers showed that cell death occurred by means of apoptosis. A significant increase in apoptotic rate (88 ± 3%) from baseline (4 ± 2%) was found in A172 cells after doxycycline induction (p < 0.005). This effect was superior to the apoptotic rate seen after treatment with recombinant TRAIL (57 ± 2%). A decrease in cell viability and an increase in the apoptotic rate were not found in TRAIL-expressing–ES cell-derived astrocytes after induction with doxycycline or in A172 cells exposed to doxycycline alone.
Engineering of transgenic hTRAIL by using ES cell–derived astrocytes induced apoptosis in human malignant glioma cells while sparing nontumor astrocytes. The apoptotic effects of transgenic hTRAIL are greater than those of recombinant hTRAIL. Analysis of these results suggests that hTRAIL-expressing–ES cell-derived astrocytes should be considered in the development of new in vivo strategies to treat malignant human gliomas.
Athanassios P. Kyritsis, Fanny Tachmazoglou, Jasti S. Rao and Vinay K. Puduvalli