| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Objectives. To investigate therapeutic efficacy and molecular mechanisms of fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) against human glioma cells.
Methods. Twelve human glioma cell lines were used in this study. Fully human anti-human TRAIL receptor mAbs (B12 : specific to DR4, Ell, H48, and KMTR2 : specific to DR5) were provided by Kirin Brewery Co. Ltd. Cytotoxicity was assessed by MTT assay. Apoptosis was detected by TUNEL assay. Clonogenic survival was assessed by colony formation efficiency assay. Cellular protein expression was analyzed with Western blot. Cell surface expression of TRAIL receptors was quantified by flow cytometry. Glioma cells were inoculated into either the frank or cerebrum of nude mice to generate glioma xenografts models.
Results. Human glioma cells were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. Anti-DR5
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mAb treatment resulted in cleavage and activation of, an initiator caspase, caspase-8, which in turn cleaved an effector caspase, caspase-3. Further cleavage of Bid, a BH3-only molecule of the Bc1-2 family members, occurred, thereby activating mitochondrial apoptosis pathways involving cleavage of caspase-9. The sensitivity of human glioma cell lines was closely associated with the expression level of DR5 at the cell surface. Cellular protein expression levels of c-FLIP_L, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAb s. Downregulation of c-FLIP_L protein expression using siRNA resulted in sensitization of human glioma cells to anti-DR5 mAbs. Furthermore, glioma cells overexpressing c-FLIP_L by transfecting the c-FLIP_L expression vector became resistant to anti-DR5 mAb treatment. Treatment of nude mice with anti-DR5 mAbs significantly suppressed growth of subcutaneous glioma xenografts compared with those treated with control non-specific antibodies. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with anti-DR5 mAbs significantly elongated life span.
Conclusions. DR5 is the predominant TRAIL receptor, which is expressed at the cell surface and mediates apoptotic signals in human glioma cells. Sensitivity of human glioma cells to anti-DR5 mAbs might be determined at least in part by expression level of c-FLIPL. Anti-DR5 mAbs exert anti-tumor effects both in vitro and in vivo. Our results suggest that specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas. Less
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