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Tumor necrosis factor mutants with selective cytotoxic activity
N Berkova1, A Lemay, V Korobko
1Centre de Recherche, Endocrinologie de la Reproduction, Pavillon Saint-François d'Assise, CHUQ, Quebec, Canada.
Abstract:
Tumor necrosis factor (TNF-alpha) has a cytotoxic or cytostatic effect when tested with various malignant cell lines. Clinical trials in cancer patients, however, revealed high systemic toxicity of TNF-alpha. The existence of two types of receptor may partially explain the pleiotropic activity of TNF-alpha. The purpose of this study was to characterize the relative cytotoxic activity of TNF-alpha and TNF mutants on the mouse fibrosarcoma L929 cells in a standard cytotoxicity test, on human larynx carcinoma HEp-2 cells, and on human monoblastoid leukemic cells U937. TNF mutants were obtained by site-directed mutagenesis. The purity of TNF-alpha was established by capillary electrophoresis. TNF-alpha and TNF mutants were analysed by Western blot analysis using monoclonal antibodies against TNF-alpha. The results show that TNF mutants can recognize the different TNF-receptors (TNF-R) selectivity. It is generally believed that activation of TNF-R75 is responsible for the systemic toxicity of TNF-alpha. Hence, the development of TNF mutants, binding selectively to TNF-R55, could lead to new option for an anticancer treatment that would be devoid of the deleterious effect of TNF-alpha.
Insights
Tumor necrosis factor-alpha (TNF-alpha) shows anti-cancer effects but causes toxicity. This study developed TNF mutants targeting specific receptors, aiming for safer anticancer therapies with reduced side effects.
Area of Science:
- Oncology
- Immunology
- Molecular Biology
Background:
- Tumor necrosis factor-alpha (TNF-alpha) exhibits cytotoxic and cytostatic effects against cancer cells.
- Clinical use of TNF-alpha is limited by high systemic toxicity.
- TNF-alpha interacts with two receptor types, contributing to its varied effects.
Purpose of the Study:
- To characterize the cytotoxic activity of TNF-alpha and its mutants on various cancer cell lines.
- To investigate the receptor selectivity of TNF mutants.
- To explore the potential of TNF mutants for targeted anticancer therapy.
Main Methods:
- Site-directed mutagenesis was used to create TNF mutants.
- Capillary electrophoresis assessed TNF-alpha purity.
- Western blot analysis with monoclonal antibodies characterized TNF-alpha and mutants.
- Cytotoxicity assays were performed on L929, HEp-2, and U937 cells.
Main Results:
- TNF mutants demonstrated selective binding to different TNF receptors (TNF-R).
- TNF-alpha and its mutants were successfully purified and analyzed.
- Differential cytotoxic activities were observed for TNF-alpha and its mutants across cell lines.
Conclusions:
- Selective TNF receptor binding by TNF mutants offers a potential strategy to mitigate TNF-alpha's systemic toxicity.
- Development of TNF mutants targeting TNF-R55 could lead to novel anticancer treatments with improved safety profiles.