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Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement
V Goossens1, J Grooten, K De Vos
1Laboratory of Molecular Biology, Ghent University, Belgium.
Abstract:
Tumor necrosis factor (TNF) is selectively cytotoxic to some types of tumor cells in vitro and exerts antitumor activity in vivo. Reactive oxygen intermediates (ROIs) have been implicated in the direct cytotoxic activity of TNF. By using confocal microscopy, flow cytometry, and the ROI-specific probe dihydrorhodamine 123, we directly demonstrate that intracellular ROIs are formed after TNF stimulation. These ROIs are observed exclusively under conditions where cells are sensitive to the cytotoxic activity of TNF, suggesting a direct link between both phenomena. ROI scavengers, such as butylated hydroxyanisole, effectively blocked the formation of free radicals and arrested the cytotoxic response, confirming that the observed ROIs are cytocidal. The mitochondrial glutathione system scavenges the major part of the produced ROIs, an activity that could be blocked by diethyl maleate; under these conditions, TNF-induced ROIs detectable by dihydrorhodamine 123 oxidation were 5- to 20-fold higher.
Insights
Tumor necrosis factor (TNF) triggers reactive oxygen intermediates (ROIs) in tumor cells, directly causing cell death. Blocking ROIs with scavengers halts TNF’s cytotoxic effect, confirming their role in cancer cell killing.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Tumor necrosis factor (TNF) exhibits selective cytotoxicity against tumor cells in vitro and in vivo.
- Reactive oxygen intermediates (ROIs) are hypothesized to mediate TNF's direct cytotoxic effects.
Purpose of the Study:
- To directly demonstrate the formation of intracellular ROIs upon TNF stimulation.
- To establish a direct link between ROI generation and TNF-induced cytotoxicity.
- To investigate the role of the mitochondrial glutathione system in scavenging TNF-induced ROIs.
Main Methods:
- Confocal microscopy
- Flow cytometry
- Dihydrorhodamine 123 probe for ROI detection
- Use of ROI scavengers (e.g., butylated hydroxyanisole) and mitochondrial glutathione system inhibitors (e.g., diethyl maleate)
Main Results:
- Intracellular ROIs were directly observed following TNF stimulation.
- ROI formation correlated directly with TNF sensitivity and cytotoxicity.
- ROI scavengers blocked both ROI formation and TNF-induced cell death.
- Inhibition of the mitochondrial glutathione system significantly increased detectable ROIs.
Conclusions:
- TNF stimulation induces intracellular ROI formation, which is directly responsible for its cytotoxic activity against tumor cells.
- The mitochondrial glutathione system plays a key role in detoxifying TNF-induced ROIs.
- Targeting ROIs represents a potential therapeutic strategy for cancer treatment.