Reactive oxygen species–dependent upregulation of death receptor, tumor necrosis factor receptor 1, is responsible for theophylline-mediated cytotoxicity in MDA-MB-231 breast cancer cells

Abstract

Theophylline, a methylxanthine drug, has been used as a therapy for respiratory diseases. Recently, it has also been shown to have a potential in treating different cancers. Also, it has shown promising results in clinical trials for AML in combination therapy. Subsequently, studies have shown theophylline to kill breast cancer cells but not normal breast cells. Therefore, in this study, we have explored the molecular mechanism underlying the cytotoxic effect of theophylline on breast cancer cells. Theophylline-treated cancer cells were analyzed for the transcript and protein expression of candidate apoptotic genes such as TNFR1, caspase-8, -9, -3 using qPCR and immunoblotting, respectively. Cell viability and apoptosis was measured in the presence or absence of TNFR1 inhibitor, R7050, using AO/EtBr staining and MTT assay, respectively. Similarly, oxidative stress was studied by analyzing ROS in the presence or absence of ROS inhibitor, NAC, using DCFDA assay. Theophylline caused reduced cell viability in cancer but not normal cells. Theophylline-treated breast cancer cells showed increased expression of death receptor, TNFR1, along with elevated levels of active caspase-8, -9 and -3. Inhibition of TNFR1 reduced caspase-dependent apoptosis even in the presence of theophylline. Theophylline further caused increased ROS generation, inhibition of which resulted in reduced TNFR1-mediated apoptosis. Theophylline also increased cathepsin activity, which was reduced on exposure of cells to TNFR1 inhibitor, R7050. We conclude that ROS-mediated activation of TNFR1 is responsible for caspase-3 and cathepsin-dependent cell death in breast cancer cells on exposure to theophylline.