Theobromine in ROS Scavenging via Nrf2 Pathway in Oxidative Stress Models

Abstract

Background: Oxidative stress contributes significantly to cancer progression and therapeutic resistance. The Nrf2 signaling pathway regulates the expression of endogenous antioxidants and detoxifying enzymes, offering a critical defense against ROS-induced cellular damage. Theobromine, a cocoa-derived methylxanthine, has been suggested to possess antioxidant potential also used as broncho dilator, vasodilator and in cardiovascular diseases. But its role in lung cancer oxidative stress models remains unclear. Objective: To evaluate the antioxidant and cytoprotective effects of Theobromine in A549 lung cancer cells and determine its involvement in Nrf2-mediated signaling. Methods: A549 cells were treated with Theobromine (32.6 µg/mL) under hydrogen peroxide-induced oxidative stress. Cell viability was assessed using the MTT assay. Intracellular ROS levels were measured using the DCFDA assay. Morphological changes were observed via phase-contrast microscopy. Gene expression of Nrf2 and its downstream targets (HO-1, NQO1, SOD1, GPx1) was analyzed by quantitative real-time PCR. Results: Theobromine significantly improved cell viability compared to the oxidative stress control. ROS levels were markedly reduced, indicating strong antioxidant activity. Morphological analysis confirmed structural preservation of cells. qRT-PCR results revealed upregulation of Nrf2 and downstream antioxidant genes, suggesting activation of the Nrf2-ARE pathway. Conclusion: Theobromine attenuates oxidative damage in A549 cells through ROS scavenging and upregulation of antioxidant defenses mediated by the Nrf2 pathway. These findings highlight its potential as a redox-regulating agent in oxidative stress-related lung cancer interventions.