Eupalinolide B exerts cytotoxic effects against KRAS-mutant NSCLC through Nrf2/HO-1-regulated ferroptosis

Abstract

Ferroptosis, an iron-dependent form of regulated cell death, is characterized by excessive reactive oxygen species (ROS) accumulation and lipid peroxidation of polyunsaturated fatty acids (PUFAs) in cellular membranes. Non-small cell lung cancer (NSCLC) harboring KRAS mutations often exhibits therapeutic resistance but may display high susceptibility to ferroptosis. Eupalinolide B (EB), a natural compound with documented anti-cancer activity, has not been thoroughly explored for its ferroptosis-inducing potential in KRAS-mutant NSCLC. In this study, we demonstrate that EB treatment significantly elevates ROS levels, intracellular iron accumulation, and lipid peroxidation in KRAS-mutant NSCLC cells, resulting in ferroptotic cell death. Molecular docking and cellular thermal shift assays reveal that EB directly binds to and activates heme oxygenase-1 (HO-1), a critical component of the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2/HO-1 oxidative stress response pathway. Genetic or pharmacological inhibition of HO-1 attenuates EB-induced ferroptosis, underscoring the pivotal role of HO-1-mediated oxidative stress in this process. Furthermore, in vivo studies using KRAS-mutant H358 xenograft models confirm the potent anti-tumor effects of EB. Collectively, our findings establish that EB triggers ferroptosis in KRAS-mutant NSCLC by activating the Keap1-Nrf2/HO-1 pathway, suggesting a promising therapeutic strategy for this challenging malignancy.

1. Introduction

Lung cancer ranks as the second most prevalent malignancy worldwide and remains the leading cause of cancer-related mortality [1,2]. Non-small cell lung cancer (NSCLC) constitutes approximately 85% of all lung cancer diagnoses [3] and has a poor overall prognosis despite advances in radiotherapy, chemotherapy, and surgical resection [4]. While these treatments provide temporary disease control, long-term outcomes for many patients remain suboptimal, particularly for those with mutations in KRAS, a common oncogenic driver in NSCLC [5]. Although targeted therapies have shown success in treating other genetic subtypes of NSCLC, KRAS-mutant NSCLC remains resistant to conventional treatments, highlighting the necessity for innovative therapeutic approaches.

Inducing tumor cell death is a promising strategy in cancer therapy. Various well-characterized forms of programmed cell death, such as apoptosis [6], necroptosis [7], and autophagy [8], have been extensively studied for their roles in cancer treatment. However, recent research has revealed the therapeutic potential of ferroptosis, a distinct form of iron-dependent cell death characterized by lipid peroxidation and iron accumulation [9,10]. Unlike other forms of cell death, ferroptosis is driven by oxidative damage to cell membranes through the peroxidation of polyunsaturated fatty acids (PUFAs) and is further amplified by reactive oxygen species (ROS) when the cell's antioxidant defenses, such as glutathione (GSH) and glutathione peroxidase 4 (GPX4), are compromised [11].

Importantly, increasing evidence suggests that ferroptosis inducers have the potential to sensitize resistant cancer cells to chemotherapy by exploiting vulnerabilities in their redox balance [12]. This phenomenon is especially important for KRAS-mutant NSCLC, a subtype known for its high levels of ROS production caused by metabolic imbalances. KRAS mutations disrupt various pathways related to oxidative stress and redox balance, resulting in elevated ROS levels that both support tumor survival and increase the susceptibility of cancer cells to ferroptosis. Therefore, ferroptosis inducers present a unique opportunity to target KRAS-mutant cancer cells by increasing ROS levels and promoting iron-dependent lipid peroxidation, thus overcoming resistance mechanisms.