Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC accounts for approximately 15–20% of all breast cancer cases and is associated with aggressive tumor behavior and poor prognosis (Ganesan et al., 2021, 2023). Treatment options for TNBC are limited compared to other breast cancer subtypes, as targeted therapies like hormonal therapy or HER2-targeted agents are not effective (Wang et al., 2023b). Chemotherapy, particularly anthracyclines like doxorubicin (DOX), is commonly used in the treatment of TNBC. However, the efficacy of chemotherapy alone is often limited due to acquired resistance and toxic side effects (Zeng et al., 2023). Therefore, there is a need to explore combination therapies that can enhance the therapeutic impact of chemotherapy while minimizing adverse effects.

Danggui Buxue Tang (DBT) is a traditional Chinese herbal formula composed of Astragali Radix (Huangqi; AR) (roots of Astragalus membranaceus (Fisch.) Bunge or Astragalus membranaceus (Fisch.) Bunge var.mongholicus (Bunge) P.K. Hsiao) and Angelicae Sinensis Radix (Danggui; ASR) (root of Angelica sinensis (Oliv.) Diels) at the ratio of 5 : 1. DBT has been used for centuries in traditional Chinese medicine to tonify blood, boost the immune system, and improve overall health (Gong et al., 2016a, 2017; Kwan et al., 2019). The pharmacological properties of DBT have been recently investigated in both in vivo and in vitro studies (Gong et al., 2015, 2016c; Zheng et al., 2012). These studies have shown that the administration of DBT in various animal models results in several effects, such as the enhancement of red and white blood cell populations, stimulation of estrogenic properties, promotion of bone regeneration, triggering of immune responses, and induction of capillary and blood vessel formation (Gong et al., 2016b, 2018; Lin et al., 2017; Zhan et al., 2014; Zhang et al., 2013).

Ferroptosis is a form of regulated cell death characterized by the accumulation of iron-dependent lipid peroxides. It is distinct from other forms of cell death, such as apoptosis or necrosis (Lei et al., 2022; Zhang et al., 2021). Emerging evidence suggests that ferroptosis can be induced as a therapeutic strategy for cancer treatment, as cancer cells often exhibit increased sensitivity to ferroptosis compared to normal cells (Yang et al., 2023a). The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway plays a crucial role in regulating cellular antioxidant responses and protecting cells from oxidative stress (Ganesan et al., 2020; Jayasuriya et al., 2021; V. Ganesh et al., 2022). However, aberrant activation of the Nrf2 pathway has been implicated in promoting cancer growth, metastasis, and chemotherapy resistance (Jayasuriya et al., 2022; Nagarajan et al., 2022). Nrf2 has been observed to be overexpressed in different types of tumors, including lung, breast, head and neck, uterine, ovary, and endometrium (Dodson et al., 2019; Feng et al., 2021; Fu et al., 2022; Shi et al., 2023; Si et al., 2020; Tang et al., 2022; Wang et al., 2022a; Yuan et al., 2021). In different types of tumors, Nrf2 and heme oxygenase 1 (HO-1) have been found to be frequently upregulated, and their elevated levels have been linked with tumor progression, aggressiveness, therapy resistance, and poor prognosis (Chang et al., 2018; Li et al., 2022b; Yang et al., 2021, 2024). HO-1, in particular, has a potent antioxidant and antiapoptotic effect that supports the growth of cancer cells and increases their resistance to therapy (Yang et al., 2021). Activation of Nrf2 triggers the upregulation of HO-1 and glutathione peroxidase 4 (GPX4), which helps reduce oxidative stress and safeguard mitochondrial integrity, potentially preventing mitochondrial dysfunction (Yang et al., 2024). Excessive oxidative stress, commonly observed in different pathological conditions, can lead to ferroptosis, a unique type of cell death. Inhibiting the activation of antioxidant pathways, like Nrf2/HO-1, may result in increased oxidative stress, accumulation of intracellular iron, and the possible induction of ferroptosis (Cheng et al., 2023; Hassannia et al., 2019; Lei et al., 2022; Tang et al., 2022; Yang et al., 2014). Therefore, targeting the Nrf2/HO-1/GPX4 pathway has emerged as a potential therapeutic strategy for cancer treatment.

In recent years, research has demonstrated the anti-cancer properties of DBT and its ability to improve the effectiveness of chemotherapy in different types of cancer. However, whether DBT can enhance the therapeutic effects of chemotherapy specifically in treating TNBC is still unknown. Therefore, the aim of this study was to explore the potential of a combination therapy using DOX and DBT for treating TNBC, focusing on the disruption of the Nrf2/HO-1/GPX4 axis through ferroptosis induction.