Liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer-related deaths [1]. According to GLOBOCAN 2020 estimates, liver cancer remains a major global health burden, with approximately 905,700 new cases and about 830,200 deaths worldwide in 2020 [1]. Its incidence has been steadily increasing in many parts of the world, particularly due to the rise in risk factors such as chronic viral hepatitis, alcohol consumption, and metabolic disorders. Despite advances in surgical techniques, locoregional therapies and chemotherapy, the prognosis of liver cancer patients remains poor [2,3]. Immunotherapy has emerged as a promising option for hepatocellular carcinoma (HCC). Immune checkpoint inhibitors such as anti-PD-1/PD-L1 antibodies (e.g., nivolumab, pembrolizumab, and atezolizumab) are now being used to treat advanced liver cancer [4,5]. However, the objective response rates of PD-1/PD-L1 inhibitors like nivolumab and pembrolizumab is approximately 15–20 % due to its unique immune-tolerant environment [5]. Their limited efficacies remain challenge.

Immunogenic cell death (ICD) is a form of regulated cell death that activates the immune system to recognize and eliminate tumor cells [6,7]. ICD promotes an adaptive immune response by releasing damage-associated molecular patterns (DAMPs) [[8], [9], [10]]. The most notable DAMPs involved in ICD include calreticulin (CRT) exposure, high-mobility group box 1 (HMGB1) release, adenosine triphosphate (ATP) secretion. These DAMPs are key to the activation of dendritic cells (DCs). Then, as antigen-presenting cells, DCs effectively process and present tumor antigens to T cells. Subsequently, the generation of cytotoxic T cells can target and kill tumor cells. Therefore, ICD effectively converts “cold” tumors, which are poorly infiltrated by immune cells, into “hot” tumors that are more immunologically active [11]. However, immunosuppressive tumor microenvironment of HCC poses significant challenges for this effective immune-based therapies [12]. The liver is an immune-tolerant organ, partly due to its exposure to gut-derived antigens and chronic inflammation. As a result, HCC develops in a tumor microenvironment that is often rich in immunosuppressive cells, such as regulatory T cells (Tregs), myeloid-derived suppressor cells, and tumor-associated macrophages (TAMs). These cells hinder the effectiveness of immune responses [13,14].

Recently, efforts have focused on identifying and screening ICD inducers to promote ICD within HCC tumor microenvironment. Agents that trigger reactive oxygen species (ROS) accumulation and endoplasmic reticulum (ER) stress have gained attention [9,15]. ER stress and oxidative stress are critical pathways in ICD induction, as they disrupt cellular homeostasis, leading to cell death and the release of DAMPs [16]. However, not all compounds that trigger ER stress or oxidative stress are effective ICD inducers [17]. Some agents, such as thapsigargin or tunicamycin, as well as the chemotherapeutic drug cisplatin, can induce oxidative stress but fail to promote the release of key DAMPs necessary for effective ICD, such as CRT exposure on the cell surface [17,18]. These findings underscore the complexity of ICD induction, where ER stress and oxidative stress may act as important endogenous stimuli but are not always sufficient to trigger the full cascade of events required for ICD [8,18,19]. Thus, it is essential to identify agents and potential targets that can effectively integrate ER stress and ROS production with the full induction of ICD to achieve optimal therapeutic efficacy.

Astragalin (ASG) is a natural flavonoid glycoside primarily found in various plants, including Astragalus membranaceus (astragalus) [20], Eriobotrya japonica (loquat) [21], and Tetrastigma hemsleyanum Diels et Gilg [22]. It has gained attention for its diverse biological activities, including anti-inflammatory, antidiabetic, neuroprotective, and anticancer effects [23]. Studies have demonstrated that ASG effectively inhibits the proliferation of cancer cells, such as liver [24], lung [25], gastric [26] kidney, skin [27] and colon cancer [28]. ASG can elevate ROS levels in specific contexts, particularly within cancer cells [25]. The increase in ROS can induce oxidative stress, leading to cell death in cancer cells while sparing normal cells. It also induces ER stress in cancer cells, activating the unfolded protein response [28]. It has been reported that ASG treatment can suppress the proliferation of liver cancer cells both in vitro and in vivo by reducing the expression of hexokinase 2, leading to metabolic reprogramming and ROS accumulation [24]. Another study also indicates that ASG can decrease the expression of Bcl-2, upregulate the expression of Bax and cleaved caspase-3, and regulate the apoptosis signaling pathway, thereby inhibiting the proliferation of liver cancer cells [29]. Although ASG has been tested in liver cancer, its underlying mechanisms of action and its potential target remain unclear.

NAD(P)H: quinone oxidoreductase 2 (NQO2) is a key enzyme that regulates cellular redox balance, particularly by reducing quinones and other reactive molecules. It has been reported that NQO2 plays a pivotal role in tumor progression, therapeutic resistance, and immune escape [30]. Its activity in liver cancer was higher than in other cancers [31]. By maintaining redox homeostasis, NQO2 helps to protect tumor cells from oxidative stress and ROS-induced DNA damages [32,33]. NQO2 activates survival signaling pathways and allows cancer cells evade apoptosis [34]. NQO2 also helps to alleviate ER stress by impairing the unfolded protein response and weakening the cell's stress response [35]. Conversely, inhibiting NQO2 could increase ROS and enhance ER stress, potentially inducing apoptosis in cancer cells [35]. Importantly, NQO2 influences immune function and promotes immune escape by modulating oxidative stress within immune cells [36]. Thus, targeting NQO2 may offer a novel therapeutic strategy for liver cancer by influencing oxidative stress, ER stress, and immune responses. Given that its multiple potentials, we want to identify whether NQO2 might be the target and mediate the antitumor effects of ASG.

To validate our hypothesis, we first assessed the growth inhibitory effects of ASG on liver cancer cell lines. ASG's impact on apoptosis, ROS accumulation, mitochondrial membrane potential, and the expression of ER stress markers were further investigated. Next, the potential targets of ASG were investigated. ASG's effects on NQO2 expression were conducted and molecular docking to confirm the binding potential between ASG and NQO2 was performed. A gene interference strategy was employed to investigate the biological role of NQO2 in promoting proliferation and inhibiting ER stress in liver cancer cells. We then explored ASG's ability to induce ICD and its role in DCs maturation using a Hepa1-6/BMDC coculture system. Lastly, the synergistic effects of ASG in combination with anti-PD-L1 antibody (αPD-L1) therapy were investigated. Our findings suggest that ASG not only serves as a promising therapeutic agent but also acts as an effective ICD inducer, by directly binding to NQO2 and mediating its degradation. Consequently, ASG inhibits the proliferation of liver cancer cells and enhances the antitumor efficacy of αPD-L1 therapy in liver cancer.