Wedelolactone induces pyroptosis to suppress retinoblastoma by inhibiting the Nrf2/Keap1 signaling pathway

Abstract

Background

Retinoblastoma is an intraocular malignancy with limited therapeutic options, imposing a severe health burden on young patients. Wedelolactone (WDL), a natural product from E. prostrata, possesses an anti-retinoblastoma activity, with the underlying regulatory mechanism remaining unknown.

Methods

Retinoblastoma cell lines and xenograft nude mouse models were treated with WDL and RTA-408, an agonist for nuclear factor-erythroid 2-related factor 2 (Nrf2). Western blotting was conducted to determine the protein expression levels of kelch-like ECH-associated protein 1 (Keap1) and Nrf2. We performed the cell counting kit-8 assay, the 5-ethynyl-2-deoxyuridine staining, and flow cytometry to detect cell viability, proliferation, and apoptosis, respectively. The tumor progression in vivo was evaluated via the measurement of volume, weight, and proliferation levels of solid tumors. Monosodium urate crystal was applied to activate pyroptosis which was assessed by the expression detection of pyroptosis-related indicators.

Results

WDL treatment elevated the expression of Keap1 and reduced the level of Nrf2. RTA-408 suppressed WDL-induced pyroptosis of retinoblastoma cells and reversed the effect of WDL on inhibiting retinoblastoma cell proliferation, promoting tumor cell apoptosis, and repressing the growth of solid tumors of the xenograft models. In addition, monosodium urate-induced pyroptosis partially restored the anti-retinoblastoma effect of WDL impaired by RTA-408.

Conclusion

WDL triggers pyroptosis by inhibiting the Nrf2/Keap1 signaling pathway to exert anti-retinoblastoma effects.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12032-025-02916-w.

Introduction

Retinoblastoma, the most common primary intraocular malignancy with an incidence of 1 in every 16,000 to 20,000 live births, mostly affects infancy and children [1]. The overall survival rate of retinoblastoma has exceeded 95% in developed countries [1, 2], whereas retinoblastoma remains a potentially deadly disease in developing countries due to late diagnosis and poor healthcare systems [3]. Currently, enucleation, radiotherapy, and systemic chemotherapy are the main therapeutic strategies for retinoblastoma. However, chemotherapy is mainly used for the treatment of early primary tumors and is often accompanied by drug resistance and cytotoxicity [4], and radiotherapy and enucleation possibly lead to the presence of cosmetic deformity [5]. Hence, it is of great significance to search for more highly efficient and safer drugs.

Natural products are an important source of innovative anti-cancer drugs, showing a positive trend in preclinical research due to their superior efficacy and safety [6]. Wedelolactone (WDL), a natural product from E. prostrata [7], exhibits numerous pharmacological activities. For example, WDL represses macrophage activation and alleviates inflammation and oxidative stress to improve acute lung injury [8]. Harkin K et al. have found that WDL mitigates retinal neurodegeneration by inhibiting the activation of Absent in melanoma 2 inflammasome [9]. In colitis, oral administration of WDL can alleviate pathological colonic damage and inflammatory infiltration [10]. In breast cancer, WDL inhibits the growth of solid tumors in vivo and represses the metastasis of breast cancer cells [11]. With the suppression of the aryl hydrocarbon receptor pathway, WDL represses the growth and migration of head and neck squamous cancer cells [12]. Our previous study reported for the first time that WDL exerted anti-tumor effects on retinoblastoma by inducing apoptosis and pyroptosis, with the underlying regulatory mechanism remaining unknown.

The nuclear factor-erythroid 2-related factor 2 (Nrf2)/kelch-like ECH-associated protein 1 (Keap1) signaling pathway is a kind of defense system for protecting cellular homeostasis [13, 14]. Nrf2 is a transcription factor, whose activity is suppressed by Keap1 [13]. The Nrf2/Keap1 signaling pathway exerts regulatory effects on the progression of different diseases, including gastric mucosal injury [15], Parkinson's disease [16], diabetes [17], and cancers [18, 19]. WDL plays a role in regulating the Nrf2/Keap1 signaling pathway. For example, WDL dose-dependently inhibits Nrf2 to repress inflammation and oxidative stress in liver injury [20]. Additionally, WDL plays a protective role in human bronchial epithelial cell injury induced by cigarette smoke extract via the Nrf2/Keap1 pathway [21]. It is unclear whether WDL exerts its anti-retinoblastoma effect by regulating the Nrf2/Keap1 pathway.

Here, to unravel the underlying mechanism of WDL in retinoblastoma, we investigated the relationship between the anti-tumor activity of WDL and the Nrf2/Keap1 signaling pathway in vitro and in vivo and found that WDL may promote pyroptosis by inhibiting the Nrf2/Keap1 signaling pathway to suppress retinoblastoma, hoping to facilitate the clinical application of WDL in retinoblastoma treatment.

Materials and methods

Cell culture and treatment

The retinoblastoma cell lines (Y79 and Weri-Rb1), purchased from iCell Bioscience Inc (Shanghai, China), were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco), 10 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco). A stock solution of WDL (50 µM) was prepared in dimethyl sulfoxide (DMSO), which was further diluted to 10, 25, and 50 µM for use in the experiments. The final concentration of DMSO in all treatment groups, including the control group, was maintained at ≤ 0.1% (v/v). RTA-408 (20 nM) was used as an agonist for Nrf2, and monosodium urate (MSU) crystal (100 µg/mL) was applied to stimulate the NLR family pyrin domain containing 3 (NLRP3), a key regulator protein of pyroptosis [22, 23]. Therefore, the cells were divided into the following 6 groups: Control (cells without any treatment), 10 µM WDL, 25 µM WDL, 50 µM WDL, WDL + RTA-408 (cells treated with 50 µM WDL and 20 nM RTA-408), and WDL + RTA-408 + MSU (cells treated with 50 µM WDL, 20 nM RTA-408, and 100 µg/mL MSU crystal) groups.

Cell viability determination

Y79 and Weri-Rb1 cells were inoculated into 96-well plates (5 × 10³ cells/well). A total of 100 µL cell medium containing 10 µL cell counting kit-8 reagents were supplemented to each well after 0, 24, 48, and 72 h of culture. After an additional 2 h of incubation, the optical density of each well was recorded at 450 nm wavelength.

The 5-ethynyl-2-deoxyuridine (Edu) staining analysis

Y79 and Weri-Rb1 cells from different groups were inoculated into 24-well plates (3 × 10⁴ cells/well) followed by 24 h of incubation. After different treatments, cells were collected on glass slides by centrifuging for 5 min at 1000 rpm using a cytocentrifuge. The cell slides were incubated with diluted Edu working solution (Beyotime, Shanghai, China) for 3 h, followed by fixing with 4% paraformaldehyde (Beyotime) for 10 min and permeabilizing with 0.3% Triton X-100 (Beyotime) for 20 min. Then the nuclear DNA was stained with 4, 6-diamino-2-phenylindole (DAPI; Beyotime) for 10 min. Finally, fluorescence microscopy was applied to record images.

Apoptosis assay

Y79 and Weri-Rb1cells were inoculated into a 6-well plate and subjected to different treatments for 24 h, followed by exposure to fluorescein isothiocyanate-conjugated anti-annexin V antibody (Annexin V; 5 µL; Beyotime) and propidium iodide (PI; 5 µL; Beyotime) for 15 min. We used a flow cytometer to measure the fluorescence intensity, which was quantified in FlowJo software (Becton Dickinson, Ashland, OR, USA). Annexin V⁺/PI⁺ cells may include late-stage apoptotic cells and pyroptotic cells undergoing secondary necrosis.

Immunofluorescence staining

NLRP3 inflammasome activation was confirmed by apoptosis-associated speck-like protein containing a CARD (ASC) immunofluorescence staining. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100, and blocked with 1% bovine serum albumin. Samples were incubated overnight at 4 °C with an anti-ASC antibody (ab283684; 1:100; Abcam, Cambridge, USA), followed by incubation with an appropriate fluorescent secondary antibody. Nuclei were counterstained with DAPI, and images were captured using a confocal microscopy system.

Western blotting

Total proteins were extracted from retinoblastoma cells (Y79 and Weri-Rb1) using RIPA lysis buffer (Beyotime) supplemented with 1% protease inhibitor cocktail (Beyotime). Lysates were centrifuged at 12,000 × g for 15 min at 4 °C, and supernatants were collected. Total protein concentrations were determined using a Bicinchoninic Acid Assay Kit (Beyotime), following the manufacturer’s instructions. Equal masses of total protein (20–30 μg per lane) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Danvers, MA, USA). The membrane with proteins was blocked with 5% skim milk and then incubated with primary antibodies, including anti-Keap1 (ab227828; 1:1,000; Abcam), anti-Nrf2 (ab62352; 1:1,000; Abcam), anti-cleaved-caspase1 (ab179515; 1:1,000; Abcam), anti-NLRP3 (ab263899; 1:1,000; Abcam), anti-cleaved-gasdermin D (cleaved-GSDMD; ab255603; 1:1,000; Abcam) and anti-β-actin (ab8227; 1:1,000; Abcam) antibodies at 4 ℃ overnight. After washing, the membrane was incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (ab288151; 1:5,000; Abcam) at room temperature. Finally, the protein exposure was conducted with a Tanon 5200 chemiluminescence imaging system (Tanon, Shanghai, China).

Measurements for lactate dehydrogenase (LDH), interleukin-1β (IL-1β) and caspase-1 activity

Determination of LDH and IL-1β levels in supernatants was conducted using corresponding enzyme-linked immunosorbent assay kits (Esebio, Shanghai, China), and the caspase-1 activity was measured by a caspase-1 Activity Assay Kit (Beyotime). All procedures were conducted in strict accordance with the instructions in the manual.

Xenograft experiments

Male BALB/c nude mice (4–6-week-old, 18–20 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China) and were randomly divided into 3 groups: Control, WDL, and WDL + RTA-408 groups (n ≥ 6). A total of 1 × 10⁷ Y79 cells (in 0.2 mL phosphate buffer saline) were subcutaneously injected into the left flank of each mouse [24]. WDL (30 mg/kg) were dissolved in 10% (w/v) hydroxypropyl β-cyclodextrin (Sigma-Aldrich, Darmstadt, Germany) aqueous solution via vortex mixing (5 min) and brief sonication (2 min, 25 °C) [8]. After Y79 cells injection for 5 days, mice in the WDL group received daily oral treatments with WDL (30 mg/kg). The mice in the WDL + RTA-408 group were administered RTA-408 at a dose of 1 mg/kg/day twice weekly by intraperitoneal injection at the same time as daily oral treatments with WDL [25]. For the Control group, an equal volume of saline was subjected to each mouse. We measured the length and width of the tumor every 5 days and calculated the volume using the following formula: length × (width)²/2. After 30 days of treatment, all mice were anesthetized by inhalation of 5% isoflurane and then sacrificed by cervical dislocation. Tumor tissues were collected for subsequent experiments. All animal experimental procedures were performed under the local ethical approval of the Animal Ethics Committee (Approval No. SYXK20230320).

Hematoxylin and Eosin (HE) staining and Ki67 staining

HE staining was performed to evaluate the toxicity of WDL in vivo. The tumor tissue samples embedded in paraffin were cut into 4 μm and were stained with hematoxylin for the nuclei and eosin for the cytoplasm. Additionally, we conducted Ki67 staining to determine the proliferation level. The sections were incubated with the rabbit anti-Ki67 antibody (ab16667; 1:100; Abcam) at 37 ℃ overnight. On the second day, anti-rabbit IgG (ab150077; 1:200; Abcam) was added after washing. Then, the sections reacted with 3.3-diaminobenzidine. All the results were observed by a microscope (Olympus, Tokyo, Japan).

Statistical analysis

Data were presented as multiple groups of repeated data or means ± standard deviation and were processed by GraphPad Prism 7.0 statistical software (GraphPad, San Diego, CA, USA). The comparisons between 2 groups were determined by student’s t-test, and those among multiple groups were determined by one-way ANOVA with Tukey's post hoc analysis. A p-value less than 0.05 was considered statistically significant.

Results

WDL inhibits the Nrf2/Keap1 signaling pathway

Prior to pharmacological assessments, we verified the biocompatibility of the solvent (0.1% DMSO) used for WDL delivery. As demonstrated in Supplementary Fig. 1, exposure of Y79 and Weri-Rb1 cells to 0.1% DMSO for 24, 48, and 72 h showed no significant difference in cell viability compared to the untreated control group (all p > 0.05), confirming the absence of cytotoxic effects from the solvent at the employed concentration. The results of western blotting displayed that compared with the Control group, the protein expression levels of Keap1 in Y79 cells of 10, 25, 50, 75, and 100 μM WDL groups were significantly increased (p < 0.05 or p < 0.01, Fig. 1A) and the levels of Nrf2 were significantly decreased (p < 0.01, Fig. 1A). Similar results were observed in Weri-Rb1 cells (p < 0.05 or p < 0.01, Fig. 1B), suggesting WDL inhibited the Nrf2/Keap1 signaling pathway in retinoblastoma cells.

Fig. 1.

Wedelolactone (WDL) inhibited the nuclear factor-erythroid 2-related factor 2 (Nrf2)/kelch-like ECH-associated protein 1 (Keap1) signaling pathway. A The protein expression levels of Keap1 and Nrf2 in Y79 cells treated with 0, 10, 25, 50, 75, and 100 μM WDL; B The protein expression levels of Keap1 and Nrf2 in Weri-Rb1 cells treated with 0, 10, 25, 50, 75, and 100 μM WDL. All data were presented as the mean ± standard deviation (SD), n ≥ 3. ^**p < 0.01, ^*p < 0.05 vs. Control group

WDL suppresses retinoblastoma cell growth through the Nrf2/Keap1 signaling pathway

RTA-408 was applied as an agonist for Nrf2. The results of western blotting showed that compared with the WDL group, the expression level of Nrf2 was significantly increased in Y79 and Weri-Rb1cells of the WDL + RTA-408 group (all p < 0.01, Fig. 2A, B), suggesting the Nrf2/Keap1 signaling pathway was activated. Compared with the Control group, the cell viability was reduced and the apoptosis rate was elevated in Y79 and Weri-Rb1cells of the WDL group (all p < 0.01, Fig. 2C–F). On the contrary, a higher level of cell viability and a lower apoptosis rate were found in the WDL + RTA-408 group compared to the WDL group (all p < 0.01, Fig. 2C–F), suggesting WDL repressed cell proliferation and induced apoptosis of retinoblastoma cells by inhibiting the Nrf2/Keap1 signaling pathway.

Fig. 2.

RTA-408 reversed the effect of WDL on retinoblastoma cells. The protein expression levels of Nrf2 in (A) Y79 and (B) Weri-Rb1 cells from different groups; The cell viability of (C) Y79 and (D) Weri-Rb1 cells from different groups; The apoptosis rate of (E) Y79 and (F) Weri-Rb1 cells from different groups. The concentration of WDL was 50 µM and RTA-408 was 20 nM. All data were presented as the mean ± SD, n ≥ 3. ^**p < 0.01 vs. Control group; ^##p < 0.01 vs. WDL group

WDL exhibits the anti-retinoblastoma effect in vivo through the Nrf2/Keap1 signaling pathway

RTA-408 was injected intraperitoneally into the xenograft nude mouse models to stimulate Nrf2. WDL displayed a good anti-retinoblastoma effect in vivo due to the reduction of tumor volume and weight (all p < 0.01, Fig. 3A–C), aggravated tumor tissue structure damage (Fig. 3D), and reduced tumor proliferation level (p < 0.01, Fig. 3E). The determination of tumor volume and weight demonstrated that RTA-408 partially reversed the inhibitory effect of WDL on tumor growth in nude mice (all p < 0.01, Fig. 3A–C). The images of HE staining showed improved tumor tissue structure damage in the WDL + RTA-408 group compared to the WDL group (Fig. 3D). In addition, the number of Ki67-positive cells was significantly higher in the WDL + RTA-408 group than in the WDL group (p < 0.05, Fig. 3E), demonstrating that RTA-408 treatment promoted the proliferation of tumors reduced by WDL.

Fig. 3.

RTA-408 reversed the anti-tumor effect of WDL in vivo. A Tumor images in different groups; B Tumor volume growth curve; C Weight of tumors; D Representative images of Hematoxylin and Eosin staining of tumor tissue sections. (400 × , scale bars = 50 μm). E Representative images of Ki67 staining of tumor tissue sections. (400 × , scale bars = 50 μm) and the quantitative results. Data were presented as repeated data or mean ± SD, n ≥ 6. ^**p < 0.01 vs. Control group; ^##p < 0.01, ^#p < 0.05 vs. WDL group

WDL activates pyroptosis through the Nrf2/Keap1 signaling pathway to exert anti-retinoblastoma effects

Consistent with our previous findings, WDL activated pyroptosis levels in retinoblastoma cells by elevating the levels of pyroptosis-related indicators and inducing ASC speck formation, a hallmark of NLRP3 inflammasome assembly (all p < 0.01, Fig. 4A–G). Compared with the WDL group, lower protein expression of cleaved-caspase1, NLRP3, and cleaved-GSDMD, attenuated ASC speck formation, and decreased levels of IL-1β, LDH release, and caspase-1 activity were found in the WDL + RTA-408 group (all p < 0.01, Fig. 4A–G), suggesting WDL promoted pyroptosis in retinoblastoma cells via the Nrf2/Keap1 signaling pathway.

Fig. 4.

The detection of pyroptosis-related indicators. The protein expression levels of cleaved-caspase1, NLR family pyrin domain containing 3 (NLRP3), and cleaved-gasdermin D (GSDMD) in (A) Y79 cells and (B) Weri-Rb1 cells. The Immunofluorescence staining of apoptosis-associated speck-like protein containing a CARD (ASC) in (C) Y79 cells and (D) Weri-Rb1 cells. The levels of (E) interleukin-1β (IL-1β), (F) lactate dehydrogenase (LDH) release, and (G) caspase-1 activity in Y79 and Weri-Rb1 cells. The concentration of WDL was 50 µM and RTA-408 was 20 nM. All data were presented as the mean ± SD, n ≥ 3. ^**p < 0.01 vs. Control group; ^##p < 0.01 vs. WDL group, ^&&p < 0.01, ^&p < 0.05 vs. WDL + RTA-408 group

MSU was administered as an agonist for NLRP3 to activate pyroptosis. The protein expression of cleaved-caspase1, NLRP3, and GSDMD, the intensity of fluorescence of ASC speck, and the levels of IL-1β, LDH release, and caspase-1 activity were significantly higher in the WDL + RTA-408 + MSU group than those in the WDL + RTA-408 group (all p < 0.01, Fig. 4A–G). Compared with the WDL + RTA-408 group, the cell viability and proliferation were repressed and the apoptosis rate was elevated in the WDL + RTA-408 + MSU group (all p < 0.01, Fig. 5A–C), showing that the promotion of pyroptosis partially restored the anti-tumor effects of WDL inhibited by RTA-408, underscoring pyroptosis as the principal anti-tumor mechanism. Therefore, we suggested that WDL may activate pyroptosis through the Nrf2/Keap1 signaling pathway to exert anti-retinoblastoma effects. Notably, pyroptosis activation also enhanced apoptosis, suggesting functional interplay between these pathways.

Fig. 5.

WDL triggered pyroptosis through the Nrf2/Keap1 signaling pathway to exert anti-retinoblastoma effects. A The cell viability of Y79 and Weri-Rb1 cells from different groups; B The proliferation levels of Y79 and Weri-Rb1 cells from different groups; C The apoptosis rates of Y79 and Weri-Rb1 cells from different groups. The concentration of WDL was 50 µM, RTA-408 was 20 nM, and monosodium urate (MSU) crystal was 100 µg/mL. All data were presented as the mean ± SD, n ≥ 3. ^**p < 0.01 vs. Control group; ^##p < 0.01 vs. WDL group, ^&&p < 0.01, ^&p < 0.05 vs. WDL + RTA-408 group

Discussion

Although the cure rate of retinoblastoma is relatively high, patients who survive will possibly lose their vision and suffer from cosmetic deformity, imposing a serious impairment on their quality of life. Hence, more efficient and safer therapeutic strategies are still urgently needed. Our previous research has reported the inhibitory effect of WDL on retinoblastoma due to the promotion of apoptosis and pyroptosis. In this study, we supplemented the underlying regulatory mechanism that WDL exerted the anti-retinoblastoma effect by triggering pyroptosis via suppression of the Nrf2/Keap1 signaling pathway.

Natural products are increasingly used in the treatment of retinoblastoma because they possess high anti-cancer activities and have fewer adverse effects. Curumin has been reported to repress retinoblastoma cell growth and migration [26]. With the up-regulation of miR-145, genistein inhibits cell proliferation and induces apoptosis of retinoblastoma cells [27]. Yin et.al have found that Gentiopicroside exhibits a good anti-retinoblastoma effect in vivo and in vitro due to the repression of cell growth and the induction of apoptosis [28]. Quercetin has been observed to cause the arrest of the G1 phase and induce apoptosis of Y79 cells [29] Accumulating studies have reported the role of WDL in oncotherapy in different kinds of tumors, including breast cancer [30], bladder cancer [31], melanoma [32], etc. We found that WDL could reduce retinoblastoma cell proliferation, induce retinoblastoma cell apoptosis, and repress the growth of solid tumors in vivo, revealing the anti-retinoblastoma effect of WDL.

The Nrf2/Keap1 signaling pathway is modulated by many natural products, involved in the process of tumorigenesis, tumor progression, and resistance to therapy. Cepharanthine induces oxidative stress with the activation of the Nrf2/Keap1 signaling pathway, exerting its anti-gastric cancer effects [33]. DihydrotanshinoneI promotes Keap1-mediated Nrf2 suppression and suppresses protein kinase C-induced Nrf2 phosphorylation to exert its inhibitory effect on gallbladder cancer growth [34]. Liao et.al have found that stigmasterol reverses the resistance of endometrial cancer cells to cisplatin by suppressing the Nrf2 level [35]. Neferine triggers apoptosis and ferroptosis of thyroid cancer cells via the inhibition of the Nrf2 pathway [36]. Eriodictyol downregulates Nrf2 phosphorylation to inhibit ovarian cancer progression and aggravate mitochondrial dysfunction [37]. In this study, we found that WDL up-regulated Keap1 and down-regulated Nrf2 in retinoblastoma cells, and RTA-408, an agonist for Nrf2, impaired the anti-tumor effect of WDL on retinoblastoma, suggesting WDL exerted anti-retinoblastoma effect via inhibiting the Nrf2/Keap1 signaling pathway.

Pyroptosis is a promising target for oncotherapy, and an increasing number of natural products exhibit the inhibitory effect on tumors via targeting pyroptosis. Neobavaisoflavone activates ROS to trigger pyroptosis in hepatocellular carcinoma, inhibiting tumor growth [38]. Alantolactone activates GSDMD-dependent pyroptosis to suppress the growth of anaplastic thyroid cancer cells and solid tumors of the subcutaneous xenograft model [39]. Ophiopogonin B triggers caspase-1/GSDMD-dependent pyroptosis to reverse the resistance of lung cancer cells to cisplatin [40]. A natural flavonoid luteolin exerts an inhibitory effect on colon cancer growth in vitro and in vivo by promoting caspase-1/GSDMD-dependent pyroptosis [41]. Accumulating studies have demonstrated that the Nrf2/Keap1 signaling pathway can modulate pyroptosis. Ba et.al have reported that triptolide decreases the levels of Keap1 and elevates the expression of Nrf2 to inhibit pyroptosis, alleviating cardiac remodeling [42]. Lycopene improves kidney injury due to the inhibition of pyroptosis through the modulation of the Nrf2/Keap1/NLRP3/Caspase-1 axis [43]. Yuan et.al have found that chrysophanol reduces oxidative stress and pyroptosis to attenuate diabetic nephropathy by activating the Nrf2/Keap1 signaling pathway [44]. In this study, retinoblastoma pyroptosis induced by WDL was inhibited by RTA-408, and the agonist of pyroptosis MSU could restore the anti-tumor effect of WDL impaired by RTA-408, suggesting WDL exerted anti-retinoblastoma effects by triggering pyroptosis through the Nrf2/Keap1 signaling pathway. Furthermore, we noted that pyroptosis activation may promote apoptosis, indicating crosstalk between these two cell death modalities. However, apoptosis induction by WDL is not wholly dependent on pyroptosis. Our previous study showed that WDL directly upregulated apoptosis-specific markers (cleaved caspase-3 and PARP) independently of pyroptosis [24]. Thus, we propose that WDL exerts its anti-retinoblastoma effects primarily through Nrf2/Keap1 pathway-mediated pyroptosis, with apoptosis acting as a complementary but mechanistically distinct pathway. Notably, while RTA-408 substantially reversed WDL’s anti-tumor effects, it did not fully restore tumor growth to control levels in vivo, suggesting residual activity independent of Nrf2 inhibition. Given that WDL is a multi-target natural compound, it is plausible that parallel mechanisms contribute to its efficacy. For instance, our in vitro data demonstrated that MSU-induced pyroptosis activation partially rescued the anti-tumor effects of WDL even under RTA-408-mediated Nrf2 activation. This implies that WDL may concomitantly engage Nrf2-independent pathways to induce pyroptosis. Such redundancy aligns with the pleiotropic actions of many phytochemicals and could explain the sustained efficacy observed in vivo. Future studies identifying these auxiliary targets will further elucidate WDL’s therapeutic potential.

Despite these findings, our study has some limitations. First, we identified that WDL could suppress the Nrf2/Keap1 pathway to trigger pyroptosis, but the precise molecular mechanism by which WDL enhances Keap1 protein levels remains incompletely resolved. Specifically, whether WDL directly binds to functional domains of Keap1, such as BTB or IVR, or regulates Keap1 through post-translational modifications was not investigated due to technical and resource constraints. Future studies employing structural biology approaches and domain-specific mutagenesis are warranted to delineate the exact binding interface and functional consequences of WDL-Keap1 interaction. Second, our study did not assess whether prolonged WDL exposure triggers compensatory Nrf2 activation—a phenomenon that could undermine long-term efficacy in clinical settings. Future work should employ extended-duration animal models to monitor Nrf2 dynamics and tumor adaptation during sustained treatment. Third, although this study established that Nrf2/Keap1 inhibition was essential for WDL-induced pyroptosis in retinoblastoma, the exact upstream mechanisms linking Nrf2 suppression to NLRP3 inflammasome activation remain unresolved. We speculate that accumulated mitochondrial ROS (due to compromised antioxidant capacity) and/or Keap1-mediated ubiquitination of NLRP3 regulators may be involved, which requires direct experimental verification in future work. Fourth, this study did not assess Nrf2/Keap1 pathway mutations in patient-derived retinoblastoma cells. Such mutations could potentially influence WDL sensitivity and merit investigation in future clinical samples. Primary tumor samples from retinoblastoma patients should be collected in the future for targeted sequencing of Nrf2 and Keap1 to identify mutations and to compare WDL responses between wild-type and mutant primary cells. Fifth, while Annexin V/PI staining indicated cell death, this assay cannot definitively distinguish between late-stage apoptosis and pyroptosis-driven secondary necrosis. Future studies employing discriminative techniques, including caspase-1/3 activity multiplex assays, live-cell imaging of gasdermin pore formation, and ultrastructural analysis, are warranted to precisely delineate these mechanisms. Sixth, due to the poor aqueous solubility of WDL, its intraocular bioavailability was not directly quantified. Efficacy was inferred from tumor response in xenograft models. Future studies will employ liquid chromatography-tandem mass spectrometry to measure WDL concentrations in plasma and retinal tissues, validating its pharmacokinetic profile and optimizing delivery strategies for clinical translation.

In conclusion, WDL triggered pyroptosis of retinoblastoma cells via suppressing the Nrf2/Keap1 signaling pathway, exerting the inhibitory effect on retinoblastoma. We unraveled the promising regulatory mechanism of WDL exerting the anti-retinoblastoma effect. Based on our findings, WDL, as a natural phytochemical, holds significant potential for clinical translation. Compared to conventional chemotherapy, WDL may offer a superior toxicity profile. Our preclinical data demonstrated that oral administration of WDL (30 mg/kg) in tumor-bearing mice did not induce obvious systemic toxicity, providing preliminary safety evidence for clinical dosing optimization. Given that current retinoblastoma therapies, such as enucleation and radiotherapy, carry risks of vision loss or disfigurement, WDL could serve as a vision-preserving adjuvant treatment, particularly for early-stage or locally advanced patients. Furthermore, its mechanism of action suggests potential synergies with targeted agents, such as NLRP3 inhibitors, offering additional options for personalized therapeutic strategies. We hope this study facilitates the clinical application of WDL on retinoblastoma and provide more insights into the development of novel therapeutic agents for retinoblastoma.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, YZJ and HJ; Methodology, YZJ and HJ; Investigation, GTW, NDP, CQN and ZPW; Formal Analysis, GTW, NDP, CQN and HBL; Writing—Original Draft, YZJ and HJ; Writing—Review & Editing, GTW, NDP, CQN and ZPW. All authors took part in the experiment. All authors read and approved the final manuscript.

Funding

This work was supported by the Guangzhou Science and Technology Program Project (Grant No. 202201020606) and Guangzhou Women and Children's Medical Center Doctoral Initiation Fund (2023BS026).

Availability of data and materials

The data and materials supporting the findings of this study are available from the corresponding authors upon request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The animal experiments were approved by the Ethics Committee of Laboratory Animal Center, Baiyun Branch of Guangzhou Women and Children's Medical Center (Approval No. SYXK20230320) in accordance with the ARRIVE guidelines.

Research involving Human and animal participants

The animal experiments were approved by the Ethics Committee of Laboratory Animal Center, Baiyun Branch of Guangzhou Women and Children's Medical Center (Approval No. SYXK20230320) in accordance with the ARRIVE guidelines. Animals with obvious anxiety, restlessness, or tumor weight exceeding 10% of the animal’s own body weight should be euthanized if the maximum diameter of the subcutaneous tumor in mice cannot exceed 20 mm and that in rats cannot exceed 40 mm. This experiment did not exceed the prescribed tumor size, which met the requirements of the Ethics Committee.

Consent for publication

Not applicable.