The ATC cell lines CAL62, C643, and BHT101 were purchased from the ATCC and preserved in our laboratory. Cells were maintained at 37 °C and 5% CO₂ in RPMI-1640 medium (Thermo Fisher Scientific, MA, USA) containing 10% fetal bovine serum (ALLBIO, Zhejiang, China). Erianin was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China), dissolved in DMSO (Beyotime, Beijing, China) and stored at −80 °C. Anlotinib was purchased from Chia Tai Tianqing Pharmaceutical Group Co., Ltd. (Jiangsu, China).

The stock solution of erianin (500 μM) was diluted in complete medium to obtain working concentrations of 2.5 nM, 5 nM, 10 nM, 25 nM, and 50 nM. BHT101, C643, and CAL62 cells were plated at a density of 500 cells per well in 96-well plates. After 24 h, different concentrations of drug-containing medium and DMSO-containing control medium were added to the respective groups, with no fewer than three replicates per concentration gradient. From Day 1 to Day 5 after drug addition, CCK-8 reagent (Beyotime, China) was added, incubated in the incubator for 1 h, and the OD values were measured at 450 nm using a microplate reader.

Cells in the logarithmic growth phase were plated at 1000 cells per well in six-well plates, followed by treatment with erianin at concentrations of 0, 25 nM, and 50 nM after 24 h. After cross-pattern mixing, the plates were incubated at 37 °C in a 5% CO₂ humidified incubator for 7 days. Colonies were fixed, stained, photographed, and counted, ensuring at least 50 cells per colony.

Drugs were prepared as serial dilution gradients for subsequent use. Cells in the logarithmic growth phase (BHT101, C643, and CAL62) were plated at 3000 cells per well in 96-well plates, followed by the addition of media containing different drug concentrations after 24 h, with triplicates for each concentration. CCK-8 reagent (Beyotime, China) was added at 48 h, 72 h, and 96 h post-treatment, incubated for 1 h, and OD values were recorded at 450 nm using a microplate reader.

Cells were treated with various concentrations of erianin for 48 h, washed with PBS, and fixed in 70% ethanol. A staining solution containing PI and RNase was added, and cells were incubated at room temperature in the dark for 30 min before analysis by flow cytometry. The Annexin V-FITC/PI dual-staining assay was used to evaluate cell death. Treated cells were centrifuged, washed with PBS, stained with Annexin V-FITC and PI, incubated for 15 min in the dark, and analyzed using a flow cytometer. Data analysis was performed with FlowJo v10.6.2 software.

BHT101, C643, and CAL62 cells were seeded in 24-well plates and treated with different drug concentrations after attachment. EdU working solution was added, and the cells were incubated at 37 °C for 2 h. After fixation with 4% paraformaldehyde for 15 min and three PBS washes, the cells were permeabilized with 0.3% Triton X-100. Staining was performed according to the instructions of the EdU detection kit (Beyotime, China). Fluorescence was observed and imaged under a fluorescence microscope, and fluorescence intensity was quantified using ImageJ software.

LDH cytotoxicity was assessed using an LDH assay kit (Beyotime, China). Cells (1000 cells/well) were seeded in 96-well plates, treated with drugs at various concentrations for 48 h (3 replicates per condition), and the supernatant was removed. LDH release reagent was added, incubated at 37 °C for 1 h, centrifuged, and transferred to new plates. LDH detection solution was added, incubated for 30 min at room temperature in the dark, and absorbance was measured at 490 nm (630 nm as reference) using a microplate reader. Cytotoxicity was calculated as: (% cell death) = (treated absorbance - control absorbance)/(maximum absorbance - control absorbance) × 100.

Proteins were extracted from cells and quantified using the BCA assay. Equal protein amounts were denatured at 95 °C for 5 min, separated by SDS-PAGE, and transferred onto PVDF membranes. Membranes were blocked with 5% skim milk, incubated with appropriately diluted primary antibodies at 4 °C overnight, and washed. HRP-conjugated secondary antibodies were applied for 1 h at room temperature. Protein bands were visualized using ECL reagents and documented with a gel imaging system. Detailed information on the antibodies utilized is provided in Supplementary Table S1.

Cells treated with erianin were fixed in 2.5% glutaraldehyde for 2 h at 4 °C, washed with PBS (3 × 10 min), and further fixed in 1% osmium tetroxide for 1–2 h. After washing, cells were dehydrated through an ethanol gradient (30%–100%) and replaced twice with 100% acetone. Samples were embedded in epoxy resin, ultrathin-sectioned to 60–80 nm, and collected on copper grids. Sections were stained with uranyl acetate and lead citrate (15 min each) and observed under a transmission electron microscope.

Microarray data ATC samples were obtained from the public GEO database, specifically from the GPL570 platform datasets GSE29265, GSE33640, GSE65144, and GSE76039. Raw data were normalized using the rma algorithm, and batch effects across datasets were corrected with the limma package. The integrated dataset included 78 normal thyroid tissues (NT), 69 papillary thyroid carcinoma (PTC), 17 poorly differentiated thyroid carcinoma (PDTC), and 52 anaplastic thyroid carcinoma (ATC) samples. Heatmaps were generated using the pheatmap package, and boxplots visualizing gene expression differences were created with the ggplot2 package.

Cells were seeded on coverslips, treated with erianin, and fixed with 4% paraformaldehyde. N-GSDME primary antibody and fluorescence-conjugated secondary antibody were applied, followed by DAPI staining for nuclei. Membrane localization of N-GSDME was observed under a fluorescence microscope (Nikon). Detailed information on antibodies is provided in Supplementary Table S1.

Total RNA was extracted from CAL62 cells using the Trizol method for lysis and purification. RNA libraries were constructed and sequenced on the BGISEQ-500 high-throughput sequencing platform. Differentially expressed genes (DEGs) were identified using the DESeq2 package. Enrichment analysis was conducted with the clusterProfiler package, and results were visualized with the cnetplot and ggplot2 packages.

Cells were seeded at 3000 cells per well in 96-well plates. Based on the individual drug IC₅₀ values, erianin and anlotinib were combined in fixed IC₅₀ ratio gradients (0, 0.25, 0.5, 1, 2, and 4× IC₅₀). Three replicates were set for each combination, along with single-drug treatment and drug-free control groups. After 72 h of treatment, CCK-8 solution was added, and cells were incubated at 37 °C in the dark for 1 h. Absorbance at 450 nm was measured. Combination index (CI) values were calculated using CompuSyn or CalcuSyn software, where CI < 0.9 indicates synergism, 0.9 ≤ CI ≤ 1.1 indicates an additive effect, and CI > 1.1 indicates antagonism (Chou, 2006). Visualization was performed using the ggplot2 package.

4–6 weeks old BALB/c nude mice were randomly divided into control, erianin, anlotinib, and combination treatment groups. Well-cultured CAL62 anaplastic thyroid cancer cells were mixed with Matrigel (Thermo Fisher Scientific, MA, USA) in a 1:1 volume ratio and subcutaneously injected into the right inguinal area of each mouse (1 × 10⁶ cells/mouse). After 7 days, mice with normally growing solid tumors were allocated to groups and treated as follows: control group received saline; erianin group received 3.5 mg/kg erianin via tail vein injection; anlotinib group received 2 mg/kg anlotinib via intraperitoneal injection; combination group received 2.6 mg/kg erianin via tail vein and 1.5 mg/kg anlotinib via intraperitoneal injection, all administered every other day. After 14 days of treatment, mice were sacrificed, tumor sizes were measured and weighed, and tumor tissues were fixed in formalin for paraffin embedding and subsequent experiments.

Paraffin sections were baked at 65 °C for 2 h, deparaffinized with xylene, rehydrated, and stained with hematoxylin and eosin (HE). After dehydration and clearing, sections were mounted with neutral resin and imaged under a microscope.

Paraffin sections were deparaffinized in xylene, rehydrated through graded ethanol, and treated with 20 μg/mL proteinase K for 15–30 min. After PBS washing, TUNEL detection solution (5 μL TdT enzyme +45 μL fluorescein label) was added, and sections were incubated at 37 °C in the dark for 60 min. After PBS washing, slides were mounted with antifade solution and observed under a fluorescence microscope (excitation: 450–500 nm, emission: 515–565 nm).

Paraffin sections were deparaffinized, rehydrated through graded ethanol, and subjected to antigen retrieval with citrate buffer under high pressure. After PBS washing, endogenous peroxidase activity was blocked with 3% H₂O₂ at room temperature, followed by 5% BSA blocking for 30 min. Primary antibodies were applied and incubated overnight at 4 °C. After PBS washing, HRP-conjugated secondary antibodies were added and incubated at 37 °C for 30 min. DAB was used for chromogenic detection, and nuclei were counterstained with hematoxylin. Sections were dehydrated through graded ethanol, cleared with xylene, mounted with neutral resin, and imaged under a microscope. The antibodies are provided in Supplementary Table S1.

Statistical analyses were performed using GraphPad Prism 9.0. Group comparisons were conducted with one-way analysis of variance (ANOVA) and t-tests, with statistical significance set at P < 0.05. Significance levels were denoted as *P < 0.05, **P < 0.01, and ***P < 0.001.

To investigate the suppressive effects of erianin on anaplastic thyroid cancer, we performed in vitro assays using ATC cell lines. The CCK-8 assay showed that erianin inhibited the proliferation of CAL62, C643, and BHT101 cells in a concentration- and time-dependent manner (Figures 1A–D). Colony formation assays confirmed this effect, with erianin (25 nM and 50 nM) significantly reducing colony-forming ability in all 3 cell lines in a concentration-dependent manner (Figure 1E). Furthermore, the half-maximal inhibitory concentration (IC50) of erianin was measured for these ATC cell lines. The IC50 values for CAL62, C643, and BHT101 cells at 72 h and 96 h were 709.9 nM, 76.27 nM, 132.7 nM and 56.23 nM, 236.5 nM, 135.6 nM, respectively (Figures 1F–H).

We further investigated the effects of erianin on the cell cycle of ATC cells. Flow cytometry revealed significant G2/M phase arrest in CAL62, C643, and BHT101 cells, with increased G2/M phase and decreased G0/G1 and S phase (Figures 2A,B). The EdU assay showed reduced EdU-positive rates in erianin-treated cells, suggesting a strong suppressive effect on DNA synthesis (Figures 2C,D). In conclusion, these results suggest that erianin effectively suppresses the proliferation of anaplastic thyroid cancer cells at nanomolar (nM) levels and induces cell cycle inhibition, leading to G2/M phase arrest, indicating its potential as a therapeutic candidate for thyroid cancer.

To evaluate the cytotoxic effects of erianin on ATC cells, Annexin V-FITC/PI staining was used to analyze cell death. Flow cytometry results demonstrated that erianin significantly induced cell death in CAL62, C643, and BHT101 cells. Compared with the DMSO control, treatment with 50 nM and 100 nM erianin significantly increased the proportion of cells in the Q2+Q3 quadrants in a concentration-dependent manner, indicating membrane rupture events (Figures 3A,B). We further measured LDH release in ATC cells following erianin treatment. At 50 nM and 100 nM, erianin markedly enhanced LDH release in CAL62, C643, and BHT101 cells, further corroborating its cytotoxicity (Figure 3C). Interestingly, forward scatter (FSC-A) and side scatter (SSC-A) analysis by flow cytometry indicated increased granularity and cell size in erianin-treated ATC cells (Figure 3D). This observation, inconsistent with the typical apoptotic characteristic of cell shrinkage, indicates the involvement of non-apoptotic mechanisms in erianin-induced cell death.

To investigate how erianin induces cell death in ATC cells, we examined the key markers of various cell death pathways. Western blot revealed that erianin significantly increased cleaved caspase-3 and N-GSDME levels (markers of apoptosis and GSDME-dependent pyroptosis), without affecting N-GSDMD (GSDMD-dependent pyroptosis), p-MLKL (necroptosis), LC3A/B (autophagy), or GPX4(ferroptosis) levels (Figure 4A). This suggests erianin induces GSDME-dependent pyroptosis, which characterized by cell swelling and membrane rupture (Liu et al., 2024a). Electron microscopy confirmed pyroptotic morphology, including cell swelling, cytoplasmic vesicle formation, and membrane rupture (Figure 4B), consistent with the cell enlargement observed in flow cytometry. Additionally, by integrating data from ATC-containing samples in the NCBI database, we found that GSDME (DFNA5) was significantly upregulated in ATC samples compared to normal thyroid (NT) and other types of thyroid cancer, whereas GSDMD did not exhibit a consistent upregulation trend (Figures 4C,D). This finding suggests that targeting GSDME-dependent pyroptosis may have greater therapeutic relevance in ATC.

Differing from GSDMD-mediated pyroptosis, GSDME-dependent pyroptosis is predominantly activated via caspase-3 cleavage (Bhat et al., 2023). Western blot further revealed that erianin treatment elevated N-GSDME and cleaved caspase-3 levels, alongside increased the caspase-3 substrate c-PARP expression. Concurrently, Bax was upregulated, while Bcl-2 was significantly downregulated (Figure 4E). These data indicate that erianin regulates the Bcl-2/Bax axis to cleave caspase-3, thereby facilitating GSDME-dependent pyroptosis. GSDME-dependent pyroptosis is initiated by the upregulation of N-GSDME and its membrane localization for pore formation (Liu et al., 2024a). Immunofluorescence analysis demonstrated a marked increase in N-GSDME fluorescence and its aggregation at the plasma membrane in erianin-treated ATC cells compared with controls (Figure 4F). Upon administration of the caspase-3 inhibitor Z-DEVD-FMK, we found that blocking caspase-3 activity partially alleviated erianin-induced cell death. At the protein level, Z-DEVD-FMK was also found to partially inhibit the expression of cleaved caspase-3 and the N-terminal fragment of GSDME (Supplementary Figure S1). Collectively, these results suggest that erianin exerts its cytotoxic effects on ATC cells by inducing apoptosis and GSDME-dependent pyroptosis through the activation of caspase-3.

To investigate how erianin inhibits ATC cells, RNA-seq was performed on CAL62 cells treated with 50 nM erianin for 48 h and controls. A total of 1364 DEGs were identified, with 836 upregulated and 528 downregulated (Figure 5A, Supplementary Material 1). KEGG enrichment analysis revealed that Erianin affects tumor-related pathways, particularly the MAPK and PI3K/AKT pathways (Figures 5B,C, Supplementary Material 2). Both the MAPK and PI3K/AKT pathways are classical oncogenic pathways that regulate caspase-3 activity. Therefore, we further validated erianin’s effects on these pathways at the protein level. Western blot analysis showed that erianin significantly reduced the phosphorylation levels of key proteins in the MAPK/ERK pathway (ERK1/2, MEK1/2) and the PI3K/AKT pathway (PI3K, AKT) in all three ATC cell lines. Additionally, the total protein expression levels of these pathway components also showed a downward trend (Figure 5D). These results suggest that Erianin inhibits MAPK/ERK and PI3K/AKT pathway activity, potentially contributing to its suppression of ATC cell malignancy.

Anlotinib is a multi-target receptor tyrosine kinase inhibitor that primarily exerts its antitumor effects by inhibiting pathways such as VEGFR in various cancers (Song et al., 2020). It also exhibits promising antitumor activity in anaplastic thyroid carcinoma (ATC), but its therapeutic efficacy remains limited (Song Y. et al., 2024). Resistance to anlotinib has been associated with aberrant activation of the PI3K/AKT signaling pathway (Chen et al., 2022a), motivating an investigation into whether erianin could potentiate its inhibitory effects.

Using CCK-8 assays, the 72-h IC50 of anlotinib was determined as 17.53 μM for CAL62, 11.18 μM for C643, and 8.21 μM for BHT101 cells (Figures 6A–C), while erianin’s IC50 values were 236.5 nM, 132.7 nM, and 727.3 nM, respectively (Figures 1F–H). To further evaluate the synergistic effects of the Erianin and anlotinib, we conducted Combination Index (CI) analysis (Chou, 2006) using fixed ratio gradients (0, 0.25, 0.5, 1, 2, 4× IC50) of the two drugs (Figure 6D). The results showed significant synergistic effects (CI < 0.9) between erianin and anlotinib at multiple concentrations, particularly at low concentrations (0.25× IC50), in all three ATC cell lines (Figure 6E). Western blot analysis demonstrated that the combination treatment markedly enhanced the suppression of p-PI3K compared to single-agent treatments (Figure 6H). Collectively, these findings indicate that erianin significantly potentiates the anti-tumor efficacy of anlotinib in ATC cells in vitro, achieving notable synergy even at low doses, thereby highlighting its potential clinical applicability.

To validate the in vitro findings, a subcutaneous CAL62 ATC xenograft model in nude mice was used to assess the in vivo effects of erianin. Tumor growth was significantly slower in both erianin and anlotinib monotherapy groups compared to controls, with the combination therapy showing the strongest inhibition (Figures 7A–D). HE and TUNEL staining revealed increased tumor cell death and apoptosis in both monotherapy groups, with the combination therapy group showing the most pronounced effects (Figures 7E,F). IHC confirmed the drugs' inhibitory effects on tumor proliferation and signaling pathways (Figure 7G). Both monotherapies reduced Ki67-positive cells and p-ERK1/2 and p-AKT levels, with the combination therapy causing further reductions. Collectively, these findings further validate the inhibitory effects of Erianin on ATC in vivo and reveal its significant synergistic anti-tumor effects with anlotinib.