The EC cell lines KYSE410 [poorly differentiated SCC, Microsatellite Stable (MSS) and TE1 (well-differentiated SCC, MSS)] were supplied by the Cell Resource Center of Biomedical Research, Institute of Development, Aging, and Cancer (Tohoku University). The identity of the cell line was confirmed by assessing the genetic and epigenetic markers. The cultures were periodically tested for mycoplasma contamination. Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc.) enriched with 10% fetal bovine serum (FBS; Biowest) and an antibiotic-antimycotic cocktail (Nacalai Tesque, Inc.). The cells were incubated at 37°C in a humidified incubator with 5% CO₂. Andrographis was kindly provided by Professor Ajay Goel (City of Hope Comprehensive Cancer Center). The compound was manufactured by EuroPharma USA. According to the manufacturer's certificate of analysis, the preparation is a purified extract of Andrographis paniculata and contains 80% Andrographolide as the principal active constituent. Chemical characterization, including the standardization of Andrographolide content and purity confirmation, was based on the manufacturer's certificate of analysis. No additional extraction or purification was performed outside EuroPharma USA for the present study. For all assays, the compound was dissolved in DMSO to prepare a stock solution, which was subsequently diluted with the culture medium to the indicated final concentrations.

For Water Soluble Tetrazolium (WST) assay, 4,000 cells/well were seeded in 96-well tissue culture plates (TPP Techno Plastic Products AG). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS and antibiotics and allowed to adhere overnight. Subsequently, ESCC cells were exposed to various concentrations of 5-FU and Andrographis for 72 h. The tested doses were 3, 3.5, 4, 4.5 and 5 µM for 5-FU, and 36, 42, 48, 54 and 60 µg/ml for Andrographis, respectively. This procedure was performed to determine the cytotoxic effects of these compounds. Cell proliferation was measured using WST 8 (Dojindo Laboratories, Inc.) according to the manufacturer's instructions. Based on the inhibitory concentration at 50% (IC₅₀) (24), a combination of 5-FU and Andrographis (3, 3.5, 4, 4.5 and 5 µM and 36, 42, 48, 54, and 60 µg/ml) was used to treat each cell line for 72 h. Absorbance values were measured for each well at a wavelength of 450 nm using SoftMax Pro microplate reader (Molecular Devices, LLC.). To maintain consistency across the experimental conditions, an identical final concentration of DMSO was applied to all treatment groups, including the control group. The interaction between 5-FU and Andrographis was quantitatively assessed by calculating the combination index (C.I.) at the IC₅₀ using the Chou-Talalay method (24). C.I. values were generated using GraphPad Prism ver. 10.0 (GraphPad Software Inc.; Dotmatics) and a C.I. <1.0 was regarded as suggestive of additive effects (24). Each experiment was independently repeated three times, with technical triplicates included in each of the biological replicates.

Colony formation assays were performed based on established protocols (25) with minor procedural adaptations. Specifically, KYSE410 and TE1 cells were seeded in 6-well tissue culture plates (TPP Techno Plastic Products AG) at a density of 500 cells/well, using an identical culture medium. The plates were incubated for 24 h. A total of 1 ml/well of 5-FU (3.5 µM), Andrographis (42 µg/ml), and a combination of 5-FU and Andrographis (3.5 µM, 42 µg/ml) were then added for 72 h. Following the initial incubation, the medium was replaced with fresh drug-free culture medium. The cells were then cultured at 37°C with 5% CO₂ in a humidified environment for 7–9 days. Colonies consisting of at least 50 cells were counted as a colony. Colony numbers were quantified using ImageJ software ver.1.54p (National Institutes of Health) (26). These counts were then compared between the control and drug treatment groups.

Apoptosis was assessed using propidium iodide/annexin V double staining and flow cytometry. Cells were inoculated into a 6-well tissue culture plate at a density of 1.6×10⁵ cells/well and pre-incubated for 24 h. Subsequently, the cultures were treated with one of the following treatments for 72 h: 5-FU (3.5 µM), Andrographis (42 µg/ml), or a combination of 5-FU and Andrographis (3.5 µM, 42 µg/ml) for 72 h. Apoptotic cells were measured using Muse^® Annexin V and Dead Cell Assay (Luminex Corporation) on a Muse™ Cell Analyzer (Merck KGaA), following the manufacturer's instructions. Data acquisition and analysis were performed using Muse Cell Analyzer software (version 1.4; Merck KGaA).

To quantify mRNA expression, cells were seeded in 6-well tissue culture plates at a density of 3.0×10⁵ cells/well. After a 24 h adherence period, the cultures were subjected to drug treatment. Specifically, the cells were exposed to 5-FU (3.5 µM), Andrographis (42 µg/ml), or a combination of 5-FU and Andrographis (3.5 µM, 42 µg/ml). Total RNA was extracted from the cells using an RNA extraction miRNeasy Mini kit (Qiagen GmbH). Complementary DNA (cDNA) was synthesized from 5.0 ng of total RNA using a Reverse Transcription kit (Toyobo Co., Ltd.) according to the manufacturer's instructions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using cDNA and the Power SYBR^® Green PCR Master Mix (Thermo Fisher Scientific Inc.). RT-qPCR analysis was conducted using a StepOne™ Real-Time PCR System (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 60 sec. The primer sequences were as follows: heme oxygenase 1 (HMOX1) forward, 5′-AAGACTGCGTTCCTGCTCAAC-3′ and reverse, 5′-AAAGCCCTACAGCAACTGTCG-3′; glutamate cysteine ligase catalytic (GCLC) forward, 5′-AGGCCAACATGCGAAAAC-3′ and reverse, 5′-CGGATATTTCTTGTTAAGGTACTGG-3′; glutamate cysteine ligase modifier (GCLM) forward, 5′-TTGGAGTTGCACAGCTGGAT-3′ and reverse, 5′-GGTTTTACCTGTGCCCACTGA-3′; glutathione peroxidase 4 (GPX4) forward, 5′-GAGGCAAGACCGAAGTAAACTAC-3′ and reverse, 5′-CCGAACTGGTTACACGGGAA-3′; solute carrier family 7 member 11 (SLC7A11) forward, 5′-TCTCCAAAGGAGGTTACCTGC-3′ and reverse, 5′-AGACTCCCCTCAGTAAAGTGAC-3′; nuclear factor kappa B subunit 1 (NFKB1) forward, 5′-AACAGAGAGGATTTCGTTTCCG-3′ and reverse, 5′-TTTGACCTGAGGGTAAGACTTCT-3′; signal transducer and activator of transcription 3 (STAT3) forward, 5′-ACCAGCAGTATAGCCGCTTC-3′ and reverse, 5′-GCCACAATCCGGGCAATCT-3′; NFE2 like bZIP transcription factor 2 (NFE2L2) forward, 5′-TCCAGTCAGAAACCAGTGGAT-3′ and reverse, 5′-GAATGTCTGCGCCAAAAGCTG-3′; acyl-CoA synthetase long chain family member 4 (ACSL4) forward, 5′-AACCCAGAAAACTTGGGCATT-3′ and reverse, 5′-GTCGGCCAGTAGAACCACT-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-GGAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-AATGAAGGGGTCATTGATGG-3′. Target gene expression levels were quantified using the 2^−ΔΔCq method (27). The resulting values were subsequently standardized to the housekeeping gene, GAPDH.

ESCC cells (7.5×10⁵ cells/well) were treated with 5-FU (3.5 µM), Andrographis (42 µg/ml), and a combination of 5-FU and Andrographis (3.5 µM, 42 µg/ml) for 72 h. The control group received RPMI-1640 medium enriched with 10% FBS and an antibiotic-antimycotic cocktail. Cells were lysed in Radioimmunoprecipitation Assay buffer (BioDynamics Laboratory, Inc.) supplemented with a proteinase inhibitor cocktail (Sigma Aldrich; Merck KGaA). Protein quantification was performed using a BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.). Subsequently, the samples were combined with the loading buffer and denatured by boiling for 5 min. Equal amounts of protein (20 µg per lane) were loaded. The samples were subjected to electrophoresis on 4–15% gradient Mini-PROTEAN^®TGX™ (Bio-Rad Laboratories, Inc.) for 50 min and transferred to PVDF membranes using Trans-Blot^®Turbo™ (Bio-Rad Laboratories, Inc.) with EB RAPID for 5 min. The membranes were initially incubated with 5% milk for 60 min at room temperature for blocking purposes. The membranes were then exposed to the indicated primary antibodies for 60 min at room temperature. mouse monoclonal anti HMOX1 (1:1,000; cat. no. sc-136960), mouse monoclonal anti-γ-GCLC (1:2,000; cat. no. sc-390811) and mouse monoclonal anti-γ-GCLM (1:5,000; cat. no. sc-55586; all from Santa Cruz Biotechnology, Inc.). The membranes were then washed three times with Tris buffer saline containing 0.05% Tween 20 at room temperature. Following washing, the membranes were probed with anti-mouse IgG (cat. no. W4028; Promega Corporation) as the secondary antibody for 30 min at room temperature. The secondary antibody was applied at the following dilutions: 1:10,000 for HMOX1, and 1:20,000 for γ-GCLC, and 1:20,000 for γ-GCLM. Mouse monoclonal β-actin antibody (691001; MP Biomedicals, LLC) was used as the loading control. Protein signals were visualized using a chemiluminescent imaging system (ATTO Corporation) after incubation with Immobilon^® Western (MilliporeSigma).

Statistical analyses were performed for each experiment as follows: Cell viability and proliferation assay: For comparisons across multiple treatment groups, one-way ANOVA followed by Tukey's post hoc test was performed. Differences among multiple treatment conditions were analyzed using one-way ANOVA with Tukey's post hoc test regarding ell colony formation assays. Percentages of apoptotic cells in cell apoptosis assay were compared using one-way ANOVA followed by Tukey's post hoc test. Relative mRNA expression levels were compared using one-way ANOVA with Tukey's post hoc test. Data are presented as the mean ± standard error of the mean (SEM). Statistically significant difference was defined as P<0.05. Analyses were conducted using JMP^® Pro ver. 18.0.2 (SAS Institute Inc.) and GraphPad Prism ver. 10.5.0 (Dotmatics).

To assess whether Andrographis has proliferation-inhibitory effects on ESCC cells and whether it additively enhances the cell proliferation inhibitory effect of 5-FU on ESCC cells, KYSE410 and TE1 were used to examine the effects of 5-FU (3, 3.5, 4, 4.5 and 5 µM) and Andrographis (36, 42, 48, 54 and 60 µg/ml) individually and in combination on cell proliferation. WST assay results indicated that Andrographis exhibited significantly greater proliferation inhibitory activity than 5-FU in both cell lines (P<0.05). Moreover, the combination of 5-FU and Andrographis significantly inhibited cell proliferation compared with individual treatments in both cell lines (P<0.05) (Fig. 1A).

Notably, both 5-FU and Andrographis inhibited the proliferation of KYSE410 and TE1 cells in a dose-dependent manner, with IC₅₀ values of 14.0 and 4.5 µM for 5-FU and 51.7 and 40.9 µg/ml for Andrographis, respectively. Moreover, when used in combination, the IC₅₀ value was further reduced, and the evaluation of the C.I. demonstrated an additive effect between 5-FU and Andrographis in both cell lines (Fig. 1B). These findings suggest that Andrographis inhibits ESCC cell proliferation, and the combination with 5-FU may enhance this inhibitory effect, following the defined criteria (28). Based on these findings, to avoid excessive cytotoxicity caused by 5-FU while enabling evaluation of whether Andrographis has an additive effect, subsequent assays were conducted using a sub-IC₅₀ concentration of 5-FU (3.5 µM).

Next, the effects of 5-FU, Andrographis, and their combination on cell viability were investigated using colony formation assays. In KYSE410 cells, both 5-FU and Andrographis significantly reduced colony formation compared with the control (P<0.001) (Fig. 2A). In TE1 cells, 5-FU did not significantly alter colony formation, whereas Andrographis significantly decreased the colony number (P<0.05) (Fig. 2B). In both cell lines, combination treatment showed a greater reduction in colony numbers than 5-FU alone. However, the extent of reduction was comparable to that observed with Andrographis alone, and no significant difference was detected between the two groups. Thus, while the combination treatment did not show a clear additional reduction compared with treatment with Andrographis alone, it exhibited a more noticeable inhibitory effect relative to 5-FU alone.

Previous studies have indicated that Andrographis induces apoptosis in various gastrointestinal adenocarcinoma cells (14–18). Next, it was assessed whether the observed suppression of cell viability following treatment with 5-FU, Andrographis, and the combination was correlated with an increase in apoptosis in ESCC cells. An Annexin V binding assay was performed to quantify apoptotic cells after individual and combination treatments. As shown in Fig. 3, Andrographis significantly elevated the apoptotic rate compared with the control, particularly in TE1 cells (P<0.05). Furthermore, the combination treatment led to a further increase in the percentage of apoptotic cells compared with the control: 20.8% vs. 14.9% in KYSE410 cells (P<0.001) (Fig. 3A) and 48.5% vs. 11.7% in TE1 cells (P<0.0001) (Fig. 3B). Consistent with the enhanced suppression of cell viability observed with combination treatment, a significantly higher percentage of apoptotic cells was observed compared with that observed with individual treatments (P<0.05) (Fig. 3A and B). These findings suggest that Andrographis not only induces apoptosis in ESCC cells but also enhances 5-FU-mediated apoptosis, potentially acting as a sensitizer or adjunct agent for chemotherapy.

Previously, it was reported that Andrographis upregulates ferroptosis-related genes, such as HMOX1, GCLC and GCLM, in gastric and colorectal adenocarcinoma cells (14,16,17). Other studies have shown that Andrographis activates ferroptosis in several malignant tumors, such as non-small cell lung cancer (29) and multiple myeloma (30) cells. To determine whether this finding is applicable to ESCC cells, RT-qPCR and western blotting assays were performed, focusing on HMOX1, GCLC and GCLM as the key target genes in ferroptosis.

As shown in Fig. 4A, HMOX1 (KYSE410 P<0.01; TE1 P<0.01), GCLC (KYSE410 P<0.001; TE1 P<0.05) and GCLM (KYSE410 P<0.001; TE1 P<0.0001) were significantly upregulated in ESCC cells after treatment with Andrographis compared with the control. Combination treatment also upregulated HMOX1 (KYSE410 P<0.0001; TE1 P<0.01), GCLC (KYSE410 P<0.001; TE1 P<0.05) and GCLM (KYSE410 P<0.001; TE1 P<0.001) at the mRNA level compared with the control. Furthermore, HMOX1 (KYSE410 P<0.0001; TE1 P<0.001), GCLC (KYSE410 P<0.001; TE1 P<0.01) and GCLM (KYSE410 P<0.001; TE1 P<0.001) were significantly upregulated by the combination treatment compared with those treated with 5-FU in both ESCC cell lines.

Western blotting was performed to validate the expression of HMOX1, GCLC and GCLM at the protein level. As shown in Fig. 4B, the expression levels of HMOX1, GCLC and GCLM at the protein level were increased in both the Andrographis and combination treatment groups compared with those in the control and 5-FU groups in both ESCC cell lines.

Regarding mRNA expressional alteration of other ferroptosis related genes such as NF-kB (NFKB1), STAT3, Nrf2 (NFE2L2) and ACSL4, these targets did not show consistent trends between untreated/individual/combination treatment groups in two ESCC cell-lines (Fig. S1). In case of GPX4, mRNA expression of GPX4 was downregulated and showed similar trends after Andrographis or combination treatment in two ESCC cell-lines (Fig. S1). On the other hand, mRNA expression of SLC7A11 were upregulated and showed similar trends after Andrographis or combination treatment (Fig. S1).