Targeting Galectin-1 with Triptolide Induces Ferroptosis in Oral Squamous Cell Carcinoma

Abstract

Background: Oral squamous cell carcinoma (OSCC) remains clinically challenging, particularly in advanced disease, where treatment resistance limits therapeutic outcomes. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a potential anticancer vulnerability. Galectin-1 (Gal-1/LGALS1), a β-galactoside-binding lectin frequently overexpressed in OSCC, is associated with tumor progression and unfavorable prognosis; however, its involvement in ferroptosis regulation remains incompletely understood. Methods: To investigate whether Triptolide (TPL) influences ferroptosis-associated responses through Gal-1 modulation, OSCC cell lines (SAS and HSC-3) were treated with TPL and analyzed for cell viability, lipid reactive oxygen species (ROS) accumulation, and glutathione peroxidase 4 (GPX4) expression. Publicly available The Cancer Genome Atlas (TCGA) datasets were examined to evaluate Gal-1 expression patterns and survival associations. An OSCC xenograft mouse model was further used to assess the antitumor effects of TPL and changes in ferroptosis-related markers in vivo. Results: TPL treatment reduced cell viability and increased lipid ROS accumulation in OSCC cells, accompanied by downregulation of GPX4 expression. Gal-1 expression was also decreased following TPL exposure in vitro and in xenograft tumors. Analysis of TCGA data revealed that elevated Gal-1 expression was significantly associated with poorer overall survival in OSCC patients. Conclusions: These findings indicate that TPL induces ferroptosis-associated responses in OSCC and suggest that this effect is partly mediated through modulation of Gal-1 expression. Gal-1 may represent a clinically relevant factor influencing ferroptosis susceptibility, and targeting this pathway warrants further investigation as a potential therapeutic strategy for OSCC.

Keywords: GPX4; Galectin-1; ferroptosis; oral squamous cell carcinoma; triptolide.

Figure 1

Galectin-1 is highly expressed in OSCC and is associated with adverse clinicopathologic features. (A) Kaplan–Meier overall survival analysis of the TCGA-HNSC cohort (n = 565) stratified by LGALS1 expression using an optimized cutpoint (TPM = 9.98), determined by maximally selected rank statistics. Survival time was truncated at 60 months (5 years). The high-expression group included 270 patients (47.8%), while the low-expression group included 295 patients (52.2%). Patients with high LGALS1 expression exhibited significantly shorter overall survival than those with low expression (log-rank p = 0.010). (B) Representative immunohistochemical (IHC) staining of Galectin-1 in human OSCC tissues and paired adjacent normal oral epithelium from a tissue microarray (TMA). Upper panels show representative tumor cores with variable staining intensities. Lower panels show two paired OSCC and adjacent normal cases demonstrating strong cytoplasmic Galectin-1 immunoreactivity in tumor tissues and weak or absent staining in normal epithelium (original magnification: ×100 and ×400). Associations between Galectin-1 IHC expression and clinicopathologic parameters are summarized in Table 1.

Figure 2

Triptolide (TPL) induces ferroptosis in OSCC cells by promoting lipid peroxidation and suppressing GPX4 expression. (A) Chemical structure of Triptolide (TPL). (B–D) Cell viability of OSCC cell lines SAS (B), SCC25 (C), and HSC-3 (D) treated with increasing concentrations of TPL (0–80 nM) for 24 and 48 h was measured using the methylene blue assay. (E) Lipid peroxidation was assessed using C11-BODIPY 581/591 staining followed by flow cytometry. SAS and HSC-3 cells were treated with TPL (20 nM, 48 h) alone or co-treated with Ferrostatin-1 (Fer-1, 10 μM). Fer-1 was added 1 h prior to TPL treatment. Right panel: quantitative analysis of lipid ROS-positive cells. Letters B denote the flow cytometric gating regions used to identify ROS-positive cell populations. (F) Western blot analysis showing GPX4 expression in SAS cells following TPL treatment (0–20 nM, 48 h). GAPDH served as the loading control. Densitometric quantification indicates a dose-dependent reduction in GPX4 expression. The GAPDH blot shown in Figure 2F is reused in Figure 3A because both panels were generated from the same experimental membrane under identical conditions. Data represent mean ± SD from three independent biological replicates (n = 3). * p < 0.05 compared with control. The original western blot figures can be found in Figure S1.

Figure 3

Triptolide suppresses Galectin-1 expression in OSCC cells. (A) Western blot analysis of Galectin-1 expression in SAS cells treated with increasing concentrations of Triptolide (TPL, 0–20 nM) for 48 h. GAPDH served as the loading control. (B) Densitometric quantification of Galectin-1 protein levels normalized to GAPDH and expressed relative to untreated control (set as 1.0). (C) Quantitative PCR (qPCR) analysis of LGALS1 mRNA expression in SAS cells following 48 h TPL treatment. Gene expression was normalized to GAPDH and calculated using the 2^−ΔΔCt method. The GAPDH loading control used in panel (A) is the same blot used in Figure 2F, as both analyses originated from the same membrane experiment. Data represent mean ± SD from three independent biological replicates (n = 3). * p < 0.05 compared with control. The original western blot figures can be found in Figure S2.

Figure 4

Galectin-1 regulates the expression of ferroptosis-associated genes in OSCC cells. (A) SAS cells were transfected with LGALS1-targeting siRNA (si-Gal-1) or scrambled control siRNA. After 48 h, mRNA expression levels of LGALS1, GPX4, SLC7A11, and FTH1 were measured using quantitative PCR (qPCR). (B) SAS cells were transfected with a pCMV-Galectin-1 overexpression plasmid. Following 48 h, the expression of LGALS1, GPX4, SLC7A11, and FTH1 was quantified by qPCR. Gene expression values were normalized to GAPDH and calculated using the 2^−ΔΔCt method. Data represent mean ± SD from three independent biological replicates (n = 3). * p < 0.05 compared with control.

Figure 5

Functional involvement of Galectin-1 in TPL-induced ferroptosis in OSCC cells. (A) SAS cells were transfected with Galectin-1–targeting siRNA (si-Gal-1) or scrambled control for 48 h, and cell viability was assessed using the methylene blue assay. (B) SAS cells were treated with Triptolide (TPL, 20 nM) for 48 h in the presence or absence of Ferrostatin-1 (Fer-1, 10 μM), and cell viability was measured. (C) Lipid reactive oxygen species (ROS) levels were determined by C11-BODIPY 581/591 staining followed by flow cytometry in TPL-treated cells (20 nM, 48 h) with or without ectopic Galectin-1 expression achieved by transfection with pCMV-Gal-1. Left panel: representative histograms; right panel: quantitative analysis of lipid ROS–positive cells. Letters B and G denote the flow cytometric gating regions used to identify ROS-positive cell populations. (D) Cell viability of TPL-treated cells (20 nM, 48 h) transfected with pCMV-Gal-1 or control vector. (E) Western blot analysis of GPX4 expression following Galectin-1 knockdown. (F) Western blot analysis of Galectin-1 and GPX4 expression in cells treated with TPL (20 nM, 48 h) alone or in combination with Fer-1 (10 μM). Densitometric analysis was performed using ImageJ software, and protein expression levels were normalized to GAPDH. Data are presented as mean ± SD from three independent biological replicates (n = 3). * p < 0.05 compared with the respective control groups. The original western blot figures can be found in Figures S3 and S4.

Figure 6

Triptolide suppresses tumor growth and modulates ferroptosis-related markers in OSCC xenograft models. (A) Representative images of SAS xenograft tumors harvested after treatment. Mice received either vehicle (PBS) or Triptolide (TPL, 0.15 mg/kg/day, intraperitoneally) for 14 days. (B) Final tumor weights at the end of treatment. (C) Tumor volumes measured every other day and calculated using the formula: Tumor volume = (length × width²)/2. (D) Body weight monitoring throughout the treatment period to assess systemic toxicity. (E) Representative immunohistochemical (IHC) staining of xenograft tumors for Galectin-1 and GPX4. Hematoxylin and eosin (H&E) staining shows tumor morphology. Magnification: 400×. Tumor growth experiments were performed with five mice per group (n = 5). Data are presented as mean ± SD. * p < 0.05 compared with vehicle control.