Digitoxin inhibits the growth of HSC-3 cells by inducing G2/M cell cycle arrest and apoptosis

Introduction

Background

Oral squamous cell carcinoma (OSCC), which accounts for over 90% of all oral cancers, is among the most common head and neck malignancies (1). In the United States alone, an estimated 54,010 new cases were projected for 2021 (2). Surgical resection followed by adjuvant radiotherapy remains a cornerstone of treatment for OSCC (3). However, the complex anatomy of the oral cavity—including functionally critical structures such as the tongue, facial nerve, and salivary glands—poses significant challenges (4). Achieving oncologically radical resection while preserving vital functions is difficult, often leading to surgical morbidity and compromising patients’ quality of life (5). Consequently, despite aggressive multimodal therapy, the long-term survival rate for OSCC patients remains unsatisfactory. Therefore, there is an urgent need to develop more effective drugs for OSCC to reduce complications and improve patient survival.

Rationale and knowledge gap

Digitoxin, a cardiac glycoside traditionally used in cardiology, has emerged as a compound with anti-cancer potential. Notably, it suppresses cancer cell proliferation at concentrations below those required to inhibit its canonical target, Na⁺/K⁺-ATPase, suggesting that its anti-tumor effects are mediated not by ion homeostasis disruption but by the activation of specific downstream signaling cascades that lead to cell death (6,7). While such mechanisms have been implicated in its efficacy against several malignancies—including lung, prostate, and breast cancers—its therapeutic potential and precise mode of action in OSCC remain to be fully elucidated (8,9).

Apoptosis is a tightly regulated and evolutionarily conserved process of programmed cell death, often initiated through mitochondrial signaling pathways (10,11). When mitochondrial apoptosis occurs in cells, the ratio of pro-apoptosis protein and anti-apoptosis protein of B-cell lymphoma-2 (Bcl-2) family is unbalanced, which leads to the change of mitochondrial membrane permeability (12). Cytochrome c (Cyt c) in mitochondria is released into cytoplasm, and combined with apoptosis activating factor-1 (Apaf-1) and cysteinyl aspartate specific proteinase 9 (Caspase-9) in cytoplasm to form a complex, which finally activates Caspase-3 and induces apoptosis (13). Therefore, the activation of caspase family proteins is one of the main ways of cancer cell apoptosis mediated by anticancer drugs.

Objective

The aim of this study was to evaluate the ability of digitoxin to inhibit the proliferation of OSCC and to elucidate the underlying molecular mechanisms, thereby providing evidence for their potential use as therapeutic agents in OSCC. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2173/rc).

Methods

Materials and reagents

HSC-3 cells [research resource identifier (RRID): cellosaurus cell line identifier (CVCL)_1288] were obtained from the Cell Bank of the Chinese Academy of Sciences. Dulbecco’s modified Eagle’s medium [1640] were purchased from Gibco (New York, USA). Foetal bovine serum (FBS) was purchased from Excell Bio (Shanghai, China). Penicillin (100 U/mL) and streptomycin (100 mg/mL) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Digitoxin, A Cell Counting Kit-8 (CCK-8) and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (Shanghai, China). A bicinchoninic acid (BCA) assay kit, Hoechst 33258 kit, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe and mitochondrial membrane potential (ΔΨ_m) assay kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd (Shanghai, China). A fluorescein isothiocyanate (FITC) Annexin V apoptosis detection assay kit and propidium iodide (PI) cycle apoptosis detection kit were purchased from BD Company (Franklin Lakes, NJ, USA). β-Actin (ZSGB-Bio Cat# TA-09, RRID: AB_2636897) goat anti-rat and goat anti-rabbit antibodies were purchased from Beijing Zhongshan Jinqiao Biotechnology Company (Beijing, China). Cyclin-dependent kinase 1 (CDK1) (Cell Signaling Technology Cat# 9116, RRID: AB_2074795), cell cycle protein B₁ (cyclinB₁) (Cell Signaling Technology Cat# 12231, RRID: AB_2783553), cell division cycle25 homolog C (Cdc25C) (Cell Signaling Technology Cat# 4688, RRID: AB_560956), Cyt c (Cell Signaling Technology Cat# 4272, RRID: AB_2090454), Caspase9 (Cell Signaling Technology Cat# 9508, RRID: AB_2068620), poly-ADP-ribose polymerase (PARP) (Cell Signaling Technology Cat# 9532, RRID: AB_659884) were purchased from Cell Signaling Technology (CST) Company (Danvers, USA). Bcl-2 associated x protein (Bax) (Boster Biological Technology Cat# A00183, RRID: AB_3080998) was purchased from Wuhan Baishi Biological Company (Wuhan, China). Bcl-2 (Abcam Cat# ab182858, RRID: AB_2715467) was purchased from Abcam (Boston, USA). Double sensitive chemiluminescence reagent was purchased from Affinity Biosciences, Cincinnati, USA. Females BALB/c nude mice (n=50, five-week-old, SP1073) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China).

Study methods

CCK-8 assay

The cells were grown at 37 °C in a humidified chamber containing 5% CO₂ in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL). CCK-8 was used to analyse the effect of Digitoxin on the proliferation of HSC-3 cells according to the manufacturer’s instructions. Briefly, 5×10³/mL cells were seeded per well of 96-well plates and incubated with digitoxin (12.5, 25, 50, 100, 200, or 400 nmol/L) for 48 h. The cells in the control plates were incubated with dimethyl sulfoxide (DMSO, 0.1%). After incubation for the specified time, 200 µL of complete culture solution and 20 µL of CCK-8 reagent were added to each well of a 96-well plate and incubated for 2 h at 37 °C. After incubation, the supernatant from each well was decanted, and the absorbance optical density (OD) value was immediately read at 450 nm with a microplate reader.

Colony formation assay

Briefly, the collected and cultured cells were diluted with 10% 1640 complete medium, and approximately 200 cells per well were added to 6-well plates. Cells were plated and cultured in a constant temperature incubator at 37 °C and 5% CO₂. The complete medium without drug was discarded, complete medium with drug concentrations of 12.5, 25 and 50 nmol/L was added to the culture, and control wells were set up. The medium was changed every two days with the corresponding drug concentration, and the medium was discarded after 10 days of incubation. Then, 500 µL of fixative was added to each well for 30 min, after which the fixative was discarded. Then, 500 µL of 0.1% crystal violet staining solution was added to each well and stained for 30 min.

PI staining assay

The cells were cultured on 6-well plates followed by digitoxin at different concentrations for 48 h. After hatching, 500 µL phenylindole (PI)/ribonuclease A (RNAseA) (100 µg/mL) was added to each tube, and the cells were gently aspirated with a pipette gun for resuspension. The cells were incubated at room temperature for 30 min in the dark, filtered with a 300-mesh sieve, and then checked by flow cytometry. Meanwhile, control group were operated by the same method.

Hoechst 33258 staining assay

The 6-well plate was placed in a constant temperature incubator at 37 °C and 5% CO₂ for 24 h. The medium was discarded, and the medium containing drugs was added to the control group. After 48 h, the cells were stained with Hoechst 33258 for 5 min. The imaging results were detected by an inverted fluorescence microscope.

Annexin V-FITC/PI assay

HSC-3 cells were divided into the negative control group, digitoxin (200 nmol/L) group, NAC (10 mmol/L) group, and NAC (10 mmol/L) + digitoxin (200 nmol/L) group. HSC-3 cells were treated for 48 h, then the samples were transferred to 1.5 mL Eppendorf (EP) tubes and centrifuged at 1,000 rpm/min for 3 min, and the supernatant was removed. Then, 5 µL Annexin V-FITC and 5 µL PI were added to each centrifuge tube according to the groups. HSC-3 cells were examined by flow cytometry after filtration with a 300-mesh screen. The operation of the group was repeated 3 times.

Detection of cellular ΔΨm

The cells reached the logarithmic growth phase. Thus, the drugs were added to the control group, then culture was continued for 48 h. In the control group, 1 µL of the apoptosis inducer carbonyl cyanide chlorophenylhydrazone (CCCP) was added, and the cells were treated for 20 min before the supernatant was removed. After washing 2 times with phosphate buffered saline (PBS), 1 mL of complete medium was added to each well, and then 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide (JC-1) staining solution was added to each well and placed in a 37 °C incubator for incubation for 20 min. After incubation, a fluorescence microscope was used to take photos and observe the cells.

ROS measurement

The drugs were added to the control group, and the NAC group and the blank group were set. And culture was continued for 48 h in a 5% CO₂ incubator at 37 °C. The final concentration of DCFH-DA was diluted 1,000 times in serum-free medium to 10 µmol/L, 1 mL in each well. DCFH-DA was incubated for 20 min at 37 °C in the dark. And the fluorescence microscope was 100× to take photos.

Western blot analysis

HSC-3 cells were treated with digitoxin and NAC for 48 h. After that, total protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and further transferred to a nitrocellulose membrane. The primary antibodies used were against CDK1 (1:1,000), cyclinB₁ (1:1,000), β-actin (1:1,000), Cdc25C (1:1,000), Bax (1:2,000), Bcl-2 (1:2,000), Caspase-9 (1:1,000), cleaved Caspase-9 (1:2000), Cyt-c (1:1,000), Caspase-3 (1:1,000), cleaved Caspase-3 (1:1,000), PARP-1 (1:1,000). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000) for 2 h, followed by enhanced chemiluminescence (ECL) detection.

Animal experiment

A total of 50 BALB/c nude mice (five-week-old, females) were used in this study. HSC-3 cells containing 5×10⁶ cells were implanted subcutaneously into the right axilla of BALB/c nude mice. The mice were randomly divided into the control, digitoxin (1 and 2 mg/kg), and cisplatin (DDP) (2 mg/kg) groups. Digitoxin was administered twice a week for four weeks and the tumour size reached 40 mm³. The body weight of the nude mice was recorded and the mice were euthanized at this point after losing more than 20% of their body weight. The major diameter of the forming tumour was measured with a micrometer and the tumour was weighed with an electronic balance.

Experiments were performed under a project license (No. GLMU-IACUC-202610055) granted by the Animal Research Committee of Guilin Medical University Science Experiment Center (Guangxi, China), in compliance with institutional guidelines for the care and use of animals.

Histology and immunohistochemistry

Formaldehyde-fixed, paraffin-embedded sections of oral cancer cell xenograft samples were subjected to haematoxylin and eosin (H&E) staining and immunohistochemistry according to standard protocols. Immunostaining of tumour samples was performed with rabbit antiKi-67 monoclonal antibody and Caspase-3 antibody. Primary antibodies were added to the sections, which were incubated overnight at 4 °C. The sections were then incubated with the secondary antibody for 1 hour at room temperature. Immunoreactivity was observed by immersing the sections in 3,3’-diaminobenzene (DAB). Samples were restained with haematoxylin and photographed.

Statistical analysis

The experimental data were analysed using GraphPad Prism software version 8. Two-tailed Student’s t-test was utilized to determine significant P values for comparison of two groups. The results are presented as the mean ± standard deviation from three independent experiments. The variance is similar between the groups. P<0.05 was considered statistically significant.

Results

Proliferation and cell cycle of HSC-3 cells

The data showed that compared with the negative control group, with increasing Digitoxin concentration, the cell survival rate decreased significantly [50% inhibitory concentration (IC₅₀) =112.98±14.56 nmol/L]. The IC₅₀ of digitoxin-treated HSC-3 cells for 48 h was 112.98 nmol/L (Figure 1A). Digitoxin inhibited the proliferation of HSC-3 cells, and the clone formation rates at 12.5, 25 and 50 nmol/L were 67.17%, 41.33%, and 22.83%, respectively (Figure 1B). The cell cycle of HSC-3 cells was blocked in G₂/M phase 48 h after digitoxin treatment (Figure 1C). The cell cycle of the control group was 4.96%±0.49% in G₂/M phase, 21.76%±1.16% in G₂/M phase for the low concentration group, 23.61%±0.52% in G₂/M phase for the medium concentration group, and 51.97%±9.31% in G₂/M phase for the high concentration group. With the increase in digitoxin concentration, the number of cells in G₀/G₁ showed a decreasing trend, while the number of cells in G₂/M phase showed an increasing trend (Figure 1D).

Figure 1 Digitoxin shows cytotoxicity towards HSC-3 cells and induce HSC-3 cell apoptosis. (A) In vitro cytotoxicity of digitoxin in HSC-3 cells for 48 h were determined by CCK-8 assay. (B) Digitoxin inhibited the cell proliferation of HSC-3 cells (0.1% Crystal Violet stained; magnification ×200). (C) After treatment with digitoxin (50, 100 and 200 nmol/L) for 48 h, digitoxin increased the cell population in the G₂/M phase. (D) Quantification of the cell population in the G₂/M phase. (E) HSC-3 cells were treated with digitoxin (50, 100 and 200 nmol/L) for 48 h, and the levels of CDK1, cyclin B1 and Cdc25C were evaluated by Western blot analysis. (F) Quantitative data of the relative protein expression. (G) Hoechst 33258 fluorescence staining results (magnification ×200). (H) Cells were harvested and assayed by annexin V-FITC/PI staining assay. Representative images show a concentration-dependent effect of digitoxin. (I) Digitoxin disrupted the interaction between Bax and Bcl-2. (J) Quantitative data of relative protein expression. (K) Digitoxin facilitates the activation of cytochrome c, Caspase-3, Caspase-9 and PARP. (L) Quantitative data of the relative protein expression. Date are shown as the mean ± SD (n=3). *, P<0.05; **, P<0.01; ***, P<0.001 vs. the control group. Bax, Bcl-2 associated x protein; Bcl-2, B-cell lymphoma-2; Caspase-3, cysteinyl aspartate specific proteinase 3; Caspase-9, cysteinyl aspartate specific proteinase 9; CCK-8, Cell Counting Kit-8; Cdc25C, cell division cycle25 homolog C; CDK1, cyclin-dependent kinase 1; cyclin B1, cell cycle protein B1; IC₅₀, 50% inhibitory concentration; PARP, poly-ADP-ribose polymerase; PI, propidium iodide; SD, standard deviation.

As shown in Figure 1E, with increasing digitoxin concentration, the expression of cyclin (CDK1) showed a decreasing trend; the expression of cell division cycle protein (Cdc25c) showed a decreasing trend; and the expression of cyclin (cyclinB₁) showed an increasing trend (Figure 1F).

Apoptosis of HSC-3 cells

Cells in the control group were stained evenly and presented a weak blue colour, while cells in the drug administration group showed a bright fluorescence signal with the solidified edge set of chromatin, and the fluorescence intensity gradually increased with increasing digitoxin concentration, indicating that digitoxin is able to induce apoptosis in HSC-3 cells (Figure 1G). The total apoptosis rates were 2.33%, 3.11%, 8.86% and 68.2% in the control group and the groups treated with different concentrations of digitoxin (50, 100, and 200 nmol/L), respectively. The total apoptosis rate of the cells gradually increased with increasing digitoxin concentration, and the apoptotic effect was concentration-dependent (Figure 1H).

To study whether digitoxin-induced apoptosis is mediated by the mitochondrial pathway, with increasing digitoxin concentration, the expression level of the pro-apoptotic protein Bax was significantly increased. The expression of the antiapoptotic protein Bcl-2 was decreased (Figure 1I,1J). The above data suggest that digitoxin can induce apoptosis of HSC-3 cells through the mitochondrial pathway by regulating the Bcl-2 family. As shown in Figure 1K, with increasing digitoxin concentration, the expression of Cyt c was upregulated. Caspase-9 and Caspase-3 expression was downregulated, cleaved Caspase-9 and cleaved Caspase-3 expression was upregulated. PARP expression levels were upregulated and then downregulated, cleaved PARP expression levels were upregulated (Figure 1L).

Changes of ΔΨm and reactive oxygen species (ROS) in HSC-3 cells

The ΔΨ_m of HSC-3 cells decreased after treatment with digitoxin for 48 h. As the concentration of digitoxin increased, the red fluorescence gradually decreased, and the green fluorescence gradually increased. The higher the concentration of digitoxin, the more obvious the decrease in ΔΨ_m. Compared with the control group, digitoxin induced ROS production in cells. The higher the concentration of digitoxin, the higher the green fluorescence and the higher the ROS expression (Figure 2).

Figure 2 Digitoxin affects mitochondrial membrane potential and ROS production in HSC-3 cells. (A) JC-1-stained HSC-3 cell lines treated with digitoxin (50, 100, and 200 nM). Images were observed using the confocal laser scanning microscope (magnification ×100). (B) DCFH-stained HSC-3 cell lines treated with digitoxin (50, 100, and 200 nM). Images were observed using the confocal laser scanning microscope (magnification, ×100). DCFH, 2’,7’-dichlorodihydrofluorescein; JC-1, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide; ROS, reactive oxygen species.

Apoptosis in HSC-3 cells is associated with the accumulation of ROS

The CCK-8 results showed that the cell survival rate of the combination group of digitoxin (200 nmol/L) and NAC (10 mmol/L) was significantly higher than the cell survival rate of the group treated with digitoxin alone (Figure 3A). Compared with the control group, 200 nmol/L digitoxin induced ROS production in HSC-3 cells, and the induction of ROS production in cells by digitoxin could be reversed by NAC (Figure 3B). As expected, the total apoptosis rate of the digitoxin treatment group was 55.6% (Figure 3C). Compared with the digitoxin treatment group, the total apoptosis rate of the digitoxin treatment group (200 nmol/L) combined with NAC (10 mmol/L) was 44.55% and significantly lower than the total apoptosis rate of the digitoxin treatment group (Figure 3D). Thus, the induction of apoptosis in HSC-3 cells by digitoxin was associated with the accumulation of ROS.

Figure 3 Effects of NAC on digitoxin-induced cytotoxicity and apoptosis in HSC-3 cells. (A) Cell proliferation was evaluated using the CCK-8 assay in HSC-3 cells treated with digitoxin (200 nM) ± NAC (10 mM) for 48 h. (B) Representative fluorescence microscopy images showing ROS levels in HSC‑3 cells following treatment with digitoxin alone or in combination with NAC.(C,D) Representative flow cytometry profiles (Annexin V‑FITC, x‑axis; PI, y‑axis) showing a concentration‑dependent effect of digitoxin. (E,F) Western blot analysis of mitochondrial apoptosis‑related proteins in HSC‑3 cells under the indicated conditions. The data are expressed as the mean ± SD of duplicate experiments. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni’s multiple comparison tests. ns, not significant; ***, P<0.001, vs. control group. ^##, P<0.01, ^###, P<0.001, vs. digitoxin treatment group. ANOVA, analysis of the variance; Bax, Bcl-2 associated x protein; Bcl-2, B-cell lymphoma-2; Caspase-3, cysteinyl aspartate specific proteinase 3; Caspase-9, cysteinyl aspartate specific proteinase 9; CCK-8, Cell Counting Kit-8; NAC, N-acetylcysteine; PARP, poly-ADP-ribose polymerase; PI, propidium iodide; ROS, reactive oxygen species; SD, standard deviation.

NAC reversed the expression of digitoxin-induced mitochondrial pathway proteins in HSC-3 cells

NAC reversed the upregulated expression of digitoxin-induced proapoptotic protein Bax and downregulated expression of antiapoptotic protein Bcl-2 (Figure 3E). Digitoxin was further verified to induce apoptosis in HSC-3 cells through the mitochondrial pathway by regulating the Bcl-2 family associated with the accumulation of ROS. As a consequence NAC (10 mmol/L) reversed digitoxin-induced Cyt c; cleaved Caspase-9, cleaved Caspase-3, and cleaved PARP expression levels were upregulated (Figure 3F). In conclusion, digitoxin induced apoptosis in HSC-3 cells by regulating mitochondrial pathway-related proteins, which may be related to digitoxin-induced intracellular ROS accumulation, which in turn caused cell damage.

Nude mouse model experiment

Digitoxin significantly inhibited subcutaneous tumour growth compared to the control group. Body weight was not affected during digitoxin treatment (Figure 4A). The results of the heterogeneous experiment showed that digitoxin inhibited tumour growth in a subcutaneous tumour model (Figure 4B). Treatment with digitoxin or DDP resulted in partial replacement of tumour tissue by calcified and necrotic tissue. Compared to digitoxin treatment, tumour tissues in the control group showed a high proliferation index as assessed by Ki67 nuclear expression. Analysis of the impact of digitoxin on the in vivo expression of Caspase-3 and Ki67 in human oral squamous carcinoma cell xenografts. H&E staining was performed on the same tumour sections for immunostaining.

Figure 4 Anticancer effect of digitoxin in vivo. (A) Digitoxin significantly inhibited the growth of subcutaneous tumours compared to the control group. Bar graph indicates tumour weight of HSC-3 xenografts. (B) H&E staining showed that as the dose of digitoxin toxin increased, the number of cancer cells decreased and the cell integrity was poor, and the number of apoptotic and necrotic cells increased. Immunohistochemical experiments verified that tumour tissues in the control group showed higher Ki67 proliferation index and high expression of Caspase-3, and that Digitoxin-treated group inhibited the growth of tumours (magnification ×400). Date are shown as the means ± standard deviation (n=8). *, P<0.05, vs. the control group. Caspase-3, cysteinyl aspartate specific proteinase 3; DDP, cisplatin; H&E, haematoxylin and eosin.

Discussion

Key findings

Digitoxin exhibits potent anti-tumor activity against OSCC through engagement of the intrinsic apoptotic cascade. In HSC-3 cells, digitoxin suppresses proliferation and induces apoptosis in a manner dependent on ROS accumulation, which serves as an upstream trigger for mitochondrial membrane permeabilization and subsequent caspase activation. These mechanistic findings are corroborated by in vivo evidence: in a murine xenograft model bearing HSC-3-derived tumours, digitoxin administration significantly suppressed tumour growth. Collectively, our study establishes digitoxin as a candidate therapeutic agent for OSCC and positions ROS-mediated mitochondrial apoptosis as a central axis of its oncoactivity.

Strengths and limitations

Although cardenolides such as digitoxin have been reported to exert anti-tumor effects in various cancer types, their activity and mechanism of action in OSCC have remained undefined. The present study addresses this gap by demonstrating that digitoxin inhibits OSCC growth through ROS-mediated mitochondrial pathway engagement, as evidenced by coordinated cell cycle arrest, apoptotic execution, and tumour suppression in xenograft models. These findings establish digitoxin as a mechanistically defined candidate for OSCC therapy and warrant its further evaluation in preclinical settings.

Explanations of findings

Digitoxin suppresses HSC-3 cell proliferation in a concentration-dependent manner, as determined by both CCK-8 viability assays and colony formation assays. The concordance between these short-term and long-term endpoints confirms that digitoxin exerts a durable anti-proliferative effect against OSCC cells, warranting further mechanistic investigation. The present study demonstrates that digitoxin induces G₂/M arrest in HSC-3 cells, as revealed by flow cytometric accumulation of cells in the G₂/M fraction. Mechanistically, digitoxin treatment reduced the abundance of the cyclin B₁-CDK1 complex and suppressed the expression of Cdc25C. Cdc25C a phosphatase that dephosphorylates CDK1 at Tyr15 to enable mitotic entry (14). These findings provide a molecular rationale for the sustained inactivation of CDK1 and the consequent G₂/M blockade. While cardenolides have previously been shown to perturb cell cycle progression in other malignancies (15), the present study identifies, for the first time, the Cdc25C-CDK1 axis as a specific target of digitoxin in OSCC. Together, these results suggest that digitoxin engages an evolutionarily conserved checkpoint machinery and point to Cdc25C as a putative node for therapeutic intervention. Aberrant proliferative signals—including replication stress, spindle checkpoint bypass, or sustained CDK activity—do not directly engage the apoptotic machinery. Rather, they sensitize cells to mitochondrial outer membrane permeabilization (MOMP) by shifting the balance between pro- and anti-apoptotic Bcl-2 family members. Thus, the cell cycle provides a permissive context in which apoptotic execution becomes conditional upon checkpoint failure (16).

In parallel to its cytostatic effects, digitoxin also engages the intrinsic apoptotic cascade. Annexin V-FITC/PI double staining revealed that the apoptotic fraction of HSC-3 cells increased in a concentration-dependent manner following digitoxin treatment. Mechanistically, digitoxin disrupts ΔΨ_m and enhances mitochondrial outer membrane permeability in HSC-3 cells. This is accompanied by the upregulation of Bax, a pro-apoptotic Bcl-2 family member, and the concomitant downregulation of the anti-apoptotic protein Bcl-2. These changes promote the release of Cyt c into the cytosol, leading to the activation of Caspase-9 and, subsequently, Caspase-3. Together, these results indicate that digitoxin triggers mitochondrial-mediated apoptosis in OSCC cells through modulation of the Bcl-2 family balance and engagement of the intrinsic apoptotic cascade. Notably, digitoxin elicited a bell-shaped Caspase-3 activation profile: cleaved Caspase-3 peaked at intermediate concentrations and declined at higher doses. This biphasic response—far from reflecting reduced apoptosis—is a hallmark of robust, synchronous MOMP (17). At higher concentrations, overwhelming MOMP accelerates cellular disintegration, leading to proteolytic degradation or extracellular leakage of activated Caspase-3. The loss of Caspase-3 immunoreactivity at high doses therefore signifies apoptotic completion, not failure. This interpretation aligns with previous reports on cardenolides and other mitochondrial toxins, and underscores a key methodological caveat: Caspase-3 immunoblotting alone, particularly at a single high dose, may substantially underestimate apoptotic activity in the absence of complementary assays such as annexin V/PI flow cytometry (18). ROS are established mediators of mitochondrial apoptosis, acting through the induction of oxidative damage and the activation of stress-sensitive signaling cascades (19). In the present study, digitoxin treatment led to a concentration-dependent increase in intracellular ROS levels in HSC-3 cells, consistent with its disruption of mitochondrial membrane integrity. Elevated ROS production is known to overwhelm cellular redox buffering capacity, resulting in lipid peroxidation, protein carbonylation, and DNA lesions—events that converge on the intrinsic apoptotic pathway via Cyt c release and Caspase-9 activation. These observations align with previous reports that cardenolides, including juglone, exert pro-oxidant effects in various cancer models, leading to PI3K/Akt pathway modulation, cell cycle arrest, and caspase-dependent apoptosis (20). Collectively, our findings suggest that oxidative stress serves as both a downstream consequence and an integral effector of digitoxin-induced mitochondrial dysfunction, contributing to the suppression of HSC-3 cell viability through the coordinated engagement of apoptotic execution. The involvement of oxidative stress in digitoxin-mediated anti-tumor activity was further supported by pharmacological intervention using the ROS scavenger NAC. NAC pretreatment significantly attenuated digitoxin-induced ROS accumulation in HSC-3 cells and, concomitantly, reversed the suppression of cell viability as determined by CCK-8 assay. Furthermore, NAC abrogated digitoxin-triggered alterations in mitochondrial apoptosis-related proteins, including the upregulation of Bax, downregulation of Bcl-2, and subsequent Caspase-9 and Caspase-3 cleavage. Collectively, these findings position ROS as an essential upstream signal that couples digitoxin exposure to mitochondrial pathway engagement and apoptotic execution in OSCC cells. Histopathological analysis of xenograft tumours reveals that digitoxin treatment markedly reduces tumour growth and suppresses the expression of the proliferation marker Ki67, while concurrently increasing the abundance of cleaved Caspase-3, a bona fide effector of apoptotic execution. These in vivo observations corroborate our in vitro findings and collectively demonstrate that digitoxin exerts anti-tumour activity against OSCC through the induction of mitochondrial apoptosis.

Implications and actions needed

The present study establishes digitoxin as a candidate therapeutic agent for OSCC and provides a mechanistic framework-centred on ROS-mediated Cdc25C-CDK1 axis suppression and Bcl-2 family modulation-to guide further translational investigation.

Conclusions

This study demonstrates that digitoxin suppresses OSCC growth through coordinated engagement of cell cycle arrest and mitochondrial apoptosis, with oxidative stress serving as an essential upstream signal. Mechanistically, digitoxin induces ROS accumulation in HSC-3 cells, leading to Cdc25C-CDK1 axis inhibition and consequent G₂/M checkpoint enforcement. Concurrently, ROS-driven disruption of ΔΨ_m shifts the Bcl-2 family balance toward a pro-apoptotic state, triggering Cyt c release and Caspase-9/3 activation. These in vitro mechanistic findings are recapitulated in vivo, where digitoxin significantly inhibits HSC-3 xenograft tumour growth and modulates Ki67 and cleaved Caspase-3 expression. Collectively, our work establishes digitoxin as a ROS-dependent, mitochondria-targeting agent with therapeutic potential in OSCC and provides a mechanistic rationale for its further translational evaluation.

Acknowledgments

Our sincere thanks go to the committee members and colleagues at Guilin Medical University for their insightful suggestions and support.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2173/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2173/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2173/prf

Funding: This work was supported by the National Natural Science Foundation of China (Nos. 81460458 and 81560574).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2173/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. GLMU-IACUC-202610055) granted by the Animal Research Committee of Guilin Medical University Science Experiment Center (Guangxi, China), in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024;74:229-63. [Crossref] [PubMed]
2. Fantozzi PJ, Bavarian R, Tamayo I, et al. The role of family history of Cancer in Oral Cavity Cancer. Head Face Med 2021;17:48. [Crossref] [PubMed]
3. Jaszkul KM, Grzybowski M, Phillips T. Otolaryngology-Head and Neck Surgery clinical electives in undergraduate medicine: a cross-sectional observational study. J Otolaryngol Head Neck Surg 2022;51:42. [Crossref] [PubMed]
4. Cannon RB, Houlton JJ, Mendez E, et al. Methods to reduce postoperative surgical site infections after head and neck oncology surgery. Lancet Oncol 2017;18:e405-13. [Crossref] [PubMed]
5. Amaral MN, Faísca P, Ferreira HA, et al. Current Insights and Progress in the Clinical Management of Head and Neck Cancer. Cancers (Basel) 2022;14:6079. [Crossref] [PubMed]
6. Lindholm H, Ejeskär K, Szekeres F. Digitoxin Affects Metabolism, ROS Production and Proliferation in Pancreatic Cancer Cells Differently Depending on the Cell Phenotype. Int J Mol Sci 2022;23:8237. [Crossref] [PubMed]
7. Lindholm H, Ejeskär K, Szekeres F. Na+/K+-ATPase subunit α3 expression is associated with the efficacy of digitoxin treatment in pancreatic cancer cells. Med Int (Lond) 2022;2:27. [Crossref] [PubMed]
8. Ayogu JI, Odoh AS. Prospects and Therapeutic Applications of Cardiac Glycosides in Cancer Remediation. ACS Comb Sci 2020;22:543-53. [Crossref] [PubMed]
9. Zhao S, Li X, Wu W, et al. Digoxin reduces the incidence of prostate cancer but increases the cancer-specific mortality: A systematic review and pooled analysis. Andrologia 2021;53:e14217. [Crossref] [PubMed]
10. Choi Y, No MH, Heo JW, et al. Resveratrol attenuates aging-induced mitochondrial dysfunction and mitochondria-mediated apoptosis in the rat heart. Nutr Res Pract 2025;19:186-99. [Crossref] [PubMed]
11. Glover HL, Schreiner A, Dewson G, et al. Mitochondria and cell death. Nat Cell Biol 2024;26:1434-46. [Crossref] [PubMed]
12. Tatarata QZ, Wang Z, Konopleva M. BCL-2 inhibition in acute myeloid leukemia: resistance and combinations. Expert Rev Hematol 2024;17:935-46. [Crossref] [PubMed]
13. Zhou XR, Wang XY, Sun YM, et al. Glycyrrhizin Protects Submandibular Gland Against Radiation Damage by Enhancing Antioxidant Defense and Preserving Mitochondrial Homeostasis. Antioxid Redox Signal 2024;41:723-43. [Crossref] [PubMed]
14. Fischer M, Schade AE, Branigan TB, et al. Coordinating gene expression during the cell cycle. Trends Biochem Sci 2022;47:1009-22. [Crossref] [PubMed]
15. Zhou M, Boulos JC, Klauck SM, et al. The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells. Cell Biol Toxicol 2023;39:2971-97. [Crossref] [PubMed]
16. Park WH. Oxidative Assault: How Pyrogallol’s Pro-Oxidant Chemistry Drives Cell Cycle Arrest and Apoptosis in Cancer. J Appl Toxicol 2026;46:434-42. [Crossref] [PubMed]
17. Maharjan PS, Bhattarai HK. Singlet Oxygen, Photodynamic Therapy, and Mechanisms of Cancer Cell Death. J Oncol 2022;2022:7211485. [Crossref] [PubMed]
18. Li X, Weng SQ, Li QH, et al. Ginsenoside Rg1 Alleviates Hydrogen Peroxide-Induced Autophagy and Apoptosis in Ovarian Granulosa Cells. Cell Biochem Biophys 2025;83:4781-92. [Crossref] [PubMed]
19. Zhao Y, Ye X, Xiong Z, et al. Cancer Metabolism: The Role of ROS in DNA Damage and Induction of Apoptosis in Cancer Cells. Metabolites 2023;13:796. [Crossref] [PubMed]
20. Zhong J, Hua Y, Zou S, et al. Juglone triggers apoptosis of non-small cell lung cancer through the reactive oxygen species -mediated PI3K/Akt pathway. PLoS One 2024;19:e0299921. [Crossref] [PubMed]