Translational cancer research · 2026
Digitoxin inhibits the growth of HSC-3 cells by inducing G2/M cell cycle arrest and apoptosis.
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Rui Ying, Wang Yan, Gong Hongfei, Zhu Xuanyu, Xu Qing, Lin Jing, Wei Jingchen
Аннотация
BACKGROUND: Oral squamous cell carcinoma (OSCC) remains a substantial global health burden. According to the latest global cancer statistics, it accounts for an estimated 300,000 new cases and 100,000 deaths annually. This study aimed to investigate the pharmacological effects of digitoxin, a cardenolide glycoside, on the modulation of proliferative capacity and apoptotic induction in human OSCC human tongue squamous carcinoma (HSC-3) cells.
METHODS: In vitro, HSC-3 cells were treated with graded concentrations of digitoxin. Cell viability and colony formation were measured by Cell Counting Kit-8 (CCK-8) and clonogenic assays, respectively. Apoptosis and cell cycle progression were analysed by flow cytometry, and mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were visualised by fluorescence microscopy. Expression of cell cycle and apoptosis-related proteins was examined by Western blotting. The involvement of oxidative stress was assessed using N-acetylcysteine (NAC) mediated ROS blockade. In vivo, HSC-3 xenograft-bearing nude mice were administered digitoxin, and tumour growth was monitored; tumour tissues were subsequently analysed for Ki67 and cleaved-cysteinyl aspartate specific proteinase 3 (cleaved Caspase-3) expression.
RESULTS: Digitoxin treatment reduced the viability of HSC-3 cells in a concentration-dependent manner and concurrently increased the apoptotic fraction. Mechanistically, digitoxin induced G2/M phase arrest, which was associated with suppression of the cell division cycle 25 homolog C (Cdc25C) phosphatase and decreased abundance of the cyclin B1-CDK1 complex, together enforcing sustained cyclin-dependent kinase 1 (CDK1) inactivation. In parallel, digitoxin triggered mitochondrial dysfunction, as evidenced by loss of ΔΨm and a marked increase in intracellular ROS accumulation. These events were accompanied by upregulation of B-cell lymphoma-2 (Bcl-2) associated x protein (Bax), downregulation of Bcl-2, and elevated levels of cytosolic cytochrome c (Cyt c) and cleaved-cysteinyl aspartate specific proteinase 9 (cleaved Caspase-9), indicating engagement of the intrinsic apoptotic pathway. Pharmacological blockade of ROS with NAC abrogated digitoxin reversed the modulation of Bcl-2 family proteins, and restored cell viability, confirming that oxidative stress functions as an essential upstream signal coordinating both cytostatic and cytotoxic arms of digitoxin's activity. In vivo, digitoxin significantly suppressed the growth of HSC-3 xenograft tumours. Together, these results establish that digitoxin exerts anti-tumour effects in OSCC through ROS-mediated mitochondrial apoptosis and cell cycle checkpoint engagement.
CONCLUSIONS: Digitoxin inhibited the growth of HSC-3 cells both in vitro and in vivo.
METHODS: In vitro, HSC-3 cells were treated with graded concentrations of digitoxin. Cell viability and colony formation were measured by Cell Counting Kit-8 (CCK-8) and clonogenic assays, respectively. Apoptosis and cell cycle progression were analysed by flow cytometry, and mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were visualised by fluorescence microscopy. Expression of cell cycle and apoptosis-related proteins was examined by Western blotting. The involvement of oxidative stress was assessed using N-acetylcysteine (NAC) mediated ROS blockade. In vivo, HSC-3 xenograft-bearing nude mice were administered digitoxin, and tumour growth was monitored; tumour tissues were subsequently analysed for Ki67 and cleaved-cysteinyl aspartate specific proteinase 3 (cleaved Caspase-3) expression.
RESULTS: Digitoxin treatment reduced the viability of HSC-3 cells in a concentration-dependent manner and concurrently increased the apoptotic fraction. Mechanistically, digitoxin induced G2/M phase arrest, which was associated with suppression of the cell division cycle 25 homolog C (Cdc25C) phosphatase and decreased abundance of the cyclin B1-CDK1 complex, together enforcing sustained cyclin-dependent kinase 1 (CDK1) inactivation. In parallel, digitoxin triggered mitochondrial dysfunction, as evidenced by loss of ΔΨm and a marked increase in intracellular ROS accumulation. These events were accompanied by upregulation of B-cell lymphoma-2 (Bcl-2) associated x protein (Bax), downregulation of Bcl-2, and elevated levels of cytosolic cytochrome c (Cyt c) and cleaved-cysteinyl aspartate specific proteinase 9 (cleaved Caspase-9), indicating engagement of the intrinsic apoptotic pathway. Pharmacological blockade of ROS with NAC abrogated digitoxin reversed the modulation of Bcl-2 family proteins, and restored cell viability, confirming that oxidative stress functions as an essential upstream signal coordinating both cytostatic and cytotoxic arms of digitoxin's activity. In vivo, digitoxin significantly suppressed the growth of HSC-3 xenograft tumours. Together, these results establish that digitoxin exerts anti-tumour effects in OSCC through ROS-mediated mitochondrial apoptosis and cell cycle checkpoint engagement.
CONCLUSIONS: Digitoxin inhibited the growth of HSC-3 cells both in vitro and in vivo.