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Published on: October 21, 2012
Digitoxin inhibits the growth of HSC-3 cells by inducing G2/M cell cycle arrest and apoptosis
Ying Rui1,2, Yan Wang3, Hongfei Gong1
1School of Pharmacy, Guilin Medical University, Guilin, China.
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.
Insights
Digitoxin effectively inhibits oral squamous cell carcinoma (OSCC) growth by inducing apoptosis and cell cycle arrest. This study demonstrates digitoxin
Area of Science:
- Pharmacology
- Oncology
- Cell Biology
Background:
- Oral squamous cell carcinoma (OSCC) presents a significant global health challenge, causing numerous annual deaths.
- Investigating novel therapeutic agents is crucial for improving OSCC patient outcomes.
Purpose of the Study:
- To evaluate the anti-cancer effects of digitoxin on human OSCC (HSC-3) cells.
- To elucidate the mechanisms underlying digitoxin's action, including its impact on cell proliferation and apoptosis.
Main Methods:
- *In vitro* studies involved treating HSC-3 cells with digitoxin and assessing viability, colony formation, apoptosis, cell cycle, mitochondrial potential, and reactive oxygen species (ROS).
- Western blotting was used to analyze key protein expressions.
- *In vivo* studies utilized a mouse xenograft model to monitor tumor growth and analyze tumor tissue markers.
Main Results:
- Digitoxin reduced HSC-3 cell viability and induced apoptosis in a dose-dependent manner.
- The drug caused G2/M phase arrest by inhibiting Cdc25C and the cyclin B1-CDK1 complex, leading to CDK1 inactivation.
- Digitoxin triggered mitochondrial dysfunction and increased ROS, activating the intrinsic apoptotic pathway, which was confirmed to be ROS-dependent.
- *In vivo*, digitoxin significantly inhibited tumor growth in xenograft models.
Conclusions:
- Digitoxin demonstrates potent anti-tumor activity against OSCC.
- The anti-cancer effects are mediated through ROS-dependent mitochondrial apoptosis and cell cycle checkpoint activation.
