Mitochondrial impairment and downregulation of Drp1 phosphorylation underlie the antiproliferative and proapoptotic effects of alantolactone on oral squamous cell carcinoma cells

by Selleckchem | Jul 28 2023

Background: Oral squamous cell carcinoma (OSCC) is one of the most prevalent and fatal oral cancers. Mitochondria-targeting therapies represent promising strategies against various cancers, but their applications in treating OSCC are limited. Alantolactone (ALT) possesses anticancer properties and also regulates mitochondrial events. In this study, we explored the effects of ALT on OSCC and the related mechanisms.

Methods: The OSCC cells were treated with varying concentrations and duration of ALT and N-Acetyl-L-cysteine (NAC). The cell viability and colony formation were assessed. The apoptotic rate was evaluated by flow cytometry with Annexin V-FITC/PI double staining. We used DCFH-DA and flow cytometry to detect reactive oxygen species (ROS) production and DAF-FM DA to investigate reactive nitrogen species (RNS) level. Mitochondrial function was reflected by mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and ATP levels. KEGG enrichment analyses determined the mitochondrial-related hub genes involved in OSCC progression. Dynamin-related protein 1 (Drp1) overexpression plasmids were further transfected into the cells to analyze the role of Drp1 in OSCC progression. Immunohistochemistry staining and western blot verified the expression of the protein.

Results: ALT exerted anti-proliferative and pro-apoptosis effects on OSCC cells. Mechanistically, ALT elicited cell injury by promoting ROS production, mitochondrial membrane depolarization, and ATP depletion, which were reversed by NAC. Bioinformatics analysis showed that Drp1 played a crucial role in OSCC progression. OSCC patients with low Drp1 expression had a higher survival rate. The OSCC cancer tissues presented higher phosphorylated-Drp1 and Drp1 levels than the normal tissues. The results further showed that ALT suppressed Drp1 phosphorylation in OSCC cells. Moreover, Drp1 overexpression abolished the reduced Drp1 phosphorylation by ALT and promoted the cell viability of ALT-treated cells. Drp1 overexpression also reversed the mitochondrial dysfunction induced by ALT, with decreased ROS production, and increased mitochondrial membrane potential and ATP level.

Conclusions: ALT inhibited proliferation and promoted apoptosis of oral squamous cell carcinoma cells via impairment of mitochondrial homeostasis and regulation of Drp1. The results provide a solid basis for ALT as a therapeutic candidate for treating OSCC, with Drp1 being a novel therapeutic target in treating OSCC.

Comments:

The study investigated the effects of alantolactone (ALT) on oral squamous cell carcinoma (OSCC) and the underlying mechanisms. ALT is a compound with anticancer properties and the ability to regulate mitochondrial events. The researchers aimed to explore the potential of ALT as a therapeutic strategy for OSCC and identify the role of dynamin-related protein 1 (Drp1) in OSCC progression.

To conduct the study, OSCC cells were treated with different concentrations and durations of ALT and N-Acetyl-L-cysteine (NAC), which is an antioxidant. The effects of ALT on cell viability, colony formation, and apoptosis were assessed. Apoptosis was evaluated using flow cytometry with Annexin V-FITC/PI double staining. Reactive oxygen species (ROS) production was measured using DCFH-DA and flow cytometry, while reactive nitrogen species (RNS) levels were investigated using DAF-FM DA. Mitochondrial function was assessed by measuring mitochondrial ROS, mitochondrial membrane potential (MMP), and ATP levels.

The study also employed KEGG enrichment analyses to identify mitochondrial-related hub genes involved in OSCC progression. Drp1, a protein associated with mitochondrial dynamics, was found to play a crucial role in OSCC progression. Immunohistochemistry staining and western blot were used to verify the expression of proteins in OSCC tissues.

The results of the study demonstrated that ALT had anti-proliferative and pro-apoptotic effects on OSCC cells. Mechanistically, ALT induced cell injury by promoting ROS production, mitochondrial membrane depolarization, and ATP depletion. These effects were reversed by NAC, indicating the involvement of oxidative stress. Bioinformatics analysis revealed that Drp1 was important in OSCC progression, and OSCC patients with low Drp1 expression had a higher survival rate. OSCC cancer tissues exhibited higher levels of phosphorylated-Drp1 and Drp1 compared to normal tissues. ALT was found to suppress Drp1 phosphorylation in OSCC cells. Furthermore, when Drp1 was overexpressed, it counteracted the reduction in Drp1 phosphorylation induced by ALT and increased the viability of ALT-treated cells. Drp1 overexpression also mitigated the mitochondrial dysfunction induced by ALT, reducing ROS production and increasing mitochondrial membrane potential and ATP levels.

In conclusion, the study demonstrated that ALT inhibited OSCC cell proliferation and promoted apoptosis by impairing mitochondrial homeostasis and regulating Drp1. These findings suggest that ALT could be a potential therapeutic candidate for treating OSCC, with Drp1 identified as a novel therapeutic target in OSCC treatment.