School and Hospital of Stomatology, Institute of Stomatology, Wenzhou Medical University, Wenzhou, China.
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China.
J Transl Med. 2023 May 18;21(1):328. doi: 10.1186/s12967-023-04188-2.
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.
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.
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.
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.
口腔鳞状细胞癌（OSCC）是最常见和最致命的口腔癌之一。线粒体靶向治疗是对抗各种癌症的有前途的策略，但它们在治疗 OSCC 中的应用有限。冬凌草甲素（ALT）具有抗癌特性，还能调节线粒体事件。在本研究中，我们探讨了 ALT 对 OSCC 的影响及其相关机制。
用不同浓度和时间的 ALT 和 N-乙酰-L-半胱氨酸（NAC）处理 OSCC 细胞。评估细胞活力和集落形成。用 Annexin V-FITC/PI 双重染色通过流式细胞术评估细胞凋亡率。我们使用 DCFH-DA 和流式细胞术检测活性氧（ROS）的产生，并使用 DAF-FM DA 检测活性氮（RNS）水平。线粒体功能通过线粒体活性氧（ROS）、线粒体膜电位（MMP）和 ATP 水平来反映。KEGG 富集分析确定了与 OSCC 进展相关的线粒体相关枢纽基因。进一步将 dynamin 相关蛋白 1（Drp1）过表达质粒转染到细胞中，分析 Drp1 在 OSCC 进展中的作用。免疫组织化学染色和 Western blot 验证了蛋白的表达。
ALT 对 OSCC 细胞表现出抗增殖和促凋亡作用。机制上，ALT 通过促进 ROS 产生、线粒体膜去极化和 ATP 耗竭来引起细胞损伤，而 NAC 则逆转了这种损伤。生物信息学分析表明，Drp1 在 OSCC 进展中起着关键作用。Drp1 低表达的 OSCC 患者的存活率更高。与正常组织相比，OSCC 癌组织中磷酸化 Drp1 和 Drp1 的水平更高。结果进一步表明，ALT 抑制了 OSCC 细胞中 Drp1 的磷酸化。此外，Drp1 过表达消除了 ALT 降低的 Drp1 磷酸化，并促进了 ALT 处理细胞的细胞活力。Drp1 过表达还逆转了 ALT 诱导的线粒体功能障碍，减少了 ROS 的产生，增加了线粒体膜电位和 ATP 水平。
ALT 通过破坏线粒体稳态和调节 Drp1 来抑制口腔鳞状细胞癌细胞的增殖并促进其凋亡。结果为 ALT 作为治疗 OSCC 的候选药物提供了坚实的基础，Drp1 是治疗 OSCC 的新的治疗靶点。
