Peng Yong-Chun, He Zhi-Jing, Yin Lun-Cai, Pi Hui-Feng, Jiang Yi, Li Ke-Yan, Tian Li, Xie Jia, Zhang Jian-Bo, Li Chen-Yao, Feng Guan-Ying, Wang Kai, Zhou Ding-Zhou, Xie Xiao-Wei, Zhang Zhi-Yuan, Fan Teng-Fei
Department of Oral and Maxillofacial Surgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China.
Department of Oncology, Affiliated Dazu Hospital of Chongqing Medical University, Chongqing 402360, China.
Phytomedicine. 2025 Jan;136:156337. doi: 10.1016/j.phymed.2024.156337. Epub 2024 Dec 18.
Oral squamous cell carcinoma (OSCC) is one of the most common malignancies. However, there is no effective treatment for OSCC.
This study aimed to identify a natural compound with significant efficacy against OSCC and elucidate its primary mechanism of action.
An FDA-approved drug library and an MCE autophagy-related molecular compound library were screened through high-throughput screening to identify an effective natural compound against OSCC. The IC50 value of sanguinarine (Sang) in OSCC cells was determined using a CCK8 assay. Immunoblotting and immunofluorescence staining were used to assess the effect of Sang on autophagic flux in OSCC cells. Changes in the acidic lysosomal environment were evaluated using RFP-GFP-LC3B and LysoSensor Green DND-189. Furthermore, limited proteolysis-coupled mass spectrometry (LiP-MS) and virtual screening techniques were utilized to identify direct binding targets of Sang, which were subsequently validated by surface plasmon resonance (SPR) and microscale thermophoresis (MST). Molecular docking combined with molecular dynamics analysis identified the binding site between the target protein and Sang. In vitro and in vivo investigations with mutant plasmids confirmed this finding.
Screening led to the identification of the naturally occurring autophagy modulator Sang as a potent inhibitor of OSCC progression. Moreover, Sang impaired lysosomal function through reducing lysosomal-associated membrane proteins, inhibiting lysosomal proteolysis, and altering the lysosomal pH. These effects contributed to defects in autophagic clearance and subsequently suppressed OSCC progression. Notably, Sang bound the phenylalanine 26 (F26) residue in pyruvate kinase M2 (PKM2) and inhibited PKM2 enzymatic activity, subsequently suppressing transcription factor EB (TFEB) expression to inhibit lysosomal function and blocking autophagic flux in OSCC cells.
Our results demonstrate for the first time that Sang can suppress the PKM2/TFEB axis, and influence lysosomal function, thereby blocking autophagy and inhibiting the progression of OSCC, making it a promising therapeutic option for the treatment of OSCC.
口腔鳞状细胞癌（OSCC）是最常见的恶性肿瘤之一。然而，目前尚无有效的OSCC治疗方法。
本研究旨在鉴定一种对OSCC具有显著疗效的天然化合物，并阐明其主要作用机制。
通过高通量筛选对美国食品药品监督管理局（FDA）批准的药物库和MCE自噬相关分子化合物库进行筛选，以确定一种有效的抗OSCC天然化合物。使用CCK8法测定血根碱（Sang）在OSCC细胞中的半数抑制浓度（IC50）值。采用免疫印迹和免疫荧光染色评估Sang对OSCC细胞自噬通量的影响。使用RFP-GFP-LC3B和溶酶体传感器绿色DND-189评估酸性溶酶体环境的变化。此外，利用有限蛋白水解偶联质谱（LiP-MS）和虚拟筛选技术鉴定Sang的直接结合靶点，随后通过表面等离子体共振（SPR）和微量热泳动（MST）进行验证。分子对接结合分子动力学分析确定了靶蛋白与Sang之间的结合位点。使用突变质粒进行的体外和体内研究证实了这一发现。
筛选鉴定出天然存在的自噬调节剂Sang是OSCC进展的有效抑制剂。此外，Sang通过减少溶酶体相关膜蛋白、抑制溶酶体蛋白水解和改变溶酶体pH来损害溶酶体功能。这些作用导致自噬清除缺陷，进而抑制OSCC进展。值得注意的是，Sang与丙酮酸激酶M2（PKM2）中的苯丙氨酸26（F26）残基结合并抑制PKM2酶活性，随后抑制转录因子EB（TFEB）表达，从而抑制溶酶体功能并阻断OSCC细胞中的自噬通量。
我们的结果首次表明，Sang可以抑制PKM2/TFEB轴，影响溶酶体功能，从而阻断自噬并抑制OSCC进展，使其成为治疗OSCC的有前景的治疗选择。
