Wang Yanhong, Mang Xinyu, Li Danni, Chen Yiliang, Cai Zhenyu, Tan Fei
Department of ORL-HNS, Shanghai Fourth People's Hospital, and School of Medicine, Tongji University, Shanghai 200432, China; Department of Pharmacy, People's Hospital of Gansu Province, Lanzhou 730000, Gansu, China.
Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, And School of Basic Medicine Peking Union Medical College, Beijing 100005, China.
Free Radic Biol Med. 2023 Nov 1;208:134-152. doi: 10.1016/j.freeradbiomed.2023.07.036. Epub 2023 Aug 4.
Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the fourth leading cause of cancer-related death worldwide. Advanced or metastatic HCC is currently managed using systemic drug therapy with unsatisfactory patient survival. Cold atmospheric plasma has emerged as a promising, physicochemical, and broad-spectrum oncotherapy. In this preclinical study, we investigated the anti-neoplastic functions and mechanism of piezoelectric direct discharge technology-based CAP, Piezo-CAP, on HCC in vitro and in vivo. Various HCC cells lines, such as SMMC7721, HepG2 and LM3, were used as in vitro cancer model for the phenotypic and mechanistic studies. Specifically, the cell counting Kit-8 and colony formation assay, flow cytometry, Transwell assay, Western blot, reactive oxygen species (ROS) assay, and glutathione to oxidized glutathione ratio (GSH/GSSG) assay were used to demonstrate plasma-induced changes in HCC cell proliferation, cell cycle progression, migration and invasion, epithelial-to-mesenchymal transition, intracellular ROS, and antioxidant capacity, respectively. In addition, the Acridine orange and ethidium bromide (AO/EB) staining and transmission electron microscopy were performed for cellular and subcellular assessment of HCC cell apoptosis. The Ad-mCherry-RFP-LC3B fluorescent double-labeled lentiviral system was used to detect autophagic flux. On the other hand, RNA-sequencing, quantitative real-time PCR, and Western blot were used to demonstrate plasma-induced metabolic and molecular disruption of tumor glycolysis and oncogenic proliferation, respectively. In vivo experiments using a human cell-line-derived xenograft model and immunohistochemistry (IHC) were utilized to investigate the mechanism. Piezo-CAP exerted anti-neoplastic functions through inhibiting cell proliferation, migration and invasion, and promote cell apoptosis and autophagy. Treatment of Piezo-CAP could suppress proliferation and induce autophagy of HCC cells through simultaneously disrupts cancer survival pathways of redox deregulation, glycolytic pathway, and PI3K/AKT/mTOR/HIF1α pathway signaling. Moreover, upon translation of these in vitro results into the tissue level, Piezo-CAP significantly suppressed in situ tumor growth. These findings collectively suggest that Piezo-CAP-induced apoptosis and autophagy of HCC cells though a multitargeted blockade of major cancer survival pathways of deregulated redox balance, glycolysis, and PI3K/AKT/mTOR/HIF-1α signaling.
肝细胞癌（HCC）是全球第六大常见癌症，也是癌症相关死亡的第四大主要原因。晚期或转移性HCC目前采用全身药物治疗，但患者生存率不尽人意。冷大气等离子体已成为一种有前景的物理化学和广谱肿瘤治疗方法。在这项临床前研究中，我们研究了基于压电直接放电技术的冷大气等离子体（Piezo-CAP）对HCC的体外和体内抗肿瘤功能及机制。各种HCC细胞系，如SMMC7721、HepG2和LM3，被用作体外癌症模型进行表型和机制研究。具体而言，分别使用细胞计数试剂盒-8和集落形成试验、流式细胞术、Transwell试验、蛋白质印迹法、活性氧（ROS）试验以及谷胱甘肽与氧化型谷胱甘肽比率（GSH/GSSG）试验，来证明等离子体诱导的HCC细胞增殖、细胞周期进程、迁移和侵袭、上皮-间质转化、细胞内ROS以及抗氧化能力的变化。此外，进行吖啶橙和溴化乙锭（AO/EB）染色以及透射电子显微镜检查，以对HCC细胞凋亡进行细胞和亚细胞评估。使用Ad-mCherry-RFP-LC3B荧光双标记慢病毒系统检测自噬通量。另一方面，分别使用RNA测序、定量实时PCR和蛋白质印迹法，来证明等离子体诱导的肿瘤糖酵解和致癌增殖的代谢及分子破坏。使用人细胞系来源的异种移植模型和免疫组织化学（IHC）进行体内实验以研究其机制。Piezo-CAP通过抑制细胞增殖、迁移和侵袭发挥抗肿瘤功能，并促进细胞凋亡和自噬。Piezo-CAP治疗可通过同时破坏氧化还原失调、糖酵解途径以及PI3K/AKT/mTOR/HIF1α途径信号传导的癌症生存途径，来抑制HCC细胞的增殖并诱导自噬。此外，将这些体外结果转化到组织水平后，Piezo-CAP可显著抑制原位肿瘤生长。这些发现共同表明，Piezo-CAP通过多靶点阻断氧化还原平衡失调、糖酵解以及PI3K/AKT/mTOR/HIF-1α信号传导等主要癌症生存途径，诱导HCC细胞凋亡和自噬。
