肝细胞癌（HCC）是全球第六常见的癌症和第四大导致癌症相关死亡的原因。目前，晚期或转移性HCC主要进行系统性药物治疗，但患者生存率不尽如人意。冷大气等离子体作为一种有希望的、物理化学的、广谱的抗癌疗法应运而生。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等离子体，即压电冷等离子体（Piezo-CAP）对HCC的体外和体内抗肿瘤功能和机制。我们使用了多种HCC细胞系，如SMMC7721、HepG2和LM3，作为体外癌症模型，用于表型和机制研究。具体来说，我们采用了细胞计数试剂盒-8和集落形成实验、流式细胞术、转移实验、蛋白质印迹、活性氧（ROS）试剂盒和还原型谷胱甘肽/氧化型谷胱甘肽比（GSH/GSSG）试剂盒来证明等离子体诱导的HCC细胞增殖、细胞周期进展、迁移和侵袭、上皮-间质转变、细胞内ROS和抗氧化能力的变化。此外，我们还进行了吖啶橙/溴乙酸酯（AO/EB）染色和透射电子显微镜观察HCC细胞凋亡的细胞和亚细胞评估。我们使用荧光双标记的Ad-mCherry-RFP-LC3B慢病毒系统来检测自噬流。另一方面，我们采用RNA-测序、定量实时PCR和蛋白质印迹技术来证明等离子体诱导的肿瘤糖酵解和致癌增殖的代谢和分子破坏。我们使用人细胞株异种移植模型和免疫组织化学（IHC）对体内实验进行机理研究。通过抑制细胞增殖、迁移和侵袭，促进细胞凋亡和自噬，压电冷等离子体发挥了抗肿瘤功能。压电冷等离子体处理可以通过同时破坏氧化还原失衡、糖酵解途径和PI3K/AKT/mTOR/HIF1α信号通路的癌症存活途径，抑制HCC细胞的增殖并诱导细胞自噬。此外，在将这些体外结果转化为组织层面时，压电冷等离子体显著抑制了原位肿瘤生长。这些发现共同表明，压电冷等离子体通过多靶向阻断调控癌症存活途径（包括失调的氧还原平衡、糖酵解和PI3K/AKT/mTOR/HIF-1α信号通路），诱导HCC细胞的凋亡和自噬。版权所有© 2023，由爱思唯尔出版。

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.Copyright © 2023. Published by Elsevier Inc.