Key Laboratory for Biorheological Science and Technology of Ministry of Education (Chongqing University), Chongqing University Cancer Hospital and Chongqing Cancer Institute and Chongqing Cancer Hospital, Chongqing 400030, P.R. China.
Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital and Chongqing Cancer Institute and Chongqing Cancer Hospital, Chongqing 400030, P.R. China.
Oncol Rep. 2019 Feb;41(2):1264-1274. doi: 10.3892/or.2018.6905. Epub 2018 Dec 6.
Reactive oxygen species (ROS) have been widely accepted as critical molecules playing regulatory roles in various biological processes, including proliferation, differentiation and apoptotic/ferroptotic/necrotic cell death. Emerging evidence suggests that ROS may be involved in the induction of epithelial‑to‑mesenchymal transition (EMT), which has been reported to promote cancer stem‑like cell (CSC) generation. Recent data indicate that altered accumulation of ROS is associated with CSC generation, EMT and hypoxia exposure, but the underlying mechanisms are poorly understood. In the present study, we derived CSCs from Panc‑1 human pancreatic cancer cells and characterized them using serial replating assays and western blot analysis. Functional identification of viable cells was performed using the CCK‑8 assay and colony formation assays. The expression of various antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GPX), was measured by western blot analysis in Panc‑1 CSCs. The role of GPX4 in regulating biological processes of Panc‑1 CSCs was assessed by proliferation, sphere formation and invasion assays with or without oxidative stress. Manipulation of GPX4 expression by siRNA knockdown or an overexpression vector was performed to assess functions including proliferation, colony formation and invasion. EMT hallmark genes were detected after GPX4 alteration by RT‑qPCR and western blot analysis. Panc‑1 CSCs displayed more resistance to hypoxia exposure. Compared with the parental Panc‑1 cells, Panc‑1 CSCs expressed an obviously higher endogenous GPX4 level, indicating their role in maintaining homeostasis. During GPX4 knockdown, ROS accumulation was promoted following oxidative stress exposure to either H2O2 or erastin. Additionally, overexpression of GPX4 eliminated ROS induction by oxidative stress exposure and thus, exerted protective effects on physiological processes in the Panc‑1 CSCs. Knockdown of GPX4 arrested cell cycle progression at the G1/G0 phase; inhibited cell proliferation, colony formation, invasion and the stemness phenotype in the Panc‑1 CSCs; and decreased the EMT phenotype. Collectively, GPX4 plays a critical role in maintaining oxidative homeostasis and regulates several biological processes, including stemness and EMT, in Panc‑1 CSCs.
活性氧 (ROS) 已被广泛认为是在各种生物过程中发挥调节作用的关键分子，包括增殖、分化和凋亡/铁死亡/坏死细胞死亡。新出现的证据表明，ROS 可能参与上皮-间充质转化 (EMT) 的诱导，已有报道称 EMT 可促进癌症干细胞 (CSC) 的产生。最近的数据表明，ROS 的积累改变与 CSC 的产生、EMT 和缺氧暴露有关，但潜在的机制尚不清楚。在本研究中，我们从人胰腺癌细胞系 Panc-1 中获得了 CSCs，并通过连续平板克隆形成实验和 Western blot 分析对其进行了表征。通过 CCK-8 测定和集落形成实验对存活细胞的功能进行了鉴定。通过 Western blot 分析测定了 Panc-1 CSCs 中各种抗氧化酶，包括超氧化物歧化酶 (SOD) 和谷胱甘肽过氧化物酶 (GPX) 的表达。通过使用或不使用氧化应激的增殖、球体形成和侵袭实验评估了 GPX4 在调节 Panc-1 CSCs 生物学过程中的作用。通过 siRNA 敲低或过表达载体操纵 GPX4 的表达，以评估包括增殖、集落形成和侵袭在内的功能。通过 RT-qPCR 和 Western blot 分析检测 GPX4 改变后 EMT 标志基因的表达。Panc-1 CSCs 对缺氧暴露表现出更强的抗性。与亲本 Panc-1 细胞相比，Panc-1 CSCs 表达明显更高的内源性 GPX4 水平，表明其在维持内稳态中的作用。在 GPX4 敲低后，Panc-1 CSCs 在受到 H2O2 或 erastin 氧化应激暴露后，ROS 积累增加。此外，过表达 GPX4 消除了氧化应激暴露诱导的 ROS 诱导，从而对 Panc-1 CSCs 中的生理过程发挥保护作用。GPX4 敲低使细胞周期停滞在 G1/G0 期；抑制 Panc-1 CSCs 的增殖、集落形成、侵袭和干细胞表型；并降低 EMT 表型。总之，GPX4 在维持氧化还原平衡方面发挥着关键作用，并调节包括 Panc-1 CSCs 中的干性和 EMT 在内的几种生物学过程。
