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Abstract：
OBJECTIVE: Resveratrol (Res) is a natural phytoestrogen with antitumor activity. This study sought to investigate the role of Res in ferroptosis in oral squamous cell carcinoma (OSCC).
METHODS: Normal human oral keratinocyte (HOK)/oral OSCC (CAL-27/SCC-9) cell lines were treated with different doses of Res. Res toxicity was determined by MTT assay, with half maximal inhibitory concentration values of Res on CAL-27 and SCC-9 cells calculated. Cell viability/colony formation efficiency/migration/invasion/cycle were assessed by CCK-8/colony formation assay/transwell assay/flow cytometry. The expression of p53 protein in the nucleus and cytoplasm, glutathione peroxidase 4 (GPX4) expression, and SLC7A11 messenger RNA (mRNA) and protein expression levels were determined by Western blot and RT-qPCR. Fe2+ content, reactive oxygen species (ROS) level, reduced glutathione (GSH), and lactate dehydrogenase (LDH) release were assessed.
RESULTS: Medium- to low-dose Res had no toxic effect on HOK cells, while high-dose Res markedly reduced HOK cell viability. Res significantly suppressed the viability of OSCC cells (CAL-27 and SCC-9). Res inhibited OSCC cell colony formation/migration/invasion, and induced G1 phase arrest. Res caused the translocation of p53 protein to the nucleus, obviously increased Fe2+ content, ROS level and LDH release, decreased GSH content and GPX4 protein expression, and induced ferroptosis. Down-regulation of p53 partially reversed the inhibitory effects of Res on CAL-27 cell malignant behaviors. Res inhibited SLC7A11 transcription by promoting p53 entry into the nucleus. SLC7A11 overexpression negated the the regulatory effects of p53 knockout on the role of Res in OSCC cell malignant behaviors and ferroptosis.
CONCLUSIONS: Res accelerated ferroptosis and inhibited malignant behaviors in OSCC cells by regulating p53/SLC7A11.
METHODS: Normal human oral keratinocyte (HOK)/oral OSCC (CAL-27/SCC-9) cell lines were treated with different doses of Res. Res toxicity was determined by MTT assay, with half maximal inhibitory concentration values of Res on CAL-27 and SCC-9 cells calculated. Cell viability/colony formation efficiency/migration/invasion/cycle were assessed by CCK-8/colony formation assay/transwell assay/flow cytometry. The expression of p53 protein in the nucleus and cytoplasm, glutathione peroxidase 4 (GPX4) expression, and SLC7A11 messenger RNA (mRNA) and protein expression levels were determined by Western blot and RT-qPCR. Fe2+ content, reactive oxygen species (ROS) level, reduced glutathione (GSH), and lactate dehydrogenase (LDH) release were assessed.
RESULTS: Medium- to low-dose Res had no toxic effect on HOK cells, while high-dose Res markedly reduced HOK cell viability. Res significantly suppressed the viability of OSCC cells (CAL-27 and SCC-9). Res inhibited OSCC cell colony formation/migration/invasion, and induced G1 phase arrest. Res caused the translocation of p53 protein to the nucleus, obviously increased Fe2+ content, ROS level and LDH release, decreased GSH content and GPX4 protein expression, and induced ferroptosis. Down-regulation of p53 partially reversed the inhibitory effects of Res on CAL-27 cell malignant behaviors. Res inhibited SLC7A11 transcription by promoting p53 entry into the nucleus. SLC7A11 overexpression negated the the regulatory effects of p53 knockout on the role of Res in OSCC cell malignant behaviors and ferroptosis.
CONCLUSIONS: Res accelerated ferroptosis and inhibited malignant behaviors in OSCC cells by regulating p53/SLC7A11.
摘要：
目的：白藜芦醇（Res）是一种具有抗肿瘤活性的天然植物雌激素。本研究旨在探讨Res在口腔鳞状细胞癌（OSCC）铁凋亡中的作用。
方法：用不同剂量的Res处理正常人口腔角质形成细胞（HOK）/口服OSCC（CAL-27/SCC-9）细胞系。用MTT法测定Res毒性，计算Res对CAL-27和SCC-9细胞的半数最大抑制浓度值。通过CCK-8/集落形成测定/transwell测定/流式细胞术评估细胞活力/集落形成效率/迁移/侵袭/周期。p53蛋白在细胞核和细胞质中的表达，谷胱甘肽过氧化物酶4（GPX4）的表达，通过Westernblot和RT-qPCR检测SLC7A11信使RNA(mRNA)和蛋白表达水平。Fe2+含量,活性氧（ROS）水平，还原型谷胱甘肽（GSH），和乳酸脱氢酶（LDH）的释放进行了评估。
结果：中低剂量Res对HOK细胞没有毒性作用，而高剂量Res显著降低HOK细胞活力。Res显著抑制OSCC细胞(CAL-27和SCC-9)的活力。Res抑制OSCC细胞集落形成/迁移/侵袭，并诱导G1期停滞。Res导致p53蛋白易位到细胞核，Fe2+含量明显增加,ROS水平和LDH释放，GSH含量和GPX4蛋白表达降低，并诱导铁性凋亡。p53的下调部分逆转了Res对CAL-27细胞恶性行为的抑制作用。Res通过促进p53进入细胞核来抑制SLC7A11转录。SLC7A11过表达否定了p53敲除对Res在OSCC细胞恶性行为和铁凋亡中的作用的调节作用。
结论：Res通过调节p53/SLC7A11促进OSCC细胞的铁凋亡并抑制其恶性行为。
方法：用不同剂量的Res处理正常人口腔角质形成细胞（HOK）/口服OSCC（CAL-27/SCC-9）细胞系。用MTT法测定Res毒性，计算Res对CAL-27和SCC-9细胞的半数最大抑制浓度值。通过CCK-8/集落形成测定/transwell测定/流式细胞术评估细胞活力/集落形成效率/迁移/侵袭/周期。p53蛋白在细胞核和细胞质中的表达，谷胱甘肽过氧化物酶4（GPX4）的表达，通过Westernblot和RT-qPCR检测SLC7A11信使RNA(mRNA)和蛋白表达水平。Fe2+含量,活性氧（ROS）水平，还原型谷胱甘肽（GSH），和乳酸脱氢酶（LDH）的释放进行了评估。
结果：中低剂量Res对HOK细胞没有毒性作用，而高剂量Res显著降低HOK细胞活力。Res显著抑制OSCC细胞(CAL-27和SCC-9)的活力。Res抑制OSCC细胞集落形成/迁移/侵袭，并诱导G1期停滞。Res导致p53蛋白易位到细胞核，Fe2+含量明显增加,ROS水平和LDH释放，GSH含量和GPX4蛋白表达降低，并诱导铁性凋亡。p53的下调部分逆转了Res对CAL-27细胞恶性行为的抑制作用。Res通过促进p53进入细胞核来抑制SLC7A11转录。SLC7A11过表达否定了p53敲除对Res在OSCC细胞恶性行为和铁凋亡中的作用的调节作用。
结论：Res通过调节p53/SLC7A11促进OSCC细胞的铁凋亡并抑制其恶性行为。
