West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China; Key Laboratory of Integrative Translational Medicine of Guangxi High Incidence Infectious Diseases, Nanning 530200, China; School of Preclinical Medicine, Guangxi University of Chinese Medicine, Nanning 530200, China.
Guangxi Institute for Food and Drug Control, Nanning 530021, China.
J Ethnopharmacol. 2024 Oct 5;332:118340. doi: 10.1016/j.jep.2024.118340. Epub 2024 May 16.
ETHNOPHARMACOLOGICAL RELEVANCE: Adinandra nitida Merr. ex Li leaves serve as a herbal tea and hold a significant role in traditional Chinese medicine, being applied to assist in tumor treatment. Flavonoids present the primary bioactive constituents in Adinandra nitida Merr. ex Li leaves. AIM OF THE STUDY: To explore the potential of total flavonoids from Adinandra nitida Merr. ex Li Leaves (TFAN) in inhibiting non-small cell lung cancer (NSCLC) and further elucidate the underlying mechanisms. MATERIALS AND METHODS: Human NSCLC cell lines and normal lung cell line were employed to assess the impact of TFAN (0-160 μg/mL for 24, 28 and 72 h) on cell proliferation in vitro. Immunofluorescence (IF) staining gauged p53 expression changes in NSCLC cells under TFAN present condition (150 μg/mL for 24 h). In vivo study utilized NSCLC cell derived xenograft tumors in nude mice, administering TFAN orally (200 and 400 mg/kg) for 14 days. Immunohistochemistry assessed Cleaved Caspase 3 expression change in A549 xenograft tumors treated with TFAN (400 mg/kg for 14 days). RNA-seq and KEGG analysis identified gene expression changes and enriched processes in A549 xenograft tumors treated with TFAN. CM-HDCFDA and metabolomics assessed ROS level and GSH/GSSG pool changes in A549 cells under TFAN present condition. Cell viability assay and IF staining assessed A549 cell proliferation and p53 expression changes under HO-induced oxidative stress (0-40 μM for 24 h) and TFAN present conditions. GSEA and N-Acetyl-L-cysteine (NAC) rescue (0-1 μM for 24 h) analyzed the impact of TFAN on GSH de novo synthesis. NADPH/NADP pool measurement and NADPH rescue (0-10 μM for 24 h) analyzed the impact of TFAN on GSH salvage synthesis. GC-FID and HPLC-MS were utilized to detect ethanol and ethyl acetate residues, and to characterize the chemical constituents in TFAN, respectively. The total flavonoid content of TFAN was determined using a 330 nm wavelength. RESULTS: TFAN significantly inhibited A549 cells (wild-type p53) but not NCI-H1299 cells (p53-deficient), NCI-H596 cells (p53-mutant) or BEAS-2B in vitro. IF staining validated p53 genotype for the cell lines and revealed an increase in p53 expression in A549 cells after TFAN treatment. In vivo, TFAN selectively inhibited A549 xenograft tumor growth without discernible toxicity, inducing apoptosis evidenced by Cleaved Caspase 3 upregulation. RNA-seq and KEGG analysis suggested ROS biosynthesis was involved in TFAN-induced p53 activation in A549 cells. Elevated ROS level in TFAN-treated A549 cells were observed. Moreover, TFAN sensitized A549 cells to HO-induced oxidative stress, with higher p53 expression. Additionally, A549 cells compensated with GSH de novo synthesis under TFAN present condition, confirmed by GSEA and NAC rescue experiment. TFAN disrupted NADPH homeostasis to impair GSH salvage biosynthesis, supported by NADPH/NADP change and NADPH rescue experiment. The chemical constituents of TFAN, with acceptable limits for ethanol and ethyl acetate residues and a total flavonoid content of 68.87%, included Catechin, Epicatechin, Quercitroside, Camellianin A, and Apigenin. CONCLUSION: The disruption of NADPH homeostasis by TFAN triggers ROS-dependent p53 activation that leads to apoptotic cell death, ultimately suppressing NSCLC growth. These findings offer potential therapeutic implications of Adinandra nitida Merr. ex Li leaves in combating NSCLC.
植物学相关内容：阿丁枫的叶子被用作草药，在传统中医中具有重要的地位，可用于辅助肿瘤治疗。黄酮类化合物是阿丁枫叶子中的主要生物活性成分。
研究目的：探索来自阿丁枫叶子的总黄酮（TFAN）抑制非小细胞肺癌（NSCLC）的潜力，并进一步阐明其潜在机制。
材料与方法：采用人类 NSCLC 细胞系和正常肺细胞系，评估 TFAN（0-160μg/ml，24、28 和 72 小时）对体外 NSCLC 细胞增殖的影响。免疫荧光（IF）染色检测 TFAN 存在条件下（24 小时，150μg/ml）p53 表达的变化。在 NSCLC 细胞衍生的裸鼠异种移植瘤模型中，口服给予 TFAN（200 和 400mg/kg）14 天。免疫组化评估 TFAN（400mg/kg，14 天）处理的 A549 异种移植瘤中 Cleaved Caspase 3 的表达变化。RNA-seq 和 KEGG 分析鉴定 TFAN 处理的 A549 异种移植瘤中的基因表达变化和富集过程。CM-HDCFDA 和代谢组学评估 TFAN 存在条件下 A549 细胞内 ROS 水平和 GSH/GSSG 池的变化。细胞活力测定和 IF 染色评估在 HO 诱导的氧化应激（0-40μM，24 小时）和 TFAN 存在条件下 A549 细胞增殖和 p53 表达的变化。GSEA 和 N-乙酰-L-半胱氨酸（NAC）解救（0-1μM，24 小时）分析 TFAN 对 GSH 从头合成的影响。NADPH/NADP 池测量和 NADPH 解救（0-10μM，24 小时）分析 TFAN 对 GSH 补救合成的影响。GC-FID 和 HPLC-MS 分别用于检测乙醇和乙酸乙酯残留，并分别对 TFAN 中的化学成分进行了表征。使用 330nm 波长测定 TFAN 的总黄酮含量。
结果：TFAN 显著抑制 A549 细胞（野生型 p53），但不抑制 NCI-H1299 细胞（p53 缺失）、NCI-H596 细胞（p53 突变）或 BEAS-2B 细胞，体外实验结果一致。IF 染色验证了细胞系的 p53 基因型，并显示 TFAN 处理后 A549 细胞中 p53 表达增加。在体内，TFAN 选择性抑制 A549 异种移植瘤生长，无明显毒性，诱导细胞凋亡，Cleaved Caspase 3 上调。RNA-seq 和 KEGG 分析表明，ROS 生物合成参与了 TFAN 诱导的 A549 细胞中 p53 的激活。在 TFAN 处理的 A549 细胞中观察到 ROS 水平升高。此外，TFAN 增敏 A549 细胞对 HO 诱导的氧化应激，p53 表达更高。此外，在 TFAN 存在条件下，A549 细胞通过 GSH 从头合成补偿，GSEA 和 NAC 解救实验得到证实。TFAN 破坏 NADPH 稳态，抑制 GSH 补救生物合成，NADPH/NADP 变化和 NADPH 解救实验得到支持。TFAN 的化学成分包括儿茶素、表儿茶素、槲皮素半乳糖苷、山茶素 A 和芹菜素，具有可接受的乙醇和乙酸乙酯残留限量和 68.87%的总黄酮含量。
结论：TFAN 破坏 NADPH 稳态，引发 ROS 依赖性 p53 激活，导致细胞凋亡，最终抑制 NSCLC 生长。这些发现为阿丁枫叶子在治疗 NSCLC 方面提供了潜在的治疗意义。
Phytomedicine. 2018-9-20
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