Bufadienolides derived from the skin of toads are often regarded as the main active components with antitumor effects. 19-Hydroxybufalin (19-HB) is a monomer of bufadienolides; however, its effects and underlying molecular mechanisms on tumor growth remain to be ascertained. In this report, we focused on the antitumor effects of 19-HB on non-small cell lung cancer to provide a scientific basis for its further development and utilization. The antitumor effects of 19-HB on the human NSCLC cell lines NCI-H1299 and NCI-H838 were examined in vitro. The cells were treated with different concentrations of 19-HB, and the inhibition of cell growth was measured by CCK-8 and colony formation assays. Furthermore, cell apoptosis was analyzed by flow cytometry, TUNEL staining, JC-1 staining, and western blotting. The effects on migration and invasion were evaluated by wound-healing assay, transwell assay, and western blotting. Finally, the antitumor effects of 19-HB were evaluated in vivo using a xenograft mouse model. 19-HB-treated NSCLC cells showed inhibited cell viability and increased apoptosis. The expression levels of cleaved caspase-3, cleaved-PARP, and Bax/Bcl-2 were upregulated, while the mitochondrial membrane potential decreased. In contrast, migration, invasion, as well as the expression of MMP2, MMP7, MMP9, the epithelial–mesenchymal transition-related proteins N-cadherin and Vimentin, and the transcription factors Snail and Slug were inhibited. Furthermore, the expression levels of the key molecules in the Wnt/β-catenin signaling pathway (CyclinD1, c-Myc, and β-catenin) were decreased. In vivo, the growth of xenograft tumors in nude mice was also significantly inhibited by 19-HB, and there were no significant changes in biochemical indicators of hepatic and renal function. 19-HB inhibited the proliferation, migration, and invasion, and promoted the apoptosis of NSCLC cells via the Wnt/β-catenin pathway. In addition, 19-HB inhibited the growth of xenograft tumors in nude mice with little toxicity to the liver and kidney. Thus, 19-HB may be a potential antitumor agent for treating NSCLC.

中文翻译：

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19-Hydroxybufalin 通过 Wnt/β-catenin 通路抑制非小细胞肺癌细胞增殖并促进细胞凋亡

蟾蜍皮肤中提取的蟾蜍二烯内酯通常被认为是具有抗肿瘤作用的主要活性成分。19-Hydroxybufalin (19-HB) 是一种蟾蜍二烯内酯的单体；然而，其对肿瘤生长的影响和潜在的分子机制仍有待确定。本报告重点研究19-HB对非小细胞肺癌的抗肿瘤作用，为其进一步开发利用提供科学依据。在体外检查了 19-HB 对人 NSCLC 细胞系 NCI-H1299 和 NCI-H838 的抗肿瘤作用。用不同浓度的 19-HB 处理细胞，并通过 CCK-8 和集落形成试验测量细胞生长的抑制。此外，通过流式细胞术、TUNEL 染色、JC-1 染色和蛋白质印迹分析细胞凋亡。通过伤口愈合试验、transwell 试验和蛋白质印迹评估对迁移和侵袭的影响。最后，使用异种移植小鼠模型在体内评估了 19-HB 的抗肿瘤作用。19-HB 处理的 NSCLC 细胞显示出细胞活力受到抑制和细胞凋亡增加。cleaved caspase-3、cleaved-PARP 和 Bax/Bcl-2 的表达水平上调，而线粒体膜电位降低。相反，迁移、侵袭以及 MMP2、MMP7、MMP9、上皮-间充质转化相关蛋白 N-钙粘蛋白和波形蛋白以及转录因子 Snail 和 Slug 的表达受到抑制。此外，Wnt/β-catenin 信号通路中的关键分子（CyclinD1、c-Myc 和 β-catenin）的表达水平降低。体内，19-HB对裸鼠移植瘤生长也有明显抑制作用，肝肾功能生化指标无明显变化。19-HB通过Wnt/β-catenin通路抑制NSCLC细胞的增殖、迁移和侵袭，促进其凋亡。此外，19-HB抑制裸鼠移植瘤的生长，对肝脏和肾脏的毒性很小。因此，19-HB 可能是治疗 NSCLC 的潜在抗肿瘤剂。19-HB抑制裸鼠移植瘤的生长，对肝肾毒性很小。因此，19-HB 可能是治疗 NSCLC 的潜在抗肿瘤剂。19-HB抑制裸鼠移植瘤的生长，对肝肾毒性很小。因此，19-HB 可能是治疗 NSCLC 的潜在抗肿瘤剂。