Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou, China; Institute of Clinical Pharmacology, Guangzhou University of Chinese Medicine, Guangzhou, China.
Department of Pharmacology & Toxicology, University of Kansas, Lawrence, KS, United States.
Phytomedicine. 2020 Mar;68:153174. doi: 10.1016/j.phymed.2020.153174. Epub 2020 Jan 18.
Wogonin has been reported to exhibit various biological activities such as anti-inflammation, anti-microbial, and anti-tumor. Previous studies have demonstrated that wogonin could down-regulate Cyclin D1 activity on multiple cancers. However, the related mechanisms have not been fully elucidated so far.
The aim of the current study was to explore whether wogonin can suppress hepatocellular carcinoma (HCC) progression and the mechanism of wogonin in inhibiting Cyclin D1 expression.
Herein, we assessed the anti-tumor activity of wogonin against hepatocellular carcinoma (HCC) by MTT assay, clonogenic assay, cell cycle analysis and orthotopic xenograft mouse models. Western blot, immunofluoscence assay, co-immunoprecipitation assay, docking program, surface plasmon resonance, site-directed mutagenesis assay and immunohistochemical assay were performed for exploring the underlying mechanisms of wogonin-induced growth inhibition in HCC.
Our results showed that non-toxic dosage of wogonin (10, 20 µM) could inhibit cells proliferation and suppress cells cycle progression in MHCC97L and HepG2 cell. Moreover, the findings from the western blot and immunofluoscence assay confirmed the inhibition action of wogonin (10, 20 µM) on Cyclin D1 expression in MHCC97L cells, and wogonin (10, 20 µM) pre-treatment was capable of promoting Cyclin D1 ubiquitination and degradation in MHCC97L cell. In addition, wogonin promoted phosphorylation of Cyclin D1 on threonine-286 site, the mutation of threonine-286 to alanine-286A blocked Cyclin D1 proteolysis induced by wogonin. Wogonin-promoted Cyclin D1 phosphorylation and subsequent proteolysis may associate with the activation of GSK3beta in cancer cells. The phosphorylated form of GSK3beta (active form) expression was significantly increased after wogonin (20 µM) exposure. Molecular docking study and Biacore SPR analysis of GSK3beta mutant further validated the high-affinity wogonin binding site on GSK3beta. Moreover, in vivo studies further confirmed that phospho-GSK3beta Tyr216 was over-expressed in HCC specimens after wogonin treatment while the amount of Cyclin D1 was significantly decreased.
In summary, our data reveal a novel molecular mechanism by which wogonin induces HCC cells cycle arrest and suppresses tumor proliferation.
研究表明，汉黄芩素具有抗炎、抗菌和抗肿瘤等多种生物学活性。先前的研究表明，汉黄芩素可以下调多种癌症中的细胞周期蛋白 D1 活性。然而，相关机制尚未得到充分阐明。
本研究旨在探讨汉黄芩素是否能抑制肝癌（HCC）的进展以及汉黄芩素抑制细胞周期蛋白 D1 表达的机制。
通过 MTT 测定、集落形成测定、细胞周期分析和原位异种移植小鼠模型，评估汉黄芩素对肝癌（HCC）的抗肿瘤活性。进行 Western blot、免疫荧光检测、免疫共沉淀检测、对接程序、表面等离子体共振、定点突变检测和免疫组织化学检测，以探索汉黄芩素诱导 HCC 生长抑制的潜在机制。
结果表明，无毒剂量的汉黄芩素（10、20 μM）可抑制 MHCC97L 和 HepG2 细胞的增殖并抑制细胞周期进程。此外，Western blot 和免疫荧光检测结果证实了汉黄芩素（10、20 μM）对 MHCC97L 细胞中细胞周期蛋白 D1 表达的抑制作用，并且汉黄芩素（10、20 μM）预处理能够促进 MHCC97L 细胞中环细胞蛋白 D1 的泛素化和降解。此外，汉黄芩素促进细胞周期蛋白 D1 在苏氨酸 286 位的磷酸化，将苏氨酸 286 突变为丙氨酸 286A 可阻断汉黄芩素诱导的细胞周期蛋白 D1 降解。汉黄芩素促进细胞周期蛋白 D1 磷酸化和随后的蛋白水解可能与癌细胞中 GSK3β的激活有关。汉黄芩素（20 μM）暴露后，磷酸化 GSK3β（活性形式）的表达显著增加。GSK3β 突变体的分子对接研究和 Biacore SPR 分析进一步验证了 GSK3β 上汉黄芩素的高亲和力结合位点。此外，体内研究进一步证实，汉黄芩素处理后 HCC 标本中磷酸化 GSK3β Tyr216 表达过度，而细胞周期蛋白 D1 的含量明显减少。
总之，本研究数据揭示了汉黄芩素诱导 HCC 细胞周期阻滞和抑制肿瘤增殖的新分子机制。
