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Wogonin inhibits cell cycle progression by activating the glycogen synthase kinase-3 beta in hepatocellular carcinoma.
Phytomedicine ( IF 7.9 ) Pub Date : 2020-01-18 , DOI: 10.1016/j.phymed.2020.153174
Ming Hong 1 , Mohammed M Almutairi 2 , Siying Li 2 , Jinke Li 2
Affiliation  

BACKGROUND 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. PURPOSE 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. METHODS 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. RESULTS 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. CONCLUSION In summary, our data reveal a novel molecular mechanism by which wogonin induces HCC cells cycle arrest and suppresses tumor proliferation.

中文翻译:

Wogonin通过激活肝细胞癌中的糖原合酶激酶3β抑制细胞周期进程。

背景技术据报道,沃戈宁具有多种生物活性,例如抗发炎,抗微生物和抗肿瘤。先前的研究表明,wogonin可能下调Cyclin D1在多种癌症上的活性。但是,到目前为止,还没有完全阐明相关的机制。目的本研究的目的是探讨沃戈宁是否可以抑制肝细胞癌(HCC)的进展以及沃戈宁抑制Cyclin D1表达的机制。方法在本文中,我们通过MTT分析,克隆形成分析,细胞周期分析和原位异种移植小鼠模型评估了wogonin对肝细胞癌(HCC)的抗肿瘤活性。Western blot,免疫荧光测定,免疫共沉淀测定,对接程序,表面等离振子共振,进行了定点诱变测定和免疫组化测定,以探索wogonin诱导的肝癌生长抑制的潜在机制。结果我们的结果表明,无毒剂量的wogonin(10,20 µM)可以抑制MHCC97L和HepG2细胞的细胞增殖并抑制细胞周期进程。此外,western blot和免疫荧光分析的结果证实了wogonin(10,20 µM)对MHCC97L细胞中Cyclin D1表达的抑制作用,并且wogonin(10,20 µM)预处理能够促进Cyclin D1泛素化和MHCC97L细胞中的降解。另外,wogonin促进了苏氨酸286位点上的细胞周期蛋白D1的磷酸化,苏氨酸286向丙氨酸286A的突变阻止了wogonin诱导的Cyclin D1蛋白水解。Wogonin促进细胞周期蛋白D1的磷酸化和随后的蛋白水解可能与癌细胞中GSK3beta的激活有关。wogonin(20 µM)暴露后,GSK3beta(活性形式)表达的磷酸化形式显着增加。GSK3beta突变体的分子对接研究和Biacore SPR分析进一步验证了GSK3beta上的高亲和力wogonin结合位点。此外,体内研究进一步证实,在用卵清蛋白处理后,HCC标本中的磷酸化-GSK3beta Tyr216过表达,而细胞周期蛋白D1的量则明显减少。结论总的来说,我们的数据揭示了一种新的分子机制,沃戈宁通过该机制诱导HCC细胞周期停滞并抑制肿瘤增殖。wogonin(20 µM)暴露后,GSK3beta(活性形式)表达的磷酸化形式显着增加。GSK3beta突变体的分子对接研究和Biacore SPR分析进一步验证了GSK3beta上的高亲和力wogonin结合位点。此外,体内研究进一步证实,在用卵清蛋白处理后,HCC标本中的磷酸化-GSK3beta Tyr216过表达,而细胞周期蛋白D1的量则明显减少。结论总的来说,我们的数据揭示了一种新的分子机制,沃戈宁通过这种机制诱导HCC细胞周期停滞并抑制肿瘤增殖。wogonin(20 µM)暴露后,GSK3beta(活性形式)表达的磷酸化形式显着增加。GSK3beta突变体的分子对接研究和Biacore SPR分析进一步验证了GSK3beta上的高亲和力wogonin结合位点。此外,体内研究进一步证实,在用卵清蛋白处理后,HCC标本中的磷酸化-GSK3beta Tyr216过表达,而细胞周期蛋白D1的量则明显减少。结论总的来说,我们的数据揭示了一种新的分子机制,沃戈宁通过该机制诱导HCC细胞周期停滞并抑制肿瘤增殖。体内研究进一步证实,wogonin处理后,HCC标本中磷-GSK3beta Tyr216过表达,而Cyclin D1的量则明显减少。结论总的来说,我们的数据揭示了一种新的分子机制,沃戈宁通过该机制诱导HCC细胞周期停滞并抑制肿瘤增殖。体内研究进一步证实,wogonin处理后,HCC标本中磷-GSK3beta Tyr216过表达,而Cyclin D1的量则明显减少。结论总的来说,我们的数据揭示了一种新的分子机制,沃戈宁通过该机制诱导HCC细胞周期停滞并抑制肿瘤增殖。
更新日期:2020-01-21
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