背景:甘草是全球常用的草药,用于治疗咳嗽,肝炎,癌症和流感在中医临床实践中的应用。现代药理研究表明异戊烯黄酮在甘草的抗肿瘤活性中起重要作用,尤其是胃中的肿瘤,肺,结肠和肝脏。Wighteone是甘草中主要的异戊烯化黄酮之一,其可能的作用和针对结直肠癌的靶点尚未被研究。
目的:本研究旨在探讨威吉特酮的抗结直肠癌作用及其机制。
方法:用SW480人结直肠癌细胞流式细胞术评价wighteone的体外抗结直肠癌活性和Akt调控作用。磷酸蛋白质组学和蛋白质印迹分析。表面等离子体共振(SPR)分析,分子对接和动力学模拟,和激酶活性测定用于研究wighteone和Akt之间的直接相互作用。使用具有SW480细胞的裸鼠异种移植模型来验证wighteone的体内抗结肠直肠癌活性。
结果:Wighteone抑制SW480细胞中Akt及其下游激酶的磷酸化,导致细胞活力下降。Wighteone与Akt的PH和激酶结构域都有直接相互作用,将Akt锁定在具有变构抑制的“封闭”构象中,Gln79,Tyr272,Arg273和Lys297由于它们的氢键和与wighteone的疏水相互作用而发挥了最关键的作用。基于Akt在SW480细胞中的过表达或激活,进一步的机制研究表明,wighteone诱导的Akt抑制导致周期停滞,SW480细胞的凋亡和自噬性死亡。此外,wighteone在裸鼠异种移植模型中发挥了体内抗结直肠癌作用和Akt抑制活性。
结论:Wighteone可通过变构抑制Akt抑制SW480细胞的生长,导致细胞周期停滞,凋亡和自噬死亡。该结果有助于了解甘草的抗肿瘤机制。并为设计用于治疗结直肠癌的新型Akt变构抑制剂提供了理论基础。
BACKGROUND: Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated.
OBJECTIVE: This study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone.
METHODS: SW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone.
RESULTS: Wighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a \"closed\" conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model.
CONCLUSIONS: Wighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.