黄腐酚通过降低Notch信号通路活性抑制前列腺癌细胞增殖
发布时间：2019-04-27 10:28
【摘要】：Notch信号通路作为一条高度保守的信号转导途径,广泛表达于从低等的无脊椎动物到高等的哺乳动物中。Notch信号通路对于调控干细胞的分化、细胞增殖、细胞凋亡等生理活性具有重要的作用。失调的Notch信号通路在多种人类癌细胞中异常表达,因此Notch信号通路已成为多种恶性肿瘤治疗和抗癌药物筛选的重要靶点之一 黄腐酚(Xanthohumol, XN)作为一种仅存在于啤酒花中的异戊二烯基黄酮类化合物,具有多种生物学活性。黄腐酚在防治糖尿病和动脉粥样硬化方面起着重要的作用,并且具有抗氧化、抗病毒、抑制乳腺癌、结肠癌、卵巢癌、前列腺癌等癌细胞生长的作用,然而其发挥作用的分子机制不详。鉴于Notch信号通路在前列腺癌细胞中呈现出高度活化的报道,推测黄腐酚抑制前列腺癌细胞的生长可能与调控细胞中Notch信号通路活性有关。 本论文旨在研究以Notch信号通路为靶点探讨黄腐酚的抗肿瘤活性及其作用机理。首先在前列腺癌细胞PC3中转染基于Notch1的荧光素酶报告质粒,构建出稳转细胞株PCNL1-2,所得细胞株可以很好地响应Notch信号的改变,能够筛选Notch信号通路抑制剂；通过此细胞株,对实验室中具有潜在抗癌活性的多种化合物进行筛选,筛选出可以抑制Notch信号通路活性的天然产物黄腐酚；MTT试验证实浓度为30μmol/L的黄腐酚在12h内无细胞毒活性,细胞活性大于80%；而细胞荧光抑制实验中浓度为30μmol/L的黄腐酚处理稳转细胞株PCNL1-2细胞12h后,细胞荧光的抑制率大于50%,表明黄腐酚可以特异性地抑制Notch信号通路活性；通过流式细胞分析术与western blot实验证实黄腐酚可以将细胞周期阻滞在Go/G1期,同时黄腐酚促进了前列腺癌细胞凋亡。通过进一步的细胞转染、western blot、RT-PCR等实验,对黄腐酚作用的分子靶点进行了探索。研究发现,Notch信号通路与GSK-3p蛋白有着密切的联系。GSK-3p可以通过降低Notch蛋白的稳定性抑制其信号通路活性,因此可以将GSK-3p看作是Notch信号通路的抑制蛋白之一。本文研究发现黄腐酚正是通过抑制GSK-3p第9位丝氨酸的磷酸化进而抑制Notch信号通路活性。 综上所述,黄腐酚通过下调前列腺癌细胞中高度活化的Notch信号通路活性而抑制前列腺癌细胞的生长。本研究阐述了黄腐酚抗肿瘤活性的分子作用机理,为黄腐酚作为Notch信号通路的抑制剂治疗前列腺癌提供了有价值的理论依据。
[Abstract]:As a highly conserved signal transduction pathway, the Notch signaling pathway is widely expressed in mammals from lower invertebrates to higher mammals. Notch signaling pathway regulates stem cell differentiation and cell proliferation. Apoptosis and other physiological activities play an important role. The dysfunctional Notch signaling pathway is expressed abnormally in a variety of human cancer cells, so the Notch signaling pathway has become one of the important targets for the treatment of many malignant tumors and screening of anticancer drugs (Xanthohumol,). As a kind of isoprene flavonoids only existed in hops, XN has many biological activities. Fulvic phenol plays an important role in the prevention and treatment of diabetes and atherosclerosis, and has anti-oxidation, anti-virus, and inhibit the growth of breast, colon, ovarian, prostate cancer, and other cancer cells. However, the molecular mechanism of its role is unknown. In view of the high activation of Notch signaling pathway in prostate cancer cells, it is suggested that the inhibition of the growth of prostate cancer cells by fulvic phenol may be related to the regulation of the activity of Notch signaling pathway in prostate cancer cells. The aim of this study was to investigate the antitumor activity and mechanism of fulvic phenol by Notch signaling pathway. Firstly, the luciferase reporter plasmid based on Notch1 was transfected into prostate cancer cell line PC3, and the stable cell line PCNL1-2, was constructed, which could respond well to the change of Notch signal and screen the inhibitor of Notch signaling pathway. Through this cell line, many kinds of compounds with potential anticancer activity in the laboratory were screened, and the natural product which could inhibit the activity of Notch signaling pathway was screened out. MTT test showed that 30 渭 mol / L of fulvic phenol had no cytotoxic activity within 12 h, and the cell activity was more than 80%. After treated with 30 渭 mol / L fulvic phenol for 12 h, the fluorescence inhibition rate of PCNL1-2 cells was more than 50%, which indicated that fulvic phenol could specifically inhibit the activity of Notch signaling pathway. Flow cytometry and western blot assay confirmed that fulvic acid could block the cell cycle in Go/G1 phase and promote apoptosis of prostate cancer cells. Further cell transfection, western blot,RT-PCR and other experiments were carried out to explore the molecular targets of the action of fulvic phenol. It has been found that Notch signaling pathway is closely related to GSK-3p protein, and GSK-3p can inhibit its signal pathway activity by reducing the stability of Notch protein, so GSK-3p can be regarded as one of the inhibitory proteins of Notch signaling pathway. In this study, it was found that fulvic phenol inhibited the activity of Notch signaling pathway by inhibiting the phosphorylation of serine at the 9th position of GSK-3p. In conclusion, fulvic phenol inhibits the growth of prostate cancer cells by down-regulating the activity of highly activated Notch signaling pathway in prostate cancer cells. In this study, the molecular mechanism of the antineoplastic activity of fulvic phenol is expounded, which provides a valuable theoretical basis for the treatment of prostate cancer with fulvic phenol as an inhibitor of Notch signaling pathway.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
本文编号：2466903
[Abstract]:As a highly conserved signal transduction pathway, the Notch signaling pathway is widely expressed in mammals from lower invertebrates to higher mammals. Notch signaling pathway regulates stem cell differentiation and cell proliferation. Apoptosis and other physiological activities play an important role. The dysfunctional Notch signaling pathway is expressed abnormally in a variety of human cancer cells, so the Notch signaling pathway has become one of the important targets for the treatment of many malignant tumors and screening of anticancer drugs (Xanthohumol,). As a kind of isoprene flavonoids only existed in hops, XN has many biological activities. Fulvic phenol plays an important role in the prevention and treatment of diabetes and atherosclerosis, and has anti-oxidation, anti-virus, and inhibit the growth of breast, colon, ovarian, prostate cancer, and other cancer cells. However, the molecular mechanism of its role is unknown. In view of the high activation of Notch signaling pathway in prostate cancer cells, it is suggested that the inhibition of the growth of prostate cancer cells by fulvic phenol may be related to the regulation of the activity of Notch signaling pathway in prostate cancer cells. The aim of this study was to investigate the antitumor activity and mechanism of fulvic phenol by Notch signaling pathway. Firstly, the luciferase reporter plasmid based on Notch1 was transfected into prostate cancer cell line PC3, and the stable cell line PCNL1-2, was constructed, which could respond well to the change of Notch signal and screen the inhibitor of Notch signaling pathway. Through this cell line, many kinds of compounds with potential anticancer activity in the laboratory were screened, and the natural product which could inhibit the activity of Notch signaling pathway was screened out. MTT test showed that 30 渭 mol / L of fulvic phenol had no cytotoxic activity within 12 h, and the cell activity was more than 80%. After treated with 30 渭 mol / L fulvic phenol for 12 h, the fluorescence inhibition rate of PCNL1-2 cells was more than 50%, which indicated that fulvic phenol could specifically inhibit the activity of Notch signaling pathway. Flow cytometry and western blot assay confirmed that fulvic acid could block the cell cycle in Go/G1 phase and promote apoptosis of prostate cancer cells. Further cell transfection, western blot,RT-PCR and other experiments were carried out to explore the molecular targets of the action of fulvic phenol. It has been found that Notch signaling pathway is closely related to GSK-3p protein, and GSK-3p can inhibit its signal pathway activity by reducing the stability of Notch protein, so GSK-3p can be regarded as one of the inhibitory proteins of Notch signaling pathway. In this study, it was found that fulvic phenol inhibited the activity of Notch signaling pathway by inhibiting the phosphorylation of serine at the 9th position of GSK-3p. In conclusion, fulvic phenol inhibits the growth of prostate cancer cells by down-regulating the activity of highly activated Notch signaling pathway in prostate cancer cells. In this study, the molecular mechanism of the antineoplastic activity of fulvic phenol is expounded, which provides a valuable theoretical basis for the treatment of prostate cancer with fulvic phenol as an inhibitor of Notch signaling pathway.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
【参考文献】
相关期刊论文 前4条
1 付亚娟;叶枫;吕卫国;谢幸;;Notch信号通路的研究现状[J];医学分子生物学杂志;2007年05期
2 周娟,邹翔,季宇彬;啤酒花的有效成分及活性研究[J];哈尔滨商业大学学报(自然科学版);2005年04期
3 姜昕;周建华;;Notch信号通路及其在肺癌发生中的作用[J];国际病理科学与临床杂志;2007年03期
4 马艳君;赵智明;刘景晶;;Notch信号通路与肿瘤的发生及其靶向抗肿瘤疗法[J];药学进展;2009年03期
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