雷公藤红素通过下调Pin1抑制Hela细胞的机制研究
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摘要
目的研究不同剂量雷公藤红素对宫颈癌Hela细胞增殖、周期、凋亡、迁移的影响及可能的作用靶点。方法将0,0.25,0.5,1,2,4μmol/L雷公藤红素处理Hela细胞12、24 h后,采用MTT试验考察细胞增殖情况;将0,0.25,0.5,1μmol/L雷公藤红素处理Hela细胞24 h后,采用流式细胞术检测细胞周期、凋亡、迁移变化情况,采用RT-qPCR、Western blot检测Pin1及Cdk2、Cdk6、Cyclin D1、Akt、P65、Jnk表达情况。结果雷公藤红素在12、24 h抑制Hela细胞增殖、迁移,且呈浓度依赖性(P<0.05)。雷公藤红素可显著降低G_1期细胞比例(P<0.05),升高G_2期细胞比例(P<0.05),降低S期细胞比率(P>0.05),且呈浓度依赖性。雷公藤红素可增加早期凋亡、晚期凋亡比例(P<0.05)。雷公藤红素在mRNA、蛋白水平显著抑制Pin1及其下游Cdk2、Cdk6、Cyclin D1、Akt、P65、Jnk的表达(P<0.05)。结论雷公藤红素可能通过下调Pin1发挥对Hela细胞的抗肿瘤作用。
Objective To investigate the influence of celastrol on the proliferation, apoptosis, cell cycle and migration, and underlying mechanism in cervical cancer Hela cells. Methods After treated with the concentrations of celastrol(0, 0.25, 0.5, 1, 2, 4 μM) for 12 h and 24 h, MTT assays were performed to determine the cell proliferation. After treated by celastrol(0, 0.25, 0.5, 1 μM) for 24 h, the cells were collected to study the cell cycle, apoptosis and migration and to research the expression of pin1, Cdk2, Cdk6, Cyclin D1, Akt, P65, Jnk by RT-qPCR and Western blot. Results Celastrol significantly suppressed the proliferation and migration in a dose-independent manner(P< 0.05). Celastrol decreased the proportion of G_1 cells(P< 0.05) and S cells(P> 0.05), increased the proportion of G_2 cells(P< 0.05). As the concentration of celastrol raised, both early and late apoptotic cells were increased. We uncovered that celastrol especially suppressed the expression of the Pin1 and thereby blocking the expression of Cdk2, Cdk6, Cyclin D1, Akt and P65. Conclusion Taken together, these findings indicated that celastrol is a potential therapeutic agent for treatment of human ovarian cancer through inhibiting Pin1.
Objective To investigate the influence of celastrol on the proliferation, apoptosis, cell cycle and migration, and underlying mechanism in cervical cancer Hela cells. Methods After treated with the concentrations of celastrol(0, 0.25, 0.5, 1, 2, 4 μM) for 12 h and 24 h, MTT assays were performed to determine the cell proliferation. After treated by celastrol(0, 0.25, 0.5, 1 μM) for 24 h, the cells were collected to study the cell cycle, apoptosis and migration and to research the expression of pin1, Cdk2, Cdk6, Cyclin D1, Akt, P65, Jnk by RT-qPCR and Western blot. Results Celastrol significantly suppressed the proliferation and migration in a dose-independent manner(P< 0.05). Celastrol decreased the proportion of G_1 cells(P< 0.05) and S cells(P> 0.05), increased the proportion of G_2 cells(P< 0.05). As the concentration of celastrol raised, both early and late apoptotic cells were increased. We uncovered that celastrol especially suppressed the expression of the Pin1 and thereby blocking the expression of Cdk2, Cdk6, Cyclin D1, Akt and P65. Conclusion Taken together, these findings indicated that celastrol is a potential therapeutic agent for treatment of human ovarian cancer through inhibiting Pin1.
引文
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[12] Lu KP,Hanes SD,Hunter T.A human peptidyl-prolyl isomerase essential for regulation of mitosis[J].Nature,1996,380(6574):544.
[13] Lu Z,Hunter T.Prolyl isomerase Pin1 in cancer[J].Cell Res,2014,24(9):1033.
[14] Wang T,Liu Z,Shi F,et al.Pin1 modulates chemo-resistance by up-regulating FoxM1 and the involvements of Wnt/beta-catenin signaling pathway in cervical cancer[J].Mol Cell Biochem,2016,413(1-2):179.
[15] Russo Spena C,De Stefano L,Palazzolo S,et al.Liposomal delivery of a Pin1 inhibitor complexed with cyclodextrins as new therapy for high-grade serous ovarian cancer[J].J Control Release,2018,281:1.
[16] Lee TH,Pastorino L,Lu KP.Peptidyl-prolyl cis-trans isomerase Pin1 in ageing,cancer and Alzheimer disease[J].Expert Rev Mol Med,2011,13:e21.
[17] Lin CH,Li HY,Lee YC,et al.Landscape of Pin1 in the cell cycle[J].Exp Biol Med (Maywood),2015,240(3):403.
[2] Wang GZ,Liu YQ,Cheng X,et al.Celastrol induces proteasomal degradation of FANCD2 to sensitize lung cancer cells to DNA crosslinking agents[J].Cancer Sci,2015,106(7):902.
[3] Kannaiyan R,Shanmugam MK,Sethi G.Molecular targets of celastrol derived from Thunder of God Vine:potential role in the treatment of inflammatory disorders and cancer[J].Cancer Lett,2011,303(1):9.
[4] Kannaiyan R,Manu KA,Chen L,et al.Celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-Jun N-terminal kinase and suppression of PI3 K/Akt signaling pathways[J].Apoptosis,2011,16(10):1028.
[5] Hu Y,Qi Y,Liu H,et al.Effects of celastrol on human cervical cancer cells as revealed by ion-trap gas chromatography-mass spectrometry based metabolic profiling[J].Biochim Biophys Acta,2013,1830(3):2779.
[6] Han X,Sun S,Zhao M,et al.Celastrol stimulates hypoxia-inducible factor-1 activity in tumor cells by initiating the ROS/Akt/p70S6K signaling pathway and enhancing hypoxia-inducible factor-1alpha protein synthesis[J].PLoS One,2014,9(11):e112470.
[7] Yang Y,Cheng S,Liang G,et al.Celastrol inhibits cancer metastasis by suppressing M2-like polarization of macrophages[J].Biochem Biophys Res Commun,2018,503(2):414.
[8] Yang H,Chen D,Cui QC,et al.Celastrol,a triterpene extracted from the Chinese "Thunder of God Vine," is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice[J].Cancer Res,2006,66(9):4758.
[9] Peng B,Gu YJ,Wang Y,et al.Mutations Y493G and K546D in human HSP90 disrupt binding of celastrol and reduce interaction with Cdc37[J].FEBS Open Bio,2016,6(7):729.
[10] Chen S,Gu C,Xu C,et al.Celastrol prevents cadmium-induced neuronal cell death via targeting JNK and PTEN-Akt/mTOR network[J].J Neurochem,2014,128(2):256.
[11] Blume-Jensen P,Hunter T.Oncogenic kinase signalling[J].Nature,2001,411(6835):355.
[12] Lu KP,Hanes SD,Hunter T.A human peptidyl-prolyl isomerase essential for regulation of mitosis[J].Nature,1996,380(6574):544.
[13] Lu Z,Hunter T.Prolyl isomerase Pin1 in cancer[J].Cell Res,2014,24(9):1033.
[14] Wang T,Liu Z,Shi F,et al.Pin1 modulates chemo-resistance by up-regulating FoxM1 and the involvements of Wnt/beta-catenin signaling pathway in cervical cancer[J].Mol Cell Biochem,2016,413(1-2):179.
[15] Russo Spena C,De Stefano L,Palazzolo S,et al.Liposomal delivery of a Pin1 inhibitor complexed with cyclodextrins as new therapy for high-grade serous ovarian cancer[J].J Control Release,2018,281:1.
[16] Lee TH,Pastorino L,Lu KP.Peptidyl-prolyl cis-trans isomerase Pin1 in ageing,cancer and Alzheimer disease[J].Expert Rev Mol Med,2011,13:e21.
[17] Lin CH,Li HY,Lee YC,et al.Landscape of Pin1 in the cell cycle[J].Exp Biol Med (Maywood),2015,240(3):403.