冬凌草甲素上调p53转录活性促进胶质瘤细胞凋亡的作用与分子机制探讨
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摘要
目的:探讨冬凌草甲素抑制胶质瘤细胞U-87 MG、A-172活性的分子机制。方法:CCK8法检测不同浓度的冬凌草甲素对胶质瘤细胞U-87 MG、A-172活力的影响。选择20μmol/L的冬凌草甲素分别处理胶质瘤细胞U-87 MG、A-172 0、6、12 h后,Western blotting法检测p53及其下游p21、Bax蛋白的表达情况,同时检测Cleaved PARP、Cleaved Caspase3的表达情况,实时荧光定量PCR法检测p53 mRNA水平的改变。应用p53转录活性抑制剂Pifithrin-α(PFT-α)预处理胶质瘤细胞U-87 MG 24 h后,Western blotting法检测冬凌草甲素对p53及其下游蛋白、Cleaved PARP、Cleaved Caspase3表达情况的影响。结果:冬凌草甲素对U-87 MG、A-172细胞的活性具有明显的抑制作用,冬凌草甲素作用24 h对胶质瘤细胞U-87 MG、A-172的IC_(50)均介于20～30μmol/L。冬凌草甲素可以上调p53及其下游p21、Bax蛋白并具有时间依赖性,而不改变p53的mRNA水平。此外,冬凌草甲素可以诱导胶质瘤细胞U-87 MG、A-172凋亡。p53转录活性抑制剂可以废除冬凌草甲素对p53及其下游p21、Bax蛋白的上调作用,同时也减弱了冬凌草甲素对胶质瘤细胞U-87 MG的促凋亡作用。结论:冬凌草甲素可能通过上调p53蛋白表达、增强其转录活性,促进胶质瘤细胞凋亡,而对胶质瘤细胞活性产生抑制效应。
Objective:To explore the molecular mechanism of oridonin inhibiting the activity of glioma U-87 MG and A-172 cells.Methods:CCK8 method was used to detect the effects of different concentration of oridonin on the activity of U-87 MG and A-172 cells.After U-87 MG and A-172 cells treated with 20 μmol/L oridonin for 0,6,12 h,Western blotting method was used to detect the expression of p53 and its downstream p21 and Bax protein,at the same time,the expression of Cleaved PARP and Cleaved Caspase3 was detected.After the pre-treatment of p53 transcriptional inhibitor Pifithrin-α(PFT-α) on U-87 MG,the effect of oridonin on the expression of p53 and its downstream protein,Cleaved PARP and Cleaved Caspase3 was detected by Western blotting method.Results:Oridonin had an obvious inhibitory effect on the activity of U-87 MG and A-172.IC_(50) of oridonin treatment for 24 h on U-87 MG and A-172 cells were between 20 and 30 μmol/L.Oridonin can up regulate p53 and its downstream p21 and Bax proteins in a time-dependent,without changing the mRNA level of p53.In addition,oridonin can induce apoptosis of U-87 MG and A-172 cells.p53 transcriptional activity inhibitor can abolish the up-regulated effect of oridonin on the p53 and its downstream p21 and Bax proteins,and also weaken the apoptosis effect of oridonin on the U-87 MG cells.Conclusion:Oridonin may promote the apoptosis of glioma cells by up-regulating the expression of p53 protein and enhancing its transcriptional activity to inhibit the activity of glioma cells.
Objective:To explore the molecular mechanism of oridonin inhibiting the activity of glioma U-87 MG and A-172 cells.Methods:CCK8 method was used to detect the effects of different concentration of oridonin on the activity of U-87 MG and A-172 cells.After U-87 MG and A-172 cells treated with 20 μmol/L oridonin for 0,6,12 h,Western blotting method was used to detect the expression of p53 and its downstream p21 and Bax protein,at the same time,the expression of Cleaved PARP and Cleaved Caspase3 was detected.After the pre-treatment of p53 transcriptional inhibitor Pifithrin-α(PFT-α) on U-87 MG,the effect of oridonin on the expression of p53 and its downstream protein,Cleaved PARP and Cleaved Caspase3 was detected by Western blotting method.Results:Oridonin had an obvious inhibitory effect on the activity of U-87 MG and A-172.IC_(50) of oridonin treatment for 24 h on U-87 MG and A-172 cells were between 20 and 30 μmol/L.Oridonin can up regulate p53 and its downstream p21 and Bax proteins in a time-dependent,without changing the mRNA level of p53.In addition,oridonin can induce apoptosis of U-87 MG and A-172 cells.p53 transcriptional activity inhibitor can abolish the up-regulated effect of oridonin on the p53 and its downstream p21 and Bax proteins,and also weaken the apoptosis effect of oridonin on the U-87 MG cells.Conclusion:Oridonin may promote the apoptosis of glioma cells by up-regulating the expression of p53 protein and enhancing its transcriptional activity to inhibit the activity of glioma cells.
引文
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[2] Tate MC,Aghi MK.Biology of angiogenesis and invasion in glioma[J].The Journal of the American Society for Experimental Neuro Therapeutics,2009,6(3):447-457.
[3] Zhang LD,Liu Z,Liu H,et al.Oridonin enhances the anticancer activity of NVP-BEZ235 against neuroblastoma cells in vitro and in vivo through autophagy[J].International Journal of Oncology,2016,49(2):657-665.
[4] Gao FH,Liu F,Wei W,et al.Oridonin induces apoptosis and senescence by increasing hydrogen peroxide and glutathione depletion in colorectal cancer cells[J].International Journal of Molecular Medicine,2012,29(4):649-655.
[5] Xu B,Shen W,Liu X,et al.Oridonin inhibits BxPC-3 cell growth through cell apoptosis[J].Acta Biochimica et Biophysica Sinica,2015,47(3):164-173.
[6] Zhang XH,Liu YX,Jia M,et al.Oridonin inhibits tumor growth in glioma by inducing cell cycle arrest and apoptosis[J].Cellular and Molecular Biology(Noisy-le-Grand,France),2014,60(6):29-36.
[7] Van Maerken T,Rihani A,Van Goethem A,et al.Pharmacologic activation of wild-type p53 by nutlin therapy in childhood cancer[J].Cancer letters,2014,344(2):157-165.
[8] Bernhart E,Damm S,Heffeter P,et al.Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and-independent pathways[J].Neuro-Oncology,2014,16(7):933-945.
[9] Tweddle DA,Pearson AD,Haber M,et al.The p53 pathway and its inactivation in neuroblastoma[J].Cancer letters,2003,197(1-2):93-98.
[10] Chen J.The cell-cycle arrest and apoptotic functions of p53 in tumor initiation and progression[J].Cold Spring Harbor perspectives in Medicine,2016,6(3):a026104.
[11] Riley T,Sontag E,Chen P,et al.Transcriptional control of human p53-regulated genes[J].Nature Reviews Molecular Cell Biology,2008,9(5):402-412.
[12] Bykov VJN,Eriksson SE,Bianchi J,et al.Targeting mutant p53 for efficient cancer therapy[J].Nature Reviews Cancer,2018,18(2):89-102.
[13] Gupta AK,Bharadwaj M,Kumar A,et al.Spiro-oxindoles as a promising class of small molecule inhibitors of p53-MDM2 interaction useful in targeted cancer therapy[J].Topics in Current Chemistry(Cham),2017,375(1):3.
[14] Saison-Ridinger M,DelGiorno KE,Zhang T,et al.Reprogramming pancreatic stellate cells via p53 activation:A putative target for pancreatic cancer therapy[J].PLoS One,2017,12(12):e0189051.
[15] Zhou R,Xu A,Gingold J,et al.Lifraumeni syndrome disease model:A platform to develop precision cancer therapy targeting oncogenic p53[J].Trends in Pharmacological Sciences,2017,38(10):908-927.
[16] Zhang HP,Li GQ,Guo WZ,et al.Oridonin synergistically enhances JQ1-triggered apoptosis in hepatocellular cancer cells through mitochondrial pathway[J].Oncotarget,2017,8(63):106833-106843.
[17] Liang J,Wang W,Wei L,et al.Oridonin inhibits growth and induces apoptosis of human neurocytoma cells via the Wnt/beta-catenin pathway[J].Oncology Letters,2018,16(3):3333-3340.
[18] Park H,Jeong YJ,Han NK,et al.Oridonin enhances radiation-induced cell death by promoting DNA damage in non-small cell lung cancer cells[J].International Journal of Molecular Sciences,2018,19(8):2378.
[19] Liu X,Kang J,Wang H,et al.Mitochondrial ROS contribute to oridonin-induced HepG2 apoptosis through PARP activation[J].Oncology Letters,2018,15(3):2881-2888.
[20] Zhang YH,Wu YL,Tashiro S,et al.Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma HeLa cells[J].Acta Pharmacologica Sinica,2011,32(10):1266-1275.
[21] Nag S,Zhang X,Srivenugopal KS,et al.Targeting MDM2-p53 interaction for cancer therapy:Are we there yet[J]?Current Medicinal Chemistry,2014,21(5):553-574.