莪术醇诱导人肝癌HepG2细胞衰老及其机制研究
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摘要
为进一步探讨莪术醇的诱导细胞衰老的机制,该研究采用荧光定量PCR技术对莪术醇处理后细胞中81个细胞衰老相关基因差异表达谱进行分析,结果发现TP53及其下游基因p16Ink4a、p21Waf1/Cip1和p27Kip1等的表达水平显著升高,伴随ABL1、ALDH1A3、CHEK2、HRAS、PTEN等多个衰老信号通路启动与效应关联基因的转录显著增强,而CyclinA2、IGFBP3、SIRT1以及TERT等细胞周期进程与衰老信号通路的负性调控基因的表达水平则显著降低。Western印迹检测结果显示,p53及其下游周期素依赖性蛋白激酶抑制物(CKI)分子p21WAF1和p16INK4水平升高,CyclinA2水平降低,与PCR结果一致,并伴野生型p53-诱导的蛋白磷酸酶1(Wip1)水平显著增高,提示莪术醇可能通过激活p53信号通路诱导HepG2细胞衰老。该研究进一步发现莪术醇能够诱导HepG2细胞发生衰老表型改变,伴G0/G1期周期阻滞。
In order to further study the antitumor mechanism of curcumol and its potential clinical application,a SYBR Green real-time polymerase chain reaction( RT-PCR) method was used to analyze the differential expression profiles of 81 human senescence-related genes in human hepatocarcinoma HepG2 cells treated with curcumol. The results showed that the expression of TP53 and its downstream genes p16 Ink4 a,p21 Waf1/Cip1 and p27 Kip1 were significantly up-regulated,accompanied by transactivation of other senescence signaling pathway related genes or senescence response genes such as ABL1,ALDH1 A3,CHEK2,HRAS,PTEN,etc.,while the expression of CyclinA2,IGFBP3,SIRT1 and TERT,the genes which negatively regulated cell cycle progression and senescence signaling,were significantly down-regulated. Western blotting verified that protein levels of p53 and its downstream CKIs,p21 WAF1 and p16 INK4 increased while Cyclin A2 decreased,consistent with the findings in PCR results. The level of wild-type p53-induced protein phosphatase 1( Wip1) was also found significantly increased,suggesting that the induction of senescence in HepG2 cells by curcumol might be through activation of p53 signaling pathway. The present study further demonstrates that curcumol is capable of inducing cellular senescent phenotype in HepG2,accompanying with cell cycle G0/G1 phase arrest.
In order to further study the antitumor mechanism of curcumol and its potential clinical application,a SYBR Green real-time polymerase chain reaction( RT-PCR) method was used to analyze the differential expression profiles of 81 human senescence-related genes in human hepatocarcinoma HepG2 cells treated with curcumol. The results showed that the expression of TP53 and its downstream genes p16 Ink4 a,p21 Waf1/Cip1 and p27 Kip1 were significantly up-regulated,accompanied by transactivation of other senescence signaling pathway related genes or senescence response genes such as ABL1,ALDH1 A3,CHEK2,HRAS,PTEN,etc.,while the expression of CyclinA2,IGFBP3,SIRT1 and TERT,the genes which negatively regulated cell cycle progression and senescence signaling,were significantly down-regulated. Western blotting verified that protein levels of p53 and its downstream CKIs,p21 WAF1 and p16 INK4 increased while Cyclin A2 decreased,consistent with the findings in PCR results. The level of wild-type p53-induced protein phosphatase 1( Wip1) was also found significantly increased,suggesting that the induction of senescence in HepG2 cells by curcumol might be through activation of p53 signaling pathway. The present study further demonstrates that curcumol is capable of inducing cellular senescent phenotype in HepG2,accompanying with cell cycle G0/G1 phase arrest.
引文
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WANG Y,XU L,2009.Advances in the researches of curcumol against tumor[J].Med Rec,15(22):3483-3486.[王耀霞,徐立春,2009.莪术醇抗肿瘤研究进展[J].医学综述,15(22):3483-3486.]
WU CM,YANG CW,LEE YZ,et al,2009.Tylophorine arrests carcinoma cells at G1 phase by downregulating cyclin A2 expression[J].Biochem Biophys Res Comm,386(1):140-145.
YANG F,HUANG L,YANG J,et al,2015.Cisplatin-induced senescence and senescence-related gene expression profiles in human nasopharyngeal carcinoma cells CNE2[J].Canc Res Prev Treat,42(1):4-8.[杨飞城,黄岚珍,阳晶,等,2015.顺铂诱导人鼻咽癌CNE2细胞衰老及其相关基因表达谱的研究[J].肿瘤防治研究,42(1):4-8.]
HUANG LZ,WANG J,LU FT,et al,2013.Mechanism study on anti-proliferative effects of curcumol in human hepatocarcinoma Hep G2 cells[J].Chin J Chin Mat Med,38(11):1812-1815.[黄岚珍,王娟,卢菲婷,等,2013.莪术醇抑制人肝癌细胞Hep G2增殖的机制[J].中国中药杂志,38(11):1812-1815.]
HUANG LZ,LI A,LIAO GZ,et al,2017.Curcumol triggers apoptosis of p53 mutant triple-negative human breast cancer MDA-MB 231 cells via activation of p73 and PUMA[J].Oncol Lett,14(1):1080-1088.
LU JJ,DANG YY,HUANG M,et al,2012.Anti-cancer properties of terpenoids isolated from Rhizoma curcumae-Areview[J].J Ethnopharmacol,143(2):406-411.
NARDELLA C,CLOHESSY JG,ALIMONTI A,et al,2011.Pro-senescence therapy for cancer treatment[J].Nat Rev Canc,11(7):503-511.
PAGANO M,PEPPERKOK R,VERDE F,et al,1992.Cyclin A is required at two points in the human cell cycle[J].Embo J,11(3):961-971.
RUFINI A,TUCCI P,CELARDO I,et al,2013.Senescence and aging:the critical roles of p53[J].Oncogene,32(43):5129-5143.
SCHOSSERER M,GRILLARI J,BREITENBACHM,2017.The dual role of cellular senescence in developing tumors and their response to cancer therapy[J].Front Oncol,7:278.
TANG QL,GUO JQ,WANG QY,et al,2015.Curcumol induces apoptosis in SPCA1 human lung adenocarcinoma cells and displays antineoplastic effects in tumor bearing mice[J].Asian Pac J Cance Prev,16(6):23072312.
TERZI MY,IZMIRLI M,GOGEBAKANB,2016.The cell fate:senescence or quiescence[J].Mol Biol Rep,43(11):1213-1220.
WANG Y,XU L,2009.Advances in the researches of curcumol against tumor[J].Med Rec,15(22):3483-3486.[王耀霞,徐立春,2009.莪术醇抗肿瘤研究进展[J].医学综述,15(22):3483-3486.]
WU CM,YANG CW,LEE YZ,et al,2009.Tylophorine arrests carcinoma cells at G1 phase by downregulating cyclin A2 expression[J].Biochem Biophys Res Comm,386(1):140-145.
YANG F,HUANG L,YANG J,et al,2015.Cisplatin-induced senescence and senescence-related gene expression profiles in human nasopharyngeal carcinoma cells CNE2[J].Canc Res Prev Treat,42(1):4-8.[杨飞城,黄岚珍,阳晶,等,2015.顺铂诱导人鼻咽癌CNE2细胞衰老及其相关基因表达谱的研究[J].肿瘤防治研究,42(1):4-8.]