Bmi-1小分子抑制剂PTC-209对肾癌细胞生物学行为的影响及机制
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摘要
目的观察Bmi-1小分子抑制剂PTC-209对肾细胞癌(简称肾癌)细胞生物学行为的影响,并探讨其机制。方法以0、1、2. 5、5、10μmol/L的PTC-209作用于肾癌786-O细胞,采用CCK-8法测算培养24、48 h时的细胞存活率,流式细胞术检测细胞凋亡及细胞周期分布情况,划痕实验观察细胞迁移能力,克隆形成实验观察细胞克隆集落形成能力,荧光定量PCR法及Western blotting法检测细胞中的Notch1及Notch信号通路下游靶基因发状分裂相关增强子1(HES1)、髓细胞组织增生蛋白(MYC)、细胞周期蛋白依赖性激酶抑制因子2A(CDKN2A)、Cyclin D1和Bcl-2。结果随着PTC-209作用浓度升高及作用时间的延长,786-O细胞存活率减少(P均<0. 05);随着PTC-209作用浓度升高,786-O细胞凋亡率、G_0/G_1期细胞比例增加,S期及G_2/M期细胞比例、细胞迁移率及克隆形成数减少(P均<0. 05);与0μmol/L比较,5μmol/L的PTC-209作用后786-O细胞中Notch1、HES1、MYC、CDKN2A、Cyclin D1、Bcl-2基因及蛋白表达均减少(P均<0. 05)。结论 PTC-209可抑制肾癌细胞增殖,促进细胞凋亡,将细胞阻滞于G_0/G_1期,减弱细胞迁移及克隆形成能力;该作用与PTC-209影响肾癌细胞中Notch信号通路相关基因表达有关。
Objective To explore the influence and mechanism of Bmi-1 small-molecule inhibitor PTC-209 on the biological behavior of renal cell carcinoma( renal carcinoma). Methods The 786-O cells were treated with 0,1,2. 5,5 and 10 μmol/L PTC-209 for 24 and 48 h. The effect of PTC-209 on 786-O cell proliferation was assessed by CCK8. Flow cytometry was applied to examine the effect of PTC-209 on 786-O cell cycle and apoptosis. Wound healing assay and colony forming assay were applied to evaluate the capacity of cell migration and colony formation,respectively. Quantitative realtime PCR and Western blotting were applied to evaluate the mRNA and protein expression of NOTCH1,HES1,MYC,CDKN2 A,CyclinD1 and Bcl-2. Results PTC-209 gradually inhibited the growth of 786-O cells with increase of drug doses and action time( P < 0. 05). PTC-209 induced both apoptosis and cell cycle arrest of 786-O cells in a dose-dependent manner. The percentages of cells in the G_0/G_1 phase increased while in the S phase and G_2/M phase decreased. The migration and colony forming ability were also inhibited by PTC-209 in a dose-dependent manner( P < 0. 05). Comparing with 0 μmol/L PTC-209,5 μmol/L PTC-209 decreased the gene and protein expression of NOTCH1,HES1,MYC,CDKN2 A,Cyclin D1 and Bcl-2( all P < 0. 05). Conclusion PTC-209 inhibits 786-O cell proliferation,induces apoptosis,blocks the cells in the G_0/G_1 phase,and significantly suppresses tumor cell migration and colony formation,and its mechanism may be related to NOTCH signaling pathway-related gene expression.
Objective To explore the influence and mechanism of Bmi-1 small-molecule inhibitor PTC-209 on the biological behavior of renal cell carcinoma( renal carcinoma). Methods The 786-O cells were treated with 0,1,2. 5,5 and 10 μmol/L PTC-209 for 24 and 48 h. The effect of PTC-209 on 786-O cell proliferation was assessed by CCK8. Flow cytometry was applied to examine the effect of PTC-209 on 786-O cell cycle and apoptosis. Wound healing assay and colony forming assay were applied to evaluate the capacity of cell migration and colony formation,respectively. Quantitative realtime PCR and Western blotting were applied to evaluate the mRNA and protein expression of NOTCH1,HES1,MYC,CDKN2 A,CyclinD1 and Bcl-2. Results PTC-209 gradually inhibited the growth of 786-O cells with increase of drug doses and action time( P < 0. 05). PTC-209 induced both apoptosis and cell cycle arrest of 786-O cells in a dose-dependent manner. The percentages of cells in the G_0/G_1 phase increased while in the S phase and G_2/M phase decreased. The migration and colony forming ability were also inhibited by PTC-209 in a dose-dependent manner( P < 0. 05). Comparing with 0 μmol/L PTC-209,5 μmol/L PTC-209 decreased the gene and protein expression of NOTCH1,HES1,MYC,CDKN2 A,Cyclin D1 and Bcl-2( all P < 0. 05). Conclusion PTC-209 inhibits 786-O cell proliferation,induces apoptosis,blocks the cells in the G_0/G_1 phase,and significantly suppresses tumor cell migration and colony formation,and its mechanism may be related to NOTCH signaling pathway-related gene expression.
引文
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[2]Giuliano S,Cormerais Y,Dufies M,et al.Resistance to sunitinib in renal clear cell carcinoma results from sequestration in lysosomes and inhibition of the autophagic flux[J].Autophagy,2015,11(10):1891-1904.
[3]Zhao Z,Chen C,Lin J,et al.Synergy between von Hippel-Lindau and p53 contributes to chemosensitivity of clear cell renal cell carcinoma[J].Mol Med Rep,2016,14(3):2785-2790.
[4]Long Q,Liu L,Xia Y,et al.High peritumoral Bmi-1 expression is an independent prognosticator of poor prognosis in renal cell carcinoma[J].Tumour Biol,2015,36(10):8007-8014.
[5]Ohtaka M,Itoh M,Tohda S.BMI1 inhibitors down-regulate NOTCH signaling and suppress proliferation of acute leukemia cells[J].Anticancer Res,2017,37(11):6047-6053.
[6]Kong Y,Ai C,Dong F,et al.Targeting of BMI-1 with PTC-209inhibits glioblastoma development[J].Cell Cycle,2018,17(10):1199-1211.
[7]Wang Q,Li Z,Wu Y,et al.Pharmacological inhibition of Bmi1by PTC-209 impaired tumor growth in head neck squamous cell carcinoma[J].Cancer Cell Int,2017,17:107.
[8]Ljungberg B,Campbell SC,Choi HY,et al.The epidemiology of renal cell carcinoma[J].Eur Urol,2011,60(4):615-621.
[9]Zhang Q,Shi J,Yuan F,et al.Higher expression of XPF is a critical factor in intrinsic chemotherapy resistance of human renal cell carcinoma[J].Int J Cancer,2016,139(12):2827-2837.
[10]Peng HX,Liu XD,Luo ZY,et al.Upregulation of the proto-oncogene Bmi-1 predicts a poor prognosis in pediatric acute lymphoblastic leukemia[J].BMC Cancer,2017,17(1):76.
[11]Srinivasan M,Bharali DJ,Sudha T,et al.Downregulation of Bmi1in breast cancer stem cells suppresses tumor growth and proliferation[J].Oncotarget,2017,8(24):38731-38742.
[12]Wang Z,Li Y,Kong D,et al.Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway[J].Cancer Res,2009,69(6):2400-2407.
[13]Mollen E,Ient J,Tjan-Heijnen V,et al.Moving breast cancer therapy up a Notch[J].Front Oncol,2018,8:518.
[14]Jin YH,Kim H,Oh M,et al.Regulation of Notch1/NICD and Hes1 expressions by GSK-3alpha/beta[J].Mol Cells,2009,27(1):15-19.
[15]Ranganathan P,Weaver KL,Capobianco AJ.Notch signalling in solid tumours:a little bit of everything but not all the time[J].Nat Rev Cancer,2011,11(5):338-351.
[16]Kreso A,van Galen P,Pedley NM,et al.Self-renewal as a therapeutic target in human colorectal cancer[J].Nat Med,2014,20(1):29-36.
[17]Ohtaka M,Itoh M,Tohda S.BMI1 inhibitors down-regulate NOTCH signaling and suppress proliferation of acute leukemia cells[J].Anticancer Res,2017,37(11):6047-6053.