敲低DNA依赖蛋白激酶催化亚基(DNA-PKcs)抑制Bel7402/5-Fu耐药肝癌细胞的细胞周期及机制
|
摘要
目的研究DNA依赖蛋白激酶催化亚基(DNA-PKcs)基因沉默后对Bel7402/5-氟尿嘧啶(5-Fu)肝癌耐药细胞的细胞周期及机制的影响,探讨DNA-PKcs影响化疗敏感性的相关机制。方法采用MTT法检测Bel7402/5-Fu细胞转染siDNA-PKcs后,对5-Fu、阿霉素(ADM)的敏感性;采用阳离子脂质体法将siDNA-PKcs寡核苷酸片段转染Bel7402/5-Fu细胞;流式细胞术检测细胞周期;Western blot法检测细胞P21、细胞周期蛋白B1(cyclin B1)、细胞分裂周期蛋白2(CDC2)的蛋白表达情况;实时定量PCR检测细胞毛细血管扩张共济失调突变基因(ATM)、p53的mRNA水平,Western blot法检测细胞ATM、P53蛋白水平。结果敲低细胞DNA-PKcs水平后,siDNA-PKcs组5-Fu、ADM的半数抑制浓度(IC50)明显降低,S期细胞减少、G2/M期细胞增多,细胞周期抑制因子P21蛋白水平增加,cyclin B1、CDC2蛋白水平降低,ATM、p53的mRNA及蛋白水平增加。结论敲低DNA-PKcs水平,增加P21蛋白水平,同时抑制cyclin B1、CDC2蛋白水平,诱导Bel7402/5-Fu细胞G2/M期阻滞,其机制可能与ATM-p53信号途径有关。
Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit( DNA-PKcs) on the cell cycle of the multidrug-resistant( MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection,the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil( 5-Fu) and adriamycin( ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile,the protein expressions of cel cycle-related proteins P21,cel cycle protein B1( cyclin B1),cel cycle division protein 2( CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated( ATM)and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclin B1 and CDC2 proteins. In addition,siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions,and finally induces G2/M phase arrest in Bel7402/5-Fu cells,which may be related to ATM-p53 signaling pathway.
Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit( DNA-PKcs) on the cell cycle of the multidrug-resistant( MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection,the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil( 5-Fu) and adriamycin( ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile,the protein expressions of cel cycle-related proteins P21,cel cycle protein B1( cyclin B1),cel cycle division protein 2( CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated( ATM)and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclin B1 and CDC2 proteins. In addition,siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions,and finally induces G2/M phase arrest in Bel7402/5-Fu cells,which may be related to ATM-p53 signaling pathway.
引文
[1]Yan J,Zhou Y,Chen D,et al.Effects of mitochondrial translocation of telomerase on drug resistance in hepatocellular carcinoma cells[J].J Cancer,2015,6(2):151-159.
[2]Li K,Li X,Tian J,et al.Downregulation of DNA-PKcs suppresses P-gp expression via inhibition of the AKT/NF-κB pathway in CD133-positive osteosarcoma MG-63 cells[J].Oncol Rep,2016,36(4):1973-1980.
[3]Handzlik J,Matys A,Kie'c-Kononowicz K.Recent advances in multi-drug resistance(MDR)efflux pump inhibitors of Gram-positive bacteria S.aureus[J].Antibiotics(Basel),2013,2(1):28-45.
[4]Cheng L,Luo S,Jin C,et al.FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/AKT signaling pathway[J/OL].Cell Death Dis,2013,4:e923.doi:10.1038/cddis.2013.450.
[5]石迪.多药耐药的研究现状及进展[J].黑龙江医学,2014,27(5):1074-1076.
[6]Davis A J,Chi L,So S,et al.BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle[J].Nucleic Acids Res,2014,42(18):11487-11501.
[7]Zhou X,Ma X,Wang Z,et al.Radiation-induced hyperproliferation of intestinal crypts results in elevated genome instability with inactive p53-related genomic surveillance[J].Life Sci,2015,143:80-88.
[8]Watanabe G,Lieber M R,Williams D.Structural step forward for NHEJ[J].Cell Res,2017 27(11):1304-1306.
[9]Enriquez-Rios V,Dumitrache L C,Downing S M,et al.DNA-PKcs,ATM,and ATR interplay maintains genome integrity during neurogenesis[J].J Neurosci,2017,37(4):893-905.
[10]Kanungo J.DNA-PK deficiency in Alzheimer’s disease[J].J Neurol Neuromedicine,2016,1(3):17-22.
[11]Fang Y,Chai Z,Wang D,et al.DNA-PKcs deficiency sensitizes the human hepatoma Hep G2 cells to cisplatin and 5-fluorouracil through suppression of the PI3K/AKT/NF-κB pathway[J].Mol Cell Biochem,2015,399(1/2):269-278.
[12]Lin Y F,Shih H Y,Shang Z,et al.DNA-PKcs is required to maintain stability of Chk1and claspin for optimal replication stress respons[J].Nucleic Acids Res,2014,42(7):4463-4473.
[13]Wayne J,Brooks T,Massey A J.Inhibition of Chk1 with the small molecule inhibitor V158411 induces DNA damage and cell death in an unperturbed S-phase[J].Oncotarget,2016,7(51):85033-85048.
[14]Shang Z F,Tan W,Liu X D,et al.DNA-PKcs negatively regulates cyclin B1 protein stability through facilitating its ubiquitination mediated by Cdh1-APC/C pathway[J].Int J Biol Sci,2015,11(9):1026-1035.
[15]Shang Z F,Tan W,Liu X D,et al.DNA-PKcs negatively regulates cyclin B1 protein stability through facilitating its ubiquitination mediated by Cdh1-APC/C pathway[J].Int J Biol Sci,2015,11(9):1026-1035.
[16]Tu W,Dong C,Konishi T,et al.G2-M phase-correlative bystander effects are co-mediated by DNA-PKcs and ATM after carbon ion irradiation[J].Mutat Res Genet Toxicol Environ Mutagen,2016,795:1-6.
[17]Zhou Y,Lee J H,Jiang W,et al.Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM[J].Mol Cell,2017,65(1):91-104.
[18]Rao F,Cha J,Xu J,et al.Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2phosphorylation of Tti1/Tel2[J].Mol Cell,2014,54(1):119-132.
[19]She T,Feng J,Lian S,et al.Sarsaparilla(Smilax Glabra Rhizome)extract activates redox-dependent ATM/ATR pathway to inhibit cancer cell growth by S phase arrest,apoptosis,and autophagy[J].Nutr Cancer,2017,69(8):1281-1289.
[20]Dong H,Shi Q,Song X,et al.Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses:The activations of NHEJ,BER and NER via ATM-p53 signaling axis[J].Toxicol Appl Pharmacol,2015,286(1):10-16.
[21]Fischer M,Quaas M,Steiner L,et al.The p53-p21-DREAM-CDE/CHR pathway regulates G2/M cell cycle genes[J].Nucleic Acids Res,2016,44(1):164-174.
[22]Demoulin B,Hermant M,Castrogiovanni C,et al.Resveratrol induces DNA damage in colon cancer cells by poisoning topoisomeraseⅡand activates the ATM kinase to trigger p53-dependent apoptosis[J].Toxicol In Vitro,2015,29(5):1156-1165.
[23]Ou X,Lu Y,Liao L,et al.Nitidine chloride induces apoptosis in human hepatocellular carcinoma cells through a pathway involving p53,p21,Bax and Bcl-2[J].Oncol Rep,2015,33(3):1264-1274.
[24]Wang Y,Xu Q,Sack L,et al.A gain-of-function senescence bypass screen identifies the homeobox transcription factor DLX2 as a regulator of ATM-p53 signaling[J].Genes Dev,2016,30(3):293-306.
[25]Li M,Fang X,Baker D J,et al.The ATM-p53 pathway suppresses aneuploidy-induced tumorigenesis[J].Proc Natl Acad Sci U S A,2010,107(32):14188-14193.
[2]Li K,Li X,Tian J,et al.Downregulation of DNA-PKcs suppresses P-gp expression via inhibition of the AKT/NF-κB pathway in CD133-positive osteosarcoma MG-63 cells[J].Oncol Rep,2016,36(4):1973-1980.
[3]Handzlik J,Matys A,Kie'c-Kononowicz K.Recent advances in multi-drug resistance(MDR)efflux pump inhibitors of Gram-positive bacteria S.aureus[J].Antibiotics(Basel),2013,2(1):28-45.
[4]Cheng L,Luo S,Jin C,et al.FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/AKT signaling pathway[J/OL].Cell Death Dis,2013,4:e923.doi:10.1038/cddis.2013.450.
[5]石迪.多药耐药的研究现状及进展[J].黑龙江医学,2014,27(5):1074-1076.
[6]Davis A J,Chi L,So S,et al.BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle[J].Nucleic Acids Res,2014,42(18):11487-11501.
[7]Zhou X,Ma X,Wang Z,et al.Radiation-induced hyperproliferation of intestinal crypts results in elevated genome instability with inactive p53-related genomic surveillance[J].Life Sci,2015,143:80-88.
[8]Watanabe G,Lieber M R,Williams D.Structural step forward for NHEJ[J].Cell Res,2017 27(11):1304-1306.
[9]Enriquez-Rios V,Dumitrache L C,Downing S M,et al.DNA-PKcs,ATM,and ATR interplay maintains genome integrity during neurogenesis[J].J Neurosci,2017,37(4):893-905.
[10]Kanungo J.DNA-PK deficiency in Alzheimer’s disease[J].J Neurol Neuromedicine,2016,1(3):17-22.
[11]Fang Y,Chai Z,Wang D,et al.DNA-PKcs deficiency sensitizes the human hepatoma Hep G2 cells to cisplatin and 5-fluorouracil through suppression of the PI3K/AKT/NF-κB pathway[J].Mol Cell Biochem,2015,399(1/2):269-278.
[12]Lin Y F,Shih H Y,Shang Z,et al.DNA-PKcs is required to maintain stability of Chk1and claspin for optimal replication stress respons[J].Nucleic Acids Res,2014,42(7):4463-4473.
[13]Wayne J,Brooks T,Massey A J.Inhibition of Chk1 with the small molecule inhibitor V158411 induces DNA damage and cell death in an unperturbed S-phase[J].Oncotarget,2016,7(51):85033-85048.
[14]Shang Z F,Tan W,Liu X D,et al.DNA-PKcs negatively regulates cyclin B1 protein stability through facilitating its ubiquitination mediated by Cdh1-APC/C pathway[J].Int J Biol Sci,2015,11(9):1026-1035.
[15]Shang Z F,Tan W,Liu X D,et al.DNA-PKcs negatively regulates cyclin B1 protein stability through facilitating its ubiquitination mediated by Cdh1-APC/C pathway[J].Int J Biol Sci,2015,11(9):1026-1035.
[16]Tu W,Dong C,Konishi T,et al.G2-M phase-correlative bystander effects are co-mediated by DNA-PKcs and ATM after carbon ion irradiation[J].Mutat Res Genet Toxicol Environ Mutagen,2016,795:1-6.
[17]Zhou Y,Lee J H,Jiang W,et al.Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM[J].Mol Cell,2017,65(1):91-104.
[18]Rao F,Cha J,Xu J,et al.Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2phosphorylation of Tti1/Tel2[J].Mol Cell,2014,54(1):119-132.
[19]She T,Feng J,Lian S,et al.Sarsaparilla(Smilax Glabra Rhizome)extract activates redox-dependent ATM/ATR pathway to inhibit cancer cell growth by S phase arrest,apoptosis,and autophagy[J].Nutr Cancer,2017,69(8):1281-1289.
[20]Dong H,Shi Q,Song X,et al.Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses:The activations of NHEJ,BER and NER via ATM-p53 signaling axis[J].Toxicol Appl Pharmacol,2015,286(1):10-16.
[21]Fischer M,Quaas M,Steiner L,et al.The p53-p21-DREAM-CDE/CHR pathway regulates G2/M cell cycle genes[J].Nucleic Acids Res,2016,44(1):164-174.
[22]Demoulin B,Hermant M,Castrogiovanni C,et al.Resveratrol induces DNA damage in colon cancer cells by poisoning topoisomeraseⅡand activates the ATM kinase to trigger p53-dependent apoptosis[J].Toxicol In Vitro,2015,29(5):1156-1165.
[23]Ou X,Lu Y,Liao L,et al.Nitidine chloride induces apoptosis in human hepatocellular carcinoma cells through a pathway involving p53,p21,Bax and Bcl-2[J].Oncol Rep,2015,33(3):1264-1274.
[24]Wang Y,Xu Q,Sack L,et al.A gain-of-function senescence bypass screen identifies the homeobox transcription factor DLX2 as a regulator of ATM-p53 signaling[J].Genes Dev,2016,30(3):293-306.
[25]Li M,Fang X,Baker D J,et al.The ATM-p53 pathway suppresses aneuploidy-induced tumorigenesis[J].Proc Natl Acad Sci U S A,2010,107(32):14188-14193.