一氧化氮对肿瘤细胞辐射敏感性的影响具有G2/M期依赖性
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摘要
目的观察不同浓度一氧化氮对携带野生型p53基因状态的人肺腺癌细胞所在不同周期中生存、凋亡、染色体畸变和DNA双链断裂的双向性影响。方法选择携带野生型p53基因的人肺腺癌细胞作为研究对象。急性X线照射(3 Gy、6 Gy)野生型p53基因的人肺腺癌细胞前6 h,使用不同浓度的一氧化氮(5μmol/L、500μmol/L)作用于细胞,或使用0. 02 Gy小剂量预照射。使用血清饥饿方法获得细胞同步化,经过离心后收集有丝分裂的细胞,有丝分裂后分别培养2. 5 h、12. 5 h、17. 5 h,依次获得G1、S、G2/M期细胞。细胞敏感性、细胞凋亡、染色体畸变和DNA双链断裂分别由克隆形成实验、TUNEL染色法、染色体显带技术和免疫荧光检测。结果主要在G2/M期细胞,在急性大剂量辐射前,先给予小剂量0. 02 Gy预照射,或使用一氧化氮供体硝酸异山梨酯(ISDN)浓度为5μmol/L,可明显观察到对于辐射的适应性反应,反应的主要外在表现为抑制细胞的凋亡,细胞内的染色体出现畸变或者DNA双链发生断裂的降低。另一方面,与之相反的是,也是在G2/M期细胞,在急性大剂量辐射前,使用高浓度的500μmol/L ISDN处理,细胞会出现明显的增敏反应,产生辐射增敏后具体的外在表现为加速了细胞的凋亡,同样染色体发生畸变以及DNA的双链断裂得到增强。结论携带野生型p53基因状态的人肺腺癌细胞在一氧化氮放射诱导下的细胞生存、凋亡、染色体畸变和DNA双链断裂的影响具有G2/M期依赖性。
Objective To observe the dual effects of the cell survival,apoptosis,chromosome aberration and DNA double strand breaks on human lung adenocarcinoma cells in different cell cycles carrying wild type p53 gene state under the action of different concentrations of nitric oxide. Methods Human lung adenocarcinoma cells carrying wild type p53 gene were selected as the research objects. The human lung adenocarcinoma cells carrying wild type p53 gene state were irradiated acute x-ray of 3 Gyor 6 Gy,after 6 hours the human lung adenocarcinoma cells using different concentrations of nitric oxide( 5μmol/L,500 μmol/L),or using a small dose of 0. 02 Gy to pre irradiation. Cell synchronization was obtained by means of serum starvation. Mitotic cells were collected in the culture medium by centrifugation. After mitosis,the cells were cultured for2. 5 hours,12. 5 hours,17. 5 hours respectively,in order to get G1,S,G2/M phase cells. Cell sensitivity,apoptosis,chromosome aberration and DNA double strand breaks were detected by clone formation,TUNEL staining,chromosome banding and immunofluorescence respectively. Results In G2/M phase cells,low dose of 0. 02 Gy pre-irradiation or 5 μmol/L isosorbide nitrate( ISDN),a nitric oxide donor,were given before acute radiation of a high dose. Adaptive responses to radiation were observed. The main external manifestations of the response were inhibition of cell apoptosis and chromosome aberration. The distortion of DNA double strand breaks was decreased. On the other hand,in G2/M phase,cells were treated with a high concentration of 500 μmol/L ISDN before acute radiation of a high dose. When we observed again,the cells showed a marked sensitization reaction,and the specific external manifestation after radiation sensitization was accelerated. Apoptosis,chromosome aberration and DNA double strand breaks were enhanced. Conclusion The survival,apoptosis,chromosome aberration and DNA double strand breaks induced by nitric oxide and radiation in human lung adenocarcinoma cells with the state of wild type p53 gene were dependent on the G2/M phase.
Objective To observe the dual effects of the cell survival,apoptosis,chromosome aberration and DNA double strand breaks on human lung adenocarcinoma cells in different cell cycles carrying wild type p53 gene state under the action of different concentrations of nitric oxide. Methods Human lung adenocarcinoma cells carrying wild type p53 gene were selected as the research objects. The human lung adenocarcinoma cells carrying wild type p53 gene state were irradiated acute x-ray of 3 Gyor 6 Gy,after 6 hours the human lung adenocarcinoma cells using different concentrations of nitric oxide( 5μmol/L,500 μmol/L),or using a small dose of 0. 02 Gy to pre irradiation. Cell synchronization was obtained by means of serum starvation. Mitotic cells were collected in the culture medium by centrifugation. After mitosis,the cells were cultured for2. 5 hours,12. 5 hours,17. 5 hours respectively,in order to get G1,S,G2/M phase cells. Cell sensitivity,apoptosis,chromosome aberration and DNA double strand breaks were detected by clone formation,TUNEL staining,chromosome banding and immunofluorescence respectively. Results In G2/M phase cells,low dose of 0. 02 Gy pre-irradiation or 5 μmol/L isosorbide nitrate( ISDN),a nitric oxide donor,were given before acute radiation of a high dose. Adaptive responses to radiation were observed. The main external manifestations of the response were inhibition of cell apoptosis and chromosome aberration. The distortion of DNA double strand breaks was decreased. On the other hand,in G2/M phase,cells were treated with a high concentration of 500 μmol/L ISDN before acute radiation of a high dose. When we observed again,the cells showed a marked sensitization reaction,and the specific external manifestation after radiation sensitization was accelerated. Apoptosis,chromosome aberration and DNA double strand breaks were enhanced. Conclusion The survival,apoptosis,chromosome aberration and DNA double strand breaks induced by nitric oxide and radiation in human lung adenocarcinoma cells with the state of wild type p53 gene were dependent on the G2/M phase.
引文
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[13]Maciag AE,Holland RJ,Kim Y,et al.Nitric oxide(NO)releasing poly ADP-ribose polymerase 1(PARP-1)inhibitors targeted to glutathione S-transferase P1-overexpressing cancer cells[J].J Med Chem,2014,57(6):2292-302.DOI:10.1021/jm401550d.
[14]Li W,Han W,Ma Y,et al.P53-dependent miRNAs mediate nitric oxide-induced apoptosis in colonic carcinogenesis[J].Free Radic Biol Med,2015,85(9):105-113.DOI:10.1016/j.freeradbiomed.2015.04.016.
[15]Yakovlev VA.Nitric oxide-dependent downregulation of BRCA1 expression promotes genetic instability[J].Cancer Res,2013,73(2):706-715.DOI:10.1158/0008-5472.CAN-12-3270.Epub 2012 Oct29.
[16]Mikhailenko VM,Diomina EA,Muzalov II,et al.Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation[J].Exp Oncol,2013,35(1):58-64.
[17]Yermilov V,Rubio J,Ohshima H.Formation of 8-nitroguanine in DNA treated with peroxynitritein vitro and its rapid removal from DNA by depurination[J].FEBS Lett,1995,376(6):207-210.
[18]Sasaki MS,Ejima Y,Tachibana A,et al.DNA damage response pathway in radioadaptive response[J].Mutat Res,2002,504(19):101-118.
[19]Koyama N,Nishida Y,Ishii T,et al.Telmisartan induces growth inhibition,DNA double-strand breaks and apoptosis in human endometrial cancer cells[J].Plos One,2014,9(3):e93050-e93051.DOI:10.1371/journal.pone.0093050.e Collection 2014.
[20]Nakahara R,Nishida K,Hashizume K,et al.Endogenous and X-rayinduced DNA double strand breaks sensitively activate apoptosis in adult neural stem cells[J].Journal of Cell Science,2015,128(19):7109-7116.DOI:10.1242/jcs.171223.
[2]Jella KK,Moriarty R,Mc Clean B,et al.Reactive oxygen species and nitric oxide signaling in bystander cells[J].PLo S One.2018,13(4):1-17.DOI:10.1371/journal.pone.0195371.
[3]Burke AJ,Sullivan FJ,Giles FJ,et al.The yin and yang of nitric oxide in cancer progression[J].Carcinogenesis,2013,34(3):503-512.DOI:10.1093/carcin/bgt034.
[4]Vahora H,Khan MA,Alalami U,et al.The Potential Role of Nitric Oxide in Halting Cancer Progression Through Chemoprevention[J].JCancer Prev,2016,21(1):1-12.DOI:10.15430/JCP.2016.21.1.1.
[5]Matsumoto H,Takahashi A,Ohnishi T.Nitric oxide radicals choreograph a radioadaptiveresponse[J].Cancer Res,2007,67(12):8574-8579.
[6]Oronsky BT,Knox SJ,Scicinski JJ.Is Nitric Oxide(NO)the Last Word in Radiosensitization?A Review[J].Transl Oncol,2012,5(2):66-71.
[7]Gao L,Williams JL.Nitric oxide-donating aspirin induces G2/Mphase cell cycle arrest in human cancer cells by regulating phase transition proteins[J].Int J Oncol,2012,41(1):325-330.DOI:10.3892/ijo.2012.1455.
[8]Jarry A,Charrier L,Bou-Hanna C,et al.Position in cell cycle controls the sensitivity of colon cancer cells to nitric oxide-dependent programmed cell death[J].Cancer Res,2004,64(12):4227-4234.
[9]Xiaoming Su,Akihisa Takahashi,GuozhenGuo,et al.Biphasic effects of NO radicals on radiation-induced lethality and chromosome aberrations in human lung cancer cells carrying different p53 gene status[J].Int J Radiat Oncol BiolPhys,2010,77(2):559-565.DOI:10.1016/j.ijrobp.2009.12.059.
[10]苏晓明,崔迪,任晔,等。一氧化氮对辐射诱导的肿瘤细胞凋亡和DNA双链断裂的双向性影响[J].疑难病杂志,2017,16(4):389-395.DOI:10.3969/j.issn.1671-6450.2017.04.018.
[11]Su XM,Takahashi A,Kondo N,et al.Nitric oxide radical-induced radioadaptation and radiosensitization are G2/M phase-dependent[J].J Radiat Res,2011,52(5):609-615.
[12]Kaplon J,van Dam L,Peeper D.Two-way communication between the metabolic and cell cycle machineries:the molecular basis[J].Cell Cycle,2015,14(13):2022-2032.DOI:10.1080/15384101.2015.1044172.
[13]Maciag AE,Holland RJ,Kim Y,et al.Nitric oxide(NO)releasing poly ADP-ribose polymerase 1(PARP-1)inhibitors targeted to glutathione S-transferase P1-overexpressing cancer cells[J].J Med Chem,2014,57(6):2292-302.DOI:10.1021/jm401550d.
[14]Li W,Han W,Ma Y,et al.P53-dependent miRNAs mediate nitric oxide-induced apoptosis in colonic carcinogenesis[J].Free Radic Biol Med,2015,85(9):105-113.DOI:10.1016/j.freeradbiomed.2015.04.016.
[15]Yakovlev VA.Nitric oxide-dependent downregulation of BRCA1 expression promotes genetic instability[J].Cancer Res,2013,73(2):706-715.DOI:10.1158/0008-5472.CAN-12-3270.Epub 2012 Oct29.
[16]Mikhailenko VM,Diomina EA,Muzalov II,et al.Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation[J].Exp Oncol,2013,35(1):58-64.
[17]Yermilov V,Rubio J,Ohshima H.Formation of 8-nitroguanine in DNA treated with peroxynitritein vitro and its rapid removal from DNA by depurination[J].FEBS Lett,1995,376(6):207-210.
[18]Sasaki MS,Ejima Y,Tachibana A,et al.DNA damage response pathway in radioadaptive response[J].Mutat Res,2002,504(19):101-118.
[19]Koyama N,Nishida Y,Ishii T,et al.Telmisartan induces growth inhibition,DNA double-strand breaks and apoptosis in human endometrial cancer cells[J].Plos One,2014,9(3):e93050-e93051.DOI:10.1371/journal.pone.0093050.e Collection 2014.
[20]Nakahara R,Nishida K,Hashizume K,et al.Endogenous and X-rayinduced DNA double strand breaks sensitively activate apoptosis in adult neural stem cells[J].Journal of Cell Science,2015,128(19):7109-7116.DOI:10.1242/jcs.171223.