过氧化物酶V在顺铂诱导HepG2肝癌细胞凋亡过程中的调控作用
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摘要
过氧化物酶V(peroxiredoxin V, Prx V)是过氧化物酶家族(peroxiredoxins, Prxs)中的一员,具有清除细胞内活性氧(reactive oxygen species, ROS)的功能。该文主要阐明了Prx V在顺铂(cisplatin, CDDP)诱导Hep G2人肝癌细胞凋亡过程中的调控作用。该研究利用顺铂处理Hep G2肝癌细胞,通过荧光显微照相、流式细胞术、蛋白质免疫印迹分析等方法检测细胞内活性氧(ROS)水平、细胞凋亡情况以及凋亡相关蛋白水平。研究结果表明,顺铂可引起细胞内的ROS水平升高导致细胞凋亡,同时造成细胞内Prx V蛋白质表达水平下降。利用慢病毒载体过量表达Prx V基因后,顺铂诱导的Prx V过量表达型HepG2细胞凋亡率明显低于Mock组,同时促凋亡蛋白cleavage-Caspase-3、Bad、cleavage-PARP表达水平也明显被下调,说明Prx V在顺铂诱导HepG2细胞凋亡过程中具有一定的抑制作用。该研究初步探究了Prx V在顺铂诱导的HepG2肝癌细胞凋亡过程中的调控作用,为肝癌的治疗研究提供了新的思路和治疗靶点。
Peroxiredoxin V(Prx V) is a member of the Peroxiredoxin families(Prxs) and plays a role on scavenging the intracellular reactive oxygen species(ROS).In the current study,we observed the regulatory role of Prx V on the apoptosis of HepG2 human liver cancer cells induced by cisplatin(CDDP).The CDDP induced cellular ROS,apoptosis and apoptosis related proteins expression levels were measured by fluorescence microscopy,flow cytometry and Western blot analysis.The results indicated that cisplatin could induce the HepG2 cell apoptosis by accumulating the intracellular ROS levels,while the protein expression level of Prx V was down-regulated.After over-expressed of the Prx V gene with lentivirus,the CDDP induced HepG2 cell apoptosis was decreased in Prx V over-expressed cells compared with that of mock cells.The pro-apototic proteins,such as cleavage-Caspase-3,Bad and cleavage-PARP expression levels were also significantly down-regulated in Prx V(O/V) cells than Mock cells.Our findings demonstrated the protective role of Prx V on CDDP induced HepG2 cell apoptosis and gave a new sight to treatment of the liver cancers.
Peroxiredoxin V(Prx V) is a member of the Peroxiredoxin families(Prxs) and plays a role on scavenging the intracellular reactive oxygen species(ROS).In the current study,we observed the regulatory role of Prx V on the apoptosis of HepG2 human liver cancer cells induced by cisplatin(CDDP).The CDDP induced cellular ROS,apoptosis and apoptosis related proteins expression levels were measured by fluorescence microscopy,flow cytometry and Western blot analysis.The results indicated that cisplatin could induce the HepG2 cell apoptosis by accumulating the intracellular ROS levels,while the protein expression level of Prx V was down-regulated.After over-expressed of the Prx V gene with lentivirus,the CDDP induced HepG2 cell apoptosis was decreased in Prx V over-expressed cells compared with that of mock cells.The pro-apototic proteins,such as cleavage-Caspase-3,Bad and cleavage-PARP expression levels were also significantly down-regulated in Prx V(O/V) cells than Mock cells.Our findings demonstrated the protective role of Prx V on CDDP induced HepG2 cell apoptosis and gave a new sight to treatment of the liver cancers.
引文
1 Bosch FX,Ribes J,Díaz M,Cléries R.Primary liver cancer:Worldwide incidence and trends.Gastroenterology 2004;127(5):S5-S16.
2 Galluzzi L,Senovilla L,Vitale I,Michels J,Martins I,Kepp O.Molecular mechanisms of cisplatin resistance.Oncogene 2012;31(15):1869.
3 Kong B,Wang Q,Fung E,Xue K,Tsang BK.p53 is required for cisplatin-induced processing of the mitochondrial fusion protein L-Opa1 that is mediated by the mitochondrial metallopeptidase Oma1 in gynecologic cancers.J Biol Chem 2011;289(39):27134-45.
4刘师兵,于洋,谢奇,赵国艳,徐冶.ABT-737通过线粒体凋亡途径增加HepG2细胞对顺铂的敏感性.中国老年学杂志(Liu Shibing,Yu Yang,Xie Qi,Zhao Guoyan,Xu Zhi.ABT-737 increased sensitivity of HepG2 cells to cisplatin through mitochondrial apoptosis.Chinese Journal of Gerontology)2018;38(6):1442-5.
5 Berndtsson M,Hflgg M,Panaretakis T,Havelka AM,Shoshan MC,Linder S.Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA.Int J Cancer 2010;120(1):175-80.
6蔡先彬,荆绪斌,胡辉,陈素钻,李映华.活性氧在顺铂诱导食管癌细胞EC-109凋亡中的作用(Cai Xianbin,Jing Xubin,Hu Hui,Chen Suzuan,Li Yinghua.The role of reactive oxygen species in cisplain-induced apoptosis of esophageal cancer cell line EC-109.Chinese Journal of Cancer)2006;25(4):427-31.
7魏燕,王蔼明,王宁,张兰梅.卵巢癌顺铂敏感株A2780及耐药株CP70细胞耐药差异与细胞内活性氧的相关性.中国临床医生杂志(Wei Yan,Wang Yiming,Wang Ning,Zhang Meilan.The correlation between the drug resistance of cisplatin sensitive ovarian cancer strain A2780 and drug resistant strain CP70 cells and intracellular reactive oxygen species.Chinese Journal of Clinicians)2015;43(10):68-70.
8 Hofmann B,Hecht HJ,FlohéL.Peroxiredoxin.Biol Chem 2002;383(3/4):347-64.
9 Kinnula VL,Lehtonen S,Sormunen R,Kaarteenaho-Wiik R,Kang SW,Rhee SG,et al.Overexpression of peroxiredoxins I,II,III,V,and VI in malignant mesothelioma.J Pathol 2002;196(3):316.
10涂青松,何剪太,陈伟.抑制过氧化物酶1表达对肝癌细胞放射敏感性的影响.中国普通外科杂志(Tu Qingsong,He Jiantai,Chen Wei.Enhancement of radiosensitivity of hepatocellular carcinoma cells by inhibition of peroxiredoxin 1 expression.Chinese Journal of General Surgery)2016;25(2):245-51.
11刘宝欣,刘英宇,魏中秋,梁婷婷,范玉磊,杨方,等.Prx-1基因沉默对TGF-β1诱导肺成纤维细胞增殖、ROS水平、p-AKT蛋白表达的影响.山东医药(Liu Baoxin,Liu Yingyu,Wei Zhongqiu,Liang Tingting,Fan Yulei,Yang Fang,et al.Effects of silencing peroxiredoxin-1 gene on proliferation,ROS and p-AKTprotein expression of pulmonary fibroblasts induced by TGF-β1.Shandong Medicine)2016;7:17-9.
12陈甫寰.同源异形框基因PRX-2调控成人表皮干细胞向汗腺细胞分化的基础研究.中国人民解放军总医院,解放军医学院,解放军总医院,军医进修学院(Chen Fuhuan.Basic research on the regulation of adult epidermal stem cell differentiation into sweat gland cells by the homologous heteromorphic frame gene PRX-2.People?s liberation army general hospital,people?s liberation army medical school,people?s liberation army general hospital),2016.
13王大宁.PrxⅢ在去甲肾上腺素诱导H9C2细胞凋亡过程中的表达变化.河北医科大学(Wang Daning.Studies on expression change of Prx III mRNA in H9C2 apoptosis process induced by Noradrenaline.Hebei Medical University),2008.
14 Choi HI,Chung KJ,Yang HY,Ren L,Sohn S,Kim PR,et al.Peroxiredoxin V selectively regulates IL-6 production by modulating the Jak2-Stat5 pathway.Free Radical Bio Med 2013;65(4):270-9.
15刘悦,焦冰洋,于楠楠,王闯,韩英浩,金成浩,等.β-拉帕醌通过下调过氧化物酶V诱导SW480结肠癌细胞凋亡.中国细胞生物学学报(Liu Yue,Jiao Bingyang,Yu Nannan,Wang Chuang,Han Yinghao,Jin Chenghao,et al.β-lapachone induced the apoptosis of SW480 colorectal cancer cell through downregulation of the peroxiredoxin V expression.Chinese Journal of Cell Biology)2016;38(12):1487-93.
16王闯,孙虎男,金成浩.Prdx V在链脲佐菌霉素诱导MIN6细胞凋亡过程中的调控作用.中国细胞生物学学报(Wang Chuang,Sun Hunan,Jin Chenghao.Regulatory effect of Prdx V on streptozotocin-induced apoptosis in MIN6 cells.Chinese Journal of Cell Biology)2018;40(2):202-9.
17焦冰洋,王闯,刘悦,宫伊希,韩英浩,金成浩,等.大黄素通过下调PrxV蛋白水平诱导AGS胃癌细胞凋亡.黑龙江八一农垦大学学报(Jiao Bingyang,Wang Chuang,Liu Yue,Gong Yixi,Han Yinghao,Jin Chenghao,et al.AGS gastric cancer cell apoptosis induced by emodin through down-regulate the peroxiredoxin V protein level.Journal of Heilongjiang Bayi Agricultural University)2018;30(1):24-8.
18冯丽,金永哲,刘磊,韩冰,吕春阳,崔玉东,等.PeroxiredoxinV保护HT22小鼠海马神经细胞抵抗NO诱导的细胞凋亡.中国细胞生物学学报(Feng Li,Jin Yongzhe,Liu Lei,Han Bing,LüChunyang,Cui Yudong,et al.Peroxiredoxin V protects the HT22cells against NO mediated apoptosis.Chinese Journal of Cell Biology)2015;37(1):53-8.
19 GN Shen,L Liu,L Feng,Jin Y,Jin MH,Han YH,et al.Knockdown of peroxiredoxin V increases glutamate-induced apoptosis in HT22 hippocampal neuron cells.Mol Med Rep2018;17:7827-34.
20陈伟庆,沈薇,沈鼎明.顺铂压力下HepG2细胞内活性氧及线粒体膜电位的变化.中华肝脏病杂志(Chen Weiqing,Shen Wei,Shen Dingming.The changes of ROS and mitochondria membrane potential in HepG2 cells on the pressure of cisplatin.Chinese journal of Hepatology)2005;13(7):531-3.
21杨晓葵,郑芳,邢辉,高庆蕾,王薇,卢运萍,等.细胞色素c介导的半胱天冬氨酸蛋白酶-3活性改变与人卵巢癌顺铂耐药的关系.中华肿瘤杂志(Yang Xiaokui,Zheng Fang,Xing Hui,Gao Qinglei,Wang Wei,Lu Yunping,et al.Relationship between cytochrome c-mediated caspase-3 activity and chemoresistance in cisplatin-resistant human ovarian cancer cell lines.Chinese Journal of Oncology)2002;24(6):544-7.
22 Siddik ZH.Cisplatin:mode of cytotoxic action and molecular basis of resistance.Oncogene 2003;22(47):7265-79.
23 Berndtsson M,Hflgg M,Panaretakis T,Havelka AM,Shoshan MC,Linder S.Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA.Int J Cancer 2010;120(1):175-80.
24刘蒙,韩峥,朱庆曦,谭洁,田霞.下调CDH17基因表达对顺铂耐药的人胃癌BGC823细胞凋亡与增殖的影响.医药导报(Liu Meng,Han Zheng,Zhu Qingxi,Tan Jie,Tian Xia.Effect of down-regulation of CDH17 gene expression on apoptosis and proliferation of cisplatin-resistant human gastric cancer BGC823cells.Herald of Medicine)2018;37(4):416-21.
25张伟星,张浩,翁伟芳.川楝素下调ATF2蛋白的表达增强顺铂对肺癌细胞的凋亡诱导活性研究.中国现代应用药学(Zhang Weixing,Zhang Hao,Weng Weifang.Toosendaninl enhances cisplatin-induced apoptosis in lung cancer through downregulating the expression of ATF2.Chinese Journal of Modern Applied Pharmacy)2018;35(4):547-51.
26符立平,顾春荣,熊佳时,孙元珏,吴敬波.SOX4抑制凋亡介导食管癌顺铂耐药的实验研究.肿瘤预防与治疗(Fu Liping,Gu Chunrong,Xiong Jiashi,Sun Yuanyu,Wu Jingbo.Experimental study of SOX4 mediated cisplatin resistance in esophagus cancer by inhibiting apoptosis.J Cancer Control Treat 2017;30(3):158-63.
27 Singh AK,Vinayak M.Resveratrol alleviates inflammatory hyperalgesia by modulation of reactive oxygen species(ROS),antioxidant enzymes and ERK activation.Inflamm Res 2017;66(10):911-21.
28 Zibara K,Zeidan A,Bjeije H,Kassem N,Badran B,El-Zein N.ROS mediates interferon gamma induced phosphorylation of Src,through the Raf/ERK pathway,in MCF-7 human breast cancer cell line.J Cell Commun Signal 2017;11(1):1-11.
29 Bragado P,Armesilla A,Silva A,Porras A.Apoptosis by cisplatin requires p53 mediated p38alpha MAPK activation through ROSgeneration.Apoptosis 2007;12(9):1733-42.
30 Conde de la Rosa L,Schoemaker MH,Vrenken TE,Buist-Homan M,Havinga R,Jansen PL,et al.Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms:involvement of JNK and ERK MAP kinases.J Hepatol 2006;44(5):918-29.
2 Galluzzi L,Senovilla L,Vitale I,Michels J,Martins I,Kepp O.Molecular mechanisms of cisplatin resistance.Oncogene 2012;31(15):1869.
3 Kong B,Wang Q,Fung E,Xue K,Tsang BK.p53 is required for cisplatin-induced processing of the mitochondrial fusion protein L-Opa1 that is mediated by the mitochondrial metallopeptidase Oma1 in gynecologic cancers.J Biol Chem 2011;289(39):27134-45.
4刘师兵,于洋,谢奇,赵国艳,徐冶.ABT-737通过线粒体凋亡途径增加HepG2细胞对顺铂的敏感性.中国老年学杂志(Liu Shibing,Yu Yang,Xie Qi,Zhao Guoyan,Xu Zhi.ABT-737 increased sensitivity of HepG2 cells to cisplatin through mitochondrial apoptosis.Chinese Journal of Gerontology)2018;38(6):1442-5.
5 Berndtsson M,Hflgg M,Panaretakis T,Havelka AM,Shoshan MC,Linder S.Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA.Int J Cancer 2010;120(1):175-80.
6蔡先彬,荆绪斌,胡辉,陈素钻,李映华.活性氧在顺铂诱导食管癌细胞EC-109凋亡中的作用(Cai Xianbin,Jing Xubin,Hu Hui,Chen Suzuan,Li Yinghua.The role of reactive oxygen species in cisplain-induced apoptosis of esophageal cancer cell line EC-109.Chinese Journal of Cancer)2006;25(4):427-31.
7魏燕,王蔼明,王宁,张兰梅.卵巢癌顺铂敏感株A2780及耐药株CP70细胞耐药差异与细胞内活性氧的相关性.中国临床医生杂志(Wei Yan,Wang Yiming,Wang Ning,Zhang Meilan.The correlation between the drug resistance of cisplatin sensitive ovarian cancer strain A2780 and drug resistant strain CP70 cells and intracellular reactive oxygen species.Chinese Journal of Clinicians)2015;43(10):68-70.
8 Hofmann B,Hecht HJ,FlohéL.Peroxiredoxin.Biol Chem 2002;383(3/4):347-64.
9 Kinnula VL,Lehtonen S,Sormunen R,Kaarteenaho-Wiik R,Kang SW,Rhee SG,et al.Overexpression of peroxiredoxins I,II,III,V,and VI in malignant mesothelioma.J Pathol 2002;196(3):316.
10涂青松,何剪太,陈伟.抑制过氧化物酶1表达对肝癌细胞放射敏感性的影响.中国普通外科杂志(Tu Qingsong,He Jiantai,Chen Wei.Enhancement of radiosensitivity of hepatocellular carcinoma cells by inhibition of peroxiredoxin 1 expression.Chinese Journal of General Surgery)2016;25(2):245-51.
11刘宝欣,刘英宇,魏中秋,梁婷婷,范玉磊,杨方,等.Prx-1基因沉默对TGF-β1诱导肺成纤维细胞增殖、ROS水平、p-AKT蛋白表达的影响.山东医药(Liu Baoxin,Liu Yingyu,Wei Zhongqiu,Liang Tingting,Fan Yulei,Yang Fang,et al.Effects of silencing peroxiredoxin-1 gene on proliferation,ROS and p-AKTprotein expression of pulmonary fibroblasts induced by TGF-β1.Shandong Medicine)2016;7:17-9.
12陈甫寰.同源异形框基因PRX-2调控成人表皮干细胞向汗腺细胞分化的基础研究.中国人民解放军总医院,解放军医学院,解放军总医院,军医进修学院(Chen Fuhuan.Basic research on the regulation of adult epidermal stem cell differentiation into sweat gland cells by the homologous heteromorphic frame gene PRX-2.People?s liberation army general hospital,people?s liberation army medical school,people?s liberation army general hospital),2016.
13王大宁.PrxⅢ在去甲肾上腺素诱导H9C2细胞凋亡过程中的表达变化.河北医科大学(Wang Daning.Studies on expression change of Prx III mRNA in H9C2 apoptosis process induced by Noradrenaline.Hebei Medical University),2008.
14 Choi HI,Chung KJ,Yang HY,Ren L,Sohn S,Kim PR,et al.Peroxiredoxin V selectively regulates IL-6 production by modulating the Jak2-Stat5 pathway.Free Radical Bio Med 2013;65(4):270-9.
15刘悦,焦冰洋,于楠楠,王闯,韩英浩,金成浩,等.β-拉帕醌通过下调过氧化物酶V诱导SW480结肠癌细胞凋亡.中国细胞生物学学报(Liu Yue,Jiao Bingyang,Yu Nannan,Wang Chuang,Han Yinghao,Jin Chenghao,et al.β-lapachone induced the apoptosis of SW480 colorectal cancer cell through downregulation of the peroxiredoxin V expression.Chinese Journal of Cell Biology)2016;38(12):1487-93.
16王闯,孙虎男,金成浩.Prdx V在链脲佐菌霉素诱导MIN6细胞凋亡过程中的调控作用.中国细胞生物学学报(Wang Chuang,Sun Hunan,Jin Chenghao.Regulatory effect of Prdx V on streptozotocin-induced apoptosis in MIN6 cells.Chinese Journal of Cell Biology)2018;40(2):202-9.
17焦冰洋,王闯,刘悦,宫伊希,韩英浩,金成浩,等.大黄素通过下调PrxV蛋白水平诱导AGS胃癌细胞凋亡.黑龙江八一农垦大学学报(Jiao Bingyang,Wang Chuang,Liu Yue,Gong Yixi,Han Yinghao,Jin Chenghao,et al.AGS gastric cancer cell apoptosis induced by emodin through down-regulate the peroxiredoxin V protein level.Journal of Heilongjiang Bayi Agricultural University)2018;30(1):24-8.
18冯丽,金永哲,刘磊,韩冰,吕春阳,崔玉东,等.PeroxiredoxinV保护HT22小鼠海马神经细胞抵抗NO诱导的细胞凋亡.中国细胞生物学学报(Feng Li,Jin Yongzhe,Liu Lei,Han Bing,LüChunyang,Cui Yudong,et al.Peroxiredoxin V protects the HT22cells against NO mediated apoptosis.Chinese Journal of Cell Biology)2015;37(1):53-8.
19 GN Shen,L Liu,L Feng,Jin Y,Jin MH,Han YH,et al.Knockdown of peroxiredoxin V increases glutamate-induced apoptosis in HT22 hippocampal neuron cells.Mol Med Rep2018;17:7827-34.
20陈伟庆,沈薇,沈鼎明.顺铂压力下HepG2细胞内活性氧及线粒体膜电位的变化.中华肝脏病杂志(Chen Weiqing,Shen Wei,Shen Dingming.The changes of ROS and mitochondria membrane potential in HepG2 cells on the pressure of cisplatin.Chinese journal of Hepatology)2005;13(7):531-3.
21杨晓葵,郑芳,邢辉,高庆蕾,王薇,卢运萍,等.细胞色素c介导的半胱天冬氨酸蛋白酶-3活性改变与人卵巢癌顺铂耐药的关系.中华肿瘤杂志(Yang Xiaokui,Zheng Fang,Xing Hui,Gao Qinglei,Wang Wei,Lu Yunping,et al.Relationship between cytochrome c-mediated caspase-3 activity and chemoresistance in cisplatin-resistant human ovarian cancer cell lines.Chinese Journal of Oncology)2002;24(6):544-7.
22 Siddik ZH.Cisplatin:mode of cytotoxic action and molecular basis of resistance.Oncogene 2003;22(47):7265-79.
23 Berndtsson M,Hflgg M,Panaretakis T,Havelka AM,Shoshan MC,Linder S.Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA.Int J Cancer 2010;120(1):175-80.
24刘蒙,韩峥,朱庆曦,谭洁,田霞.下调CDH17基因表达对顺铂耐药的人胃癌BGC823细胞凋亡与增殖的影响.医药导报(Liu Meng,Han Zheng,Zhu Qingxi,Tan Jie,Tian Xia.Effect of down-regulation of CDH17 gene expression on apoptosis and proliferation of cisplatin-resistant human gastric cancer BGC823cells.Herald of Medicine)2018;37(4):416-21.
25张伟星,张浩,翁伟芳.川楝素下调ATF2蛋白的表达增强顺铂对肺癌细胞的凋亡诱导活性研究.中国现代应用药学(Zhang Weixing,Zhang Hao,Weng Weifang.Toosendaninl enhances cisplatin-induced apoptosis in lung cancer through downregulating the expression of ATF2.Chinese Journal of Modern Applied Pharmacy)2018;35(4):547-51.
26符立平,顾春荣,熊佳时,孙元珏,吴敬波.SOX4抑制凋亡介导食管癌顺铂耐药的实验研究.肿瘤预防与治疗(Fu Liping,Gu Chunrong,Xiong Jiashi,Sun Yuanyu,Wu Jingbo.Experimental study of SOX4 mediated cisplatin resistance in esophagus cancer by inhibiting apoptosis.J Cancer Control Treat 2017;30(3):158-63.
27 Singh AK,Vinayak M.Resveratrol alleviates inflammatory hyperalgesia by modulation of reactive oxygen species(ROS),antioxidant enzymes and ERK activation.Inflamm Res 2017;66(10):911-21.
28 Zibara K,Zeidan A,Bjeije H,Kassem N,Badran B,El-Zein N.ROS mediates interferon gamma induced phosphorylation of Src,through the Raf/ERK pathway,in MCF-7 human breast cancer cell line.J Cell Commun Signal 2017;11(1):1-11.
29 Bragado P,Armesilla A,Silva A,Porras A.Apoptosis by cisplatin requires p53 mediated p38alpha MAPK activation through ROSgeneration.Apoptosis 2007;12(9):1733-42.
30 Conde de la Rosa L,Schoemaker MH,Vrenken TE,Buist-Homan M,Havinga R,Jansen PL,et al.Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms:involvement of JNK and ERK MAP kinases.J Hepatol 2006;44(5):918-29.
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