衣霉素诱导的内质网应激对人食管癌细胞自噬和凋亡的影响及其与奥沙利铂化疗耐药的关系
|
摘要
目的研究衣霉素(TM)诱导人食管癌细胞TE-1发生内质网应激(ERS)及其介导的自噬和凋亡作用,并为化疗耐药的发生提供线索。方法 TM作用于人食管癌细胞系TE-1,建立内质网应激模型。Western blot检测Tm作用于TE-1细胞后ERS标志性蛋白GRP78、内质网应激通路相关蛋白(eIF2α、CHOP)、AKT/mTOR通路相关蛋白、自噬相关蛋白(LC3-I/LC3-II、Beclin-1、p62)和凋亡相关蛋白(Bcl-2、Bax、Caspase 3)的表达。MTT法测定不同浓度TM和奥沙利铂对TE-1细胞存活率的影响。单丹酞戊二胺(MDC)实验检测细胞自噬。FCM法检测细胞凋亡。结果 TM可诱导ERS标志蛋白GRP78的表达量增加。Tm作用后PERK及eIF2α的磷酸化水平升高,CHOP的表达量增加。TM作用后,细胞内自噬小泡数量呈现浓度—时间依赖性增加;自噬相关蛋白LC3-I/LC3-II、Beclin-1、p62的表达量随TM作用浓度增加而上调;Bax、Caspase 3表达上调,Bcl-2表达下调;AKT/mTOR通路相关蛋白磷酸化水平降低。TM联合奥沙利铂可增强TE-1细胞对奥沙利铂的敏感性;BEZ235可促进细胞发生自噬和凋亡,并增强TE-1细胞对奥沙利铂的敏感性。结论 TM作用于人食管癌细胞TE-1后可诱导ERS并导致细胞发生自噬和凋亡,同时增强细胞对奥沙利铂的敏感性,可能与AKT/mTOR相关通路调控密切相关。
Objective To investigate the effects of Tunicamycin(TM)-induced endoplasmic reticulum stress(ERS) on autophagy and apoptosis in human esophageal cancer(EC) cell line TE-1, and to provide clues for the occurrence of chemotherapy resistance. Methods ERS model was established in TE-1 cells with TM. Western blot analysis was used to detect the expression of ERS marker protein GRP78, ERS pathway-related proteins(eIF2α, CHOP), AKT/mTOR pathway-related proteins, autophagy-related proteins(LC3-I/LC3-II,Beclin-1, p62) and apoptosis-related proteins(Bcl-2, Bax, Caspase 3). The effects of different concentrations of TM and oxaliplatin on TE-1 cell survival rate were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay. The autophagy was detected by Monodansylcadaverine(MDC), and apoptosis by flow cytometry. Results TM induced the expression of GRP78 increasing. After TM treatment, the phosphorylation levels of PERK and eIF2α increased, and the CHOP expression increased. The number of autophagic vesicles increased in a concentration-time dependent manner. Along with the increase of the concentration of TM, the expressions of LC3-I/LC3-II, Beclin-1, p62, Bax and Caspase 3 were up-regulated, while the Bcl-2 expression was down-regulated, and the phosphorylation of AKT/m TOR pathway-related proteins was decreased. TM combined with oxaliplatin enhanced the sensitivity of TE-1 cells to oxaliplatin.BEZ235 promoted the incidence of autophagy and apoptosis and elevated the sensitivity of TE-1 cells to oxaliplatin. Conclusion Taken together, TM induced ERS in EC cells and promoted autophagy and apoptosis, and increased the sensitivity of TE-1 to oxaliplatin through inhibiting AKT/mTOR signaling pathway.
Objective To investigate the effects of Tunicamycin(TM)-induced endoplasmic reticulum stress(ERS) on autophagy and apoptosis in human esophageal cancer(EC) cell line TE-1, and to provide clues for the occurrence of chemotherapy resistance. Methods ERS model was established in TE-1 cells with TM. Western blot analysis was used to detect the expression of ERS marker protein GRP78, ERS pathway-related proteins(eIF2α, CHOP), AKT/mTOR pathway-related proteins, autophagy-related proteins(LC3-I/LC3-II,Beclin-1, p62) and apoptosis-related proteins(Bcl-2, Bax, Caspase 3). The effects of different concentrations of TM and oxaliplatin on TE-1 cell survival rate were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay. The autophagy was detected by Monodansylcadaverine(MDC), and apoptosis by flow cytometry. Results TM induced the expression of GRP78 increasing. After TM treatment, the phosphorylation levels of PERK and eIF2α increased, and the CHOP expression increased. The number of autophagic vesicles increased in a concentration-time dependent manner. Along with the increase of the concentration of TM, the expressions of LC3-I/LC3-II, Beclin-1, p62, Bax and Caspase 3 were up-regulated, while the Bcl-2 expression was down-regulated, and the phosphorylation of AKT/m TOR pathway-related proteins was decreased. TM combined with oxaliplatin enhanced the sensitivity of TE-1 cells to oxaliplatin.BEZ235 promoted the incidence of autophagy and apoptosis and elevated the sensitivity of TE-1 cells to oxaliplatin. Conclusion Taken together, TM induced ERS in EC cells and promoted autophagy and apoptosis, and increased the sensitivity of TE-1 to oxaliplatin through inhibiting AKT/mTOR signaling pathway.
引文
[1]张喜平,吕艳.胸腹腔镜微创手术与传统开胸手术对食管癌患者术后肺部感染及肺功能的影响[J].中国实用医刊, 2018, 45(1):71-73. doi:10.3760/cma. j. issn.1674-4756.2018.01.025.
[2]陈万青,孙可欣,郑荣寿,等.2014年中国分地区恶性肿瘤发病和死亡分析[J].中国肿瘤, 2018, 27(1):1-14. doi:10.11735/j. issn.1004-0242.2018.01. A001.
[3]刘亮,左静,武中林,等.肺耐药相关蛋白在食管鳞状细胞癌中的表达及其与多药耐药的相关性[J].中国老年学, 2016, 36(17):4141-4142. doi:10.3969/j. issn.1005-9202.2016.17.002.
[4]傅剑华,谭子辉.食管癌外科治疗的现状与未来展望[J].中国肿瘤临床, 2016, 43(12):507-510. doi:10.3969/j.issn.1000-8179.2016.12.144.
[5]何荣琦,许荣誉,李旭,等.内质网应激在二十二碳六烯酸提高食管癌EC9706细胞顺铂化疗敏感性中的作用[J].中华临床营养杂志, 2017, 26(6):372-377.
[6]Galmiche A, Sauzay C, Chevet E, et al. Role of the unfolded protein response in tumor cell characteristics and cancer outcome[J]. Curr Opin Oncol, 2017, 29(1):41-47.
[7]Lindholm D, Korhonen L, Eriksson O, et al. Recent Insights into the Role of Unfolded Protein Response in ER Stress in Health and Disease[J]. Front Cell Dev Biol, 2017, 5:48.doi:10.3389/fcell.2017.00048.
[8]唐夏莉,焦德敏,陈君,等. miRNA-126对肺癌A549细胞的增殖、迁移、侵袭及EGFR/AKT/mTOR信号通路的影响[J].中国病理生理杂志, 2016, 32(3):458-463. doi:10.3969/j. issn.1000-4718.2016.03.012.
[9]Zou H, Li L, Garcia Carcedo I, et al. Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis[J]. Int J Nanomedicine, 2016, 11:1947-1958.DOI:10.2147/IJN. S100744.
[10]Yu H, Shi N, Pan Z, et al. Abstract 1253:Dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with AKT inhibitor MK2206 in esophageal carcinoma cells[J]. Cancer Res, 2014,74(19 Suppl):1253. DOI:10.1158/1538-7445. AM2014-1253.
[11]黄玲.低剂量奥沙利铂联合紫杉醇诱导人卵巢癌细胞A2780凋亡的内质网应激机制研究[J].海南医学院学报,2015, 21(3):299-302. doi:46.1049. R.20141217.1050.001.
[12]董传芳,刘雪平,徐松,等.糖基化终末产物对SH-SY5Y细胞内质网应激的影响[J].卒中与神经疾病, 2012,19(2):67-71. doi:10.3969/j. issn.1007-0478.2012.02.001.
[13]Bohnert KR, Gallot YS, Sato S, et al. Inhibition of ER stress and unfolding protein response pathways causes skeletal muscle wasting during cancer cachexia[J]. FASEB J, 2016,30(9):3053-3068. doi:10.1096/fj.201600250RR.
[14]Hsu HS, Liu CC, Lin JH, et al. Involvement of ER stress,PI3K/AKT activation, and lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis[J]. Sci Rep, 2017,7(1):14272. doi:10.1038/s41598-017-14612-5.
[15]Jiang S, Zhang E, Zhang R, et al. Altered activity patterns of transcription factors induced by endoplasmic reticulum stress[J]. BMC Biochem, 2016, 17:8. doi:10.1186/s12858-016-0060-2.
[16]袁爱红,查必祥,吴吉萍,等.针刺对糖尿病大鼠胰腺内质网应激PERK-CHOP途径与Bax/Bcl-2基因表达的影响[J].中华中医药杂志, 2017, 29(3):1291-1294.
[17]汪应红,王欢,左龙泉,等.自噬在内质网应激诱导的肝星状细胞凋亡中的作用研究[J].安徽医科大学学报,2016, 51(8):1115-1119.
[18]Blagosklonny MV. Hypoxia, MTOR and autophagy:converging on senescence or quiescence[J]. Autophagy, 2013,9(2):260-262. doi:10.4161/auto.22783.
[19]蔡方雨,邹志田,朱晓峰.自噬促进肺腺癌细胞对培美曲塞耐药的实验研究[J].黑龙江医药科学, 2017, 46(2):79-81. doi:10.3969/j. issn.1008-0104.2017.02.034.
[20]孙王宏,王海峰,杨德林.内质网-自噬途径在肿瘤耐药中的研究现状[J].国际肿瘤学杂志, 2017,44(8):590-593. doi:10.3760/cma. j. issn.1673-422X.2017.08.008.
[21]栾阳,刘彼得. PI3K/mTOR双重抑制剂NVP-BEZ235在肾癌治疗中的研究进展[J].国际泌尿系统杂志, 2016,36(1):130-133. doi:10.3760/cma. j. issn.1673-4416.2016.01.039.
[22]王鹤儒,于洋,陈丽艳. PI3K/mTOR抑制剂NVP-BEZ235抑制人乳腺癌细胞增殖及促进细胞自噬[J].基础医学与临床, 2016, 36(8):1139-1141. doi:10.16352/j. issn.1001-6325.2016.08.021.
[23]Venkannagari S, Fiskus W, Peth K, et al. Superior efficacy of co-treatment with dual PI3K/mTOR inhibitor NVPBEZ235 and pan-histone deacetylase inhibitor against human pancreatic cancer[J]. Oncotarget, 2012, 3(11):1416-27.
[2]陈万青,孙可欣,郑荣寿,等.2014年中国分地区恶性肿瘤发病和死亡分析[J].中国肿瘤, 2018, 27(1):1-14. doi:10.11735/j. issn.1004-0242.2018.01. A001.
[3]刘亮,左静,武中林,等.肺耐药相关蛋白在食管鳞状细胞癌中的表达及其与多药耐药的相关性[J].中国老年学, 2016, 36(17):4141-4142. doi:10.3969/j. issn.1005-9202.2016.17.002.
[4]傅剑华,谭子辉.食管癌外科治疗的现状与未来展望[J].中国肿瘤临床, 2016, 43(12):507-510. doi:10.3969/j.issn.1000-8179.2016.12.144.
[5]何荣琦,许荣誉,李旭,等.内质网应激在二十二碳六烯酸提高食管癌EC9706细胞顺铂化疗敏感性中的作用[J].中华临床营养杂志, 2017, 26(6):372-377.
[6]Galmiche A, Sauzay C, Chevet E, et al. Role of the unfolded protein response in tumor cell characteristics and cancer outcome[J]. Curr Opin Oncol, 2017, 29(1):41-47.
[7]Lindholm D, Korhonen L, Eriksson O, et al. Recent Insights into the Role of Unfolded Protein Response in ER Stress in Health and Disease[J]. Front Cell Dev Biol, 2017, 5:48.doi:10.3389/fcell.2017.00048.
[8]唐夏莉,焦德敏,陈君,等. miRNA-126对肺癌A549细胞的增殖、迁移、侵袭及EGFR/AKT/mTOR信号通路的影响[J].中国病理生理杂志, 2016, 32(3):458-463. doi:10.3969/j. issn.1000-4718.2016.03.012.
[9]Zou H, Li L, Garcia Carcedo I, et al. Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis[J]. Int J Nanomedicine, 2016, 11:1947-1958.DOI:10.2147/IJN. S100744.
[10]Yu H, Shi N, Pan Z, et al. Abstract 1253:Dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with AKT inhibitor MK2206 in esophageal carcinoma cells[J]. Cancer Res, 2014,74(19 Suppl):1253. DOI:10.1158/1538-7445. AM2014-1253.
[11]黄玲.低剂量奥沙利铂联合紫杉醇诱导人卵巢癌细胞A2780凋亡的内质网应激机制研究[J].海南医学院学报,2015, 21(3):299-302. doi:46.1049. R.20141217.1050.001.
[12]董传芳,刘雪平,徐松,等.糖基化终末产物对SH-SY5Y细胞内质网应激的影响[J].卒中与神经疾病, 2012,19(2):67-71. doi:10.3969/j. issn.1007-0478.2012.02.001.
[13]Bohnert KR, Gallot YS, Sato S, et al. Inhibition of ER stress and unfolding protein response pathways causes skeletal muscle wasting during cancer cachexia[J]. FASEB J, 2016,30(9):3053-3068. doi:10.1096/fj.201600250RR.
[14]Hsu HS, Liu CC, Lin JH, et al. Involvement of ER stress,PI3K/AKT activation, and lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis[J]. Sci Rep, 2017,7(1):14272. doi:10.1038/s41598-017-14612-5.
[15]Jiang S, Zhang E, Zhang R, et al. Altered activity patterns of transcription factors induced by endoplasmic reticulum stress[J]. BMC Biochem, 2016, 17:8. doi:10.1186/s12858-016-0060-2.
[16]袁爱红,查必祥,吴吉萍,等.针刺对糖尿病大鼠胰腺内质网应激PERK-CHOP途径与Bax/Bcl-2基因表达的影响[J].中华中医药杂志, 2017, 29(3):1291-1294.
[17]汪应红,王欢,左龙泉,等.自噬在内质网应激诱导的肝星状细胞凋亡中的作用研究[J].安徽医科大学学报,2016, 51(8):1115-1119.
[18]Blagosklonny MV. Hypoxia, MTOR and autophagy:converging on senescence or quiescence[J]. Autophagy, 2013,9(2):260-262. doi:10.4161/auto.22783.
[19]蔡方雨,邹志田,朱晓峰.自噬促进肺腺癌细胞对培美曲塞耐药的实验研究[J].黑龙江医药科学, 2017, 46(2):79-81. doi:10.3969/j. issn.1008-0104.2017.02.034.
[20]孙王宏,王海峰,杨德林.内质网-自噬途径在肿瘤耐药中的研究现状[J].国际肿瘤学杂志, 2017,44(8):590-593. doi:10.3760/cma. j. issn.1673-422X.2017.08.008.
[21]栾阳,刘彼得. PI3K/mTOR双重抑制剂NVP-BEZ235在肾癌治疗中的研究进展[J].国际泌尿系统杂志, 2016,36(1):130-133. doi:10.3760/cma. j. issn.1673-4416.2016.01.039.
[22]王鹤儒,于洋,陈丽艳. PI3K/mTOR抑制剂NVP-BEZ235抑制人乳腺癌细胞增殖及促进细胞自噬[J].基础医学与临床, 2016, 36(8):1139-1141. doi:10.16352/j. issn.1001-6325.2016.08.021.
[23]Venkannagari S, Fiskus W, Peth K, et al. Superior efficacy of co-treatment with dual PI3K/mTOR inhibitor NVPBEZ235 and pan-histone deacetylase inhibitor against human pancreatic cancer[J]. Oncotarget, 2012, 3(11):1416-27.