Background: Radiotherapy is one of the main therapeutic approaches for non–small cell lung cancer(NSCLC). However, radioresistant cancer cells can eventually cause tumor relapse and even fatal metastasis. It is thoug...Background: Radiotherapy is one of the main therapeutic approaches for non–small cell lung cancer(NSCLC). However, radioresistant cancer cells can eventually cause tumor relapse and even fatal metastasis. It is thought that radioresistance and metastasis could be potentially linked by epithelial?mesenchymal transition(EMT). In this study, we established radioresistant NSCLC cells to investigate the potential relationship among radioresistance, EMT, and enhanced metastatic potential and the underlying mechanism involving liver kinase B1(LKB1)?Salt?inducible kinase 1(SIK1) signaling.Methods: The radioresistant cell lines A549 R and H1299 R were generated by dose?gradient irradiation of the paren?tal A549 and H1299 cells. The radioresistance/sensitivity was evaluated by Cell Counting Kit?8 assay, apoptosis analysis, and/or clonogenic cell survival assay. The EMT phenotype and the signaling change were assessed by Western blot?ting. The abilities of invasion and migration were evaluated by transwell assays and wound healing assays.Results: The radioresistant cell lines A549 R and H1299 R displayed mesenchymal features with enhanced invasion and migration. Mechanistically, A549 R and H1299 R cells had attenuated LKB1?SIK1 signaling, which leaded to the up?regulation of Zinc?finger E?box?binding homeobox factor 1(ZEB1)—a transcription factor that drives EMT. Re?expression of LKB1 in A549 R cells reversed the EMT phenotype, whereas knockdown of LKB1 in H1299 R cells further promoted the EMT phenotype. Moreover, re?expression of LKB1 in A549 cells increased the radiosensitivity, whereas knockdown of LKB1 in H1299 cells decreased the radiosensitivity.Conclusions: Our findings suggest that attenuated LKB1?SIK1 signaling promotes EMT and radioresistance of NSCLC cells, which subsequently contributes to the enhanced metastatic potential. Targeting the LKB1?SIK1?ZEB1 pathway to suppress EMT might provide therapeutic benefits.展开更多
目的胃癌放疗抵抗是绝大多数胃癌患者在治疗过程中所面临的一大难题,探讨其关键基因靶点与潜在机制,对指导临床胃癌患者放射治疗及预后分析有着深远意义。本研究旨在分析丝裂原活化蛋白激酶14(mitogen-activated protein kinase 14,MAPK...目的胃癌放疗抵抗是绝大多数胃癌患者在治疗过程中所面临的一大难题,探讨其关键基因靶点与潜在机制,对指导临床胃癌患者放射治疗及预后分析有着深远意义。本研究旨在分析丝裂原活化蛋白激酶14(mitogen-activated protein kinase 14,MAPK14)在胃癌放疗抵抗患者及胃癌放疗抵抗细胞株中的表达,探究其在胃癌放疗抵抗中的潜在作用。方法采用the Cancer Genome Atlas(TCGA)数据库获得416例胃癌患者MAPK14的mRNA表达水平,Kaplan Meier生存曲线分析MAPK14表达与胃癌患者生存的关联性;建立胃癌放疗抵抗细胞株(放抗株)SGC-7901-R,通过克隆形成实验鉴定其放射线抗性,流式细胞术检测其细胞凋亡、细胞周期改变,蛋白质印迹分析放疗抵抗细胞株(放抗株)SGC-7901-R与亲本胃癌细胞株SGC-7901的MAPK14蛋白表达差异;免疫组化技术验证胃癌放疗抵抗患者与胃癌放疗敏感患者的MAPK14蛋白表达差异。结果 Kaplan-Meier生存曲线分析显示,MAPK14低表达组患者的总生存期优于MAPK14高表达组,中位生存期分别为57.39和19.32个月,两者的预后差异有统计学意义(χ2=14.96,P=0.000 1)。克隆形成实验表明,在4Gy放射线的作用下,放抗株SGC-7901-R的克隆形成能力高于亲本胃癌细胞株SGC-7901,差异有统计学意义,t=41.99,P<0.001。流式检测显示,经4Gy放射线处理后,SGC-7901-R的细胞凋亡率低于SGC-7901细胞,差异有统计学意义,t=8.88,P=0.000 9;与SGC-7901细胞相比,SGC-7901-R的细胞周期阻滞在G2/M期,差异有统计学意义,t=3.011,P=0.039 5。蛋白质印迹结果证实,放抗株SGC-7901-R的MAPK14蛋白表达量高于亲本SGC-7901细胞(1.09±0.07 vs 0.59±0.11),差异有统计学意义,t=3.869,P=0.018。免疫组化结果显示,与胃癌放疗敏感患者相比,胃癌放疗抵抗患者肿瘤组织中MAPK14蛋白表达水平上调,差异有统计学意义,t=26.27,P<0.001。结论 MAPK14异常高表达介导胃癌放疗抵抗且与胃癌患者不良预后�展开更多
Background Radiotherapy plays a critical role in the management of non-small cell lung cancer （NSCLC）. This study was conducted to identify gene expression profiles of acquired radioresistant NSCLC cell line establi...Background Radiotherapy plays a critical role in the management of non-small cell lung cancer （NSCLC）. This study was conducted to identify gene expression profiles of acquired radioresistant NSCLC cell line established by fractionated ionizing radiation （FIR） by cDNA microarray. Methods The human lung adenocarcinoma cell line Anip973 was treated with high energy X-ray to receive 60 Gy in 4 Gy fractions. The radiosensitivity of Anip973R and its parental line were measured by clonogenic assay. Gene expression profiles of Anip973R and its parental line were analyzed using cDNA microarray consisting of 21 522 human genes. Identified partly different expressive genes were validated by quantitative reverse transcription-polymerase chain reaction （Q-RT-PCR）. Results Fifty-nine upregulated and 43 downregulated genes were identified to radio-resistant Anip973R. Up-regulated genes were associated with DNA damage repair （DDB2）, extracellular matrix （LOX）, cell adhesion （CDH2）, and apoptosis （CRYAB）. Down-regulated genes were associated with angiogenesis （GBP-1）, immune response （CD83）, and calcium signaling pathway （TNNC1）. Subsequent validation of selected eleven genes （CD24, DDB2, IGFBP3, LOX, CDH2, CRYAB, PROCR, ANXA1 DCN, GBP-1 and CD83） by Q-RT-PCR was consistent with microarray analysis. Conclusions Fractionated ionizing radiation can lead to the development of radiation resistance. Altered gene profiles of radioresistant cell line may provide new insights into mechanisms underlying clinical radioresistance for NSCLC.展开更多
Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncol- ogy. The hypoxia-inducible factor （HIF）-lα plays a crucial role in radiosensitivity, but the detailed mechanisms remain...Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncol- ogy. The hypoxia-inducible factor （HIF）-lα plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by ir- radiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving frac- tion. EGFP-LC3 plasmid was stably transfected into cells to monitor autopbagic processes. Western blotting was used to evaluate the protein expression levels of HIF-lα, c-Jun, phosphorylated c-Jun, Be- clin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phos- phorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Be- clin 1 expression, instead of HIF-1α, could be blocked by SP600125 （a specific inhibitor of c-Jun NH2- terminal kinase）, followed by suppression of autophagy. Under hypoxia, combined treatment of irradia- tion and chloroquine （a potent autophagy inhibitor） significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclinl expression may be considered as a target to reverse the radioresistance in cancer cells.展开更多
背景:瘢痕疙瘩是一种创伤后成纤维细胞大量增殖和胶原过度沉积的良性皮肤肿瘤。瘢痕疙瘩组织内血管闭塞、炎症和成纤维细胞的增殖及胶原分泌等高代谢行为均导致组织乏氧,组织乏氧在瘢痕疙瘩的发病和预后中起重要作用。目的:就缺氧诱导因...背景:瘢痕疙瘩是一种创伤后成纤维细胞大量增殖和胶原过度沉积的良性皮肤肿瘤。瘢痕疙瘩组织内血管闭塞、炎症和成纤维细胞的增殖及胶原分泌等高代谢行为均导致组织乏氧,组织乏氧在瘢痕疙瘩的发病和预后中起重要作用。目的:就缺氧诱导因子1α在瘢痕疙瘩肿瘤特性中的研究进展作一综述。方法:检索知网数据库(CNKI)、维普数据库及万方数据平台,PubMed数据库、Biosos Preview数据库及Web of Science数据库从建库至2020年11月的相关文献,中英文检索词分别为"瘢痕疙瘩;缺氧诱导因子1;放疗抵抗""keloids;HIF-lα;Radioresistance"。经过阅读文题、摘要和全文的逐步筛选,最终纳入104篇符合入选标准的文献。结果与结论:乏氧微环境抑制缺氧诱导因子1α的降解,增多的缺氧诱导因子1α可诱导瘢痕疙瘩产生上皮间质转化、血管生成、糖酵解、放疗抵抗等类似于恶性肿瘤的生物学行为,而这些病理生理过程又加重组织乏氧,使得缺氧诱导因子1α呈指数增加,形成恶性循环。目前,瘢痕疙瘩的治疗仍以手术切除联合放疗为主。以缺氧诱导因子1α为靶点的治疗,如高压氧治疗治疗,可以显著改善瘢痕疙瘩组织的乏氧环境,促进伤口的愈合,逆转瘢痕疙瘩成纤维细胞的肿瘤特性,但目前关于高压氧治疗辅助手术联合放疗治疗瘢痕疙瘩的研究并不多,靶向瘢痕疙瘩缺氧诱导因子1α的临床治疗仍在探索中。展开更多
基金supported by grants from the Jiangsu Provincial Department of Human Resources and Social Security under the“Six Talent Peaks”Project(No.2013-WSN-082)the Jiangsu Provincial Commission of Health and Family Planning under the Project of Science and Technology(No.H201426)
文摘Background: Radiotherapy is one of the main therapeutic approaches for non–small cell lung cancer(NSCLC). However, radioresistant cancer cells can eventually cause tumor relapse and even fatal metastasis. It is thought that radioresistance and metastasis could be potentially linked by epithelial?mesenchymal transition(EMT). In this study, we established radioresistant NSCLC cells to investigate the potential relationship among radioresistance, EMT, and enhanced metastatic potential and the underlying mechanism involving liver kinase B1(LKB1)?Salt?inducible kinase 1(SIK1) signaling.Methods: The radioresistant cell lines A549 R and H1299 R were generated by dose?gradient irradiation of the paren?tal A549 and H1299 cells. The radioresistance/sensitivity was evaluated by Cell Counting Kit?8 assay, apoptosis analysis, and/or clonogenic cell survival assay. The EMT phenotype and the signaling change were assessed by Western blot?ting. The abilities of invasion and migration were evaluated by transwell assays and wound healing assays.Results: The radioresistant cell lines A549 R and H1299 R displayed mesenchymal features with enhanced invasion and migration. Mechanistically, A549 R and H1299 R cells had attenuated LKB1?SIK1 signaling, which leaded to the up?regulation of Zinc?finger E?box?binding homeobox factor 1(ZEB1)—a transcription factor that drives EMT. Re?expression of LKB1 in A549 R cells reversed the EMT phenotype, whereas knockdown of LKB1 in H1299 R cells further promoted the EMT phenotype. Moreover, re?expression of LKB1 in A549 cells increased the radiosensitivity, whereas knockdown of LKB1 in H1299 cells decreased the radiosensitivity.Conclusions: Our findings suggest that attenuated LKB1?SIK1 signaling promotes EMT and radioresistance of NSCLC cells, which subsequently contributes to the enhanced metastatic potential. Targeting the LKB1?SIK1?ZEB1 pathway to suppress EMT might provide therapeutic benefits.
文摘目的胃癌放疗抵抗是绝大多数胃癌患者在治疗过程中所面临的一大难题,探讨其关键基因靶点与潜在机制,对指导临床胃癌患者放射治疗及预后分析有着深远意义。本研究旨在分析丝裂原活化蛋白激酶14(mitogen-activated protein kinase 14,MAPK14)在胃癌放疗抵抗患者及胃癌放疗抵抗细胞株中的表达,探究其在胃癌放疗抵抗中的潜在作用。方法采用the Cancer Genome Atlas(TCGA)数据库获得416例胃癌患者MAPK14的mRNA表达水平,Kaplan Meier生存曲线分析MAPK14表达与胃癌患者生存的关联性;建立胃癌放疗抵抗细胞株(放抗株)SGC-7901-R,通过克隆形成实验鉴定其放射线抗性,流式细胞术检测其细胞凋亡、细胞周期改变,蛋白质印迹分析放疗抵抗细胞株(放抗株)SGC-7901-R与亲本胃癌细胞株SGC-7901的MAPK14蛋白表达差异;免疫组化技术验证胃癌放疗抵抗患者与胃癌放疗敏感患者的MAPK14蛋白表达差异。结果 Kaplan-Meier生存曲线分析显示,MAPK14低表达组患者的总生存期优于MAPK14高表达组,中位生存期分别为57.39和19.32个月,两者的预后差异有统计学意义(χ2=14.96,P=0.000 1)。克隆形成实验表明,在4Gy放射线的作用下,放抗株SGC-7901-R的克隆形成能力高于亲本胃癌细胞株SGC-7901,差异有统计学意义,t=41.99,P<0.001。流式检测显示,经4Gy放射线处理后,SGC-7901-R的细胞凋亡率低于SGC-7901细胞,差异有统计学意义,t=8.88,P=0.000 9;与SGC-7901细胞相比,SGC-7901-R的细胞周期阻滞在G2/M期,差异有统计学意义,t=3.011,P=0.039 5。蛋白质印迹结果证实,放抗株SGC-7901-R的MAPK14蛋白表达量高于亲本SGC-7901细胞(1.09±0.07 vs 0.59±0.11),差异有统计学意义,t=3.869,P=0.018。免疫组化结果显示,与胃癌放疗敏感患者相比,胃癌放疗抵抗患者肿瘤组织中MAPK14蛋白表达水平上调,差异有统计学意义,t=26.27,P<0.001。结论 MAPK14异常高表达介导胃癌放疗抵抗且与胃癌患者不良预后�
文摘Background Radiotherapy plays a critical role in the management of non-small cell lung cancer （NSCLC）. This study was conducted to identify gene expression profiles of acquired radioresistant NSCLC cell line established by fractionated ionizing radiation （FIR） by cDNA microarray. Methods The human lung adenocarcinoma cell line Anip973 was treated with high energy X-ray to receive 60 Gy in 4 Gy fractions. The radiosensitivity of Anip973R and its parental line were measured by clonogenic assay. Gene expression profiles of Anip973R and its parental line were analyzed using cDNA microarray consisting of 21 522 human genes. Identified partly different expressive genes were validated by quantitative reverse transcription-polymerase chain reaction （Q-RT-PCR）. Results Fifty-nine upregulated and 43 downregulated genes were identified to radio-resistant Anip973R. Up-regulated genes were associated with DNA damage repair （DDB2）, extracellular matrix （LOX）, cell adhesion （CDH2）, and apoptosis （CRYAB）. Down-regulated genes were associated with angiogenesis （GBP-1）, immune response （CD83）, and calcium signaling pathway （TNNC1）. Subsequent validation of selected eleven genes （CD24, DDB2, IGFBP3, LOX, CDH2, CRYAB, PROCR, ANXA1 DCN, GBP-1 and CD83） by Q-RT-PCR was consistent with microarray analysis. Conclusions Fractionated ionizing radiation can lead to the development of radiation resistance. Altered gene profiles of radioresistant cell line may provide new insights into mechanisms underlying clinical radioresistance for NSCLC.
基金supported by the National Natural Science Foundation of China(No.81201779)
文摘Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncol- ogy. The hypoxia-inducible factor （HIF）-lα plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by ir- radiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving frac- tion. EGFP-LC3 plasmid was stably transfected into cells to monitor autopbagic processes. Western blotting was used to evaluate the protein expression levels of HIF-lα, c-Jun, phosphorylated c-Jun, Be- clin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phos- phorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Be- clin 1 expression, instead of HIF-1α, could be blocked by SP600125 （a specific inhibitor of c-Jun NH2- terminal kinase）, followed by suppression of autophagy. Under hypoxia, combined treatment of irradia- tion and chloroquine （a potent autophagy inhibitor） significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclinl expression may be considered as a target to reverse the radioresistance in cancer cells.
文摘背景:瘢痕疙瘩是一种创伤后成纤维细胞大量增殖和胶原过度沉积的良性皮肤肿瘤。瘢痕疙瘩组织内血管闭塞、炎症和成纤维细胞的增殖及胶原分泌等高代谢行为均导致组织乏氧,组织乏氧在瘢痕疙瘩的发病和预后中起重要作用。目的:就缺氧诱导因子1α在瘢痕疙瘩肿瘤特性中的研究进展作一综述。方法:检索知网数据库(CNKI)、维普数据库及万方数据平台,PubMed数据库、Biosos Preview数据库及Web of Science数据库从建库至2020年11月的相关文献,中英文检索词分别为"瘢痕疙瘩;缺氧诱导因子1;放疗抵抗""keloids;HIF-lα;Radioresistance"。经过阅读文题、摘要和全文的逐步筛选,最终纳入104篇符合入选标准的文献。结果与结论:乏氧微环境抑制缺氧诱导因子1α的降解,增多的缺氧诱导因子1α可诱导瘢痕疙瘩产生上皮间质转化、血管生成、糖酵解、放疗抵抗等类似于恶性肿瘤的生物学行为,而这些病理生理过程又加重组织乏氧,使得缺氧诱导因子1α呈指数增加,形成恶性循环。目前,瘢痕疙瘩的治疗仍以手术切除联合放疗为主。以缺氧诱导因子1α为靶点的治疗,如高压氧治疗治疗,可以显著改善瘢痕疙瘩组织的乏氧环境,促进伤口的愈合,逆转瘢痕疙瘩成纤维细胞的肿瘤特性,但目前关于高压氧治疗辅助手术联合放疗治疗瘢痕疙瘩的研究并不多,靶向瘢痕疙瘩缺氧诱导因子1α的临床治疗仍在探索中。
