背景与目的已有的研究证明低氧可诱导肿瘤细胞自噬发生,自噬水平与肿瘤放疗敏感性相关,因而调控自噬信号通路是增强放疗敏感性极具潜力的治疗策略。本研究旨在探讨联合应用自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)下调低氧环境中...背景与目的已有的研究证明低氧可诱导肿瘤细胞自噬发生,自噬水平与肿瘤放疗敏感性相关,因而调控自噬信号通路是增强放疗敏感性极具潜力的治疗策略。本研究旨在探讨联合应用自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)下调低氧环境中人肺腺癌A549细胞的自噬水平后对其放疗敏感性的影响。方法实验设低氧对照组及低氧+3-MA(自噬抑制剂)组,分别采用电镜检测自噬体变化,Western blot检测自噬标记蛋白-微管相关蛋白1轻链3(microtubule-associated protein 1 lightchain3,LC3)蛋白表达,分析LC3II/LC3I比值变化。随后每组再给予直线加速器(0Gy、2Gy、4Gy、6Gy、8Gy、10Gy)照射后采用M法检测细胞增殖活性。结果与低氧对照组相比,低氧+3-MA组自噬体数量、LC3II/LC3I比值均下降。给予照射后,与低氧对照组比较,低氧+3-MA组细胞增殖活性下降。结论在低氧环境中,A549细胞保护性自噬增加,抑制自噬可增强A549细胞的放疗敏感性。展开更多
Our previous findings have demonstrated that autophagy regulation can alleviate the decline of learning and memory by eliminating deposition of extracellular beta-amyloid peptide （Aβ） in the brain after stroke, but...Our previous findings have demonstrated that autophagy regulation can alleviate the decline of learning and memory by eliminating deposition of extracellular beta-amyloid peptide （Aβ） in the brain after stroke, but the exact mechanism is unclear. It is presumed that the regulation of beta-site APP-deaving enzyme 1 （BACE1）, the rate-limiting enzyme in metabolism of Aβ, would be a key site. Neuro-2a/amyloid precursor protein 695 （APP695） cell models of cerebral isch- emia were established by oxygen-glucose deprivation to investigate the effects of Rapamycin （an autophagy inducer） or 3-methyladenine （an autophagy inhibitor） on the expression of BACE1. Either oxygen-glucose deprivation or Rapamycin down-regulated the expression of BACE1 while 3-methyladenine up-regulated BACE1 expression. These results confirm that oxygen-glucose deprivation down-regulates BACE1 expression in Neuro-2a/APP695 cells through the introduction of autophagy.展开更多
This study aimed to elucidate the potential mechanisms through which bone marrow-derived mesenchymal stem cells(BM-MSCs)may be effective in alleviating experimental colitis induced by treatment with 2,4,6-trinitrobenz...This study aimed to elucidate the potential mechanisms through which bone marrow-derived mesenchymal stem cells(BM-MSCs)may be effective in alleviating experimental colitis induced by treatment with 2,4,6-trinitrobenzene-sulfonate acid(TNBS),specifically through autophagy modulation.Methods:BM-MSCs were collected from BALB/c mice for subsequent experiments.The study employed cell counting kits(CCK-8)to investigate the impact of the MSC-conditioned medium(M medium)on the proliferation of RAW264.7 macrophages.The GFP-mRFP-LC3 adenovirus was transfected into RAW264.7 to detect autophagic flux.The gene expression of cytokines was assessed through quantitative reverse transcription polymerase chain reaction(qRT-PCR).Western blot analysis was employed to determine the presence of a binding interaction between NOD-like receptor protein 3(NLRP3)and autophagy.Furthermore,a colitis mouse model was established by TNBS induction.Clinical disease activity score was assessed regularly,and histological and morphometric analyses were performed on colonic tissues.Inflammatory serum cytokines were identified using an enzyme-linked immunosorbent assay.Results:BM-MSCs significantly promoted the proliferation of RAW264.7.In vitro lipopolysaccharide(LPS)-stimulated RAW264.7 cells,treated with BM-MSCs,triggered autophagy and inhibited cytokine mRNA expression.Additionally,in LPS-induced RAW264.7,BM-MSCs enhanced the Beclin1 protein expression and the microtubule-associated protein 1 light chain 3(LC3)-II to LC3-I ratio while suppressing the protein levels of NLRP3 and apoptosis-associated speck-like protein(ASC).Nevertheless,3-methyladenine(3-MA),an inhibitor of autophagy,prevented the impact of BM-MSCs by reducing the levels of NLRP3 and ASC proteins,suggesting that autophagy triggered the inhibition of the NLRP3 inflammasome.In comparison to the mice in the TNBS group,the mice in the TNBS+MSC group displayed a more acute form of colitis,and the IL1βand IL18 cytokines in their serum were lowered as well.In the meantime,3-MA raised IL展开更多
C16 saturated fatty acid (Palmitic acid) is one of the most common dietary fatty acids which played an important role in the cellular biological functions. Palmitic acid (PA) was tested for potential inhibition of DNA...C16 saturated fatty acid (Palmitic acid) is one of the most common dietary fatty acids which played an important role in the cellular biological functions. Palmitic acid (PA) was tested for potential inhibition of DNA topoisomerase I (topo I) and it exhibited inhibitory activity in the nanomolar range. Treatment of lung adenocarcinoma cell line A549 with PA resulted in a decrease in cell viability in a concentration-dependent manner, and PA showed cytotoxicity with an IC50 value of 150 μM. DNA fragmentation assay and caspase activity indicated that PA does not induce apoptotic cell death in A549 cells. Finally, we found that PA was able to cause an increase in autophagic flux in a time-dependent manner, evidenced by the accumulation of LC3 through monodansylcadaverine (MDC) staining. More importantly, inhibition of autophagy by blocking autophagosome formation via the inhibition of type III Phosphatidylinositol 3-kinases (PI-3K), by 3-Methyladenine (3-MA) was able to effectively suppress PA-induced autophagy. We showed that inhibition of autophagy sensitized the cells signal to PA-induced apoptosis, suggesting the pro-survival function of autophagy induced by PA. Taken together, results from this study reveal that PA as a topo I inhibitor induced autophagic cell death in A549 cells.展开更多
文摘背景与目的已有的研究证明低氧可诱导肿瘤细胞自噬发生,自噬水平与肿瘤放疗敏感性相关,因而调控自噬信号通路是增强放疗敏感性极具潜力的治疗策略。本研究旨在探讨联合应用自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)下调低氧环境中人肺腺癌A549细胞的自噬水平后对其放疗敏感性的影响。方法实验设低氧对照组及低氧+3-MA(自噬抑制剂)组,分别采用电镜检测自噬体变化,Western blot检测自噬标记蛋白-微管相关蛋白1轻链3(microtubule-associated protein 1 lightchain3,LC3)蛋白表达,分析LC3II/LC3I比值变化。随后每组再给予直线加速器(0Gy、2Gy、4Gy、6Gy、8Gy、10Gy)照射后采用M法检测细胞增殖活性。结果与低氧对照组相比,低氧+3-MA组自噬体数量、LC3II/LC3I比值均下降。给予照射后,与低氧对照组比较,低氧+3-MA组细胞增殖活性下降。结论在低氧环境中,A549细胞保护性自噬增加,抑制自噬可增强A549细胞的放疗敏感性。
基金supported by the National Natural Science Foundation of China,No.31171014,31371065a grant from Shanghai Municipal Health Bureau,China,No.20134125a grant from Shanghai Pudong District Health Bureau of China,No.PDZz2013-10
文摘Our previous findings have demonstrated that autophagy regulation can alleviate the decline of learning and memory by eliminating deposition of extracellular beta-amyloid peptide （Aβ） in the brain after stroke, but the exact mechanism is unclear. It is presumed that the regulation of beta-site APP-deaving enzyme 1 （BACE1）, the rate-limiting enzyme in metabolism of Aβ, would be a key site. Neuro-2a/amyloid precursor protein 695 （APP695） cell models of cerebral isch- emia were established by oxygen-glucose deprivation to investigate the effects of Rapamycin （an autophagy inducer） or 3-methyladenine （an autophagy inhibitor） on the expression of BACE1. Either oxygen-glucose deprivation or Rapamycin down-regulated the expression of BACE1 while 3-methyladenine up-regulated BACE1 expression. These results confirm that oxygen-glucose deprivation down-regulates BACE1 expression in Neuro-2a/APP695 cells through the introduction of autophagy.
文摘This study aimed to elucidate the potential mechanisms through which bone marrow-derived mesenchymal stem cells(BM-MSCs)may be effective in alleviating experimental colitis induced by treatment with 2,4,6-trinitrobenzene-sulfonate acid(TNBS),specifically through autophagy modulation.Methods:BM-MSCs were collected from BALB/c mice for subsequent experiments.The study employed cell counting kits(CCK-8)to investigate the impact of the MSC-conditioned medium(M medium)on the proliferation of RAW264.7 macrophages.The GFP-mRFP-LC3 adenovirus was transfected into RAW264.7 to detect autophagic flux.The gene expression of cytokines was assessed through quantitative reverse transcription polymerase chain reaction(qRT-PCR).Western blot analysis was employed to determine the presence of a binding interaction between NOD-like receptor protein 3(NLRP3)and autophagy.Furthermore,a colitis mouse model was established by TNBS induction.Clinical disease activity score was assessed regularly,and histological and morphometric analyses were performed on colonic tissues.Inflammatory serum cytokines were identified using an enzyme-linked immunosorbent assay.Results:BM-MSCs significantly promoted the proliferation of RAW264.7.In vitro lipopolysaccharide(LPS)-stimulated RAW264.7 cells,treated with BM-MSCs,triggered autophagy and inhibited cytokine mRNA expression.Additionally,in LPS-induced RAW264.7,BM-MSCs enhanced the Beclin1 protein expression and the microtubule-associated protein 1 light chain 3(LC3)-II to LC3-I ratio while suppressing the protein levels of NLRP3 and apoptosis-associated speck-like protein(ASC).Nevertheless,3-methyladenine(3-MA),an inhibitor of autophagy,prevented the impact of BM-MSCs by reducing the levels of NLRP3 and ASC proteins,suggesting that autophagy triggered the inhibition of the NLRP3 inflammasome.In comparison to the mice in the TNBS group,the mice in the TNBS+MSC group displayed a more acute form of colitis,and the IL1βand IL18 cytokines in their serum were lowered as well.In the meantime,3-MA raised IL
文摘C16 saturated fatty acid (Palmitic acid) is one of the most common dietary fatty acids which played an important role in the cellular biological functions. Palmitic acid (PA) was tested for potential inhibition of DNA topoisomerase I (topo I) and it exhibited inhibitory activity in the nanomolar range. Treatment of lung adenocarcinoma cell line A549 with PA resulted in a decrease in cell viability in a concentration-dependent manner, and PA showed cytotoxicity with an IC50 value of 150 μM. DNA fragmentation assay and caspase activity indicated that PA does not induce apoptotic cell death in A549 cells. Finally, we found that PA was able to cause an increase in autophagic flux in a time-dependent manner, evidenced by the accumulation of LC3 through monodansylcadaverine (MDC) staining. More importantly, inhibition of autophagy by blocking autophagosome formation via the inhibition of type III Phosphatidylinositol 3-kinases (PI-3K), by 3-Methyladenine (3-MA) was able to effectively suppress PA-induced autophagy. We showed that inhibition of autophagy sensitized the cells signal to PA-induced apoptosis, suggesting the pro-survival function of autophagy induced by PA. Taken together, results from this study reveal that PA as a topo I inhibitor induced autophagic cell death in A549 cells.
