新藤黄酸诱导脑胶质瘤细胞U87自噬的研究
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摘要
目的观察不同浓度新藤黄酸(gambogenic acid,GNA)对脑胶质瘤细胞U87增殖及自噬的影响。方法倒置显微镜下观察细胞形态变化,采用噻唑蓝法检测细胞的存活率,单丹磺酰尸胺(monodansylcadaverine,MDC)染色法检测自噬泡的形成,吖啶橙(acridine orange,AO)染色流式细胞仪观察酸性囊泡细胞器的数量变化,Westernblot法检测自噬相关蛋白微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)-Ⅱ/LC3-Ⅰ以及Beclin-1的表达变化。结果 GNA在1～32μmol/L浓度范围抑制U87细胞的增殖;MDC染色表明GNA促进U87细胞内自噬泡的形成;AO染色表明GNA增加U87细胞内酸性囊泡细胞器的数量;Western blot结果显示,GNA作用U87细胞后,LC3-Ⅱ/LC3-Ⅰ比值增大,Beclin-1的表达增加,提示细胞内自噬活性增强。结论 GNA在一定剂量和时间范围内抑制脑胶质瘤细胞U87细胞的增殖可能与诱导U87细胞自噬的发生有关。
Objective To investigate the effect of different concentrations of gambogenic acid(GNA)on the proliferation and autophagy of glioma U87 cells.Methods An inverted microscope was used to observe cell morphological changes.MTT assay was used to measure cell viability.Monodansylcadaverine(MDC)staining was used to detect the formation of autophagic vacuoles.Acridine orange(AO)staining and flow cytometry were used to observe the change in the number of acidic vesicular organelles.Western blot was used to measure the expression of the autophagy-related proteins microtubule-associated protein 1 light chain 3-II(LC3-II),microtubule-associated protein 1 light chain 3-I(LC3-I),and Beclin-1.Results GNA inhibited the proliferation of U87 cells at a concentration of 1-32μmol/L.MDC staining showed that GNA promoted the formation of autophagic vacuoles in U87 cells.AO staining showed that GNA increased in the number of acidic vesicular organelles in U87 cells.Western blot showed that after U87 cells were treated with GNA,there were significant increases in LC3-II/LC3-I ratio and the expression of Beclin-1,suggesting that there was an increase in autophagy activity in these cells.Conclusion GNA can effectively inhibit the proliferation of glioma U87 cells within a certain range of time and concentration,possibly by inducing the autophagy of U87 cells.
Objective To investigate the effect of different concentrations of gambogenic acid(GNA)on the proliferation and autophagy of glioma U87 cells.Methods An inverted microscope was used to observe cell morphological changes.MTT assay was used to measure cell viability.Monodansylcadaverine(MDC)staining was used to detect the formation of autophagic vacuoles.Acridine orange(AO)staining and flow cytometry were used to observe the change in the number of acidic vesicular organelles.Western blot was used to measure the expression of the autophagy-related proteins microtubule-associated protein 1 light chain 3-II(LC3-II),microtubule-associated protein 1 light chain 3-I(LC3-I),and Beclin-1.Results GNA inhibited the proliferation of U87 cells at a concentration of 1-32μmol/L.MDC staining showed that GNA promoted the formation of autophagic vacuoles in U87 cells.AO staining showed that GNA increased in the number of acidic vesicular organelles in U87 cells.Western blot showed that after U87 cells were treated with GNA,there were significant increases in LC3-II/LC3-I ratio and the expression of Beclin-1,suggesting that there was an increase in autophagy activity in these cells.Conclusion GNA can effectively inhibit the proliferation of glioma U87 cells within a certain range of time and concentration,possibly by inducing the autophagy of U87 cells.
引文
[1] YUN C W.The roles of autophagy in cancer[J].International Journal of Molecular Sciences,2018,19(11):3466.
[2] LIMPERT A S,LAMBERT L J,BAKAS N A,et al.Autophagy in cancer:regulation by small molecules[J].Trends in Pharmacological Sciences,2018,39(12):1021-1032.
[3] HUANG F,WANG B R.Role of autophagy in tumorigenesis,metastasis,targeted therapy and drug resistance of hepatocellular carcinoma[J].World Journal of Gastroenterology,2018,24(41):4643-4651.
[4] DAVIS M E.Epidemiology and overview of gliomas[J].Seminars in oncology nursing,2018,34(5):420-429.
[5] OSTROM Q T,GITTLEMAN H,XU J,et al.CBTRUS statistical report:primary brain and other central nervous system tumors diagnosed in the United States in 2009-2013[J].Neuro-oncology,2016,18(suppl 5):1-75.
[6] 何烨,王科明.新藤黄酸抗肿瘤作用的研究进展[J].现代肿瘤医学,2015,23(1):126-128.
[7] 刘修树,周娟,王效山.藤黄中新藤黄酸提取分离工艺改进[J].安徽中医学院学报,2012,31(3):69-71.
[8] YAN F,WANG M,LI J,et al.Gambogenic acid induced mitochondrial-dependent apoptosis and referred to phospho-Erk1/2 and phospho-p38 MAPK in human hepatoma HepG2 cells[J].Environmental Toxicology and Pharmacology,2012,33(2):181-190.
[9] 陆莹,李庆林.新藤黄酸诱导黑色素瘤B16细胞线粒体自噬的机制研究[J].现代中西医结合杂志,2016,25(19):2079-2082.
[10] CHENG H,SU J J,PENG J Y,et al.Gambogenic acid inhibits proliferation of A549 cells through apoptosis inducing through up-regulation of the p38 MAPK cascade[J].Journal of Asian Natural Products Research,2011,13(11):993-1002.
[11] CHEN H B,ZHOU L Z,MEI L,et al.Gambogenic acid-induced time- and dose-dependent growth inhibition and apoptosis involving Akt pathway inactivation in U251 glioblastoma cells[J].Journal of Natural Medicines,2012,66(1):62-69.
[12] YUAN G J,DENG J J,CAO D D,et al.Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells[J].World Journal of Gastroenterology,2017,23(30):5530-5537.
[13] YAO C J,CHOW J M,CHUANG S E,et al.Induction of forkhead class box O3a and apoptosis by a standardized ginsenoside formulation,KG-135,is potentiated by autophagy blockade in A549 human lung cancer cells[J].Journal of Ginseng Research,2017,41(3):247-256.
[14] LIU F,GAO S,YANG Y,et al.Curcumin induced autophagy anticancer effects on human lung adenocarcinoma cell line A549[J].Oncology Letters,2017,14(3):2775-2782.
[15] RAI S,ARASTEH M,JEFFERSON M,et al.The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independ ently of the WD domain required for LC3-associated phagocytosis[J].Autophagy,2018:1-14[2018-11-7].https://doi.org/10.1080/15548627.2018.1534507.
[16] WANG J,YU Y,LU K,et al.Silica nanoparticles induce autophagy dysfunction via lysosomal impairment and inhibition of autophagosome degradation in hepatocytes[J].International Journal of Nanomedicine,2017,12:809-825[2016-12-8].https://doi.org/10.2147/IJN.S123596.
[17] LI Z,LI Q,LV W,et al.The interaction of Atg4B and Bcl-2 plays an important role in Cd-induced crosstalk between apoptosis and autophagy through disassociation of Bcl-2-Beclin1 in A549 cells[J].Free Radical Biology & Medicine,2018[2018-11-16].https://doi.org/10.1016/j.freeradbiomed.2018.11.020.
[2] LIMPERT A S,LAMBERT L J,BAKAS N A,et al.Autophagy in cancer:regulation by small molecules[J].Trends in Pharmacological Sciences,2018,39(12):1021-1032.
[3] HUANG F,WANG B R.Role of autophagy in tumorigenesis,metastasis,targeted therapy and drug resistance of hepatocellular carcinoma[J].World Journal of Gastroenterology,2018,24(41):4643-4651.
[4] DAVIS M E.Epidemiology and overview of gliomas[J].Seminars in oncology nursing,2018,34(5):420-429.
[5] OSTROM Q T,GITTLEMAN H,XU J,et al.CBTRUS statistical report:primary brain and other central nervous system tumors diagnosed in the United States in 2009-2013[J].Neuro-oncology,2016,18(suppl 5):1-75.
[6] 何烨,王科明.新藤黄酸抗肿瘤作用的研究进展[J].现代肿瘤医学,2015,23(1):126-128.
[7] 刘修树,周娟,王效山.藤黄中新藤黄酸提取分离工艺改进[J].安徽中医学院学报,2012,31(3):69-71.
[8] YAN F,WANG M,LI J,et al.Gambogenic acid induced mitochondrial-dependent apoptosis and referred to phospho-Erk1/2 and phospho-p38 MAPK in human hepatoma HepG2 cells[J].Environmental Toxicology and Pharmacology,2012,33(2):181-190.
[9] 陆莹,李庆林.新藤黄酸诱导黑色素瘤B16细胞线粒体自噬的机制研究[J].现代中西医结合杂志,2016,25(19):2079-2082.
[10] CHENG H,SU J J,PENG J Y,et al.Gambogenic acid inhibits proliferation of A549 cells through apoptosis inducing through up-regulation of the p38 MAPK cascade[J].Journal of Asian Natural Products Research,2011,13(11):993-1002.
[11] CHEN H B,ZHOU L Z,MEI L,et al.Gambogenic acid-induced time- and dose-dependent growth inhibition and apoptosis involving Akt pathway inactivation in U251 glioblastoma cells[J].Journal of Natural Medicines,2012,66(1):62-69.
[12] YUAN G J,DENG J J,CAO D D,et al.Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells[J].World Journal of Gastroenterology,2017,23(30):5530-5537.
[13] YAO C J,CHOW J M,CHUANG S E,et al.Induction of forkhead class box O3a and apoptosis by a standardized ginsenoside formulation,KG-135,is potentiated by autophagy blockade in A549 human lung cancer cells[J].Journal of Ginseng Research,2017,41(3):247-256.
[14] LIU F,GAO S,YANG Y,et al.Curcumin induced autophagy anticancer effects on human lung adenocarcinoma cell line A549[J].Oncology Letters,2017,14(3):2775-2782.
[15] RAI S,ARASTEH M,JEFFERSON M,et al.The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independ ently of the WD domain required for LC3-associated phagocytosis[J].Autophagy,2018:1-14[2018-11-7].https://doi.org/10.1080/15548627.2018.1534507.
[16] WANG J,YU Y,LU K,et al.Silica nanoparticles induce autophagy dysfunction via lysosomal impairment and inhibition of autophagosome degradation in hepatocytes[J].International Journal of Nanomedicine,2017,12:809-825[2016-12-8].https://doi.org/10.2147/IJN.S123596.
[17] LI Z,LI Q,LV W,et al.The interaction of Atg4B and Bcl-2 plays an important role in Cd-induced crosstalk between apoptosis and autophagy through disassociation of Bcl-2-Beclin1 in A549 cells[J].Free Radical Biology & Medicine,2018[2018-11-16].https://doi.org/10.1016/j.freeradbiomed.2018.11.020.