目的探讨姜黄素(curcumin)对乳腺癌细胞中顺铂(Cisplatin,CDDP)治疗敏感性的影响及其可能机制。方法 CCK-8检测、Hoechst染色观察CDDP、姜黄素单独及联合用药48 h对MCF7细胞增殖抑制和细胞凋亡的影响;Western blot分析MCF7细胞在CDDP(0...目的探讨姜黄素(curcumin)对乳腺癌细胞中顺铂(Cisplatin,CDDP)治疗敏感性的影响及其可能机制。方法 CCK-8检测、Hoechst染色观察CDDP、姜黄素单独及联合用药48 h对MCF7细胞增殖抑制和细胞凋亡的影响;Western blot分析MCF7细胞在CDDP(0、1.25、2.5、5、10、20μg/m L)、姜黄素(0、1、5、20、30、50、100μmol/L)单独及联合处理后FEN1(flap endonuclease 1)表达水平的变化;CCK-8检测沉默FEN1对MCF7细胞中CDDP敏感性的影响。结果 CDDP、姜黄素均可以剂量依赖方式抑制MCF7细胞增殖,其IC50分别为5、30μmol/L;与单独2μg/m L CDDP处理组比较,2μg/m L CDDP联合20μmol/L姜黄素、30μmol/L姜黄素处理组对细胞增殖的抑制效应明显增强[分别为(84.1±0.8)%、(51.1±0.5)%、(29.4±0.3)%,P<0.05];与单独20μmol/L姜黄素处理组比较,20μmol/L姜黄素联合2μg/m L CDDP、5μg/m L CDDP处理组对细胞增殖的抑制效应也明显增强[分别为(76.9±0.7)%、(42.4±0.3)%、(31.6±0.4)%,P<0.05];2μg/m L CDDP联合20μmol/L姜黄素组的细胞凋亡明显增强(P<0.05);与Control siRNA组比较,FEN1 siRNA+CDDP组可显著增强CDDP抑制MCF7细胞增殖的敏感性[(25.4±0.3)%vs(18.7±0.2)%,P<0.05];CDDP增殖抑制无效浓度或低浓度时,FEN1蛋白的表达随CDDP的处理剂量依赖性上调,而姜黄素可剂量依赖性下调FEN1蛋白表达;与单独CDDP和姜黄素处理组比较,2μg/m L CDDP联合20μmol/L姜黄素组可显著降低FEN1蛋白表达(P<0.05)。结论姜黄素可增强CDDP对乳腺癌细胞的敏感性,其机制与降低细胞FEN1的表达有关。展开更多
Completion of lagging strand DNA synthesis requires processing of up to 50 million Okazaki fragments per cell cycle in mammalian cells.Even in yeast,the Okazaki fragment maturation happens approximately a million time...Completion of lagging strand DNA synthesis requires processing of up to 50 million Okazaki fragments per cell cycle in mammalian cells.Even in yeast,the Okazaki fragment maturation happens approximately a million times during a single round of DNA replication.Therefore,efficient processing of Okazaki fragments is vital for DNA replication and cell proliferation.During this process,primase-synthesized RNA/DNA primers are removed,and Okazaki fragments are joined into an intact lagging strand DNA.The processing of RNA/DNA primers requires a group of structure-specific nucleases typified by flap endonuclease 1(FEN1).Here,we sum-marize the distinct roles of these nucleases in different pathways for removal of RNA/DNA primers.Recent findings reveal that Okazaki fragment maturation is highly coordinated.The dynamic interactions of polymerase d,FEN1 and DNA ligase I with prolif-erating cell nuclear antigen allow these enzymes to act sequentially during Okazaki fragment maturation.Such protein–protein interactions may be regulated by post-translational modifications.We also discuss studies using mutant mouse models that suggest two distinct cancer etiological mechanisms arising from defects in different steps of Okazaki fragment maturation.Mutations that affect the efficiency of RNA primer removal may result in accumulation of unligated nicks and DNA double-strand breaks.These DNA strand breaks can cause varying forms of chromosome aberrations,contributing to development of cancer that associates with aneuploidy and gross chromosomal rearrangement.On the other hand,mutations that impair editing out of polymerase a incorporation errors result in cancer displaying a strong mutator phenotype.展开更多
文摘目的探讨姜黄素(curcumin)对乳腺癌细胞中顺铂(Cisplatin,CDDP)治疗敏感性的影响及其可能机制。方法 CCK-8检测、Hoechst染色观察CDDP、姜黄素单独及联合用药48 h对MCF7细胞增殖抑制和细胞凋亡的影响;Western blot分析MCF7细胞在CDDP(0、1.25、2.5、5、10、20μg/m L)、姜黄素(0、1、5、20、30、50、100μmol/L)单独及联合处理后FEN1(flap endonuclease 1)表达水平的变化;CCK-8检测沉默FEN1对MCF7细胞中CDDP敏感性的影响。结果 CDDP、姜黄素均可以剂量依赖方式抑制MCF7细胞增殖,其IC50分别为5、30μmol/L;与单独2μg/m L CDDP处理组比较,2μg/m L CDDP联合20μmol/L姜黄素、30μmol/L姜黄素处理组对细胞增殖的抑制效应明显增强[分别为(84.1±0.8)%、(51.1±0.5)%、(29.4±0.3)%,P<0.05];与单独20μmol/L姜黄素处理组比较,20μmol/L姜黄素联合2μg/m L CDDP、5μg/m L CDDP处理组对细胞增殖的抑制效应也明显增强[分别为(76.9±0.7)%、(42.4±0.3)%、(31.6±0.4)%,P<0.05];2μg/m L CDDP联合20μmol/L姜黄素组的细胞凋亡明显增强(P<0.05);与Control siRNA组比较,FEN1 siRNA+CDDP组可显著增强CDDP抑制MCF7细胞增殖的敏感性[(25.4±0.3)%vs(18.7±0.2)%,P<0.05];CDDP增殖抑制无效浓度或低浓度时,FEN1蛋白的表达随CDDP的处理剂量依赖性上调,而姜黄素可剂量依赖性下调FEN1蛋白表达;与单独CDDP和姜黄素处理组比较,2μg/m L CDDP联合20μmol/L姜黄素组可显著降低FEN1蛋白表达(P<0.05)。结论姜黄素可增强CDDP对乳腺癌细胞的敏感性,其机制与降低细胞FEN1的表达有关。
基金supported by the National Cancer Institute grants R01CA073764 and R01CA085344.
文摘Completion of lagging strand DNA synthesis requires processing of up to 50 million Okazaki fragments per cell cycle in mammalian cells.Even in yeast,the Okazaki fragment maturation happens approximately a million times during a single round of DNA replication.Therefore,efficient processing of Okazaki fragments is vital for DNA replication and cell proliferation.During this process,primase-synthesized RNA/DNA primers are removed,and Okazaki fragments are joined into an intact lagging strand DNA.The processing of RNA/DNA primers requires a group of structure-specific nucleases typified by flap endonuclease 1(FEN1).Here,we sum-marize the distinct roles of these nucleases in different pathways for removal of RNA/DNA primers.Recent findings reveal that Okazaki fragment maturation is highly coordinated.The dynamic interactions of polymerase d,FEN1 and DNA ligase I with prolif-erating cell nuclear antigen allow these enzymes to act sequentially during Okazaki fragment maturation.Such protein–protein interactions may be regulated by post-translational modifications.We also discuss studies using mutant mouse models that suggest two distinct cancer etiological mechanisms arising from defects in different steps of Okazaki fragment maturation.Mutations that affect the efficiency of RNA primer removal may result in accumulation of unligated nicks and DNA double-strand breaks.These DNA strand breaks can cause varying forms of chromosome aberrations,contributing to development of cancer that associates with aneuploidy and gross chromosomal rearrangement.On the other hand,mutations that impair editing out of polymerase a incorporation errors result in cancer displaying a strong mutator phenotype.
