OBJECTIVE Low dose of actinomycin D(LDAct D)was reported as a potent P53 activator and protected normal proliferating cells during anti-mitotic chemotherapy.However,the mechanism of LDAct D on P53 activation is still ...OBJECTIVE Low dose of actinomycin D(LDAct D)was reported as a potent P53 activator and protected normal proliferating cells during anti-mitotic chemotherapy.However,the mechanism of LDAct D on P53 activation is still undetermined.In this study,the mechanism of LDAct D on the synergistic antitumor effect for cisplatin(CDDP)and P53 reactivation in KB cells was studied in detail.METHODS Cell viability was determined by MTT and LDH release.Apoptosis was determined by AnnexinⅤ-FITC/PI staining.Mitochondrial membrane potential(MMP)was detected by JC-1 stain-ing.Expression of P53,PARP,BAX,BCL-XL,PUMA,MDM2 and MDMX was detected by Western blotting(WB)and/or immunofluorescence(IF).P53-MDM2 complex was detected by ELISA.Molecular docking of receptor MDM2 and MDMX with actinomycin D(ACTD)was analyzed by Discovery Studio.RESULTS Compared with CDDP alone,P53 expression and the cytotoxicity on KB cells was significantly increased by the combination therapy.P53 regulatory proteins were increased while MMP was decreased.Meanwhile,knockdown of PUMA(P53 upregulated modulator of apoptosis)efficiently blocked the synergistic effect of LDAct D to CDDP.P53 activation was found to be accompanied with the increase of MDMX but not MDM2.Meanwhile,MDM2-P53 complex in KB cells was significantly decreased by LDAct D.Docking of both receptor MDM2 and MDMX with ACTD exhibited well established bonds with nearby amino acid residues.CONCLUSION LDAct D was probably an inhibitor of both MDM2 and MDMX.The synergistic effects of LDAct D for CDDP on KB cells depended on its effect on reactivating P53 and PUMA mediated mitochondrial apoptosis.展开更多
Natural product biosynthesis is controlled at multiple levels.Characterization of naturally occurring promoters has facilitated the study of the synthetic biology of natural products.Herein,we report the discovery of ...Natural product biosynthesis is controlled at multiple levels.Characterization of naturally occurring promoters has facilitated the study of the synthetic biology of natural products.Herein,we report the discovery of two highyield actinomycin D(ActD)-producing streptomycetes and the identification of a strong bidirectional acmN2p promoter from the ActD gene clusters and its application in heterologous expression of three core genes involved in the bacterial alkaloid bohemamine biosynthesis,providing a good example for identification of new promoters for synthetic biological applications.展开更多
Actinomycin D (AMD) is an anticancer antibiotic that can bind selectively to both double-stranded and single-stranded DNA, and this binding greatly enhances DNA photosensitization. Using electron paramagnetic resonanc...Actinomycin D (AMD) is an anticancer antibiotic that can bind selectively to both double-stranded and single-stranded DNA, and this binding greatly enhances DNA photosensitization. Using electron paramagnetic resonance (EPR) in combination with spin trapping techniques, a systematic study was carried out on the reactive oxygen species generated in the photosensitization process of AMD. It was found that 1O2 and $O_2^{ - \cdot } $ are important reactive intermediates either in solution or in DNA complexes, and the generation of these species is in competition. This finding suggests that the photodynamic action of AMD proceeds via two pathways: energy transfer (type I mechanism) and electron transfer (type II mechanism). 1O2 is the main product formed via energy transfer reaction in solution while electron transfer between the excited states of AMD and DNA becomes the predominant pathway in DNA complexes.展开更多
Actinomycin D(AMD) is well known for its specific inhibition of DNA transcription, and has been used clinically as an antitumor drug for the treatment of some highly malignant tumors. Based on the former research, two...Actinomycin D(AMD) is well known for its specific inhibition of DNA transcription, and has been used clinically as an antitumor drug for the treatment of some highly malignant tumors. Based on the former research, two [D-Phe 2] 2AMD analogs with L-MeVal(the fifth amino acid residue in the cyclic depsipeptide of AMD) substituted by D-MeVal and D-MePhe were designed to reduce the toxicity and increase the antitumor activity. Another analog in which the D-Val residue replaced with D-MeVal was designed to eliminate or to weaken the hydrogen bonds of D-Val residues between α and β rings. All three novel compounds were prepared from C terminal to N terminal in solution phase to form linear pentapeptides, and cyclized by BOP-Cl/Et 3N in DCM. Condensation of pentapeptide lactone with BMNBCA, followed by catalytic reduction, controlling oxidation by K 3Fe(CN) 6 and purification afforded the analogs as red solid. The spectrum data of all three analogs including HR-MS, 1H NMR and [α] D were given.展开更多
本实验考察从链霉菌发酵产生的放线菌素D粗提物中分离制备高纯度放线菌素D的工艺,以聚合物纳米微球为固相载体,对聚合物纳米微球型号、洗脱条件进行优化。最终确立放线菌素D的分离纯化工艺为:采用PS40-300(以聚苯乙烯/二乙烯基苯聚合物...本实验考察从链霉菌发酵产生的放线菌素D粗提物中分离制备高纯度放线菌素D的工艺,以聚合物纳米微球为固相载体,对聚合物纳米微球型号、洗脱条件进行优化。最终确立放线菌素D的分离纯化工艺为:采用PS40-300(以聚苯乙烯/二乙烯基苯聚合物为基质,粒径40μm,孔径300)纳米微球作为层析填料;以链霉菌发酵产生的放线菌素D粗提物为上样样品,用适量95%乙醇(V/V)充分溶解,上样量为0.5 g/100 m L,控制洗脱速度为5.0 m L/min,以65%乙醇水(V/V)洗脱,收集纯度95%以上的洗脱液,真空浓缩得到高纯度的放线菌素D。结果表明该分离纯化工艺,纯度与收率都较高,流程简单、可行,为该产品产业化开发奠定基础。展开更多
基金The project supported by Ministry of Science and Technology Project of International Cooperation(2011DFR31240)National Science and Technology Major Projects″Major Drug Discovery(″2012ZX09301002001001)
文摘OBJECTIVE Low dose of actinomycin D(LDAct D)was reported as a potent P53 activator and protected normal proliferating cells during anti-mitotic chemotherapy.However,the mechanism of LDAct D on P53 activation is still undetermined.In this study,the mechanism of LDAct D on the synergistic antitumor effect for cisplatin(CDDP)and P53 reactivation in KB cells was studied in detail.METHODS Cell viability was determined by MTT and LDH release.Apoptosis was determined by AnnexinⅤ-FITC/PI staining.Mitochondrial membrane potential(MMP)was detected by JC-1 stain-ing.Expression of P53,PARP,BAX,BCL-XL,PUMA,MDM2 and MDMX was detected by Western blotting(WB)and/or immunofluorescence(IF).P53-MDM2 complex was detected by ELISA.Molecular docking of receptor MDM2 and MDMX with actinomycin D(ACTD)was analyzed by Discovery Studio.RESULTS Compared with CDDP alone,P53 expression and the cytotoxicity on KB cells was significantly increased by the combination therapy.P53 regulatory proteins were increased while MMP was decreased.Meanwhile,knockdown of PUMA(P53 upregulated modulator of apoptosis)efficiently blocked the synergistic effect of LDAct D to CDDP.P53 activation was found to be accompanied with the increase of MDMX but not MDM2.Meanwhile,MDM2-P53 complex in KB cells was significantly decreased by LDAct D.Docking of both receptor MDM2 and MDMX with ACTD exhibited well established bonds with nearby amino acid residues.CONCLUSION LDAct D was probably an inhibitor of both MDM2 and MDMX.The synergistic effects of LDAct D for CDDP on KB cells depended on its effect on reactivating P53 and PUMA mediated mitochondrial apoptosis.
基金supported in parts by National Natural Science Foundation of China grants(82173688 and 82373772)The science and technology innovation Program of Hunan Province(2021RC4067)+2 种基金a research fund from Institute of Health and Medicine(IHM),Hefei Comprehensive National Science Center(to Y.H.)the Chinese Ministry of Education 111 Project(BP0820034)(to Y.D.)the Fundamental Research Funds for the Central Universities of Central South University 2020zzts247(to D.T.).
文摘Natural product biosynthesis is controlled at multiple levels.Characterization of naturally occurring promoters has facilitated the study of the synthetic biology of natural products.Herein,we report the discovery of two highyield actinomycin D(ActD)-producing streptomycetes and the identification of a strong bidirectional acmN2p promoter from the ActD gene clusters and its application in heterologous expression of three core genes involved in the bacterial alkaloid bohemamine biosynthesis,providing a good example for identification of new promoters for synthetic biological applications.
文摘Actinomycin D (AMD) is an anticancer antibiotic that can bind selectively to both double-stranded and single-stranded DNA, and this binding greatly enhances DNA photosensitization. Using electron paramagnetic resonance (EPR) in combination with spin trapping techniques, a systematic study was carried out on the reactive oxygen species generated in the photosensitization process of AMD. It was found that 1O2 and $O_2^{ - \cdot } $ are important reactive intermediates either in solution or in DNA complexes, and the generation of these species is in competition. This finding suggests that the photodynamic action of AMD proceeds via two pathways: energy transfer (type I mechanism) and electron transfer (type II mechanism). 1O2 is the main product formed via energy transfer reaction in solution while electron transfer between the excited states of AMD and DNA becomes the predominant pathway in DNA complexes.
文摘Actinomycin D(AMD) is well known for its specific inhibition of DNA transcription, and has been used clinically as an antitumor drug for the treatment of some highly malignant tumors. Based on the former research, two [D-Phe 2] 2AMD analogs with L-MeVal(the fifth amino acid residue in the cyclic depsipeptide of AMD) substituted by D-MeVal and D-MePhe were designed to reduce the toxicity and increase the antitumor activity. Another analog in which the D-Val residue replaced with D-MeVal was designed to eliminate or to weaken the hydrogen bonds of D-Val residues between α and β rings. All three novel compounds were prepared from C terminal to N terminal in solution phase to form linear pentapeptides, and cyclized by BOP-Cl/Et 3N in DCM. Condensation of pentapeptide lactone with BMNBCA, followed by catalytic reduction, controlling oxidation by K 3Fe(CN) 6 and purification afforded the analogs as red solid. The spectrum data of all three analogs including HR-MS, 1H NMR and [α] D were given.
基金Science and Technology Planning Project of Fujian Province(2014R1009-10)
文摘本实验考察从链霉菌发酵产生的放线菌素D粗提物中分离制备高纯度放线菌素D的工艺,以聚合物纳米微球为固相载体,对聚合物纳米微球型号、洗脱条件进行优化。最终确立放线菌素D的分离纯化工艺为:采用PS40-300(以聚苯乙烯/二乙烯基苯聚合物为基质,粒径40μm,孔径300)纳米微球作为层析填料;以链霉菌发酵产生的放线菌素D粗提物为上样样品,用适量95%乙醇(V/V)充分溶解,上样量为0.5 g/100 m L,控制洗脱速度为5.0 m L/min,以65%乙醇水(V/V)洗脱,收集纯度95%以上的洗脱液,真空浓缩得到高纯度的放线菌素D。结果表明该分离纯化工艺,纯度与收率都较高,流程简单、可行,为该产品产业化开发奠定基础。
