Mutactimycin,a new anthracycline antibiotic,consists of 5 components: mutac-timycin A,B,C,D and 7-deoxymutactimycinone A. Mutactimycin components differ from each other in suppressing proliferation and inducing differ...Mutactimycin,a new anthracycline antibiotic,consists of 5 components: mutac-timycin A,B,C,D and 7-deoxymutactimycinone A. Mutactimycin components differ from each other in suppressing proliferation and inducing differentiation of human erythroleukemia K-562 cells. Determined by benzidine staining, the percentage of K-562 cells with hemoglobin expression was86% by mutactimycin C. The peak percentage of benzidine positive cells appeared on day 5 or 6.The differentiated cells were partially reversed after drug removal.The induced cells lost the clonogenicity in soft-agar. The kinetics of differentiation induced by mutactimycin C, in comparison with other known anthracycline antibiotics,was similar to that by doxorubicin, epirubicin anddaunorubicin. Mutactimycin C was more active than aclarubicin A. The structure-activity relationship for mutactimycin components suggested that the substituting groups on C_3, C_11, and the saccharide on C_7 might be important for their activities.展开更多
Objective The whole process of vaccine preparation is time-consuming and technically challenging. Here the hGM-CSF-engineered K-562 cell line was constructed to simplify tumor vaccine preparation process. Methods The ...Objective The whole process of vaccine preparation is time-consuming and technically challenging. Here the hGM-CSF-engineered K-562 cell line was constructed to simplify tumor vaccine preparation process. Methods The eukaryocyte expressing plasmid pcDNA3.1/GM-CSF was first constructed and its accuracy was verified through sequencing. The pcDNA3.1/GM-CSF was transfected into COS-7 cells to verify GM-CSF expression and cytokine activity using TF-1 cell line. Then the plasmid was transfected into K-562 cell line using liposome method, and was selected under G-418 and sub-cloned by limiting dilution. GM-CSF product from the monoclone GM-CSF-K-562 cell lines was quantified using ELISA method. Results We successfully constructed the hGM-CSF eukaryocyte expressing plasmid and hGM-CSF expressing K-562 cell line. Conclusion The construction of K-562/GM-CSF line will simplify the preparation of tumor vaccine, thus facilitating the application of tumor vaccination therapy in clinical application.展开更多
Background:Circular RNAs(circR NAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)comple...Background:Circular RNAs(circR NAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)complex subunit2(CNOT2),which was found to regulate tumor cell apoptosis through caspase pathway.Methods:Potential circR NA.0007127 target microRNAs(miRNAs)were analyzed by miRanda,TargetScan,and RNAhybrid software,and the miRNAs with binding sites for apoptosis-related genes were screened.The roles of circR NA.0007127 and its downstream target,microR NA(miR)-513a-5p,were validated by quantitative real-time polymerase chain reaction(qPCR),flow cytometry,mitochondrial membrane potential,immunofluorescence,western blot,and caspase-8(CASP8)protein activity in vitro in HO-induced K-562 cells.The circRNA.0007127-miR-513a-5p and CASP8-miR-513a-5p interactions were verified by luciferase reporter assays.Results:Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells.Compared with the control group,the expression of CASP8 was reduced by 50%and the 43-kD fragment of CASP8 protein was significantly reduced(P≤0.05).The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8,with extremely significant differences(P≤0.001).The overexpression of miR-513a-5p inhibited the gene expression level of CASP8in a human myeloid leukemia cell model(75%change)and the level of a 43-kD fragment of CASP8 protein(P small-interfering RNA(siRNA)and the miR-≤0.01).The rescue experiment showed that cotransfection with circRNA.0007127513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate,suggesting that the miR-513a-5p inhibitor is a circRNA.0007127siRNA antagonist.Conclusions:CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis,which can serve as a novel powerful molecular target for K-562 cells.展开更多
文摘Mutactimycin,a new anthracycline antibiotic,consists of 5 components: mutac-timycin A,B,C,D and 7-deoxymutactimycinone A. Mutactimycin components differ from each other in suppressing proliferation and inducing differentiation of human erythroleukemia K-562 cells. Determined by benzidine staining, the percentage of K-562 cells with hemoglobin expression was86% by mutactimycin C. The peak percentage of benzidine positive cells appeared on day 5 or 6.The differentiated cells were partially reversed after drug removal.The induced cells lost the clonogenicity in soft-agar. The kinetics of differentiation induced by mutactimycin C, in comparison with other known anthracycline antibiotics,was similar to that by doxorubicin, epirubicin anddaunorubicin. Mutactimycin C was more active than aclarubicin A. The structure-activity relationship for mutactimycin components suggested that the substituting groups on C_3, C_11, and the saccharide on C_7 might be important for their activities.
文摘Objective The whole process of vaccine preparation is time-consuming and technically challenging. Here the hGM-CSF-engineered K-562 cell line was constructed to simplify tumor vaccine preparation process. Methods The eukaryocyte expressing plasmid pcDNA3.1/GM-CSF was first constructed and its accuracy was verified through sequencing. The pcDNA3.1/GM-CSF was transfected into COS-7 cells to verify GM-CSF expression and cytokine activity using TF-1 cell line. Then the plasmid was transfected into K-562 cell line using liposome method, and was selected under G-418 and sub-cloned by limiting dilution. GM-CSF product from the monoclone GM-CSF-K-562 cell lines was quantified using ELISA method. Results We successfully constructed the hGM-CSF eukaryocyte expressing plasmid and hGM-CSF expressing K-562 cell line. Conclusion The construction of K-562/GM-CSF line will simplify the preparation of tumor vaccine, thus facilitating the application of tumor vaccination therapy in clinical application.
基金supported by the Guangzhou Science and Technology Plan Project(No.201904010027)the Key Clinical Technology Program of Guangzhou(No.2019ZD18),China。
文摘Background:Circular RNAs(circR NAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)complex subunit2(CNOT2),which was found to regulate tumor cell apoptosis through caspase pathway.Methods:Potential circR NA.0007127 target microRNAs(miRNAs)were analyzed by miRanda,TargetScan,and RNAhybrid software,and the miRNAs with binding sites for apoptosis-related genes were screened.The roles of circR NA.0007127 and its downstream target,microR NA(miR)-513a-5p,were validated by quantitative real-time polymerase chain reaction(qPCR),flow cytometry,mitochondrial membrane potential,immunofluorescence,western blot,and caspase-8(CASP8)protein activity in vitro in HO-induced K-562 cells.The circRNA.0007127-miR-513a-5p and CASP8-miR-513a-5p interactions were verified by luciferase reporter assays.Results:Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells.Compared with the control group,the expression of CASP8 was reduced by 50%and the 43-kD fragment of CASP8 protein was significantly reduced(P≤0.05).The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8,with extremely significant differences(P≤0.001).The overexpression of miR-513a-5p inhibited the gene expression level of CASP8in a human myeloid leukemia cell model(75%change)and the level of a 43-kD fragment of CASP8 protein(P small-interfering RNA(siRNA)and the miR-≤0.01).The rescue experiment showed that cotransfection with circRNA.0007127513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate,suggesting that the miR-513a-5p inhibitor is a circRNA.0007127siRNA antagonist.Conclusions:CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis,which can serve as a novel powerful molecular target for K-562 cells.
