Objective: B-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined...Objective: B-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined with miR-15a oligonucleotides (ODN) could enhance methotrexate (MTX)-induced apoptosis in Raji cells. Methods: Chemically synthesized miR-15a ODN and Bcl-2 siRNA were transfected in Raji cells by using a HiPerFect Transfection Reagent and then combined with MTX. Expression levels of Bcl-2 protein were detected by Western blot. Cell proliferation was determined by CCK8 assay. The rate of cell apoptosis was determined by Annexin V/PI double staining. The morphology of apoptotic cells was observed by Hoechst-33 258 staining. Results: After the cells were transfected with miR-15a ODN combined with Bcl-2 siRNA, Bcl-2 protein levels were evidently decreased. CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl- 2 siRNA with MTX group, and single MTX group (P<0.05). Hoechst 33258 staining revealed numerous apoptotic cells. AnnexinV/PI double staining showed that the apoptotic rates were (13.13±1.60)%, (34.47±2.96)%, (32.87±3.48)%, and (45.47±2.16)% in MTX, Bcl-2 siRNA plus MTX, miR-15a ODN plus MTX, and miR-15a ODN combined with Bcl- 2 siRNA plus MTX groups, respectively. Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05). Conclusions: Bcl-2 siRNA combined with miR-15a ODN could enhance MTX-induced apoptosis in Raji cells. Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.展开更多
Multiple drug resistance(MDR)is a tough problem in developing hepatocellular carcinoma(HCC)therapy.Here,we developed TPGS-coated cationic liposomes with Bcl-2 siRNA corona to load doxorubicin(Dox)i.e.,Bcl-2 siRNA/Dox-...Multiple drug resistance(MDR)is a tough problem in developing hepatocellular carcinoma(HCC)therapy.Here,we developed TPGS-coated cationic liposomes with Bcl-2 siRNA corona to load doxorubicin(Dox)i.e.,Bcl-2 siRNA/Dox-TPGS-LPs,to enhance anticancer effect of Dox in HCC-MDR.TPGS i.e.,d-α-tocopheryl polyethylene glycol 1000 succinate,inhibited Pglycoprotein(P-gp)efflux pump and Bcl-2 siRNA suppressed anti-apoptotic Bcl-2 protein.The Bcl-2 siRNA loaded in the liposomal corona was observed under transmission electron microscopy.The stability and hemolysis evaluation demonstrated Bcl-2 siRNA/Dox-TPGSLPs had good biocompatibility and siRNA-corona could protect the liposomal core to avoid the attachment of fetal bovine serum.In drug-resistant cells,TPGS effectively prolonged intracellular Dox retention time and siRNA-corona did improve the internalization of Dox from liposomes.In vitro and in vivo anticancer effect of this dual-functional nanostructure was examined in HCC-MDR Bel7402/5-FU tumor model.MTT assay confirmed the IC50 value of Dox was 20–50 fold higher in Bel7402/5-FU MDR cells than that in sensitive Bel7402 cells.Bcl-2 siRNA corona successfully entered the cytosol of Bel7402/5-FU MDR cells to downregulate Bcl-2 protein levels in vitro and in vivo.Bcl-2 siRNA/Dox-TPGS-LPs showed superior to TPGS-(or siRNA-)linked Dox liposomes in cell apoptosis and cytotoxicity assay in Bel7402/5-FU MDR cells,and 7-fold greater effect than free Dox in tumor growth inhibition of Bel7402/5-FU xenograft nude mice.In conclusion,TPGS-coated cationic liposomes with Bcl-2 siRNA corona had the capacity to inhibit MDR dual-pathways and subsequently improved the anti-tumor activity of the chemotherapeutic agent co-delivered to a level that cannot be achieved by inhibiting a MDR single way.展开更多
Objective: To study whether the small-interference RNA (siRNA) targeting against Bcl-2 gene can enhance sensi-tivity of HL-60 cell to arsenic trioxide. Methods: SiRNA was transferred into the HL-60 cells. At 6 h after...Objective: To study whether the small-interference RNA (siRNA) targeting against Bcl-2 gene can enhance sensi-tivity of HL-60 cell to arsenic trioxide. Methods: SiRNA was transferred into the HL-60 cells. At 6 h after transfection, the cells were cultured with arsenic trioxide. The cell growth of the HL-60 cells was detected using MTT at 24, 48 and 72 h, respectively. The levels of the Bcl-2 protein and reactive oxygen species (ROS), as well as of the membrane potential of the mitochondrion were determined by flow cytometry. Results: The Bcl-2 siRNA significantly increased the inhibitory action of arsenic trioxide on growth of HL-60 cells. The combination of siRNA with arsenic trioxide resulted in decrease of the Bcl-2 protein level and increase of the ROS level, as well as significant descending of the membrane potential of mitochondrion of HL-60 (P < 0.05). Conclusion: The siRNA targeting Bcl-2 can increase the sensitivity of the HL-60 leukemia cells to arsenic trioxide by inhibiting the expression of Bcl-2 protein.展开更多
OBJECTIVE To study whether siRNA targeting against the Bcl-2 gene can enhance sensitivity of HL-60 cells to all trans retinoic acid(ATRA). METHODS siRNA,which is a leading sequence selected by previous experiments,was...OBJECTIVE To study whether siRNA targeting against the Bcl-2 gene can enhance sensitivity of HL-60 cells to all trans retinoic acid(ATRA). METHODS siRNA,which is a leading sequence selected by previous experiments,was transferred into HL-60 cells.At 6 h after transfection,the cells were cultured with ATRA.The cell growth of the HL-60 cells was measured by the MTT assay at 24, 48,72 h.The level of the Bcl-2 protein and ROS(reactive oxygen species)as well as membrane potential of the mitochondria were determined by flowcytometry. RESULTS siRNA significantly increased the inhibitory effect of ATRA on growth of the HL-60 cells.The combination of siRNA with ATRA resulted in a decrease in the Bcl-2 protein level and an increase in the ROS level as well as significantly lowering the mitochondrial membrane potential of the HL-60 cells(P<0.05). CONCLUSION Effective siRNA targeting of Bcl-2 increases the sensitivity of HL-60 leukemic cells to ATRA by inhibiting the expression of the Bcl-2 protein.展开更多
基金supported by the Overseas Chinese Affairs Office of the State Council Key Discipline Construction Fund (Grant No. 51205002)the Guangdong Province Key Foundation of Science and Technology Program (Grant No.2009B0507000029)+1 种基金Major Research Plan of the National Natural Science Foundation of China (Cultivating project, Grant No. 91129720)the National Natural Science Foundation of China (Grant No. 81170496)
文摘Objective: B-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined with miR-15a oligonucleotides (ODN) could enhance methotrexate (MTX)-induced apoptosis in Raji cells. Methods: Chemically synthesized miR-15a ODN and Bcl-2 siRNA were transfected in Raji cells by using a HiPerFect Transfection Reagent and then combined with MTX. Expression levels of Bcl-2 protein were detected by Western blot. Cell proliferation was determined by CCK8 assay. The rate of cell apoptosis was determined by Annexin V/PI double staining. The morphology of apoptotic cells was observed by Hoechst-33 258 staining. Results: After the cells were transfected with miR-15a ODN combined with Bcl-2 siRNA, Bcl-2 protein levels were evidently decreased. CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl- 2 siRNA with MTX group, and single MTX group (P<0.05). Hoechst 33258 staining revealed numerous apoptotic cells. AnnexinV/PI double staining showed that the apoptotic rates were (13.13±1.60)%, (34.47±2.96)%, (32.87±3.48)%, and (45.47±2.16)% in MTX, Bcl-2 siRNA plus MTX, miR-15a ODN plus MTX, and miR-15a ODN combined with Bcl- 2 siRNA plus MTX groups, respectively. Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05). Conclusions: Bcl-2 siRNA combined with miR-15a ODN could enhance MTX-induced apoptosis in Raji cells. Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.
基金financially supported by National Basic Research Program of China(973 Program,2015CB931802)Natural Science Foundation of China(31470968 and 81627901)。
文摘Multiple drug resistance(MDR)is a tough problem in developing hepatocellular carcinoma(HCC)therapy.Here,we developed TPGS-coated cationic liposomes with Bcl-2 siRNA corona to load doxorubicin(Dox)i.e.,Bcl-2 siRNA/Dox-TPGS-LPs,to enhance anticancer effect of Dox in HCC-MDR.TPGS i.e.,d-α-tocopheryl polyethylene glycol 1000 succinate,inhibited Pglycoprotein(P-gp)efflux pump and Bcl-2 siRNA suppressed anti-apoptotic Bcl-2 protein.The Bcl-2 siRNA loaded in the liposomal corona was observed under transmission electron microscopy.The stability and hemolysis evaluation demonstrated Bcl-2 siRNA/Dox-TPGSLPs had good biocompatibility and siRNA-corona could protect the liposomal core to avoid the attachment of fetal bovine serum.In drug-resistant cells,TPGS effectively prolonged intracellular Dox retention time and siRNA-corona did improve the internalization of Dox from liposomes.In vitro and in vivo anticancer effect of this dual-functional nanostructure was examined in HCC-MDR Bel7402/5-FU tumor model.MTT assay confirmed the IC50 value of Dox was 20–50 fold higher in Bel7402/5-FU MDR cells than that in sensitive Bel7402 cells.Bcl-2 siRNA corona successfully entered the cytosol of Bel7402/5-FU MDR cells to downregulate Bcl-2 protein levels in vitro and in vivo.Bcl-2 siRNA/Dox-TPGS-LPs showed superior to TPGS-(or siRNA-)linked Dox liposomes in cell apoptosis and cytotoxicity assay in Bel7402/5-FU MDR cells,and 7-fold greater effect than free Dox in tumor growth inhibition of Bel7402/5-FU xenograft nude mice.In conclusion,TPGS-coated cationic liposomes with Bcl-2 siRNA corona had the capacity to inhibit MDR dual-pathways and subsequently improved the anti-tumor activity of the chemotherapeutic agent co-delivered to a level that cannot be achieved by inhibiting a MDR single way.
基金Supported by the key Subject Foundation of Overseas Chinese Affairs Office of the State Council (No. 2005).
文摘Objective: To study whether the small-interference RNA (siRNA) targeting against Bcl-2 gene can enhance sensi-tivity of HL-60 cell to arsenic trioxide. Methods: SiRNA was transferred into the HL-60 cells. At 6 h after transfection, the cells were cultured with arsenic trioxide. The cell growth of the HL-60 cells was detected using MTT at 24, 48 and 72 h, respectively. The levels of the Bcl-2 protein and reactive oxygen species (ROS), as well as of the membrane potential of the mitochondrion were determined by flow cytometry. Results: The Bcl-2 siRNA significantly increased the inhibitory action of arsenic trioxide on growth of HL-60 cells. The combination of siRNA with arsenic trioxide resulted in decrease of the Bcl-2 protein level and increase of the ROS level, as well as significant descending of the membrane potential of mitochondrion of HL-60 (P < 0.05). Conclusion: The siRNA targeting Bcl-2 can increase the sensitivity of the HL-60 leukemia cells to arsenic trioxide by inhibiting the expression of Bcl-2 protein.
基金The work was supported by a grant from the Key Subject Foundation of Overseas Chinese Affairs Office of the State Council(No.51205002)
文摘OBJECTIVE To study whether siRNA targeting against the Bcl-2 gene can enhance sensitivity of HL-60 cells to all trans retinoic acid(ATRA). METHODS siRNA,which is a leading sequence selected by previous experiments,was transferred into HL-60 cells.At 6 h after transfection,the cells were cultured with ATRA.The cell growth of the HL-60 cells was measured by the MTT assay at 24, 48,72 h.The level of the Bcl-2 protein and ROS(reactive oxygen species)as well as membrane potential of the mitochondria were determined by flowcytometry. RESULTS siRNA significantly increased the inhibitory effect of ATRA on growth of the HL-60 cells.The combination of siRNA with ATRA resulted in a decrease in the Bcl-2 protein level and an increase in the ROS level as well as significantly lowering the mitochondrial membrane potential of the HL-60 cells(P<0.05). CONCLUSION Effective siRNA targeting of Bcl-2 increases the sensitivity of HL-60 leukemic cells to ATRA by inhibiting the expression of the Bcl-2 protein.
