OBJECTIVE To investigate the effects of survivin antisense oligodeoxy-nucleotid (ASODN) on proliferation and apoptosis in the chronic myeloid leukemia cell line K562. METHODS Different concentrations of an antisense o...OBJECTIVE To investigate the effects of survivin antisense oligodeoxy-nucleotid (ASODN) on proliferation and apoptosis in the chronic myeloid leukemia cell line K562. METHODS Different concentrations of an antisense oligodeoxy-nucleotid and control sequence (scrambled ODN) targeting the survivin gene were transferred into K562 by a lipofectin reagent. The MTT assay was used to measure the growth inhibitory rate, IC50, and to observe the cytotoxicity of survivin ASODN in the K562 cells. The morphologic changes in the nucleus and the apoptotic rate were observed by Hoechst33342/PI staining. Caspase-3 activity was evaluated by a kinase activity assay. The changes of survivin protein expression after transfection were detected by Western blots. RESULTS Eight hours after transfection, fluorescence in the K562 cells was well distributed. Treatment of the cells for 44 h with different concentrations of survivin ASODN produced a IC50 of 800 nmol/L. The growth inhibitory rate with 200, 400, 600 and 1000 nmol/L of survivin ASODN was 15.8±1.6%, 23.8±5.9%, 37.1±5.6% and 77.3±2.5% respectively. After 36 h of of survivin ASODN treatment, distinct morphologic changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were observed by Hoechst33342/PI staining. Caspase-3 activity increased significantly after treatment of the cells with different concentrations of survivin ASODN(P<0.01)and following treatment with 800 nmol/L survivin ASODN, survivin expression decreased significantly. CONCLUSION Survivin ASODN exerts an anti-cancer effect by inducing apoptosis in K562 leukaemia cells. Up-regulated expression of caspase-3 may play a role in this process.展开更多
OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI)...OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.展开更多
文摘OBJECTIVE To investigate the effects of survivin antisense oligodeoxy-nucleotid (ASODN) on proliferation and apoptosis in the chronic myeloid leukemia cell line K562. METHODS Different concentrations of an antisense oligodeoxy-nucleotid and control sequence (scrambled ODN) targeting the survivin gene were transferred into K562 by a lipofectin reagent. The MTT assay was used to measure the growth inhibitory rate, IC50, and to observe the cytotoxicity of survivin ASODN in the K562 cells. The morphologic changes in the nucleus and the apoptotic rate were observed by Hoechst33342/PI staining. Caspase-3 activity was evaluated by a kinase activity assay. The changes of survivin protein expression after transfection were detected by Western blots. RESULTS Eight hours after transfection, fluorescence in the K562 cells was well distributed. Treatment of the cells for 44 h with different concentrations of survivin ASODN produced a IC50 of 800 nmol/L. The growth inhibitory rate with 200, 400, 600 and 1000 nmol/L of survivin ASODN was 15.8±1.6%, 23.8±5.9%, 37.1±5.6% and 77.3±2.5% respectively. After 36 h of of survivin ASODN treatment, distinct morphologic changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were observed by Hoechst33342/PI staining. Caspase-3 activity increased significantly after treatment of the cells with different concentrations of survivin ASODN(P<0.01)and following treatment with 800 nmol/L survivin ASODN, survivin expression decreased significantly. CONCLUSION Survivin ASODN exerts an anti-cancer effect by inducing apoptosis in K562 leukaemia cells. Up-regulated expression of caspase-3 may play a role in this process.
基金Shanghai Municipal Health Bureau:Traditional Chinese Medicine in Treating with Advanced Hepatocellular Carcinoma(No.ZYSNXD-CC-ZDYJ032)
文摘OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.
