| Objective: To observe the effects of nilotinib, arsenic trioxide, Imatinib of different concentrations alone and both combined on proliferation, apoptosis and gene expression of Imatinib-resistant K562 cells.Methods : Human leukemia cell line K562G cells were cultivated in vitro. K562G cells were seperated into control group without any drug and experimental groups with STI571,ATO,AMN107 of different concentrations and combination of STI571+ATO,AMN107+ATO,STI571+ AMN107.MTT was used to detect their effect on cell proliferation. Flow cytometry was operated to detect apoptosis by using PI single staining. Fluorescence quantitative PCR was detected to observe the levels of bcr/abl gene expression.Results: 1.The result by MTT assay: (1) K562G cells was inhibited on resistant concentration of STI571. (2) K562G cells was inhibited by ATO with dose dependent. (3) K562G cells was inhibited by AMN107 with dose dependent. (4) K562G cells was inhibited by combination STI571+ATO with dose dependent,stronger(P<0.05) inhibition than single ATO(except 1.0μmol/L). (5) K562G cells was inhibited by combintion AMN107+ATO with dose dependent,the combintion AMN107+ATO of all concentration showed stronger (P<0.05) inhibition than single AMN107 or ATO. (6) K562G cells was inhibited by combintion AMN107+STI571 with dose dependent, the combintion AMN107+STI571 of all concentration showed stronger (P<0.05) inhibition than single AMN107 orSTI571.2. The result of percentage of apoptisis cells by Flow cytometry: The rate of control groups cell apoptosis: 9.86±1.02. 1) The rate of STI571 (0.25,2.5,10.0μmol / L) groups cell apoptosis: 10.54±1.21,10.62±1.40,11.20±1.81, no significant cell apoptosis with all concentrations. 2) The rate of ATO (1.0,5.0,10.0μmol / L) groups cell apoptosis : 19.12±1.10,60.98±2.28,52.28±1.21, the highest cell apoptosis happened with concentration of 5.0μmol / L. 3) The rate of AMN107 (0.25,2.5μ, 5.0μmol / L) groups cell apoptosis : 17.54±2.21,30.41±2.22,60.98±2.64, showing a dose dependent. 4) The rate of STI571 + ATO [(0.25 +1.0), (2.5 +5.0), (10.0 +10.0)μmol / L] groups cell apoptosis: 31.2±1.10,70.98±2.28,62.98±1.21, the highest cell apoptosis happened with concentration of (5.0 +2.5 )μmol / L , and singificant differences between combintion STI571+ATO groups and single ATO groups (P<0.05). 5) The rate of AMN107 + ATO [(0.25 +1.0) , (2.5 +5.0), (5.0 +10.0)μmol / L] groups cell apoptosis: 29.5±1.10,86.65±2.28,71.65±1.21, of which (2.5 +5.0)μmol / L concentration of the highest rates of apoptosis, it was singificant difference combintion AMN107+ATO than single AMN107 or ATO group (P<0.05). 6) The rate of AMN107 + STI571 [(0.25 +0.25), (2.5 +2.5), (5.0 +10.0)]μmol / L groups cell apoptosis: 13.37±1.10,59.64±2.28,71.19±1.21, with a dose dependent. There was singificant differences between the combintion AMN107+STI571groups and single AMN107 groups (P<0.05).3. The levels of bcr/abl gene expression by Fluorescence quantitative PCR: 1) The bcr/abl gene expression levels of ATO (1.0,5.0,10.0μmol / L) groups were 1.18×106 copies / ml, 3.28×104 copies / ml, 5.23×104 copies / ml. 2) The bcr/abl gene expression levels of AMN107 (0.25,2.5,5.0μmol / L) groups were 8.13×104 copies / ml, 1.58×103 copies / ml, negative. 3) The bcr/abl gene expression levels of ATO + STI571 [(1.0 +0.25), (5.0 +2.5), (10.0 +10.0)μmol / L] groups were 1.10×105 copies / ml, 5.06×103 copies / ml, 6.43×103 copies / ml. 4) The bcr/abl gene expression levels of ATO + AMN107 [(1.0 +0.25), (5.0 +2.5), (10.0 +10.0)μmol / L] groups were 2.54×104 copies / ml, double negatives. 5) The bcr/abl gene expression levels of AMN107 + STI571 [(0.25 +0.25), (2.5 +2.5), (5.0 +10.0)μmol / L] groups were 3.13×103 copies / ml, double negatives.Conclusion: 1.K562G cells showed drug-resistance to imatinib with concentrations not higher than 10μmol / L.2. The growth of K562G cells could be inhibitid by ATO and led to the cells apoptosis, showing a dependent of drug doses,the stongest apoptosis happened with the concentration of 5.0μmol/L.3. The growth of K562G cells could be inhibitid by AMN107 and led to the cells apoptosis, showing a dependent of drug doses.4. As to cell inhibition rate and apoptosis rate, a better effect result showed with the combination of STI571 and ATO on K562G cells than single ATO . 5. As to cell inhibition rate and apoptosis rate, a better effect with the combination of AMN107 and ATO on K562G cells than single ATO, showing a synergistic effect.6. As to cell inhibition rate and apoptosis rate, a better effect with the combination of STI571 and AMN107 on K562G cells than single AMN107 or STI571,showing a synergistic effect.7. The decrease of bcr/abl gene expression level on K562G cells happened in AMN107,ATO and combination groups.
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