Study On Synergistically Inducing Cell Apoptosis And Its Mechanism By Arsenic Trioxide Combined With Triptolide In Myelodysplastic Syndrome Cell Line SKM-1

Part I Study on inducing cell apoptosis and its mechanism with arsenic trioxide in myelodysplastic syndrome cell line SKM-1Objective To investigate the mechanism of cell apoptosis in myelodysplastic syndrome cell line SKM-1 induced with arsenic trioxide?As₂O₃?.Methods MDS SKM-1 cells were cultured with different concentration As₂O₃ for 24 h and 48 h.Cell morphology was observed by light microscope.Cell viability,cell apoptosis,intracellular reactive oxygen species?ROS?level,anti-apoptotic gene Bcl-2,pro-apoptotic gene Bax and caspase-3 were determined by the MTT assay,flow cytometry analysis of annexin V-FITC/propidium iodide?PI?double-stained cells,flow cytometry analysis of intracellular 2?,7?-dichlorodihydrofluorescein diacetate?DCFH-DA?fluorescence,and Real-time quantitative reverse transcription polymerase chain reaction?RQ-RT-PCR?,respectively.Results The treatment with As₂O₃ of 2,8,32 ?M concentration substantially suppressed SKM-1 cell growth and induced its apoptosis compared with control group?all P<0.01?,but not As₂O₃ of 0.25,0.5 ?M concentration??>0.05?.The rates of proliferation inhibition and apoptosis of SKM-1 cells were increased gradually with the increase of concentration and prolonging of action time with AS₂O₃?all P<0.01?.The SKM-1 cells were showed typical cell apoptotic morphology under the microscope,including chromatin condensation,marginalization,nuclear pyknosis,nuclear fragmentation,segmented blocks of chromatin and apoptotic bodies of some cells,etc.With the increase of concentration and prolonging of action time wirh AS₂O₃,the intracellular ROS levels were increased gradually?P<0.01?,the expression levels of pro-apoptotic gene Bax and caspase-3-m RNA were increased gradually?P<0.01,?<0.05?,but the expression levels of anti-apoptotic gene Bcl-2-m RNA were decreased gradually?P<0.01?.Conclusion The treatment with As₂O₃ of 2,8,32 ?M concentration substantially suppressed SKM-1 cell growth and induced its apoptosis,but not As₂O₃ of low concentration?0.25,0.5 ?M?,and there are dose and time dependent manner.The treatment with As₂O₃ suppressed SKM-1 cell growth and induced its apoptosis maybe via increasing intracellular ROS level,downregulating the expression of anti-apoptotic gene Bcl-2,upregulating the expression of pro-apoptotic gene Bax and caspase-3.Part II Study on inducing cell apoptosis and its mechanism with triptolide in myelodysplastic syndrome cell line SKM-1Objective To investigate the mechanism of cell apoptosis in myelodysplastic syndrome cell line SKM-1 induced with triptolide?TL?.Methods MDS SKM-1 cells were cultured with different concentration triptolide for 24 h and 48 h.Cell morphology was observed by light microscope.Cell viability,cell apoptosis,intracellular reactive oxygen species?ROS?level,anti-apoptotic gene Bcl-2,pro-apoptotic gene Bax and caspase-3 were determined by the MTT assay,flow cytometry analysis of annexin V-FITC/propidium iodide?PI?double-stained cells,flow cytometry analysis of intracellular 2?,7?-dichlorodihydrofluorescein diacetate?DCFH-DA?fluorescence,and Real-time quantitative reverse transcription polymerase chain reaction?RQ-RT-PCR?,respectively.Results The treatment with TL of 40,60,80 ng/ml concentration substantially suppressed SKM-1 cell growth and induced its apoptosis compared with control group?all P<0.01?,but not TL of 10,20 ng /ml concentration??>0.05?.The rates of proliferation inhibition and apoptosis of SKM-1 cells were increased gradually with the increase of concentration and prolonging of action time with TL?all P<0.01?.The SKM-1 cells were showed typical cell apoptotic morphology under the microscope,including chromatin condensation,marginalization,nuclear pyknosis,nuclear fragmentation,segmented blocks of chromatin and apoptotic bodies of some cells,etc.With the increase of concentration and prolonging of action time with TL,the intracellular ROS levels were increased gradually?P<0.01?,the expression levels of pro-apoptotic gene Bax and caspase-3-m RNA were increased gradually?all P<0.01?,but the expression levels of anti-apoptotic gene Bcl-2-m RNA were decreased gradually?P<0.01?.Conclusion The treatment with TL of 40,60,80 ng/ml concentration substantially suppressed SKM-1 cell growth and induced its apoptosis,but not TL of low concentration?10,20 ng/ml?,and there are dose and time dependent manner.The treatment with TL suppressed SKM-1 cell growth and induced its apoptosis maybe via increasing intracellular ROS level,downregulating the expression of anti-apoptotic gene Bcl-2,upregulating the expression of pro-apoptotic gene Bax and caspase-3.Part III Study on synergistically inducing cell apoptosis and its mechanism with arsenic trioxide in combination with triptolide in myelodysplastic syndrome cell line SKM-1Objective To investigate the mechanism of cell apoptosis in myelodysplastic syndrome cell line SKM-1 synergistically induced with arsenic trioxide?As₂O₃?in combination with triptolide?TL?.Methods MDS SKM-1 cells were cultured with different concentration As₂O₃,TL,or As₂O₃+TL for 48 h.Cell morphology was observed by light microscope.Cell viability,cell apoptosis,intracellular reactive oxygen species?ROS?level,anti-apoptotic gene Bcl-2,pro-apoptotic gene Bax and caspase-3 were determined by the MTT assay,flow cytometry analysis of annexin V-FITC/propidium iodide?PI?double-stained cells,flow cytometry analysis of intracellular 2?,7?-dichlorodihydrofluorescein diacetate?DCFH-DA?fluorescence,and Real-time quantitative reverse transcription polymerase chain reaction?RQ-RT-PCR?,respectively.Combination index?CI?was calculated,respectively.Results Except for low concentration 0.25umol/L AS₂O₃ in combination with 10 ng /ml TL?? > 0.05?,The treatment with As₂O₃ in combination with TL of other concentration substantially suppressed SKM-1 cell growth and induced its apoptosis compared with As₂O₃ or TL alone?all P<0.01?.The rates of proliferation inhibition and apoptosis of SKM-1 cells were increased gradually with the concentration increase of AS₂O₃ in combination with TL?all P<0.01?,all CI<1.The SKM-1 cells were showed typical cell apoptotic morphology under the microscope,including chromatin condensation of some cells,marginalization,nuclear pyknosis,nuclear fragmentation,segmented blocks of chromatin and apoptotic bodies of some cells,etc.With the concentration increase of AS₂O₃ in combination with TL,the intracellular ROS levels were increased gradually?P<0.01?,CI<1,the expression levels of pro-apoptotic gene Bax and caspase-3-m RNA were increased gradually?all P<0.01?,CI<1,but the expression levels of anti-apoptotic gene Bcl-2-m RNA were decreased gradually?P<0.01?,CI<1.Conclusion The treatment with As₂O₃ in combination with TL substantially synergistically suppressed SKM-1 cell growth and induced its apoptosis maybe via increasing intracellular ROS level,downregulating the expression level of anti-apoptotic gene Bcl-2,upregulating the expression level of pro-apoptotic gene Bax and caspase-3.