Chen Nai-Yao, Wang Jing, Wang Xue-Ming, Zhang Hai-Xia, Yan Zhen-Yu, Wang Ying-Man, Zhang Song, Yan Bing
Department of Hematology, North China Coal Medical College Affiliated Hospital, Tangshan 063000, Hebei Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2010 Aug;18(4):882-6.
This study was aimed to explore the effects of STI571 alone or with As₂O₃ on proliferation, apoptosis and caspase 3, bcl-xL mRNA expression of K562 cells, and the molecular mechanism of As₂O₃ enhancing the anti-leukemia effect of STI571 so as to provide the scientific basis for clinical treatment of chronic myeloid leukemia. The effect of drugs on proliferation of K562 cells was assayed by MTT method, the apoptosis rate of K562 cells was detected by flow cytometry with Annexin V/PI double staining, the caspase 3, bcl-xL mRNA expressions of K562 cells were determined by real time quantitative PCR. The results showed that STI571 alone or with As₂O₃ both could inhibit the proliferation of K562 cells. OD value in test groups reduced along with prolonging of action times, the OD values between different time points were significantly different (p < 0.05), furthermore the OD values at 72 hours in test groups were lowest, while as compared with control group, OD values at same time points in test groups all gradually decreased, among which decrease of OD value in test 5 group was most significant. The flow cytometric detection indicated that along with time prolonging, the apoptotic rate in control group not obviously changed, but the apoptotic rate in test groups gradually increased, the difference between time points was significant (p < 0.05), moreover apoptotic rate increased most obviously at 72 hours, while as compared with control group, apoptotic rate at same time points in test groups was gradually enhanced (p < 0.05), among which the apoptotic rate in test 5 group was highest. The real time qPCR assay revealed that as compared with control group, the bcl-xL mRNA expression in test groups reduced with decrease of 2-ΔΔCT value, furthermore the decrease of expression level in test 3 group was higher than that in test 2 group (p < 0.05), while the caspase 3 mRNA expression in test groups was enhanced with increase of 2-ΔΔCT value, moreover the increase of expression level in test 3 group was higher than that in test 2 group (p < 0.05). It is concluded that the STI571 can inhibit the proliferation of K562 cells, accelerate the apoptosis of K562 cells. The STI571 combined with As₂O₃ can enhance these two effects, increase the expression of caspase-3 mRNA and decrease the expression of bcl-xL mRNA. Therefore, the effect of STI571 combined with As₂O₃ on expression of caspase 3 and bcl-xL mRNA may be one of molecular mechanisms underlying their synergic antileukemia efficacy.
本研究旨在探讨STI571单独或与As₂O₃联合应用对K562细胞增殖、凋亡及半胱天冬酶3（caspase 3）、bcl-xL mRNA表达的影响，以及As₂O₃增强STI571抗白血病作用的分子机制，为慢性髓性白血病的临床治疗提供科学依据。采用MTT法检测药物对K562细胞增殖的影响，应用Annexin V/PI双染法通过流式细胞术检测K562细胞凋亡率，采用实时定量PCR法检测K562细胞caspase 3、bcl-xL mRNA表达。结果显示，STI571单独或与As₂O₃联合应用均能抑制K562细胞增殖。各试验组光密度（OD）值随作用时间延长而降低，不同时间点的OD值差异有统计学意义（p<0.05），且各试验组72小时时OD值最低；与对照组比较，各试验组同一时间点OD值均逐渐降低，其中试验5组OD值降低最显著。流式细胞术检测表明，随着时间延长，对照组凋亡率无明显变化，而各试验组凋亡率逐渐升高，各时间点差异有统计学意义（p<0.05），且72小时时凋亡率升高最明显；与对照组比较，各试验组同一时间点凋亡率逐渐升高（p<0.05），其中试验5组凋亡率最高。实时定量PCR检测显示，与对照组比较,各试验组bcl-xL mRNA表达随2-ΔΔCT值降低而减少，且试验3组表达水平降低幅度高于试验2组（p<0.05），而各试验组caspase 3 mRNA表达随2-ΔΔCT值升高而增强，且试验3组表达水平升高幅度高于试验2组（p<0.05）。结论：STI571能抑制K562细胞增殖，促进K562细胞凋亡。STI571与As₂O₃联合应用可增强这两种作用，增加caspase-3 mRNA表达，降低bcl-xL mRNA表达。因此，STI571与As₂O₃联合应用对caspase 3和bcl-xL mRNA表达的影响可能是其协同抗白血病效应的分子机制之一。
