Objective: To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism.
Methods: The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT.
Results: Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G1 phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells.
Conclusion: TPA induces NB4 cell cycle arrest in G1 phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.
题目: 佛波酯化合物TPA诱导急性早幼粒细胞白血病细胞NB4增殖与凋亡的机制研究.
目的: 探讨佛波酯化合物TPA对急性早幼粒细胞白血病细胞NB4增殖、凋亡的影响及其分子机制。.
方法: CCK-8法检测不同浓度TPA在不同时间点对NB4细胞增殖的影响；瑞氏-吉姆萨染色观察NB4细胞形态学变化；流式细胞术检测TPA处理后NB4细胞周期、凋亡情况；高通量微阵列分析和实时荧光定量PCR法分析TPA处理后NB4细胞mRNA的表达；Western blot检测CDKN1A、CDKN1B、CCND1、MYC、Bax、Bcl-2、c-Caspase 3、c-Caspase 9、PIK3R6、AKT和p-AKT的蛋白表达。.
结果: 与对照组相比，TPA能抑制NB4细胞增殖，诱导细胞向成熟粒-单核系分化；诱导细胞G1期阻滞及凋亡；差异表达的mRNA显著富集在PI3K/AKT通路；TPA能提高NB4细胞中CCND1、CCNA1、CDKN1A的mRNA水平，降低MYC的mRNA水平；TPA能上调NB4细胞中CDKN1A、CDKN1B、CCND1、Bax、c-Caspase 3、c-Caspase 9、PIK3R6蛋白水平，下调MYC、Bcl-2、p-AKT蛋白水平。.
结论: TPA通过调控PIK3/AKT信号通路诱导NB4细胞周期阻滞于G1期并促进其凋亡。.
Keywords: NB4 cell; acute promyelocytic leukemia; apoptosis; phorbol-12-myristate-13-ace-tate; proliferation.