阿卡宁下调Trx/Akt通路诱导急性髓系白血病细胞凋亡的作用研究

Effect of alkannin on apoptosis of acute myeloid leukemia cells by down-regulating Trx/Akt pathway

目的 探讨新疆紫草Lithospermum erythrorhizon代表成分阿卡宁对急性髓系白血病细胞HL-60的抑制作用及机制。方法 取对数生长期HL-60细胞,采用台盼蓝法检测阿卡宁、紫草素处理后的细胞增殖抑制率;Hoechst 33258染色观察细胞形态;流式细胞仪检测细胞凋亡率;通过系统药理学方法筛选出阿卡宁以及急性髓系白血病(acute myeloid leukemia,AML)的共同靶点并分析得到关键靶点。通过邻苯二甲醛(o-phthalaldehyde,OPA)荧光探针法、差示扫描荧光分析实验在体外分子水平验证化合物与靶点的结合能力。通过Western blotting检测凋亡通路标志分子半胱氨酸天冬氨酸蛋白酶-3(cysteinasparate protease-3,Caspase-3)、cleaved Caspase-3、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2associated X protein,Bax)蛋白表达。结果 阿卡宁呈剂量相关性抑制HL-60细胞增殖。Hoechst染色后荧光显微镜下可见阿卡宁处理后的HL-60细胞呈现细胞缩小、染色质颜色加深、染色质逐渐碎片化等的凋亡形态;与对照组比较,阿卡宁诱导的细胞凋亡率显著升高(P<0.001),Bcl-2蛋白表达水平显著下调(P<0.01),Bax、cleaved Caspase-3蛋白表达水平均显著上调(P<0.01、0.001);通过系统药理学筛选得到阿卡宁和AML共有靶点96个,对共有靶点分析得到蛋白质-蛋白质相互作用网络(protein-protein interaction,PPI)中度值较高的靶点是蛋白激酶B(protein kinase B,Akt)、半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,CASP3)、细胞色素C(cytochrome C,CYCS)、硫氧还蛋白还原酶(thioredoxin reductase,TXNRD)等。基因本体(gene ontology,GO)功能和京都基因与基因百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路分析表明阿卡宁治疗AML主要涉及RNA聚合酶II启动子转录的正调控、细胞凋亡过程的正调控及信号转导等生物学过程,涉及的分子功能有蛋白结合、氧化还原酶活性,信号通路主要包括癌症相关信号通路。共价对接结果表明,阿卡宁与硫氧还蛋白(thioredoxin,Trx)活性位点半胱氨酸形成共价键。体外分子水平结果显示阿卡宁可以与谷胱甘肽(glutathione,GSH)、Trx结合。Western blotting结果显示阿卡宁可显著下调Trx、Akt、p-Akt的表达(P<0.05、0.001)。结论 阿卡宁诱导HL-60细胞凋亡,其机制与下调Trx/Akt通路有关。

Objective To investigate the inhibitory effect and mechanism of alkannin(ALK), a representative component of Lithospermum erythrorhizon, on HL-60 cells of acute myeloid leukemia. Methods HL-60 cells in logarithmic proliferation phase were selected, and inhibitory rate of cell proliferation after treatment with ALK and shikonin(SK) was detected by trypan blue method;Hoechst 33258 staining was used to observe the cell morphology;Apoptosis rate was detected by flow cytometry;Systematic pharmacological methods was used to screen the common targets of ALK and acute myeloid leukemia(AML) and the key targets were analyzed. The binding ability of compound with target was verified at the molecular level by an o-phthalaldehyde(OPA) fluorescence probe and differential scanning fluorescence analysis in vitro. The protein expressions of apoptosis pathway marker cystein-asparate protease-3(Caspase-3), cleaved Caspase-3, B-cell lymphoma-2(Bcl-2) and Bcl-2 associated X protein(Bax) were detected by Western blotting. Results ALK inhibited the proliferation of HL-60 cells in a dose-dependent manner. Under fluorescence microscope after Hoechst staining, HL-60 cells treated with ALK showed apoptosis morphology, such as cell shrinkage, chromatin color deepening,chromatin fragmentation, etc;Compared with control group, apoptosis rate induced by ALK was significantly increased(P < 0.001),Bcl-2 protein expression was significantly decreased(P < 0.01), Bax and cleared Caspase-3 protein expressions were significantly increased(P < 0.01, 0.001);A total of 96 common targets of ALK and AML were obtained through systematic pharmacological screening, and the higher degree targets in protein-protein interaction(PPI) network were protein kinase B(Akt), cystein-asparate protease-3(CASP3), cytochrome C(CYCS), thioredoxin reductase(TXNRD), etc. Gene ontology(GO) function and Kyoto encyclopedia of genes and genomes(KEGG) pathway analysis showed that the treatment of AML with ALK was mainly related to the positive regulation of RNA polymerase II promoter transcription, apoptosis, and signal transduction regulation, involved molecular functions include protein binding and oxidoreductase activity, signal pathways mainly including cancer-related signal pathways. The results of covalent docking showed that ALK formed a covalent bond with cysteine at the active site of thioredoxin(Trx). Molecular results showed that ALK could bind to glutathione(GSH) and Trx. Western blotting results showed that ALK could significantly downregulate the expressions of Trx, Akt, and p-Akt(P < 0.05, 0.001). Conclusion ALK induced apoptosis of HL-60 cells, which is related to the inhibition of the Trx/Akt pathway.