PDF(Pubmed)
Abstract：
BACKGROUND: Indigofera suffruticosa Mill. is used as a folk medicine for treating patients with leukemia, however very little is known regarding the molecular mechanism of its anti-leukemic activity and the chemical profile of the active extract. The present study aimed to reveal the molecular effect of I. suffruticosa aerial parts extract (ISAE) on leukemia cells and its chemical constituents.
METHODS: Cytotoxicity of ISAE were determined by resazurin viability assay, multitox - Glo multiplex cytotoxicity assay, and Annexin V staining assay. Cell cycle profiles were revealed by propidium iodide staining assay. The effects of ISAE on G2/M arrest signaling and DNA damage were evaluated by Western blot assay and phospho-H2A.X staining assay. The chemical profile of ISAE were determined by tandem mass spectroscopy and molecular networking approach.
RESULTS: We showed that the acute lymphoblastic leukemia cell line Jurkat cell was more responsive to ISAE treatment than other leukemia cell lines. In contrast, ISAE did not induce cytotoxic effects in normal fibroblast cells. Cell cycle analysis revealed that ISAE triggered G2/M arrest in Jurkat cells in dose- and time-dependent manners. Elevation of annexin V-stained cells and caspase 3/7 activity suggested ISAE-induced apoptosis. Furthermore, ISAE alone could increase the phosphorylation of CDK1 at Y15 and activate the ATR/CHK1/Wee1/CDC25C signaling pathway. However, the addition of caffeine, a widely used ATR inhibitor to ISAE, reduced the phosphorylation of ATR, CHK1, and CDK1, as well as G2/M arrest in Jurkat cells. Moreover, increased phospho-H2A.X stained cells indicated the involvement of DNA damage in the anti-leukemic effect of ISAE. Finally, qualitative analysis using UPLC-tandem mass spectroscopy and molecular networking revealed that tryptanthrin was the most abundant organoheterocyclic metabolite in ISAE. At equivalent concentrations to ISAE, tryptanthrin induced G2/M arrest of Jurkat cells, which can be prevented by caffeine.
CONCLUSIONS: ISAE causes G2/M arrest via activating ATR/CHK1/CDK1 pathway and tryptanthrin is one of the active components of ISAE. Our findings provide subtle support to the traditional use of I. suffruitcosa in leukemia management in folk medicine.
METHODS: Cytotoxicity of ISAE were determined by resazurin viability assay, multitox - Glo multiplex cytotoxicity assay, and Annexin V staining assay. Cell cycle profiles were revealed by propidium iodide staining assay. The effects of ISAE on G2/M arrest signaling and DNA damage were evaluated by Western blot assay and phospho-H2A.X staining assay. The chemical profile of ISAE were determined by tandem mass spectroscopy and molecular networking approach.
RESULTS: We showed that the acute lymphoblastic leukemia cell line Jurkat cell was more responsive to ISAE treatment than other leukemia cell lines. In contrast, ISAE did not induce cytotoxic effects in normal fibroblast cells. Cell cycle analysis revealed that ISAE triggered G2/M arrest in Jurkat cells in dose- and time-dependent manners. Elevation of annexin V-stained cells and caspase 3/7 activity suggested ISAE-induced apoptosis. Furthermore, ISAE alone could increase the phosphorylation of CDK1 at Y15 and activate the ATR/CHK1/Wee1/CDC25C signaling pathway. However, the addition of caffeine, a widely used ATR inhibitor to ISAE, reduced the phosphorylation of ATR, CHK1, and CDK1, as well as G2/M arrest in Jurkat cells. Moreover, increased phospho-H2A.X stained cells indicated the involvement of DNA damage in the anti-leukemic effect of ISAE. Finally, qualitative analysis using UPLC-tandem mass spectroscopy and molecular networking revealed that tryptanthrin was the most abundant organoheterocyclic metabolite in ISAE. At equivalent concentrations to ISAE, tryptanthrin induced G2/M arrest of Jurkat cells, which can be prevented by caffeine.
CONCLUSIONS: ISAE causes G2/M arrest via activating ATR/CHK1/CDK1 pathway and tryptanthrin is one of the active components of ISAE. Our findings provide subtle support to the traditional use of I. suffruitcosa in leukemia management in folk medicine.
摘要：
背景：印度。被用作治疗白血病患者的民间药物，然而，关于其抗白血病活性的分子机制和活性提取物的化学特征知之甚少。本研究旨在揭示木薯地上部分提取物（ISAE）对白血病细胞及其化学成分的分子效应。
方法：采用利天青活力测定法测定ISAE的细胞毒性，Multitox-Glo多重细胞毒性试验，和膜联蛋白V染色测定。通过碘化丙啶染色测定揭示细胞周期谱。通过蛋白质印迹测定和磷酸-H2A评估ISAE对G2/M阻滞信号传导和DNA损伤的影响。X染色测定。通过串联质谱和分子网络方法确定ISAE的化学概况。
结果：我们表明，急性淋巴细胞白血病细胞系Jurkat细胞比其他白血病细胞系对ISAE治疗更敏感。相比之下,ISAE在正常成纤维细胞中不诱导细胞毒性作用。细胞周期分析显示ISAE以剂量和时间依赖性方式在Jurkat细胞中触发G2/M阻滞。膜联蛋白V染色的细胞和半胱天冬酶3/7活性的升高表明ISAE诱导的细胞凋亡。此外，单独的ISAE可以增加Y15处CDK1的磷酸化并激活ATR/CHK1/Wee1/CDC25C信号通路。然而,添加咖啡因，一种广泛使用的ISAEATR抑制剂，减少ATR的磷酸化，CHK1和CDK1，以及Jurkat细胞中的G2/M阻滞。此外，增加磷酸-H2A。X染色的细胞表明DNA损伤参与了ISAE的抗白血病作用。最后,使用UPLC串联质谱和分子网络的定性分析表明，色胺酮是ISAE中最丰富的有机杂环代谢物。在与ISAE相当的浓度下，色胺酮诱导Jurkat细胞G2/M期阻滞,这可以通过咖啡因来预防。
结论：ISAE通过激活ATR/CHK1/CDK1途径引起G2/M期阻滞，色胺酮是ISAE的活性成分之一。我们的发现为民间医学在白血病管理中的传统使用I.suffruitcosa提供了微妙的支持。
方法：采用利天青活力测定法测定ISAE的细胞毒性，Multitox-Glo多重细胞毒性试验，和膜联蛋白V染色测定。通过碘化丙啶染色测定揭示细胞周期谱。通过蛋白质印迹测定和磷酸-H2A评估ISAE对G2/M阻滞信号传导和DNA损伤的影响。X染色测定。通过串联质谱和分子网络方法确定ISAE的化学概况。
结果：我们表明，急性淋巴细胞白血病细胞系Jurkat细胞比其他白血病细胞系对ISAE治疗更敏感。相比之下,ISAE在正常成纤维细胞中不诱导细胞毒性作用。细胞周期分析显示ISAE以剂量和时间依赖性方式在Jurkat细胞中触发G2/M阻滞。膜联蛋白V染色的细胞和半胱天冬酶3/7活性的升高表明ISAE诱导的细胞凋亡。此外，单独的ISAE可以增加Y15处CDK1的磷酸化并激活ATR/CHK1/Wee1/CDC25C信号通路。然而,添加咖啡因，一种广泛使用的ISAEATR抑制剂，减少ATR的磷酸化，CHK1和CDK1，以及Jurkat细胞中的G2/M阻滞。此外，增加磷酸-H2A。X染色的细胞表明DNA损伤参与了ISAE的抗白血病作用。最后,使用UPLC串联质谱和分子网络的定性分析表明，色胺酮是ISAE中最丰富的有机杂环代谢物。在与ISAE相当的浓度下，色胺酮诱导Jurkat细胞G2/M期阻滞,这可以通过咖啡因来预防。
结论：ISAE通过激活ATR/CHK1/CDK1途径引起G2/M期阻滞，色胺酮是ISAE的活性成分之一。我们的发现为民间医学在白血病管理中的传统使用I.suffruitcosa提供了微妙的支持。
