Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification

Abstract

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.

Isorhamnetin induces ROS-dependent cycle arrest at G2/M phase and apoptosis in human hepatocarcinoma Hep3B cells

“…Therefore, it was first examined whether the growth inhibitory effect of isorhamnetin in Hep3B cells was associated with cell cycle arrest. The results of flow cytometry analysis showed that isorhamnetin caused G2/M phase arrest, similar to the results of previous studies in several human cancer cell lines (Li et al 2014;Wu et al 2016;Wei et al 2018;Zhang et al 2018), suggesting that G2/M arrest is one of the mechanisms of isorhamnetin-mediated growth inhibitory effect in Hep3B cells. The progression of the cell cycle in eukaryotic cells is strictly controlled by the interaction of cyclins and Cdks with their inhibitory factors.…”

“…Because isorhamnetin-mediated cell cycle arrest at G2/M phase has been reported to be related to apoptosis in some cancer cell lines (Li et al 2014;Zhang et al 2015;Wu et al 2016;Wei et al 2018;Zhang et al 2018), it was evaluated whether G2/M arrest by isorhamnetin was associated with Figure 4. Effects of isorhamnetin (ISO) on the expression of Bcl-2 family proteins and cytochrome c, ROS generation and MMP in Hep3B cells.…”

“…Isorhamnetin exhibits many biological properties due to its anti-inflammatory, antioxidant and metabolic properties (Luo et al 2015;Seo et al 2016;Yang et al 2016;Abdallah and Esmat 2017;Qi et al 2018), and is also considered to have the potential of anti-cancer agents in the results of various cancer cell models. According to previous studies, isorhamnetin has been reported to prevent the proliferation of human leukemia, breast cancer, colon cancer and cervical cancer cells through G2/M phase arrest (Li et al 2014;Wu et al 2016;Wei et al 2018;Zhang et al 2018), and to induce mitotic block in non-small cell lung carcinoma cells, thus enhancing cisplatin-and carboplatin-induced G2/M arrest (Zhang et al 2015). On the other hand, isorhamnetin induced S phase arrest in some cancer cells (Wang et al 2018;Wu et al 2018), indicating that cell cycle arrest by isorhamnetin depends on the type of cancer cell line.…”

Insight into the secret of how indigo naturalis works: in vitro and in vivo validation of the critical role of bile­

“…Due to its manufacturing process, the material composition of IN is about 10% organic and about 90% inorganic matter [6]. Its organic components are mainly indole alkaloids, such as indigo, indirubin [7], etc. The inorganic components contain more than 70% calcium carbonate and about 10% silica.…”

Screening anti‐allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method

“…Cell membrane chromatography (CMC), developed by He and colleagues, represents a new type of biological affinity chromatography and is recognized as an effective method to screen bioactive components of complex systems (He & Geng, ; He et al ., ; Wang, Sun, Zhang, Du, & He, ). However, although CMC has been used to screen various components acting on various cells from complex systems (Chen et al ., ; Wu et al ., ), it still has the disadvantage of low sensitivity. Multiple techniques, like nuclear magnetic resonance spectroscopy (NMR) and gas or liquid chromatography mass spectrometry (GC–MS or LC–MS), have been used instead to analyze and identify unknown compounds, with LC–MS being the most widely used technique due to its high sensitivity, convenience and other advantages (Han, Zhang, Wei, & Wang, 2012; Han, Zhang, Huang, & Wang, 2012).…”