Minor Hydroxylated Triterpenoids Produced in Engineered Yeast by the Enzymes OSC and CYP716s from the Plant Enkianthus chinensis and Their Anti-Inflammatory and Hepatoprotective Activities.

Triterpenoids are a type of specialized metabolites that exhibit a wide range of biological activities. However, the availability of some minor triterpenoids in nature is limited, which has hindered our understanding of their pharmacological potential. To overcome this limitation, heterologous biosynthesis of triterpenoids in yeast has emerged as a promising and time-efficient production platform for obtaining these minor compounds. In this study, we analyzed the transcriptomic data of Enkianthus chinensis to identify one oxidosqualene cyclase (EcOSC) gene and four CYP716s. Through heterologous expression of these genes in yeast, nine natural pentacyclic triterpenoids, including three skeleton products (1-3) produced by one multifunctional OSC and six minor oxidation products (4-9) catalyzed by CYP716s, were obtained. Of note, we discovered that CYP716E60 could oxidize ursane-type and oleanane-type triterpenoids to produce 6ß-OH derivatives, marking the first confirmed C-6ß hydroxylation in an ursuane-type triterpenoid. Compound 9 showed moderate inhibitory activity against NO production and dose-dependently reduced IL-1ß and IL-6 production at the transcriptional and protein levels. Compounds 1, 2, 8, and 9 exhibited moderate hepatoprotective activity with the survival rates of HepG2 cells from 61% to 68% at 10 µM.

Bufalin suppresses esophageal squamous cell carcinoma progression by activating the PIAS3/STAT3 signaling pathway.

Background: Esophageal cancer, especially esophageal squamous cell carcinoma (ESCC), is a common malignant tumor of the digestive tract. Bufalin is an effective anti-tumor compound. However, little is known about the regulatory mechanisms of Bufalin in ESCC. To investigate the effect and molecular mechanism of Bufalin on the proliferation, migration and invasion of ESCC cells will provide a more reliable basis for the application of Bufalin in clinical tumor therapy. Methods: First, the half-inhibitory concentration (IC50) of Bufalin was evaluated by Cell Counting Kit-8 (CCK-8) assays. In vitro, the effects of Bufalin on the proliferation of the ECA109 cells was measured using CCK-8 and 5-ethynyl-2'-deoxyuridine assays. Wound-healing and transwell assays were used to evaluate the effects of Bufalin on the migration and invasion of the ECA109 cells. Further, to determine the mechanisms underlying the Bufalin-mediated suppression of cell progression in ESCC, total RNA was extracted from negative control (NC) and Bufalin treated cells to perform RNA-sequencing (RNA-seq) to screen for abnormally expressed genes. In vivo, the ECA 109 cells were subcutaneously injected into BALB/c nude mice to determine the effects of Bufalin on tumor cell proliferation. The protein inhibitor of activated signal transducer and activator of transcription 3 (PIAS3), signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 (p-STAT3) protein expression levels in the ECA109 cells were detected by Western blot. Results: The CCK-8 assays showed that the IC50 of Bufalin was 200 nM. The proliferation, migration, and invasion ability of the ECA109 cells was significantly inhibited in the Bufalin group in a concentration-dependent manner. In vivo, the Xenograft tumor model showed that Bufalin decreased the tumor volume and weight of the subcutaneous tumors. The RNA-seq results showed that the expression of PIAS3 was upregulated in the Bufalin group. Additionally, down-regulation of PIAS3 decreased the inhibition of STAT3, thereby increasing p-STAT3 expression. Finally, PIAS3 knockdown reversed the inhibitory effects of Bufalin on the proliferation, migration, and invasion of the ECA109 cells. Conclusions: Bufalin may inhibit the proliferation, migration, and invasion of the ECA109 cells through the PIAS3/STAT3 signaling pathway.

The toxic natural product tutin causes epileptic seizures in mice by activating calcineurin.

Tutin, an established toxic natural product that causes epilepsy in rodents, is often used as a tool to develop animal model of acute epileptic seizures. However, the molecular target and toxic mechanism of tutin were unclear. In this study, for the first time, we conducted experiments to clarify the targets in tutin-induced epilepsy using thermal proteome profiling. Our studies showed that calcineurin (CN) was a target of tutin, and that tutin activated CN, leading to seizures. Binding site studies further established that tutin bound within the active site of CN catalytic subunit. CN inhibitor and calcineurin A (CNA) knockdown experiments in vivo proved that tutin induced epilepsy by activating CN, and produced obvious nerve damage. Together, these findings revealed that tutin caused epileptic seizures by activating CN. Moreover, further mechanism studies found that N-methyl-D-aspartate (NMDA) receptors, gamma-aminobutyric acid (GABA) receptors and voltage- and Ca2+- activated K+ (BK) channels might be involved in related signaling pathways. Our study fully explains the convulsive mechanism of tutin, which provides new ideas for epilepsy treatment and drug development.