ABSTRACT
The purpose of this study was to select the active compounds targeting Hsp90 protein in pancreatic cancer cells through a new dual "target + activity" rapid discovery technique. We combined an in vitro anti-cancer activity screening method with a dual-luciferase reporter gene and multi-chromatography separation technology, for rapid discovery of potential Hsp90 inhibitors from the Chinese herbal medicine Physalis angulata L. The anti-proliferation activity of those compounds was assessed in pancreatic cancer cell line BxPC-3 by MTT assays. The molecular mechanisms of Hsp90 inhibition were explored by Western blot and shRNA knockdown assays. As a result, two withanolides, withanolide E (WE) and 4β-hydroxywithanolide E (HWE), were identified from Physalis angulata L. The half maximal inhibitory concentration (IC50) of WE and HWE were 0.71±0.03 and 1.23±0.10 μmol·L-1 for the growth of BxPC-3 cells in 48 h. Luciferase reporter assay demonstrated that WE and HWE significantly induced heat shock element (HSE) activity in a dose- and time-dependent manner. The molecular mechanism study showed that after exposing to 5 μmol·L-1 WE or HWE for 48 h, the aggregation of Hsp90 dimer was upregulated to 6.5±1.3 and 11.8±2.0 fold, while the expression of Hsp90 client protein Akt was downregulated to 21.7%±2.8% and 9.8%±1.4% of the control group. Moreover, the Hsp90 inhibitory activity of WE or HWE was canceled by shRNA mediated Hsp90 knockdown. Overall, based on the dual "target + active" rapid discovery technique, two new Hsp90 inhibitors WE and HWE were found from Physalis angulata L. The Hsp90 inhibitory mechanism of WE and HWE may be mediated by induction of Hsp90 aggregate dimer and inhibition of Hsp90 client protein Akt expression.
ABSTRACT
Objective: To investigate the chemical constituents from whole herb of Physalis angulata and test their cytotoxic activity. Methods: Compounds were isolated by chromatography methods and LC-MS guided analysis, structures were determined by spectroscopic techniques of 1D- and 2D-NMR (HSQC, HMBC, 1H-1H COSY, and NOESY), cytotoxic activity experiments were conducted by MTT method. Results: Twelve compounds were identified as physalins A (1), B (2), C (3), D (4), F (5), H (6), I (7), O (9), 5α-ethoxy-6β-hydroxy-5,6-dihydrophysalin B (8), β-sitosterol (10), 2-carboxyoxanilic acid methyl ester (11), and acetaminobenzoic acid (12). Conclusion: Among these compounds, compound 11 is a new natural product. Compounds 1-3 and 9 are firstly obtained from P. angulata. All compounds are tested for their inhibitory activities against human lung adenocarcinoma strain A-549, with IC50 values of 1.9-20.2 μmol/L, which indicates good cytotoxic activity of these physalins.
ABSTRACT
OBJECTIVE: To study the chemical constituents of whole plant of Physalis angulata L. METHODS: The constituents were isolated and purified by chromatographic methods and their structures were elucidated by spectroscopic methods and physio-chemical analysis. RESULTS: Thirteen known compounds was isolated and identified as physalin A (1), physalin B (2), physalin E (3), physalin P(4), stigmast-5-en-3β-ol (5), ergost-5, 24 (28) -diene-3β-ol (6), brassicaaterol (7), stigmasta-22-en-3, 6-dione (8), pregn-5-en-3-ol-20-carboxylic acid (9), ergost-5, 24 (28)-diene-3β, 23S-diol (10), ergosta-5, 25 (26)-diene-3, 24ξ-diol(11), n-hexade-canoic acid (12), and n-heptadecanoic acid (13). CONCLUSION: All of the compounds except 2 and 3 are isolated from this plant for the first time, and compounds 5-10 and 12-13 are isolated from Physalis for the first time.