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.

This research aimed at the key issue that chemical drugs and Chinese medicine hydrophilic small molecule anti-tumor drugs were difficult to break through the dense interstitial permeability barrier of pancreatic cancer to achieve the key problem of drug efficacy in the deep part of tumor tissue. To solve this problem, the lipophilic molecule squalene (SQ) and the hydrophilic anti-tumor drug chidamide (CHI) were linked by a trypsin responsive bond to form a prodrug (SQ-CHI) and a folic acid modified prodrug self-assembled nanoparticles (FA-SQ-CHI NPs) were further developed. The feature of prodrug molecules and nanoparticles were characterized. The in vitro release characteristics and cytotoxicity of blank vector were investigated. The efficacy and permeability of the prodrug nanoparticles in the PSN-1 monolayer cell and PSN-1/HSPC co-cultured tumor spheroids model was evaluated. The results showed that SQ-CHI prodrug molecules and FA-SQ-CHI NPs were successfully developed. The nanoparticles were regular spherical, well-dispersed, with a particle size of (173.3 ± 1.5) nm, a drug load of (59.02 ± 0.8) % and showed trypsin responsive release ability. The prodrug nanoparticles can significantly enhance the penetration and anti-proliferation effects of CHI in the PSN-1/HSPC tumor spheroids. In conclusion, the construction of folic acid-modified SQ-CHI prodrug self-assembled nanoparticles can significantly enhance the penetration of CHI in the pancreatic cancer microenvironment in vitro. This research would provide a new idea for the construction of targeted drug delivery system for chemical drugs and Chinese medicine hydrophilic small molecule drugs in the application of anti-pancreatic cancer.