Background
In our previous study, Hibiscus syriacus leaf tissue was successfully cultivated in an optimized callus culture system, and subsequently extracted with 70% ethanol to prepare H. syriacus callus extract (HCE). The previous study suggested that the callus culture is useful method for obtaining the anti-inflammatory ingredients from H. syriacus.
Conclusion
Our findings can serve as a starting point for developing the novel anti-CRC agent using HCE, as a targeted medicine acting on regulating Notch signaling and cholesterol synthesis.
Methods
The cytotoxicity of HCE was investigated by MTT and colonies formation. The underling mechanism by which HCE regulates specific proteins in HT-29 cells was evaluated by the proteomic analysis. These putative proteins were validated using qRT-PCR and immunoblotting analyses. Subsequently, oral administration of HCE for 15 days further evaluating the anti-tumor activity by mRNA and protein expressions levels and tumor histopathology.
Purpose
In the present study, we aimed to investigate the effect of HCE on the colorectal cancer (CRC) and its underlying mechanism of action using HT-29 cells and thymus-deficient mice bearing HT-29 xenografts.
Results
Results of cell viability and colony formation assays revealed a significant cytotoxic effect of HCE at doses below 100 μg/ml against HT-29 cells, but not against normal cells. Through differential protein expression analysis, signaling pathways underlying anti-CRC activity were predicted in HCE-treated HT-29 cells: Notch signaling, cholesterol biosynthesis, and AMPK signaling pathways. qRT-PCR and immunoblotting analyses indicated that the cytotoxic effect of HCE against HT-29 cells might be associated with the suppression of Notch signaling, which positively contributes to cholesterol biosynthesis. To our knowledge, this can be presented as the first study to demonstrate the detailed relationship between Notch signaling and cholesterol-AMPK signaling. Our in vivo result further corroborated the in vitro finding that 100 and 200 mg/kg HCE for 15 days exerts its anti-cancer effect via Notch signaling-mediated suppression of cholesterol synthesis without systemic toxicity.