Saikosaponin-d simultaneously induces apoptotic and paraptosis-like cell death in hepatoma cells / 西安交通大学学报(医学版)

ABSTRACT

Objective: To investigate the cell death-inducing effect of saikosaponin-d (SSD) on human hepatoma Hep3B cells and its potential mechanism. Methods: Hepatoma Hep3B cells were divided into five groups: blank control group, DMSO (vehicle) group, and three SSD treatment groups treated with various doses of SSD (5, 10, and 15 μmol/L). MTT assay was used to evaluate cell viability. Flow cytometer was employed to quantitatively detect the percentage of dead cells after Annexin V-FITC/PI double staining. Apoptosis was detected morphologically after Hoechst 33258 staining. The activity of caspase-3 apoptotic protease was determined by spectrophotometry. Cell morphologic changes were observed with an inverted microscope. Western blotting and Real-time PCR were employed to evaluate the expression levels of C/EBP homology (CHOP) protein and mRNA, respectively. Results: MTT assay showed that SSD inhibited the viability of human hepatoma Hep3B cells in concentration- and time-dependent manners. Sinomenine hydrochloride induced the death of Hep3B cells in a concentration-dependent manner indicated by flow cytometry. After staining with Hoechst 33258, the nuclei of SSD-treated cells showed nucleosomal agglutination, nucleosomal shrinkage and fragmentation under the fluorescence microscope, which are the characteristics of apoptotic cells. SSD significantly activated the key apoptotic executor caspase-3. The occurrence of paraptosis, characterized by extensive cytoplasmic vacuoles, was observed in SSD-treated cells under an inverted microscope. The pretreatment of a pancaspase inhibitor Z-VAD-FMK completely inhibited caspase-3 activity triggered by SSD, but only partially suppressed cell death and could not reduce the cytoplasmic vacuolation in SSD-treated cells. The protein and mRNA expressions of CHOP, a stress-inducible molecule, were upregulated by SSD, which could not be inhibited by Z-VAD-FMK. Conclusion: SSD can simultaneously induce caspase-dependent apoptosis and caspase-independent paraptosis in human hepatoma Hep3B cells. The upregulated expression of CHOP may be the mechanism involved in SSD-induced paraptosis.