Lariciresinol induces apoptosis in HepG2 cells via mitochondrial-mediated apoptosis pathway.

Lariciresinol (LA) is one of the main active ingredients in many traditional medicinal plants such as Patrinia, and has the role of anti-liver cancer. However, the precise mechanisms are unclear. This study investigated the molecular mechanisms of LA against HepG2 cells. LA anti-tumor activity was assessed with the CCK-8, Ki-67, and immunofluorescence staining. Cells apoptotic ratio was evaluated by Annexin V/PI double-staining assay. A proteomic approach was used to identify differentially expressed proteins after LA treatment. JC-1 staining was carried out to detect the mitochondrial membrane potential (ΔΨm), and the Western blot analysis was used to analyse the apoptosis-associated proteins. Our results suggested that LA significantly suppressed the viability of HepG2 cells. The CCK-8 and Ki-67 expression indicated dose-dependent decreases in cell proliferation. Flow cytometry analysis showed that LA exhibited a apoptosis-inducing effect. The proteomic study observed the presence of apoptosis-associated proteins and mitochondrial dysfunction in HepG2 cells after LA-treatment. Further analysis showed that LA could trigger the mitochondrial-mediated apoptosis pathway, based on a decrease in ΔΨm; deliver of cytochrome c; activation of caspase-9/-3 and poly(ADP-ribose) polymerase; and decrease of the proportion of Bcl-2/Bax. Collectively, our studies found that LA exhibits significant cytotoxic effects by inhibiting cell proliferation, inducing apoptosis, possibly via activation of the mitochondrial-mediated apoptosis pathway.

[Expression and analysis of recombinant pIL-18 in Pichia pastoris].

The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR, then the pPIC9K-IL-18 of fusion expression vector was constructed by inserting IL-18 fragment,and was transformed to GS115 by electroporation, multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol, SDS-PAGE was used to analyze expression product, fusion protein was purified by Sephadex G-100 column, bioactivity of IL-18 was measured by MTT assays. Experiment results show fusion protein of pIL-18 secreted by GS115,expression reaches the secretion peak of 160 mg/L at 72 h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in Pichia pastoris.