Effects Of Autophagy On Adenanthin-induced Cell Death In Hepatoma Cells

Autophagy is a basic degradation process of intracellular substances including cytoplasmic components, senescent organelles and long-life proteins by lysosomes. During this process, targeted cytoplasmic constituents are isolated by double membrane-like vehicles and transported to lysosomes for degradation. Autophagy is often presented in normal life activities in eukaryotic cells and plays important roles in the adaptation of metabolic stress, maintaining genomic stability and keeping homeostasis. A series of studies show that autophagy participates in the occurrence and development of cell death process, in which it may have promoting, hindering or no obvious effect. Recently, our group have first reported that adenanthin, a natural diterpenoid extracted from the leaves of Rabdosia adenantha, inhibits the activity of peroxiredoxin I/II and accumulates intracellular ROS, which eventually induces selective killing effect on hepatocellular carcinoma(HCC) cells. Current studies show that ROS usually enhances cellular autophagic activity. In this work, we will focus on whether autophagy impacts adenanthin-induced HCC cell death. The results from western blot and immunofluorescence technique showed that adenanthin can significantly increase autophagic activity in Bel-7402 cells, as determined by an important autophagy index LC3-II protein accumulation, GFP-LC3 and endogenous LC3 protein aggregation, the latter presenting obvious dot-like structures within cytoplasm. Silencing Beclin-1 expression with RNA interfering technology does not influence adenanthin-induced autophagic activity and cell death, suggesting that adenanthin-enhanced autophagy is Beclin-1 independent. Further, CCK-8 results showed that compared with wild type mouse embryonic fibroblasts(WT MEFs), ATG5 knock out(ATG5-/-) MEFs were more resistant to adenanthin-induced cell growth inhibition. Similarly, knock down of ATG5 also significantly antagonizes adenanthin-induced cell growth inhibition of Bel-7402 cells. These results suggested that adenanthin-enhanced autophagy activity promotes its cell death induction of HCC cells. With the work going on, we will deeply understand the potential mechanism of adenanthin-induced selective killing effect on HCC cells, and also provide more information of therapeutic strategy for specific targeting and killing cancer cells.