Discovery Of Some Anti-cancer Small Molecule Compound Inducing Autophagy And Mechanism Study

Autophagy is an important physiological processes occuring in eukaryotic cells by primary lysosome treating cell endogenous substrate, by which life maintains protein metabolism balancing and intracellular environment stable. Autophagy not only help the cells removes waste, reconstructs the structures, but also plays an important role in growth , development, apoptosis, proteasome degradation and other important biological courses. At the same time, it's closely related with tumor occurrence and development. Finding small molecule anti-tumor compounds to promote cell autophagy can help to research on antineoplastic agents.pGFP-LC3 plasmid were transfected into HCT-116, HeLa cells, in which microtubulin light chain 3 and green fluorescent protein were fused expressing. In shaping the course of cell cloning, selecting the fluorescent semi-monoclonal cell cultivated again after vaccination into 96 well medium plate to form a real monoclonal. After continuous propagation, cells were detected with vinblastine, evaluating the signal stability of the strain. During autophagy,the loacation of LC3 change from the cytoplasm by diffusion into the distribution around the autophagysome, meanwhile the detection the change of LC3 from LC3I into LC3II. by Western blot We have successfully established screening model on autophagy in HCT-116 cells.Using the above model, 28 compounds were chosed from more than 100 with anti-tumor activity,which IC50 in HCT-116 cell is below 5.5μ?g/ml, incubation with HCT-116 cells for 24 hours by the quality of 3 times the IC50 or 9 times the IC50 of the compounds.Collectting cells to detect by western-blot. The first screening got 11 compounds having autophagy activity. In the second screening, 6 of 11 compounds had been identified as possessing distinct autophagy activity. Meanwhile,the six compounds were also detected on the inhibiton of proteasome and activation of CHOP.The compound is named physalin B, which induce HCT-116 cell autophagy in obvious time and concentration-dependent manner. At the same time the compound caused autophagy signaling pathway-related protein phosphorylation of AKT reduction, ERK phosphorylation level up regulation and mTOR phosphorylation extent down regulation. However, the beclin-1 expression of autophagy-related protein did not increase, and autophagy could not be blocked by a typical autophagy inhibitor 3-MA, but can be reversed by PD98059, an inhibitor of ERK. The apoptosis caused by the compound could be reversed by caspase inhibitor Z-VAD-FMK.The compound cause to cell autophagy dependent of I-type PI3K- rather than III-type PI3K.