Aurora kinase-B silencing promotes apoptosis of osteosarcoma 143B cells by ULK1 phosphorylation-induced autophagy / 南方医科大学学报

OBJECTIVE@#To investigate the effect of Aurora kinase B (AURKB) silencing-induced autophagy on apoptosis of osteosarcoma 143B cells and the underlying molecular mechanisms.@*METHODS@#Human osteosarcoma 143B cells were transfected with Lv/shAURKB or the negative control vector Lv/shScrambled followed by treatment with chloroquine (CQ) for 24 h. Western blotting was performed to detect the protein expression levels of AURKB, P62, LC3, cleaved caspase-3, Bcl-2, and P-ULK1. Transmission electron microscopy and LC3 dual-label fluorescence method were used to trace the autophagosomes in 143B cells to assess cell autophagy, and the cell apoptosis was detected using flow cytometry and TUNEL assay. Co-immunoprecipitation assay was used to detect the interaction between AURKB and ULK1.@*RESULTS@#The ratio of autophagy-related proteins LC3 II/I and the number of autophagosomes were significantly increased in 143B cells after transfection with Lv/shAURKB ( < 0.05), which significantly increased the expression of cleaved caspase-3 and reduced the expression of Bcl-2 ( < 0.05). Combined treatment of the cells with Lv/shAURKB and the autophagy inhibitor chloroquine obviously restored the expressions of caspase-3 and Bcl-2 ( < 0.05). Transfection with Lv/shAURKB significantly increased the apoptosis rate of 143B cells ( < 0.05), and this effect was significantly antagonized by combined treatment with chloroquine ( < 0.05). AURKB silencing strongly activated the phosphorylation of the autophagy-initiating protein ULK1 in 143B cells ( < 0.05). The results of co-immunoprecipitation assay confirmed when AURKB was immunoprecipitated, ULK1 also precipitated.@*CONCLUSIONS@#Silencing AURKB can induce autophagy by activating ULK1 phosphorylation to promote apoptosis in 143B cells.

Molecules and their functions in autophagy

Autophagy is a self-degradation system of cellular components through an autophagosomal-lysosomal pathway. Over the last 15 yr, yeast genetic screens led to the identification of a number of genes involved in the autophagic pathway. Most of these autophagy genes are present in higher eukaryotes and regulate autophagy process for cell survival and homeostasis. Significant progress has recently been made to better understand the molecular mechanisms of the autophagy machinery. Especially, autophagy process, including the regulation of autophagy induction through mTOR and the nucleation and elongation in autophagosome formation through class III phosphatidylinositol 3-kinase complex and ubiquitin-like conjugation systems, became evident. While many unanswered questions remain to be answered, here, we summarize the recent process of autophagy with emphasis on molecules and their protein complexes along with advanced molecular mechanisms that regulate the autophagy machinery.