Intracellular AFP Blocks All-trans retinoic acid induced ATG7 expression and Autophagy in Hepatocellular carcinoma cells
Abstract Background: Retinoic acid and retinoid acid receptor (RA-RAR) signaling exhibits suppressive functions in the progression of hepatocellular carcinoma (HCC) through multiple mechanisms. However, whether RA-RAR signaling induces autophagy that contributes its anti-tumor activity in HCC remains elusive.Methods: The effects of RA-RAR pathway were investigated in two HCC cell lines: AFP positive PLC/PRF/5 cells and AFP negative HLE cells. Cell autophagy, apoptosis and proliferation were analyzed by Western blotting, co-immunoprecipitation (CoIP), Immunofluorescence staining, chromatin immunoprecipitation (ChIP), Caspase-3 activity and Cell Counting Kit (CCK)-8.Results: ATRA dosage-dependently induced high levels of cell autophagy through its specific nuclear receptor RAR in both the PLC/PRF/5 and HLE cells. ChIP assay showed that RAR bind to response elements of key autophagy-initiated gene autophagy-relevant protein (ATG) 7 gene in the 5’-flanking region. Analyses based on CoIP further revealed that AFP formed complex with RAR in PLC/PRF/5 cells. Knockdown of AFP reduced the AFP and RAR combination, and thus up-regulated the expression of ATG7 gene and cell autophagy. Interestingly, in the HLE cells, AFP overexpressed and combination with RAR resulted in down-regulated ATG7 gene expression and reduction of cell autophagy. In both cell lines, ATRA inhibited cell proliferation and induced cell apoptosis, which was impacted by AFP action.Conclusion: The current study indicated that autophagy participated in the functionality of ATRA on HCC cells and AFP is a key regulator of ATRA induced autophagy through forming complexes with RAR in HCC cells.