All-trans retinoic acid inhibits malignant behavior of hepatocellular car-cinoma cells via regulation of autophagy by JNK/Bcl-2 signaling
AIM:This study aims to investigate the effect of all-trans retinoic acid(ATRA)in promoting cel-lular autophagy through JNK/Bcl-2 signaling pathway to inhibit the malignant biological behaviors of hepatocarcinoma cells.METHODS:Mouse Hepa1-6 hepatocarcinoma cells were used as the research subjects,five groups were set up as follows:control group,ATRA group,ATRA+Ad-GFP group,ATRA+Ad-Bcl-2 group,and ATRA+SP600125 group.Western blot and co-immunoprecipitation(Co-IP)were conducted to detect the expression of Bcl-2,c-Jun N-terminal ki-nase(JNK),and autophagy-related marker proteins in each group.Autophagy flow was observed by using laser confocal microscopy,and the number and morphology of autophagosomes in cells were examined by electron microscopy.Scratch assay and Transwell assay were employed to assess the migration and invasion capabilities of cells,while indocyanine green(ICG)and periodic acid-Schiff(PAS)staining were performed to evaluate cellular metabolic synthesis ability.RE-SULTS:Compared to the control group,ATRA enhanced the expression of phosphorylated bcl-2(P＜0.05),inducing cellular autophagy.Overexpression of Bcl-2 suppressed the expression of autophagy markers LC3-Ⅱ/LC3-Ⅰ and beclin-1(P＜0.01),and protein-protein interaction of Bcl-2 and beclin-1 was detectable.The highly selective JNK inhibitor SP600125 reduced the expression of ATRA-induced phosphorylated JNK and Bcl-2,while inhibiting the expression of au-tophagy marker proteins LC3-Ⅱ/LC3-Ⅰ and beclin-1,thus downregulating the level of ATRA-induced autophagy(P＜0.05).ATRA treatment significantly reduced the number of migrating and invading cells and increased the number of ICG uptake and PAS-stained positive cells(P＜0.05),while in ATRA+Ad-Bcl-2 group and ATRA+SP600125 group,the num-ber of migrating and invading cells exhibited a significant increase(P＜0.05),the number of ICG uptake and PAS-stained positive cells was significantly decreased(P＜0.05).CONCLUSION:ATRA may enhance the level of cellular autopha-gy through the JNK/Bcl-2 signaling pathway,thereby inhibiting the malignant biological behaviors of hepatocarcinoma cells.
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