Abstract
Hepatocellular carcinoma is the fourth leading cause of cancer-related deaths due to its high rate of recurrence and metastasis. All-trans-retinoic acid (ATRA) can inhibit the malignant behaviors of hepatocarcinoma cells. Autophagy is reportedly involved in the migration and metastasis of various cancer cells. This study aimed to investigate the effect of autophagy on the function of ATRA on hepatocarcinoma cells, and to explore its possible underlying mechanism. Hepatocarcinoma cell lines, Hepa1-6 and HepG2, were treated with ATRA and autophagy inhibitors, including 3-methyladenine (3-MA) and Bafilomycin (Baf). Transmission electron microscopy, laser scanning, western blot, and real-time PCR demonstrated that ATRA induces autophagy in hepatocarcinoma cells. Trypan blue staining, a wound healing assay, and a transwell assay showed that 3-MA and Baf reverses the inhibitory functions of ATRA on the proliferation, migration, and invasion of hepatocarcinoma cells. Flow cytometry, Hoechst staining, periodic acid-Schiff staining, and indocyanine green uptake validated that 3-MA and Baf reverses the function of ATRA on apoptosis and the differentiation of hepatocarcinoma cells. Real-time PCR, western blot, and an immunofluorescence assay demonstrated that the reversal of the epithelial-mesenchymal transition (EMT) process by ATRA is weakened when autophagy is inhibited. Additionally, we confirmed that Bcl-2 is associated with the induction of ATRA-induced autophagy instead of the PI3K/Akt/mTOR pathway. These findings suggest that ATRA induces autophagy and autophagic cell death through the Bcl-2/Beclin1 pathway. Furthermore, ATRA-induced autophagy is involved in the inhibitory effect of ATRA on the malignant behaviors of hepatocarcinoma cells by reversing the EMT process.