Fang Shuyu, Hu Chaoqun, Xu Lei, Cui Jiejie, Tao Li, Gong Mengjia, Wang Yi, He Yun, He Tongchuan, Bi Yang
Department of Pediatric Research Institute of Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics Chongqing, P.R. China.
Department of Microbiology, Chongqing Medical University Chongqing, P.R. China.
Am J Transl Res. 2020 Oct 15;12(10):6793-6810. eCollection 2020.
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.
肝细胞癌因其高复发率和转移率，是癌症相关死亡的第四大主要原因。全反式维甲酸（ATRA）可抑制肝癌细胞的恶性行为。据报道，自噬参与各种癌细胞的迁移和转移。本研究旨在探讨自噬对ATRA作用于肝癌细胞功能的影响，并探索其可能的潜在机制。用ATRA和自噬抑制剂（包括3-甲基腺嘌呤（3-MA）和巴弗洛霉素（Baf））处理肝癌细胞系Hepa1-6和HepG2。透射电子显微镜、激光扫描、蛋白质免疫印迹和实时荧光定量PCR结果表明，ATRA可诱导肝癌细胞发生自噬。台盼蓝染色、伤口愈合实验和Transwell实验表明，3-MA和Baf可逆转ATRA对肝癌细胞增殖、迁移和侵袭的抑制作用。流式细胞术、Hoechst染色、过碘酸希夫染色和吲哚菁绿摄取实验证实，3-MA和Baf可逆转ATRA对肝癌细胞凋亡和分化的作用。实时荧光定量PCR、蛋白质免疫印迹和免疫荧光实验表明，当自噬被抑制时，ATRA对上皮-间质转化（EMT）过程的逆转作用减弱。此外，我们证实Bcl-2与ATRA诱导的自噬有关，而非PI3K/Akt/mTOR通路。这些发现表明，ATRA通过Bcl-2/Beclin1通路诱导自噬和自噬性细胞死亡。此外，ATRA诱导的自噬通过逆转EMT过程参与了ATRA对肝癌细胞恶性行为的抑制作用。
