Crocin induces autophagic apoptosis in hepatocellular carcinoma by inhibiting Akt/mTOR activity

Chong Yao,1,2 Bing-Bing Liu,3 Xiao-Dong Qian,2 Li-Qin Li,4 Heng-Bin Cao,2 Qiao-Sheng Guo,1 Gui-Fen Zhou5 1Institute of Chinese Medicinal Materials, Nanjing Agricultural University, Nanjing 210095, China; 2Pharmaceutical Department, Huzhou Central Hospital, Huzhou 313003, China; 3Pharmaceutical Department, TCM Hospital of Changxin, Huzhou 313100, China; 4Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, Huzhou 313000, China; 5College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 310053, China Background: Autophagy induction is a common mechanism for antitumor chemicals in induction of cancer cell death. However, the role of autophagy in crocin-induced apoptosis is barely studied in hepatocellular carcinoma (HCC).Materials and methods: The influence of crocin on growth, apoptosis, and autophagy and its mutual relations were analyzed by Cell Counting Kit-8 assay, flow cytometer, EGFP-LC3 puncta analysis, and Western blot in HCC cells. The activities of Akt/mTOR axis and its roles in autophagy regulation were also detected by Western blot in HCC cells treated with crocin. Finally, the roles of Akt/mTOR axis in crocin-induced autophagic apoptosis were analyzed by Western blot and flow cytometer in HCC cells.Results: The results showed that crocin can induce growth inhibition in a does- and time-dependent pattern by apoptosis. Increased LC3 puncta and upregulated LC3-II accumulation was observed as early as at 6 hours in HepG2 and HCCLM3 cells treated with 3 mg/mL crocin. Moreover, apoptosis analysis using flow cytometer and cleaved poly (ADP-ribose) polymerase detection revealed that autophagy initiation was prior to apoptosis activation in HCC cells treated with crocin. When autophagy was blocked with 3-methyladenine, crocin-induced apoptosis was inhibited in HCC cells. Furthermore, crocin treatment constrained the activities of key proteins in Akt/mTOR signaling, such as p-Akt (S473), p-mTOR (S2448), and p-p70S6K (T389), suggesting that crocin could induce autophagic apoptosis in HCC cells in an Akt/mTOR-dependent mechanism. Indeed, when autophagy was suppressed by forced expression of Akt, the crocin-induced apoptosis was also impaired in HCC cells.Conclusion: The results suggested that crocin could induce autophagic apoptosis in HCC cells by inhibiting Akt/mTOR activity. This study originally revealed that autophagic apoptosis is a novel cytotoxic function of crocin, which lays the theoretical foundation for clinical application of crocin in HCC. Keywords: crocin, apoptosis, autophagy, Akt/mTOR, autophagic apoptosi