Tanshinone I Inhibits Metastasis of Cervical Cancer Cells by Inducing BNIP3/NIX-mediated Mitophagy and Reprogramming Mitochondrial Metabolism

Background: Cervical cancer is the most common malignancy of the female lower genital tract. Tanshinone I (Tan I) is one of the crucial lipid-soluble components of red sage (Salvia miltiorrhiza). While its mode of action against cervical cancer is unclear. Purpose: Our study aimed to explore the role of Tan I on cervical cancer in vitro. Study design and methods: Effects of Tan I on cervical cancer cells viability, migration and mitochondrial function were investigated by Cell Counting Kit-8, Transwell and Fluorescence laser confocal microscope assays respectively. The potential mechanism of Tan I was uncovered by an integrative approach combining RNA profiling and hydrogen nuclear magnetic resonance-based metabolic analysis, molecular docking and Western blot. Results: Tan I significantly inhibited the growth and colony formation of HeLa and SiHa cells. It induced apoptosis and cell cycle S phase arrest at low (12.5-25 mu M) but not high (50 mu M) concentrations. It also altered the HeLa cell ultrastructure, decreased the membrane potential and increased the total mitochondrial content. Further, Tan I induced autophagic flux and the colocalization of mitochondria with lysosomes, led to decreased adhesion, invasion, and migration of cervical cancer cells. Transcriptomic analysis revealed that Tan I altered the RNA profile and signal processing in HeLa cells. Tan I significantly impacted "central carbon metabolism in cancer" and "mitophagy-animal" processes. A global metabolic analysis identified 25 metabolites affected by Tan I treatment in HeLa cells. Changes in the metabolic profile indicated that Tan I affected such processes as protein digestion and absorption, central carbon metabolism in cancer, and aminoacyl-tRNA biosynthesis in cervical cancer cells. Furthermore, Tan I significantly induced the expression of mitophagy-related proteins BNIP3, NIX and Optineurin and the conversion from LC3-I to LC3-II, inhibited the NDP52 and P62 level in a concentration-dependent manner. While CQ further increased the conversion of LC3-I to LC3-II and the expression of P62. Moreover, Tan I interacted with BNIP3 and NIX through hydrogen bond. Tan I induce mitophagy could be prevented by BNIP3 and NIX siRNA transfection. Conclusion: Tan I induced the BNIP3/NIX-mediated mitophagy, and reprogrammed the mitochondrial metabolism in cervical cancer cells, thus inhibiting metastasis.