Chrysophanol localizes in mitochondria to promote cell death through upregulation of mitochondrial cyclophilin D in HepG2 cells
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Abstract:
Objective: Chrysophanol (Chry) displays potent anticancer activity in human cancer cells and animal models, but the cellular targets of Chry have not been fully defined. Herein, we speculated whether mitochondria were a target involved in Chry-induced cytotoxicity. Methods: Human liver cancer cell line HepG2 was incubated. The cytotoxicity was evaluated by MTT assay. Mitochondria localization was evaluated by a confocal microscopy. Mitochondrial membrane potential ΔΨm was detected by TMRE staining and determined by the flow cytometer. The levels of ATP, mitochondrial superoxide anions, and GSH/GSSG were determined according to the assay kits. The apoptosis were evaluated through Hoechst33342/PI and Annexin V/PI staining, respectively. The expression of cyclophilin D (CyPD) was determined by immunoblot method, and the interaction between CyPD and Chry was analyzed by molecule docking procedure. Results: Chry itself mainly localized in mitochondria to cause mitochondrial dysfunction and cell death in HepG2 cells. As regard to the mechanism, cyclosporin A as the inhibitor for the formation of mitochondrial permeability transition pore (mPTP) moderately suppressed cell death, indicating mPTP involved in the process of cell death. Further, Chry enhanced the protein expression of Cyclophilin D (CyPD) which is a molecular componentry and a modulator of mPTP, while antioxidant N-acetyl-L-cysteine inhibited the expression of CyPD. Molecule docking procedure disclosed two hydrogen-bonds existed in CyPD-Chry complex with -11.94 kal/mol of the binding affinity value. Besides, the mtDNA-deficient HepG2-ρ0 cells were much resistant to Chry-induced cell death, indicating mtDNA at least partly participated in cell death. A combination of Chry and VP-16 produced the synergism effect toward cell viability and ΔΨm, while Chry combined with Cis-Pt elicited the antagonism effect. Conclusion: Taken together, enrichment in mitochondria and actions on mPTP, CyPD and mtDNA provides an insight into the anticancer mechanism of Chry. The combination therapy for Chry with clinical drugs may deserve to further explore.
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This work was supported by the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2018-131), the National Natural Sciences Foundation of China (Grant No. 21302079).
Yu Xie, Ling Zhang, Yuan-yuan Li, Dian He, Li-fang Zheng. Chrysophanol localizes in mitochondria to promote cell death through upregulation of mitochondrial cyclophilin D in HepG2 cells[J]. Chinese Herbal Medicines (CHM),2021,13(2):221-227
