Role Of Mitophagy In Arsenic Trioxide-and Metformin-induced Apoptosis Of Cervical Cancer HeLa Cells

Background Cervical cancer is one of the most common cancer in women.Mitochondria are essential organelles that regulate cellular energy homeostasis,proliferation and cell death.The removal of damaged mitochondria through autophagy,a process called mitophagy,is closely related to the occurrence and development of malignant tumors.Arsenic trioxide?ATO?is an effective chemotherapeutic drug for the treatment of leukemia and various solid tumors.Metformin is the most commonly prescribed drug for type 2 diabetes mellitus.In recent years,in addition to glucose lowering,several studies have presented evidences suggesting some potential roles of metformin,as antitumor,neuroprotection effect and immunity enhances.Whether ATO and Met have a combined anti-cancer effect,and whether Parkin-mediated mitophagy is involved in the mechanisms of anti-tumor induced by ATO and Met is unclear.Objective The aim of this study is to explore the effects of ATO combined with Met on the proliferation inhibition and the apoptosis induction of cervical cancer cells,then explore the role of mitophagy mediated by Pink1-Parkin signaling pathway in the anti-tumor effects induced by ATO and Met.Lastly,we want to provide a basis for the possibility of using ATO and Met in the treatment of cervical cancer and other cancer treatment.Methods Human cervical cancer Hela cells and Parkin gene-transduced HeLa cells were used as target cells.MTT assays and flow cytometry?FCM?analysis were applied to examine cell proliferation and apoptosis.Cell morphology was observed by light microscope?LM?after Wright-Giemssa staining;We used transmission electron microscopy?TEM?to observe the ultrastructure of cells.The cell cycle and apoptosis rate were detected by FCM,Western-blotting method was used to detect the expression level of apoptosis-related and autophagy-related proteins.The formation of autophagosomes,co-localization of mitochondria and autophagosomes were observed by laser confocal microscopy?LSCM?and fluorescence microscopy.ResultsThe results of MTT assay indicated that different concentrations of ATO and Met could inhibit cell proliferation in a time-and concentration-dependent manner in HeLa cells.The IC₅₀0 of ATO-treated cells at 24,48 and 72 h were 8.36±0.81,2.73±0.31,and 1.76±0.15mmol/L,respectively.The IC₅₀0 of Met-treated cells at 24,48and 72 h were 64.8±1.2 mmol/L,33.25±2.45 mmol/L and 26.5±0.7 mmol/L respectively.After treatment with 2?mol/L and 4?mol/L ATO in combination with 20mmol/L Met for 24-72 h,The inhibition rate was significantly increased compared with the single treatment,but the combined treatment of ATO and Met has no synergistic or additive effect on the inhibition activity of HeLa cells;The results observed by LM after Wright-Gemsa staining showed that some cell nuclear chromatin dense deep staining,nuclear fragmentation and other typical apoptotic morphology after 4mmol/L ATO treatment.The ultrastructure of cells observed by TEM showed that 4mmol/L ATO and 40 mmol/L Met alone or in combination could cause typical apoptotic morphological changes in HeLa cells,such as vacuolar degeneration,chromatin compaction,edge collection,nuclear fragmentation and apoptotic body formation;Flow cytometry analysis showed that the apoptotic rate of HeLa cells was increased and the proportion of G2/M phase cells was also increased after 4?mol/L ATO and 40 mmol/L Met treatment for 24 h.The change was more obvious after the combination of these two drugs.It is suggested that both ATO and Met could induce apoptosis in Hela cells by G2/M phase arrest,but they had no synergistic or additive effect;Western-blotting assay showed that the expression level of anti-apoptotic protein Bcl-2 was down-regulated and the expression level of Bax was up-regulated after treatment of HeLa cells with 2?mol/L and 4?mol/L ATO and40 mmol/L Met alone or in combination for 24 h.The ratio of Bax/Bcl2 was significantly increased,and the expression of PI3K and Akt and p-Akt were also significantly decreased,especially when the two drugs were combined,but no synergistic or additive effect was observed.When the cells treated with 4?mol/L ATO for 3 h,the expression level of LC3 II increased significantly.After 6 h and 12 h,the expression of LC3 II were 2.32 and2.39 times compared to the control group,respectively.At the same time,the the expression level of autophagic lysosomal substrate protein P62 was significantly reduced.By 9 h,the level of P62 was only 16%of the control group.Treatment of HeLa cells with 40 mmol/L of Met for 3-12 h also resulted in an increase in the amount of LC3 II and a significant decrease in P62 protein levels.However,2?mol/L and 4?mol/L ATO combined with Met did not cause further increase of LC3 I to LC3II transformation,or a more significant decrease of P62 level,indicating that ATO and Met inhibited HeLa cell proliferation activity.At the same time,the cells were induced to undergo autophagy,but these two drugs showed no additive or synergistic effect.HeLa cells transfected with GFP-LC3 were used as the target cells.The results of LSCM indicated that the GFP-LC3 was evenly distributed throughout the cells in the control group.After treatment with 4?mol/L ATO and 40 mmol/L Met,a large number of spotted green fluorescence formed by GFP-LC3 could be found in the cytoplasm.After the mitochondria of the above cells were labeled with Mito tracker,a large number of cord-like mitochondria were observed in the cells,suggesting that both ATO and Met could induce mitophagy in HeLa cells.After the treatment of 2?mol/L and 4?mol/L ATO alone or in combination,the level of mitochondrial membrane protein Tom 20 in HeLa cells was decreased,while the mitochondrial fusion proteins Mfn1 and Mfn2 were also significantly down-regulated,demonstrating that both of ATO and Met could cause autophagic degradation of mitochondria in HeLa cells.The results of the fluorescence microscope showed that GFP-Parkin could co-localize with the mitochondrial activity probe Mito tracker in ATO and Met treated GFP-Parkin-HeLa cells.The two kinds of fluorescence-overlapping granular orange fluorescence indicated that the mitochondria were damaged and the mitochondrial autophagy was activated after the treatment of ATO and Met.Western-blotting assay showed that the content of Pink1 was increased,and the level of Parkin protein was down-regulated after 2 and 4?mol/L ATO was applied to GFP-Parkin-HeLa cells for 6 h,suggesting that the Pink1-Parkin signaling pathway was involved in controlling the ATO-and Met-indcued mitophagy and mitophagic apoptosis in Hela cells.Conclusions1 ATO and Met are effectively able to inhibit the proliferation activity of HeLa cells and induce the apoptosis of HeLa cells by regulating Bax/Bcl-2 genes and PI3K/Akt pathway,and the ATO-Met combination has no obvious synergistic effect.2 Both ATO and Met can induce mitophagy,in tune with cellular apoptosis,in HeLa cells,which suggests that ATO-and Met-induced apoptosis in HeLa cells is mitophagic apoptosis.3 The Pink1-Parkin pathway is involved in controlling the ATO-and Met-induced mitophagy and mitophagic apoptosis in HeLa cells.