Intracellular ATP Concentration Determines The Sensitivity Of Cell Death Induced By Proteasome Inhibition

Background and Objective: The ubiquitin-proteasome system (UPS) regulates the vital process of cell by degradation of a variety of proteins, and proteasome is an important target for anti-cancer, the sensitivity of cell death induced by proteasome inhibition is different. After our research group has discovered for the first time that the 26S proteasome activity is regulated bidirectionally by ATP concentrationins in vitro, the 26S Proteasome Activity-ATP Concentration Model is then created. ATP is a kind of endogenous small molecule, and different organs in animal have different ATP concentrations. Therefor, it's assumed that the sensitivity of cell death induced by protesome inhibition may be regulated by intracellular ATP concentration. The aim of this work is to prove that the intracellular ATP concentration can determine the sensitivity of cell death induced-by proteasome inhibition, thus providing evidencs for the regulation of proteasome function by intracellular ATP concentration.Methods and Results:1. The regulation of the intracellular ATPK562 cell and H460 cell were chosen because their pathway of ATP generation had been disclosed by our previous works. ATP generation in K562 is mainly by glycolytic pathway and partially by aerobic oxidation, while that in H460 is dominantly by aerobic oxidation. Various glucose contents, Adenosine (Ade), Oligomycin (OLIG), 2-Deoxy-D-Glucose (2DG) were used to regulate the intracellular ATP concentration. OLIG is an oxidative phosphorylation inhibitor that blocking mitochondrial/glycolytic ATP generation, and 2DG is aglycolytic inhibitor that blocking glycolytic pathway. Previou works had proved that the intracellular ATP concentration can be control.2. The study of effects intracellular ATP concentration on the sensitivity of cell death induced by proteasome inhibition.2.1 The effects on sensitivity of cell death induced by proteasome inhibition.in K562 by regulating the intracellular ATP concentration.2.1.1 The effects on the sensitivity of cell death induced by proteasome inhibition between culture mediums with D-glucose and without glucoseD-glucose in normal culture medium, is the the main source of intracellular ATP. Thus intrcellular ATP concentration in medium with D-glucose is higher than without glucose.①Different concentrations of MG132 (0 ~ 20μM) and MG262 (0 ~ 1μM) were respectively added to cells in culture medium with D-glucose and without glucose for different hours. Then Annexin V-FITC flow cytometry showed that in D-glucose the cell death induced by MG132 or MG262 was more serious than in no glucose, and was in a dose-dependent manner, the difference of cell death rate being 24.8% with MG132 (10μM) for 12 hours, 59.8% with MG132 (20μM) for 24 hours, and 59.8% with MG262 (1μM) for 12 hours.②MG262(1μM) was respectively added to the cells in culture medium with D-glucose and withou glucose, and then inverted fluorescence microscope was used to observe the changes in cell morphology (ph 100X) dynamically for 12 hours. Cells in culture medium with D-glucose showed significant morphological changes of cell death, such as "budding", apoptotic body formation and cell rupture, in glucose-free medium the integrity of cell membrane were reserved.Therefore, it's suggested that when intracellular ATP concentration is higher,the cell death induced-by protesome inhibition will be more serious.2.1.2 The effects on sensitivity of cell death induced by proteasome inhibition between culture medium with D-glucose and L-glucoseL-glucose, the enantiomer of D-glucose, cannot generate energy. It was used for the balance of medium osmotic pressure. ①MG132 (5μM) and MG262 (1μM) were respectively added to D-glucose culture mediums with D-glucose and L-glucose for 9 hours and 18 hours. By flow cytometry, it was shown that the cell death in culture medium with D-glucose was more serious than that in culture medium with L-glucose. Difference of cell death rate was about 6% for 9h, about 30% for 18 hours, being statistically significant (p <0.01).②MG132 (5μM) was added to culture mediums with D-glucose and L-glucose 12 hours, and transmission electron microscope (TEM) was used to observe the changes in cell ultrastructure, serious cell death changes in medium with D-glucose, including nuclear enrichment, chromatin presented as crescent-shaped, or clumping and gathered in the nuclear membrane, nuclear fragmentation, cytoplasmic vacuolization and cell membrane rupture; while cells in medium with L-glucose showed slight lethal changes: such as apoptotic bodies, a few cytoplasmic vacuoles and plasma membrane kept integrity.Base on these results, the effect of medium osmotic pressure on cell death could be excluded, and therefore it could be concluded higher intracellular ATP concentration might cause more serious cell death induced by proteasome inhibition.2.1.3 The effects on sensitivity of cell death induced by proteasome inhibition by using Ade to upregulate intracellular ATP①Experiments were performed both in medium with D-glucose and withou glucose,and cells were divided into control group, Adegroup, MG262 group and Ade + MG262 group, with each drug added for 24 hours. Flow cytometry showed that the cell death rate in medium with D-glucose was 50.7% ~ 82%, while 15.8% ~ 36.3% in medium withou glucose. In glucose-free mediu, cell death rate was higher in Ade + MG262 group than MG262 group (p<0.01),while in D-glucose medium, the cell death rates were the same, accompanied by images of varing from early apoptosis to late apoptosis.②Experiment were performed in D-glucose culture medium .Cells were divided into control group, Ade group, MG262 group, Ade + MG262 group, MG132 group and Ade + MG132 group, with each drug added for 18 hours, The LDH values were 431.31 U / L in MG262 group, 939.3 U / L in Ade + MG262group, 718.85 U / L in MG132 group, 1098.85 U / L in Ade + MG132 group. Differences of LDH values between Ade + MG132 group and MG132 group, and between Ade + MG262 group and MG262 group were statistically significant (p <0.01).It is implied that when Ade upregulate the intracellular ATP, cell death induced by proteasome inhibition is more serious.2.1.4 The effect on sensitivity of cell death induced by proteasome inhibition by using OLIG to downregulate intracellular ATP①The effect on sensitivity of cell death induced by proteasome inhibition by using OLIG to downregulate intracellular ATP in glucose-free medium.Experiment was performed in glucose-free medium. Cells were divided into control group, OLIG group, Ade group, OLIG + Ade group, MG132 group, MG132 + OLIG group , with each drug were for 12 hours. By flow cytometry, it was shown that cell death rate in OLIG group was about 40 %, about zero in OLIG + Ade group, and 35.3% in MG132 group, the cell death rate in OLIG + MG132 group was the same as OLIG group.②The effect on sensitivity of cell death induced by proteasome inhibition by using OLIG to downregulate intracellular ATP in D-glucose medium.Experiment was performed with D-glucose medium, cells were divide into control group, OLIG group, MG262 group, OLIG + MG262 group, MG132 group and OLIG + MG132 group, with each drug added for 15 hours. By flow cytometry, it was shown that the cell death rate was 33.63% in OLIG + MG262 group, 63.7% in MG262 group, the difference between the two groups was statistically significant (p <0.01). Images of cell death in OLIG + MG132 group was mainly in early apoptosis, while in MG132 group mainly in late apoptosis.It is implied that downregulating intracellular ATP by OLIG in glucose-free had no effect on the sensitivity of cell death induced by proteasome inhibition, while in D-glucose medium had alleviated the cell death.2.1.5 The effect on sensitivity of cell death induced by proteasome inhibition by using 2DG to downregulate the intracellular ATPExperiment was performed in D-glucose medium. Cells were divided to control group, Ade Group, 2DG group, 2DG + MG132 group and 2DG + Ade + MG132 group , with each drug added for 12 hours. By flow cytometry, it was shown that cell death was 56.15% in MG132 group, 24.75% in 2DG + MG132 group, 13.7% in 2DG + MG132 group, and 49.8% in 2DG + Ade + MG132 group.It was there implied that downregulating the intracellular ATP in D-glucose medium by 2DG can reduce cell death induced by proteasome inhibition, while upwnregulating the intracellular ATP in D-glucose medium by Ade can increase the cell death. The sensitivity of cell death induced by proteasome inhibition was different accompanied by the different intracellular ATP concentration.2.2 The effect on sensitivity of cell death induced by proteasome inhibition in H460 by regulating the intracellular ATP concentration2.2.1 The effect on sensitivity of cell death induced by proteasome inhibition by using OLIG to downregulate the intracellular ATP in D-glucose mediumExperiments were perfoemed in D-glucose medium. Cells were divided into control group, OLIG group, MG132 group and OLIG + MG132 group, with each drug added for 6 hours. By flow cytometry, it was shown that the cell death rate was10.23% in MG132 group, 30.27% in OLIG + MG132 group. There was significant difference between the two group cell death rate (p<0.05).2.2.2 the effect on sensitivity of cell death induced by proteasome inhibition by using 2DG to downregulate the intracellular ATP①Experiments were performed in D-glucose medium. Cells were divided into control group, 2DG group, MG132 group and 2DG + MG132 group, with each drug added for 12 hours. By flow cytometry, it was shown that the cell death rate was 20.27% in MG132 group, 15% in 2DG + MG132 group. There was significant difference between the two group cell death rate (p<0.05).②Experiments were performed in D-glucose medium. Cells were divided into control group, 2DG group, MG132 group and 2DG + MG132 group, with each drug added for 12 hours. The LDH values were 373U / L in MG132 group, 124U / L in 2DG + MG132 group. Difference of LDH values between 2DG + MG132 group and MG132 group was statistically significant (p <0.05). It was shown inconsistent changes existed when downregulating intracellular ATP, that is,since the ATP generation pathway of H460 is dominantly by aerobic oxidation, the intracellular ATP concentration was significantly lower when OLIG was used to block oxidative phosphorylation, resulting in increased cell death induced by protesome inhibition; but 2DG slightly lowered the ATP concentration, and cell death induced by proteasome inhibition was reduced.Conclusions:1. Intracellular ATP concentration determines the sensitivity of cell death induced by proteasome inhibition.2. Intracellular ATP bidirectionally regulates proteasome inhibition-induced cell death.