Pyocyanin Induces NK92 Cell Apoptosis Via Mitochondrial Damage And Elevated Intracellular Ca²⁺

Background:Pseudomonas aeruginosa?P.aeruginosa?-derived redox toxin pyocyanin?PCN?has been proved to induce cell apoptosis mediated by the generation of reactive oxygen species?ROS?,which has been studied mainly in epithelial cells and neutrophils.However,we previously found that the PCN-producing strain PA14induced cell apoptosis in human NK cell line NK92 more effectively than in PCN-deficient strain PA14-phZ1/2 via a yet undetermined mechanism.Objective:This study tried to explore the molecular mechanism of apoptosis of NK92 cells induced by PCN through stimulating NK92 cells at different PCN concentration and incubation time,using Fluorescence activated Cell Sorting?FACS?,Luciferase Assays and Western Blotting to determine whether PCN caused mitochondrial damage and change of ROS in NK92 cells,explore the role of[Ca²⁺]_i and pro-apoptotic Bcl-2 family proteins and clarify the molecular mechanism in PCN-induced apoptosis.Methods:First,we stimulated NK92 cells by PCN at different conditions or DMSO?vehicle control?.The growth behavior of the cells was observed directly under microscope and photographed.Cells were collected for FACS to get the percentage of Annexin V in order to determine the apoptosis of NK92 cells.We used molecular probe JC-1 to detect mitochondrial membrane potential of NK92 cells to measure mitochondrial damage and detected ATP levels and Caspase-3,8,9 protein levels with Luciferase Assay and Western Blotting respectively,which was to reveal the mitochondrial apoptotic pathway.Then,we measured the level of intracellular ROS by FACS.NK92 cells were pretreated with diphenyleneiodonium?DPI?,a NADPH oxidase inhibitor,as a negative control.Furthermore,we measured the Ca²⁺/Fluo-4fluorescence intensity by FACS and treated the Ca²⁺-chelating agent ethylene glycol tetra-acetic acid?EGTA?as a negative control.Finally,Western Blotting was used to detect the levels of pro-apoptotic Bcl-2 family proteins,such as Bim,BID,Bik,Bak,Bad,Phospho-Bad and so on.Meanwhile,EGTA was used as negative control,and FACS was used to detect the mitochondrial membrane potential level to determine whether[Ca²⁺]_i was correlated with mitochondrial damage.Results:The state of the cells changed and the percentage of Annexin V detected by FACS increased in a concentration-and-time-dependent manner after PCN treated in NK92 cells.By detecting the intracellular mitochondrial membrane potential levels,we found that the green fluorescence of JC-1 monomers increased in a time-and concentration-dependent manner,with a decrease of ATP and ROS and an increase of Caspase-9 and Caspase-3,while the expression of Caspase-8 did not change significantly,which suggested that PCN-induced NK92 cell apoptosis was responsible to mitochondrial damage despite inhibiting intracellular ROS generation.PCN treatment increased[Ca²⁺]_i in NK92 cells more than twofold after 2 h stimulation,whereas EGTA inhibited apoptosis.PCN triggered the activation of Bim,Bid,Bik,Bak,and phospho-Bad in NK92 cells in a concentration-dependent manner without the effect of EGTA.NK92 cells were pretreated with EGTA,FACS was used to detect mitochondrial membrane potential,and found that the stability of mitochondria was not affected by EGTA.Conclusion:Through this study,we found a new mechanism of PCN-induced apoptosis,and got the following conclusions:1)PCN induced mitochondrial-dependent apoptosis in NK92 cells;2)PCN induced NK92 cell apoptosis did not involve oxidative stress;3)PCN caused an increase of[Ca²⁺]_i in NK92 cells;4)PCN activated pro-apoptotic Bcl-2 family proteins.