Study On NAD~+ Depletion Abolishes 2-DG Induced Metabolic Adaptation,Inducing Enhanced Cell Killing And Radiosensitivity Of U-87MG Glioma Cell

Objective:We aimed to contirm NAD(?) s function in 2-DG induced metabolic adapation including ATP preserving, maintainance of oxidation-reduction balance and inhibition of caspase 3. We further attempted to verify NAD+ depletion induced cell killing and radiosensitization under 2-DG treatment, providing evidences for usage of NAD+ depletion with combination of 2-DG in treating gliomas.Methods:NAMPT(key enzyme of NAD+ salvage synthesis), caspase 3 protein expression was detected by Western blot, ATP, NAD+ or NADPH content was quantified by ATP, NAD+ or NADPH content detection kits, intracellular ROS level was detected by flow cytometry after DCFH incubation.Cell death was counted by flow cytometry after Annexin-V-FITC and PI staining.4Gy X ray was used in group of control,2-DG,siNAMPT,2-DG+siNAMPT.Number of clony formation was counted to evaluate radiosensitivity, number of foci formation was counted after γH2AX incubation by Confocal Laser Scanning Microscope to evaluate the extent of DNA damage.Results:Under 2-DG treatment, Western blot showed NAMPT protein increased in time and concentration dependent manner, NAD+ content presented the same trend as NAMPT protein; ROS level (mean Value of DCFH fluorescence) decreased in group of 2-DG compared to control; cleaved caspase 3 protein decreased in 2-DG concentration dependent manner.Under siNAMPT or exNAD treatment, ATP content, NADPH content,caspase 3 alteration was firmly in accordance with NAD+content alteration. ROS level showed consistency with NAD+ content alteration.Under siNAMPT+2-DG treatment,Western blot showed caspase 3 protein significiantly increased in group of siNAMPT+2-DG compared to group of 2-DGalone; furthermore, group of siNAMPT+2-DG+exNAD showed partial decreased cleaved caspase 3 compared to group of siNAMPT+2-DG, but more than group of 2-DG and untreated.Annexin-V-FITC and PI stained cell negativity indicated group of siNAMPT +2-DG inducing significant cell death compared to group of si-scramble RNA+2-DG alone,and its cell death could be reduced by addition of exNAD. Clony formation counts indicated group of siNAMPT+2-DG showing significant decreased clony formation than group of si-scramble RNA, siNAMPT and si-scrambleRNA +2-DG. Laser Scanning Confocal Microscopy detecting DNA foci formation showed foci number of all groups made no Statistical differences at 4 and 10 h, group of siNAMPT +2-DG possessed the most foci number at 24 h when group of si-scramble RNA had returned to the basal foci Level,which indicated siNAMPT +2-DG treatment significantly interfered the process of DNA repair after irradiation.Conclusions:We confirmed 2-DG induced NAD+ augment were involved in modulating ATP complement, attenuating ROS level and caspase 3 cleavage,which indicated NAD+ is the major blockage preventing 2-DG killing cells, meanwhile we verified NAD+ depletion with combination of 2-DG showed potential in inducing glioma cell death and raidosensitization of glioma.