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. 2019 Sep:26:101220.
doi: 10.1016/j.redox.2019.101220. Epub 2019 May 16.

Sodium sulfide selectively induces oxidative stress, DNA damage, and mitochondrial dysfunction and radiosensitizes glioblastoma (GBM) cells

Adam Y Xiao  1 , Matthew R Maynard  2 , Cortt G Piett  3 , Zachary D Nagel  3 , J Steven Alexander  1 , Christopher G Kevil  4 , Michael V Berridge  5 , Christopher B Pattillo  1 , Lane R Rosen  2 , Sumitra Miriyala  6 , Lynn Harrison  7
Affiliations

Sodium sulfide selectively induces oxidative stress, DNA damage, and mitochondrial dysfunction and radiosensitizes glioblastoma (GBM) cells

Adam Y Xiao et al. Redox Biol. 2019 Sep.

Abstract

Glioblastoma (GBM) has a poor prognosis despite intensive treatment with surgery and chemoradiotherapy. Previous studies using dose-escalated radiotherapy have demonstrated improved survival; however, increased rates of radionecrosis have limited its use. Development of radiosensitizers could improve patient outcome. In the present study, we report the use of sodium sulfide (Na2S), a hydrogen sulfide (H2S) donor, to selectively kill GBM cells (T98G and U87) while sparing normal human cerebral microvascular endothelial cells (hCMEC/D3). Na2S also decreased mitochondrial respiration, increased oxidative stress and induced γH2AX foci and oxidative base damage in GBM cells. Since Na2S did not significantly alter T98G capacity to perform non-homologous end-joining or base excision repair, it is possible that GBM cell killing could be attributed to increased damage induction due to enhanced reactive oxygen species production. Interestingly, Na2S enhanced mitochondrial respiration, produced a more reducing environment and did not induce high levels of DNA damage in hCMEC/D3. Taken together, this data suggests involvement of mitochondrial respiration in Na2S toxicity in GBM cells. The fact that survival of LN-18 GBM cells lacking mitochondrial DNA (ρ0) was not altered by Na2S whereas the survival of LN-18 ρ+ cells was compromised supports this conclusion. When cells were treated with Na2S and photon or proton radiation, GBM cell killing was enhanced, which opens the possibility of H2S being a radiosensitizer. Therefore, this study provides the first evidence that H2S donors could be used in GBM therapy to potentiate radiation-induced killing.

Keywords: DNA damage; DNA repair; Glioblastoma; Hydrogen sulfide; Ionizing radiation; Mitochondria; Reactive oxygen species.

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Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Sodium sulfide is cytotoxic to GBM but not human cerebral microvascular endothelial cells. Cells were treated with Na2S for 4 h with media change and replacement at 2 h (A). The dose response of T98G and U87 cells to Na2S was determined using clonogenic survival. The dashed green line indicates the position of Surviving Fraction = 1 on the graph. Data was analyzed using a one-way ANOVA with Tukey's post-hoc analysis (B). Free sulfide levels were measured after the 4 h treatment with 476 μM Na2S using HPLC. Data was analyzed using a Student's t-test comparing 0 and 476 μM for each cell line (C). The effect of 476 μM Na2S on hCMEC/D3 was also determined using clonogenic survival. Data was analyzed using a Student's t-test comparing 0 and 476 μM (D). All data are from 3 independent experiments. Error bars represent SD and * represents P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Fig. 2
Sodium sulfide induces DNA double-strand break formation in GBM. Representative images of γH2AX foci following treatment with 476 μM Na2S or degassed water for 4 h are shown (A). The number of foci per cell was quantified with JCount Pro using the same parameters for all cell types and replicates (B). Analysis was performed on 150 cells pooled from 3 independent experiments and error bars represent standard error of the mean (SEM). * represents P < 0.05 using a Student's t-test.
Fig. 3
Fig. 3
Sodium sulfide increases oxidative DNA base damage. The alkaline comet assay with FPG treatment was used to detect oxidative base damage following 0 or 476 μM Na2S treatment for 4 h (A) and the tail moment was measured using OpenComet (B). Analysis was performed on 3 independent experiments with at least 50 cells in each experiment. Error bars represent SD and * represents P < 0.05 using a Student's t-test.
Fig. 4
Fig. 4
Sodium sulfide does not affect DSB repair and OGG1-directed BER. T98G cells were pretreated with 476 μM Na2S or degassed water for 2 h, transfected with reporter plasmid, and treated for an additional 2 h. For non-homologous end joining, repaired plasmids encode functional GFP and fluorescent expression was detected 24 and 48 h post-transfection using flow cytometry. Data is expressed as GFP+/RFP+ to normalize for transfection efficiency (A). To assess BER, mOrange plasmid containing an 8-oxoguanine was co-transfected with pMaxBFP into T98G cells. Transcriptional mutagenesis of 8-oxoguanine results in functional mOrange that was detected 17 h after transfection via flow cytometry. Data is expressed as mOrange+ x MFI normalized to BFP+ x MFI as a transfection control (B). Error bars represent SD. Data was analyzed using a Student's t-test to compare treated and untreated cells at a particular time point.
Fig. 5
Fig. 5
Sodium sulfide increases oxidative stress and ROS formation in GBM. GSH and GSSG levels were measured using HPLC to calculate the GSH: GSSG ratio after 4 h of treatment with 0 or 476 μM Na2S (A). ROS was measured using CM-H2DCFDA. Cells were pretreated with TEMPOL for 1 h and then 476 μM Na2S or degassed water was also added to the cells. New media with only 476 μM Na2S or degassed water was added to the cells after 2 h. Data are expressed as a fold change relative to respective controls. N = 3 for U87, N = 4 for T98G and hCMEC/D3 cells (B). Error bars represent SD and * represents P < 0.05 using a paired Student's t-test.
Fig. 6
Fig. 6
Sodium sulfide alters mitochondrial function and inhibits complex III activity. Oxygen consumption rate (OCR) was measured in cells treated with 476 μM Na2S or degassed water for 4 h using a Seahorse Bioscience XF24 Extracellular Flux Analyzer (A). Basal respiration is the OCR prior to addition of oligomycin (B). The spare capacity was calculated as the FCCP OCR minus the basal OCR (C). Complex I activity was measured as the rotenone-sensitive oxidation of NADH at 340 nm in cell lysate (D). Complex III activity was measured as the antimycin A-sensitive oxidation of cytochrome c at 550 nm in cell lysate (E). All data are from 3 independent experiments. Error bars represent SD and * represents P < 0.05 using a Student's t-test. Refer to the web version for easier identification of cell lines in Figure 6A.
Fig. 7
Fig. 7
GBM sensitivity to sodium sulfide is dependent on the electron transport chain. U87 and LN-18 ρ0 were confirmed by PCR using mitochondria specific cytochrome b primers and β-actin as a control (A). Survival of LN18 ρ+ and ρ0 following treatment with Na2S or degassed water for 4 h was determined using clonogenic assay (B). U87 ρ0 cells did not form colonies. All data are from 3 independent experiments. Error bars represent SD and * represents P < 0.05 using a one-way ANOVA with Tukey's post-hoc analysis.
Fig. 8
Fig. 8
Sodium sulfide selectively radiosensitizes GBM to ionizing radiation. T98G cells were treated with 476 μM Na2S or degassed water for a total of 4 h and irradiated during the 4th hr of Na2S treatment. Surviving fraction at each radiation dose was normalized to their respective controls to account for Na2S induced killing (A). Data were curve fit to the linear quadratic (LQ) equation to calculate RBE10 and DEF10 at 10% surviving fraction (SF10). Protons had an RBE10 = 1.2. Photons and protons had a DEF10 = 1.34. hCMEC/D3 cells were similarly irradiated using a clinically relevant dose of 2 Gy (B) and data analyzed by two-way ANOVA with Tukey's post-hoc for multiple comparison.
Fig. 9
Fig. 9
DNA Damage Induction by Ionizing Radiation and Sodium Sulfide. The synergistic effect of 476 μM Na2S on IR-induced DNA damage was assessed using the alkaline comet assay with FPG treatment (A) or γH2AX foci/cell (B, C, D). T98G cells were treated with 0 or 476 μM Na2S for a total of 4 h and irradiated with either 1.9 Gy photon or 2 Gy proton during the 4th hour of Na2S treatment. The comet assay was performed immediately after this 4 h treatment schedule. Analysis was performed on 3 independent experiments with at least 50 cells in each experiment using a one-way ANOVA with Tukey's post-hoc analysis. Error bars represent SD (A). γH2AX foci was quantified immediately after the 4 h treatment schedule (0 h repair time, B) and at 2 (C) and 24 h (D) repair time to determine DSB repair kinetics. Analysis was performed on 150 cells pooled from 3 independent experiments and error bars represent SEM (B, C, D). For all experiments, * represents P < 0.05. Each time point was compared using a one-way ANOVA with Tukey's post-hoc analysis but only differences between 0 and 476 μM Na2S at each IR dose are indicated for simplicity. See text for explanation of the whole analysis.
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