Hydropersulfides (RSSH) attenuate doxorubicin-induced cardiotoxicity while boosting its anticancer action

Fig. 1. Cytoprotection by exogenous H2S, other RSS, and dexrazoxane against DOX-induced toxicity. (a) chemical structures of the RSS and dexrazoxane used in this study. (b) Cell viability of H9c2 cardiac myoblasts pretreated with 25 μM of each RSS for 4 h followed by DOX (5 μM) exposure for 24 h. (c) Dose-dependent protection by RSSH donor AST against DOX (5 μM). (d) Dose-dependent protection by dexrazoxane (DRZ) against DOX (5 μM). Cell viability is assessed using CCK-8. Data are shown as the mean ± SEM (n = 4–5 per group). ***P < 0.005 and ****P < 0.001 vs. DOX group.

Fig. 2. RSSH donor AST reduces DOX-induced ROS levels in H9c2 cardiac myoblasts via activation of Nrf2 and downstream NQO1. Representative EPR spectra showing RSSH-mediated scavenging of (a) whole cell and (b) mitochondrial ROS emission. H9c2 cells were pretreated with AST (25 μM) for 4 h followed by treatment with or without DOX (5 μM) for 4 h for whole cell ROS measurement and for 24 h for mitochondrial ROS measurement. (c) Whole-cell, and (d) mitochondrial ROS emission measured by EPR spectroscopy. The relative expression level of (e) nuclear Nrf2; (f) cytosolic Nrf2; and (g) whole cell NQO1 in H9c2 cells. Cells were pretreated with 0, 25, and 50 μM AST for 24 h, followed by treatment with either vehicle or 0.5 μM DOX for 24 h. Expression levels of these proteins were measured by immunoblotting. Data are shown as the mean ± SEM (n = 3 per group (e–g) and n = 4 per group (a–d)). *P < 0.05, **P < 0.01 and ****P < 0.001 between respective groups.

Fig. 3. RSSH donor AST restores DOX-mediated decreases in expression of nuclear PGC-1α and its downstream product, cytosolic TFAM. (a) Relative expression level of nuclear PGC-1α, and (b) cytosolic TFAM of H9c2 cells. H9c2 cells were pretreated with 0, 25, and 50 μM AST for 24 h followed by treatment with vehicle or 0.5 μM DOX 24 h. Protein expression levels were determined by immunoblotting. (c) Caspase 3-like activity of H9c2 measured using a colorimetric assay. Data are shown as the mean ± SEM (n = 3–4 per group). *P < 0.05, **P < 0.01 and ****P < 0.001 vs. control groups and between respective groups.

Fig. 4. RSSH donor AST increases the efficacy of DOX in cancer cells. Cell viability of (a) MDA-MB-468, (b) MCF-7, and (c) HepG2 cells that were pretreated with AST (6.3–100 μM) for 4 h followed by DOX (5 μM) for 24 h. Cell viability is assessed using CCK-8 assay. Data are shown as the mean ± SEM (n = 3 per group). *P < 0.05, and **P < 0.01 vs. DOX group.

Fig. 5. Cancer cells have higher basal level of H2S, sulfane sulfur and reducing equivalents. RSSH donor AST restores the reduction of GSH in H9c2 cells while depleting GSSG in cancer cells, leading to increased ROS emission in the mitochondria of cancer cells. Basal level of (a) H2S; (b) sulfane sulfur; and reducing equivalents (c) NADH/NAD+ of H9c2 and cancer cells. Absolute GSH, absolute GSSG and the GSH/GSSG ratio change of (d) H9c2 and (e) MDA-MB-468 cells. (f) whole cell, and (g) mitochondrial ROS emissions are measured by EPR spectroscopy. (h) Schematic illustration of ROS generation following reductive stress due to lowered mitochondrial GSSG, which normally acts as a primary electron acceptor. Cells were pretreated with 0 or 25 μM AST for 4 h followed by co-treatment with 5 μM DOX for 24 h H2S and sulfane sulfur species levels were measured using fluorescent probes. NADH/NAD+ and GSH/GSSG ratio were measured using colorimetric assays. Absolute GSH, absolute GSSG, and the GSH/GSSG ratio were measured using colorimetric assays. Data are shown as the mean ± SEM (n = 3 per group (a–e), n = 6–8 per group (f), and n = 6–7 per group (g)). *P < 0.05, **P < 0.01 and ****P < 0.001 vs.…

Fig. 6. Schematic representation of the opposing effects of RSSH in cancer cells vs. cardiac cells. Cancer cells have significantly higher levels of basal RSS compared with cardiac cells, with both cells within the redox buffering capacity. DOX treatment induces oxidative stress in both cell types, ultimately leading to cell death. RSSH administration alleviates DOX-induced oxidative stress in cardiac cells, but induces reductive stress in cancer cells that already have higher levels of RSS.