Hydrogen Sulfide Oxidation: Adaptive Changes in Mitochondria of SW480 Colorectal Cancer Cells upon Exposure to Hypoxia

Abstract

Hydrogen sulfide (H₂S), a known inhibitor of cytochrome c oxidase (CcOX), plays a key signaling role in human (patho)physiology. H₂S is synthesized endogenously and mainly metabolized by a mitochondrial sulfide-oxidizing pathway including sulfide:quinone oxidoreductase (SQR), whereby H₂S-derived electrons are injected into the respiratory chain stimulating O₂ consumption and ATP synthesis. Under hypoxic conditions, H₂S has higher stability and is synthesized at higher levels with protective effects for the cell. Herein, working on SW480 colon cancer cells, we evaluated the effect of hypoxia on the ability of cells to metabolize H₂S. The sulfide-oxidizing activity was assessed by high-resolution respirometry, measuring the stimulatory effect of sulfide on rotenone-inhibited cell respiration in the absence or presence of antimycin A. Compared to cells grown under normoxic conditions (air O₂), cells exposed for 24 h to hypoxia (1% O₂) displayed a 1.3-fold reduction in maximal sulfide-oxidizing activity and 2.7-fold lower basal O₂ respiration. Based on citrate synthase activity assays, mitochondria of hypoxia-treated cells were 1.8-fold less abundant and displayed 1.4-fold higher maximal sulfide-oxidizing activity and 2.6-fold enrichment in SQR as evaluated by immunoblotting. We speculate that under hypoxic conditions mitochondria undergo these adaptive changes to protect cell respiration from H₂S poisoning.

Figure 1

Stimulation of O₂ consumption by sulfide. Representative oxygen consumption traces (blue) and corresponding O₂ consumption rate (OCR, red traces) acquired with normoxic (a) or hypoxia-treated SW480 cells (b), following the addition of cells (4 × 10⁶), rotenone (Rot., 5 μM) either alone (top traces) or plus antimycin A (Ant. A, 5 μM, bottom traces), and subsequent injection of a sulfide solution (3-5 mM) at increasing rates (10 nL·s⁻¹, 20 nL·s⁻¹, 40 nL·s⁻¹, 80 nL·s⁻¹, and 160 nL·s⁻¹, corresponding, respectively, to injections #1 to #5). (c, d) OCR values obtained from the oxygraphic traces, respectively shown in (a) and (b), measured at basal condition and upon sulfide injection at increasing rates after addition of rotenone alone (full symbols) or rotenone plus antimycin A (hollow symbols). Mitochondrial H₂S consumption in normoxic cells was calculated by determining the OCR measured at the highest non-inhibitory H₂S injection rate (highlighted with grey bar in (c)) and subtracting the OCR measured after the addition of rotenone (horizontal dashed line in (c)), yielding ΔOCR_(-Ant). Then, the ΔOCR at the corresponding sulfide injection in the antimycin A-containing measurement was calculated in the same manner, yielding ΔOCR_(+Ant). By calculating ΔOCR_(‐Ant) − ΔOCR_(+Ant), the genuine mitochondrial H₂S-dependent OCR (OCR_mitH2S) was determined. Finally, OCR_mitH2S was multiplied by 1.33 to account for the number of H₂S molecules consumed per O₂ molecule, yielding an estimated sulfide oxidizing activity of 12.7 nM H₂S·s⁻¹·10⁶ cells⁻¹. Employing the same procedure for cells exposed to hypoxia (b, d), an activity of 9.5 nM H₂S·s⁻¹·10⁶ cells⁻¹ was estimated.

Figure 2

Effect of hypoxia on mitochondrial sulfide consumption. (a) Mean values of maximal estimated sulfide consumption activity (calculated as described in the legend of Figure 1(c)), measured in normoxic (n = 9, blue bar) and hypoxia-treated (n = 8, red bar) cells. (b) Citrate synthase activity in normoxic (n = 13, blue bar) and hypoxia-treated (n = 10, red bar) cell lysates. (c) Maximal sulfide consumption activity normalized to the citrate synthase activity, as measured in normoxic (blue bar) and hypoxia-treated (red bar) cells. ^∗P ≤ 0.05; ^∗∗P ≤ 0.01; ^∗∗∗P ≤ 0.001.

Figure 3

Effect of hypoxia on SQR expression. Representative Western blot analyzing SQR expression in normoxic and hypoxia-exposed SW480 cells (a), with the corresponding total protein load quantitation by stain-free imaging technology (see Materials and Methods). SQR levels in normoxic (n = 4 in triplicate, blue bars) and hypoxia-treated cells (n = 4 in triplicate, red bars), as normalized to total protein (b) or citrate synthase activity (c). ^∗∗∗P ≤ 0.001.

Figure 4

Adaptive changes occurring in mitochondria in response to hypoxia. Upon prolonged exposure to hypoxia, mitochondria become less abundant, but enriched in sulfide:quinone oxidoreductase (SQR). Consistently, their maximal sulfide-oxidizing activity increases, while overall decreasing in the cell. These changes are proposed to occur to prevent H₂S inhibition of cytochrome c oxidase (CcOX) and thus protect cell respiration from H₂S poisoning.