Introduction Recent data indicate that hydrogen sulfide (H2S) has dual effects on cellular bioenergetics: promotion of electron transport at low (physiological) concentrations, and inhibition of mitochondrial respiration at higher (cytotoxic) concentrations. Based on recent data from our group, demonstrating the upregulation of the H2S-producing enzyme cystathionine-β-synthase (CBS) in surgical specimens from human colorectal cancer patients, and in human colon cancer cell lines, we have investigated the subcellular localization of CBS in HCT116 colon cancer cell line and explored the potential role of CBS-derived H2S in the regulation of their bioenergetic function in vitro. Methods HCT116 cells were cultured in McCoy’s 5A medium. For shRNA-mediated silencing of CBS and CSE, HCT116 cells were transduced with either a lentiviral vector containing shRNA sequences targeting CBS or CSE. A non-targeting control shRNA sequence (shNT) was used to control of off-target effects. In studies investigating the cellular localization of CBS, HCT116 cells were subjected subcellular fractionation by centrifugation. Mitochondrial and cytosolic fractions were analyzed by western blotting; membranes were probed overnight with anti-CBS (60 kDa), anti-β-actin (43 kDa), anti-LDH (35 kDa), anti-Tom20 (20 kDa) and anti-Complex IV (17 kDa) antibodies. In a separate set of experiments, the mitochondrial outer membrane was subjected to limited trypsin digestion, followed by Western blotting. The XF24 Extracellular Flux Analyzer (Seahorse) was used to measure bioenergetic function. Oxygen consumption rate (OCR) after oligomycin (1.5 μg/ml) was used to assess ATP production rate and OCR after FCCP (0.5 μM) to assess maximal mitochondrial respiratory capacity. 2-deoxyglucose (100 mM) was used to estimate cellular glycolytic dependency and antimycin A (2 μg/ml) and rotenone (2 μM) were used to inhibit the flux of electrons through complex III and I, to detect residual non-mitochondrial activity. For the measurement of glycolytic parameters, the changes in Proton Production Rate (PPR) were monitored in response to the sequential administration of D-glucose (10 mM), oligomycin (1.5 μM) and 2-deoxyglucose (100 mM), to assess glycolysis, maximal glycolytic capacity, glycolytic reserve capacity, and non-glycolytic acidification rate, respectively. GAPDH activity was determined by a kinetic assay. Bioenergetic measurements in mitochondria isolated from shNT and shCBS HCT116 cells were conducted in the absence or presence of L-cysteine (30 nM). Basal respiration (State 2) in the presence of succinate and rotenone was measured, followed by State 3 (phosphorylating respiration), in the presence of ADP. State 4o was determined after the addition of oligomycin, and maximal uncoupler-stimulated respiration (State 3u) was assessed in the presence of FCCP. Results Cell fractionation studies showed that a significant portion of the total amount of cellular CBS was associated with the mitochondria in HCT116 cells. A trypsin digestion assay on isolated mitochondria showed that CBS was primary associated with the outer mitochondrial membrane. In agreement with the physiological role of endogenous H2S in promoting cellular bioenergetics in various cell types including colonocytes, low concentrations of H2S caused a stimulation of mitochondrial function in mitochondria isolated from HCT116 cells. Moreover, shRNA-mediated silencing of CBS (but not of CSE) expression, or CBS inhibition with AOAA reduced basal cellular respiration, suppressed the calculated ATP synthesis, and attenuated the spare respiratory capacity. CBS silencing or CBS inhibition also reduced glycolytic functions, while CSE silencing was without significant effect. This latter effect may be attributed, at least in part, to inhibition of GAPDH activity: GAPDH activity was quantified in HCT116 cells, and it was reduced by CBS silencing. (GAPDH activity in wild-type and CBS silenced cells amounted to 6.2 ± 0.1 U/ml and 4.0 ± 0.1 U/ml, respectively, p Conclusions Prior studies have demonstrated that H2S donors can stimulate mitochondrial electron transport and ATP generation in various cell lines in vitro. Furthermore, H2S, via sulfhydration, has been shown to increase the catalytic activity of the glycolytic enzyme GAPDH. Consistent with these findings, our results show that both oxidative and glycolytic tumor cell metabolism is suppressed by shRNA-mediated silencing of CBS, or by treating cells with AOAA. Tumor bioenergetics relies both on oxidative phosphorylation, as well as glycolysis. Because proliferation, migration and invasion are energetically demanding processes, we hypothesize that CBS inhibition/silencing reduces the intracellular levels of H2S, which, in turn, contributes to the energy starvation of the tumor, impairing its growth. In addition to bioenergetic effects, we hypothesize that endogenous H2S may also contribute to tumor growth by a variety of mechanisms including activation of phosphoinositide-3-kinase and Akt kinase, inhibition of phosphatases and the regulation of cell cycle genes. Together with additional in vitro and in vivo data presented at the current meeting, these data support the view that CBS-derived H2S serves as an endogenous cancer cell bioenergetic factor, and identify CBS as a potential future antitumor drug target.
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Open Access
Jan 1, 2015
Christopher Holmes 
