Role of Cystathionine ß-Synthase and 3-Mercaptopyruvate Sulfurtransferase in the Regulation of Proliferation, Migration, and Bioenergetics of Murine Breast Cancer Cells.

Cystathionine ß-synthase (CBS), CSE (cystathionine γ-lyase) and 3-mercaptopyruvate sulfurtransferase (3-MST) have emerged as three significant sources of hydrogen sulfide (H2S) in various forms of mammalian cancer. Here, we investigated the functional role of CBS' and 3-MST's catalytic activity in the murine breast cancer cell line EO771. The CBS/CSE inhibitor aminooxyacetic acid (AOAA) and the 3-MST inhibitor 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE) were used to assess the role of endogenous H2S in the modulation of breast cancer cell proliferation, migration, bioenergetics and viability in vitro. Methods included measurements of cell viability (MTT and LDH assays), cell proliferation and in vitro wound healing (IncuCyte) and cellular bioenergetics (Seahorse extracellular flux analysis). CBS and 3-MST, as well as expression were detected by Western blotting; H2S production was measured by the fluorescent dye AzMC. The results show that EO771 cells express CBS, CSE and 3-MST protein, as well as several enzymes involved in H2S degradation (SQR, TST, and ETHE1). Pharmacological inhibition of CBS or 3-MST inhibited H2S production, suppressed cellular bioenergetics and attenuated cell proliferation. Cell migration was only inhibited by the 3-MST inhibitor, but not the CBS/CSE inhibitor. Inhibition of CBS/CSE of 3-MST did not significantly affect basal cell viability; inhibition of 3-MST (but not of CBS/CSE) slightly enhanced the cytotoxic effects of oxidative stress (hydrogen peroxide challenge). From these findings, we conclude that endogenous H2S, generated by 3-MST and to a lower degree by CBS/CSE, significantly contributes to the maintenance of bioenergetics, proliferation and migration in murine breast cancer cells and may also exert a minor role as a cytoprotectant.

Effects of the PARP Inhibitor Olaparib on the Response of Human Peripheral Blood Leukocytes to Bacterial Challenge or Oxidative Stress.

Prior studies demonstrate the activation of poly-(ADP-ribose) polymerase 1 (PARP1) in various pathophysiological conditions, including sepsis. We have assessed the effect of olaparib, a clinically used PARP1 inhibitor, on the responses of human peripheral blood leukocytes (PBMCs) obtained from healthy volunteers in response to challenging with live bacteria, bacterial lipopolysaccharide (LPS), or oxidative stress (hydrogen peroxide, H2O2). The viability of PBMCs exposed to olaparib or to the earlier generation PARP inhibitor PJ-34 (0.1-1000 µM) was monitored using Annexin V and 7-aminoactinomycin D. To evaluate the effects of olaparib on the expression of PARP1 and its effects on protein PARylation, PBMCs were stimulated with Staphylococcus aureus with or without olaparib (1-10 µM). Changes in cellular levels of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP), as well as changes in mitochondrial membrane potential (MMP), were measured in PBMCs exposed to H2O2. Bacterial killing was evaluated in PBMCs and polymorphonuclear leukocytes (PMNs) incubated with S. aureus. Cytokine production was measured in supernatants using a cytometric bead array. Reactive oxygen species (ROS), nitric oxide (NO) production, and phagocytic activity of monocytes and neutrophils were measured in whole blood. For ROS and NO production, samples were incubated with heat-killed S. aureus; phagocytic activity was assessed using killed Escherichia coli conjugated to FITC. Olaparib (0.1-100 µM) did not adversely affect lymphocyte viability. Olaparib also did not interfere with PARP1 expression but inhibits S. aureus-induced protein PARylation. In cells challenged with H2O2, olaparib prevented NAD+ and ATP depletion and attenuated mitochondrial membrane depolarization. LPS-induced production of TNF-α, MIP-1α, and IL-10 by PBMCs was also reduced by olaparib. Monocytes and neutrophils displayed significant increases in the production of ROS and NO after stimulation with S. aureus and phagocytic (E. coli) and microbicidal activity, and these responses were not suppressed by olaparib. We conclude that, at clinically relevant concentrations, olaparib exerts cytoprotective effects and modulates inflammatory cytokine production without exerting adverse effects on the cells' ability to phagocytose or eradicate pathogens. The current data support the concept of repurposing olaparib as a potential experimental therapy for septic shock.

Repurposing of Clinically Approved Poly-(ADP-Ribose) Polymerase Inhibitors for the Therapy of Sepsis.

ABSTRACT: Sepsis' pathogenesis involves multiple mechanisms that lead to a dysregulation of the host's response. Significant efforts have been made in search of interventions that can reverse this situation and increase patient survival. Poly (ADP-polymerase) (PARP) is a constitutive nuclear and mitochondrial enzyme, which functions as a co-activator and co-repressor of gene transcription, thus regulating the production of inflammatory mediators. Several studies have already demonstrated an overactivation of PARP1 in various human pathophysiological conditions and that its inhibition has benefits in regulating intracellular processes. The PARP inhibitor olaparib, originally developed for cancer therapy, paved the way for the expansion of its clinical use for nononcological indications. In this review we discuss sepsis as one of the possible indications for the use of olaparib and other clinically approved PARP inhibitors as modulators of the inflammatory response and cellular dysfunction. The benefit of olaparib and other clinically approved PARP inhibitors has already been demonstrated in several experimental models of human diseases, such as neurodegeneration and neuroinflammation, acute hepatitis, skeletal muscle disorders, aging and acute ischemic stroke, protecting, for example, from the deterioration of the blood-brain barrier, restoring the cellular levels of NAD+, improving mitochondrial function and biogenesis and, among other effects, reducing oxidative stress and pro-inflammatory mediators, such as TNF-α, IL1-ß, IL-6, and VCAM1. These data demonstrated that repositioning of clinically approved PARP inhibitors may be effective in protecting against hemodynamic dysfunction, metabolic dysfunction, and multiple organ failure in patients with sepsis. Age and gender affect the response to PARP inhibitors, the mechanisms underlying the lack of many protective effects in females and aged animals should be further investigated and be cautiously considered in designing clinical trials.

Immunophenotyping of Monocytes During Human Sepsis Shows Impairment in Antigen Presentation: A Shift Toward Nonclassical Differentiation and Upregulation of FCγRi-Receptor.

Monocytes and macrophages are pivotal in the host response to sepsis, recognizing the infecting microorganism and triggering an inflammatory response. These functions are, at least in part, modulated by the expression of cell surface receptors. We aimed to characterize the monocyte phenotype from septic patients during an ongoing sepsis process and its association with clinical outcomes. Sixty-one septic patients and 31 healthy volunteers (HVs) were enrolled in the study. Samples were obtained from patients at baseline (D0, N = 61), and after 7 (D7, N = 36) and 14 days of therapy (D14, N = 22). Monocytes from septic patients presented decreased expression of CD86, HLA-DR, CD200R, CCR2, CXCR2, and CD163 compared with HV monocytes. In contrast, the PD-1, PD-L1, CD206, CD64, and CD16 expression levels were upregulated in patients. HLA-DR, CD64, PD-1, and PD-L1 expression levels were higher in survivors than in nonsurvivors. Increased CD86, HLA-DR, and CXCR2 expression levels were observed in follow-up samples; in contrast, CD64 and CD16 GMFI decreased over time. In conclusion, monocytes from septic patients show antigen presentation impairment as characterized by decreased HLA-DR and costimulatory CD86 expression and increased PD-1 and PD-L1 expression. On the contrary, increased monocyte inflammatory and phagocytic activities may be inferred by the increased CD16 and CD64 expression. We found conflicting results regarding differentiation toward the M2 phenotype, with increased CD206 expression and decreased CD163 expression on monocytes from septic patients, whereas the subset of nonclassical monocytes was demonstrated by increased CD16.

Effect of natural porcine surfactant in Staphylococcus aureus induced pro-inflammatory cytokines and reactive oxygen species generation in monocytes and neutrophils from human blood.

Surfacen® is a clinical surfactant preparation of porcine origin. In the present study, we have evaluated the effect of Surfacen® in the modulation of oxidative burst in monocytes and neutrophils in human blood and pro-inflammatory cytokine production in peripheral blood mononuclear cells (PBMC). Reactive oxygen species (ROS) level was measured in monocytes and neutrophils by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) as substrate, while, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were estimated in PBMC supernatant by enzyme-linked immunosorbent assays (ELISA). Our results show that Staphylococcus aureus-induced ROS level was slightly affected by Surfacen® added to whole blood monocytes and neutrophils. The time course experiments of pre-incubation with Surfacen® showed no significant increase of ROS level at 2h; however, the ROS level decreased when pre incubated for 4h and 6h with Surfacen®. Pre-incubation of PBMC cells with Surfacen® at 0.125 and 0.5mg/mL showed a dose-dependent suppression of TNF-α levels measured after 4h of S. aureus stimulation, an effect less impressive when cells were stimulated for 24h. A similar behavior was observed in IL-6 release. In summary, the present study provides experimental evidence supporting an anti-inflammatory role of Surfacen® in human monocytes and neutrophils in vitro.

Generation of nitric oxide and reactive oxygen species by neutrophils and monocytes from septic patients and association with outcomes.

In this study, our aims were to evaluate the reactive oxygen species (ROS) and nitric oxide (NO) generation by monocytes and neutrophils from septic patients and to correlate their levels with clinical outcomes. Forty-nine septic patients and 19 healthy volunteers were enrolled in the study. The ROS and NO production was quantified in monocytes and neutrophils in whole blood by flow cytometry, constitutively, and after stimulation with Staphylococcus aureus and Pseudomonas aeruginosa. Nitric oxide production by monocytes was higher in septic patients compared with healthy volunteers for all conditions and by neutrophils at baseline, and ROS generation in monocytes and neutrophils was higher in septic patients than in healthy volunteers for all conditions. Nitric oxide production by monocytes and neutrophils was decreased at day 7 compared with that at admission (day 0) in survivors at baseline and after stimulation with S. aureus. Reactive oxygen species production by the monocytes and neutrophils was decreased in survivors at day 7 compared with day 0 under all conditions, except by neutrophils at baseline. No difference was found in NO and ROS generation by monocytes and neutrophils between day 7 and day 0 in nonsurvivors. Generation of NO and ROS by neutrophils and monocytes is increased in septic patients, and their persistence is associated with poor outcome.