Effects of Sulforaphane-Induced Cell Death upon Repeated Passage of Either P-Glycoprotein-Negative or P-Glycoprotein-Positive L1210 Cell Variants.

The expression of the membrane ABCB1 transporter in neoplastic cells is one of the most common causes of reduced sensitivity to chemotherapy. In our previous study, we investigated the effect of a single culture of ABCB1-negative (S) and ABCB1-positive variants of L1210 cells (R and T) in the presence of sulforaphane (SFN). We demonstrated that SFN induces the onset of autophagy more markedly in S cells than in R or T cells. In the current study, we focused on the effect of the repeated culture of S, R and T cells in SFN-containing media. The repeated cultures increased the onset of autophagy compared to the simple culture, mainly in S cells and to a lesser extent in R and T cells, as indicated by changes in the cellular content of 16 and 18 kDa fragments of LC3B protein or changes in the specific staining of cells with monodansylcadaverine. We conclude that SFN affects ABCB1-negative S cells more than ABCB1-positive R and T cells during repeated culturing. Changes in cell sensitivity to SFN appear to be related to the expression of genes for cell-cycle checkpoints, such as cyclins and cyclin-dependent kinases.

Cell Death Effects Induced by Sulforaphane and Allyl Isothiocyanate on P-Glycoprotein Positive and Negative Variants in L1210 Cells.

Variants of L1210 leukemia cells-namely, parental P-glycoprotein-negative S cells and R and T cells expressing P-glycoprotein, due to selection with vincristine and transfection with the human p-glycoprotein gene, respectively-were used. The responses of these cell variants to two naturally occurring isothiocyanates-sulforaphane (SFN, from cruciferous vegetables) and allyl isothiocyanate (AITC, from mustard, radish, horseradish and wasabi)-were studied. We obtained conflicting results for the cell death effects induced by isothiocyanates, as measured by i. cell counting, which showed inhibited proliferation, and ii. cell metabolic activity via an MTS assay, which showed an increased MTS signal. These results indicated the hyperactivation of cell metabolism induced by treatment with isothiocyanates. In more detailed study, we found that, depending on the cell variants and the isothiocyanate used in treatment, apoptosis and necrosis (detected by annexin-V cells and propidium iodide staining), as well as autophagy (detected with monodansylcadaverine), were involved in cell death. We also determined the cell levels/expression of Bcl-2 and Bax as representative anti- and pro-apoptotic proteins of the Bcl-2 family, the cell levels/expression of members of the canonical and noncanonical NF-κB pathways, and the cell levels of 16 and 18 kDa fragments of LC3B protein as markers of autophagy.

Low molecular thiols, pH and O2 modulate H2S-induced S-nitrosoglutathione decomposition - •NO release.

We studied the involvement of O2, pH and low molecular thiols in H2S-induced decomposition of S-nitrosoglutathione (GSNO). The GSNO decomposition - •NO release was evaluated by UV-VIS spectroscopy and Griess assay. The H2S donor Na2S was used. O2 slightly increased, but was not necessary for the H2S-induced GSNO decomposition. The rate of GSNO decomposition depended on pH; the maximum rate was observed at pH 7.4-8.0, and this decreased with lowering pH (6.4-4.5) as well as with increasing pH at 9.0-12.0. H2S-induced GSNO decomposition was slowed by the presence of other thiols, such as L-cysteine (Cys), N-acetyl-L-cysteine (NAC) and L-glutathione (GSH), but not in the presence of L-methionine (Met) or oxidized glutathione (GSSG). In sharp contrast, at pH 6.0, H2S-induced GSNO decomposition was negligible, yet the presence of Cys, NAC and GSH induced the H2S-driven GSNO decomposition (whilst Met and GSSG were inactive). In conclusion we postulate an involvement of low molecular thiols and pH in •NO signaling, by modulating the interactions of H2S with nitroso compounds, and hence in part they also appear to control H2S-triggered •NO release. The interaction of H2S and/or its derivatives with the thiol group may be responsible for the observed effects.

The aqueous garlic, onion and leek extracts release nitric oxide from S-nitrosoglutathione and prolong relaxation of aortic rings.

Garlic, onion and leek have beneficial effects in treatment of numerous health disorders. The aim of the present study was to investigate underlying molecular mechanisms. To test the potency of the aqueous garlic, onion and leek extracts to release NO from GSNO we have measured NO oxidation product, NO(2)-, by the Griess reagent method. Further, we studied the ability of garlic extract to relax noradrenaline-precontracted rat aortic rings in the presence of GSNO and effects of garlic extract on electrical properties of rat heart intracellular chloride channels. We have observed that: i) garlic, onion and leek extracts released NO from GSNO in the order: garlic > onion > leek; ii) the ability of garlic extract to release NO was pH-dependent (8.0 > 7.4 > 6.0) and potentiated by thiols (Cys >> GSH = N-acetyl-cysteine > oxidized glutathione) at concentration 100 µmol/l; iii) the garlic extract (0.045 mg/ml) prolonged relaxation time of aortic rings induced by GSNO (50 nmol/l) and inhibited intracellular chloride channels. We suggest that NO-releasing properties of the garlic, onion and leek extracts and their interaction with Cys and GSH are involved in NO-signalling pathway which contributes to some of its numerous beneficial biological effects.

On the involvement of H2S in nitroso signaling and other mechanisms of H2S action.

Both endogenously produced and exogenously administered H2S exert numerous biological effects. However, the molecular mechanisms underlying these effects are not fully understood. This review surveys the biological effects of H2S and summarizes the molecular mechanisms of H2S action. It focuses on the role of H2S/HS--induced NO release from nitroso compounds, modulation of ion channels and the antioxidant and radical properties of H2S in the molecular mechanism of its effects. The potential involvement of H2S in nitroso signaling underlying its diverse biological effects is also discussed.

The hypothesis of the main role of H2S in coupled sulphide-nitroso signalling pathway.

As a part of the nitroso signalling pathway, nitroso-compounds serve as stores and carriers of NO; as part of the sulphide signalling pathway, bound sulfane-sulphur compounds serve as stores and carriers of H2S. Here we hypothesise a coupled sulphide-nitroso signalling pathway, in which H2S plays a main role. H2S releases NO from the endogenous S-nitroso-compounds nitroso-cysteine, nitroso-acetylcysteine and nitroso-albumin. Relaxation of noradrenaline-precontracted aortic rings by H2S is also enhanced in the presence of nitroso-albumin, which may implicate the involvement of the nitroso signalling pathway. Pretreatment of albumin, cysteine, N-acetylcysteine and lipids with H2S results in binding of sulphur to these compounds creating thus new-modified sulphur compounds that release NO from nitroso-compounds directly and/or through released H2S, which suggests sulphide-nitroso signalling pathway participation. This hypothesis is supported by the observation that the pretreatment of noradrenaline-precontracted aortic rings with H2S significantly enhanced relaxation induced by nitroso-glutathione in the absence of H2S. We assume that the NO release from nitroso-compounds directly by H2S or indirectly by the H2S-induced sulphur-bound compounds represents coupled sulphide-nitroso signalling, which may explain some of the numerous biological effects of H2S that are shared with NO.