Lih-Wen Deng

The Slow-Releasing Hydrogen Sulfide Donor, GYY4137, Exhibits Novel Anti-Cancer Effects In Vitro and In Vivo

The slow-releasing hydrogen sulfide (H2S) donor, GYY4137, caused concentration-dependent killing of seven different human cancer cell lines (HeLa, HCT-116, Hep G2, HL-60, MCF-7, MV4-11 and U2OS) but did not affect survival of normal human lung fibroblasts (IMR90, WI-38) as determined by trypan blue exclusion. Sodium hydrosulfide (NaHS) was less potent and not active in all cell lines. A structural analogue of GYY4137 (ZYJ1122) lacking sulfur and thence not able to release H2S was inactive. Similar results were obtained using a clonogenic assay. Incubation of GYY4137 (400 µM) in culture medium led to the generation of low (<20 µM) concentrations of H2S sustained over 7 days. In contrast, incubation of NaHS (400 µM) in the same way led to much higher (up to 400 µM) concentrations of H2S which persisted for only 1 hour. Mechanistic studies revealed that GYY4137 (400 µM) incubated for 5 days with MCF-7 but not IMR90 cells caused the generation of cleaved PARP and cleaved caspase 9, indicative of a pro-apoptotic effect. GYY4137 (but not ZYJ1122) also caused partial G2/M arrest of these cells. Mice xenograft studies using HL-60 and MV4-11 cells showed that GYY4137 (100–300 mg/kg/day for 14 days) significantly reduced tumor growth. We conclude that GYY4137 exhibits anti-cancer activity by releasing H2S over a period of days. We also propose that a combination of apoptosis and cell cycle arrest contributes to this effect and that H2S donors should be investigated further as potential anti-cancer agents.

Structural basis for the inhibition mechanism of human cystathionine gamma-lyase, an enzyme responsible for the production of H(2)S

Impairment of the formation or action of hydrogen sulfide (H2S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine β-synthase and cystathionine γ-lyase (CSE) are two pyridoxal-5′-phosphate (PLP)-dependent enzymes largely responsible for the production of H2S in mammals. Inhibition of CSE by dl-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP·PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H2S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE·PLP·PAG complex highlights the particular importance of Tyr114 in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

Regulation of Heart Function by Endogenous Gaseous Mediators—Crosstalk Between Nitric Oxide and Hydrogen Sulfide

Both nitric oxide (NO) and hydrogen sulfide (H(2)S) are two important gaseous mediators regulating heart function. The present study examined the interaction between these two biological gases and its role in the heart. We found that l-arginine, a substrate of NO synthase, decreased the amplitudes of myocyte contraction and electrically induced calcium transients. Sodium hydrogen sulfide (an H(2)S donor), which alone had minor effect, reversed the negative inotropic effects of l-arginine. The effect of l-arginine + sodium hydrogen sulfide was abolished by three thiols (l-cysteine, N-acetyl-cysteine, and glutathione), suggesting that the effect of H(2)S + NO is thiol sensitive. The stimulatory effect on heart contractility was also induced by GYY4137, a slow-releasing H(2)S donor, when used together with sodium nitroprusside, an NO-releasing donor. More importantly, enzymatic generation of H(2)S from recombinant cystathionine-γ-lyase protein also interacted with endogenous NO generated from l-arginine to stimulate heart contraction. In summary, our data suggest that endogenous NO may interact with H(2)S to produce a new biological mediator that produces positive inotropic effect. The crosstalk between H(2)S and NO also suggests an intriguing potential for the endogenous formation of a thiol-sensitive molecule, which may be of physiological significance in the heart.

Molecular basis for chromatin binding and regulation of MLL5

The human mixed-lineage leukemia 5 (MLL5) protein mediates hematopoietic cell homeostasis, cell cycle, and survival; however, the molecular basis underlying MLL5 activities remains unknown. Here, we show that MLL5 is recruited to gene-rich euchromatic regions via the interaction of its plant homeodomain finger with the histone mark H3K4me3. The 1.48-Å resolution crystal structure of MLL5 plant homeodomain in complex with the H3K4me3 peptide reveals a noncanonical binding mechanism, whereby K4me3 is recognized through a single aromatic residue and an aspartate. The binding induces a unique His–Asp swapping rearrangement mediated by a C-terminal α-helix. Phosphorylation of H3T3 and H3T6 abrogates the association with H3K4me3 in vitro and in vivo, releasing MLL5 from chromatin in mitosis. This regulatory switch is conserved in the Drosophila ortholog of MLL5, UpSET, and suggests the developmental control for targeting of H3K4me3. Together, our findings provide first insights into the molecular basis for the recruitment, exclusion, and regulation of MLL5 at chromatin.

Delineating the Site of Interaction on the pIII Protein of Filamentous Bacteriophage fd with the F-pilus of Escherichia coli

The minor coat protein pIII at one end of the filamentous bacteriophage fd, mediates the infection of Escherichia coli cells displaying an F-pilus. pIII has three domains (D1, D2 and D3), terminating with a short hydrophobic segment at the C-terminal end. Domain D2 binds to the tip of F-pilus, which is followed by retraction of the pilus and penetration of the E. coli cell membrane, the latter involving an interaction between domain D1 and the TolA protein in the membrane. Surface residues on the D2 domain of pIII were replaced systematically with alanine. Mutant virions were screened for D2-pilus interaction in vivo by measuring the release of infectious virions from E. coli F(+) cells infected with the mutants. A competitive ELISA was developed to measure in vitro the ability of mutant phages to bind to purified pili. This allowed the identification of amino acid residues involved in binding to F and to EDP208 pili. These residues were found to cluster on the outer rim of the 3D structure of the D2 domain, unexpectedly identifying this as the F-pilus binding region on the pIII protein.

Hydrogen sulfide donors in research and drug development

Hydrogen sulfide (H2S) has recently emerged as an important biological gasomediator as a result of numerous insightful studies. The use of H2S releasing compounds has attracted much attention as they can exert crucial effects on a wide range of cellular signaling processes. Some of these effects are potentially exploitable in terms of anti-inflammatory and anti-tumor effects, as well as precise ion-channel regulation, cardiovascular protection and oxidation resistance. Unfortunately, the potential therapeutic effects of H2S are controversial due to conflicting published results regarding its effects on cellular activities arising, perhaps in part, from the use of different H2S donors. Therefore it is essential to review the most commonly used H2S releasing compounds, some of which are currently in clinical trials along with their associated in vitro and/or in vivo biological effects.

Nickel(II) Dithiocarbamate Complexes Containing Sulforhodamine B as Fluorescent Probes for Selective Detection of Nitrogen Dioxide

We synthesized complexes of Ni(II) with dithiocarbamate ligands derived from the ortho and para isomers of sulforhodamine B fluorophores and demonstrated they are highly selective in reactions with nitrogen dioxide (NO2). Compared with the para isomer, the ortho isomer showed a much greater fluorescence increase upon reaction with NO2, which led to oxidation and decomplexation of the dithiocarbamate ligand from Ni(II). We applied this probe for visual detection of 1 ppm NO2 in the gas phase and fluorescence imaging of NO2 in macrophage cells treated with a nitrogen dioxide donor.

RNA interference against mixed lineage leukemia 5 resulted in cell cycle arrest

Mixed lineage leukemia 5 (MLL5) encodes a mammalian trithorax group (TrxG) protein located within chromosome band 7q22, which is a frequently deleted region found in acute myeloid malignancies. Trithorax and polycomb (PcG) group proteins are evolutionarily conserved transcriptional regulators that maintain the expression of Homeobox (HOX) genes at the epigenetic level during development. Recently, the emerging roles of TrxG and PcG group proteins in cell cycle regulation have begun to be elucidated. In this study, we demonstrated that the mammalian trxG protein MLL5 is involved in multiple cell cycle regulation. Knockdown of MLL5 by small interfering RNA resulted in the retarded cell growth and attenuated intake of BrdU in multiple tumor and normal diploid cells. The cell cycle arrest induced by knockdown of MLL5 took place at both the G1 and G2/M phases. This growth-inhibitory effect and dual-phase arrest were also found in p53-knockout cell lines, suggesting that the transactivation activity of p53 was dispensable for the MLL5-knockdown-mediated cell cycle arrest. In addition, up-regulation of cyclin-dependent kinase inhibitor p21 and de-phosphorylation of retinoblastoma protein were observed in all cell lines tested regardless of their p53 status. Taken together, our data suggest that silencing of MLL5 leads to up-regulation of p21 and dephosphorylation of pRb, which at least partially contributes to the G1 phase and G2/M phase arrest. These findings provide evidence that MLL5 might be an important cell cycle regulator, participating in cell cycle regulatory network machinery at multiple cell cycle stages.

Gene profiling reveals hydrogen sulphide recruits death signaling via the N-methyl-D-aspartate receptor identifying commonalities with excitotoxicity.

Recently the role of hydrogen sulphide (H2S) as a gasotransmitter stimulated wide interest owing to its involvement in Alzheimers disease and ischemic stroke. Previously we demonstrated the importance of functional ionotropic glutamate receptors (GluRs) by neurons is critical for H2S‐mediated dose‐ and time‐dependent injury. Moreover N‐methyl‐D‐aspartate receptor (NMDAR) antagonists abolished the consequences of H2S‐induced neuronal death. This study focuses on deciphering the downstream effects activation of NMDAR on H2S‐mediated neuronal injury by analyzing the time‐course of global gene profiling (5, 15, and 24 h) to provide a comprehensive description of the recruitment of NMDAR‐mediated signaling. Microarray analyses were performed on RNA from cultured mouse primary cortical neurons treated with 200 µM sodium hydrosulphide (NaHS) or NMDA over a time‐course of 5–24 h. Data were validated via real‐time PCR, western blotting, and global proteomic analysis. A substantial overlap of 1649 genes, accounting for over 80% of NMDA global gene profile present in that of H2S and over 50% vice versa, was observed. Within these commonly occurring genes, the percentage of transcriptional consistency at each time‐point ranged from 81 to 97%. Gene families involved included those related to cell death, endoplasmic reticulum stress, calcium homeostasis, cell cycle, heat shock proteins, and chaperones. Examination of genes exclusive to H2S‐mediated injury (43%) revealed extensive dysfunction of the ubiquitin‐proteasome system. These data form a foundation for the development of screening platforms and define targets for intervention in H2S neuropathologies where NMDAR‐activated signaling cascades played a substantial role. J. Cell. Physiol. 226: 1308–1322, 2011.

Highly Selective and Sensitive Near‐Infrared‐Fluorescent Probes for the Detection of Cellular Hydrogen Sulfide and the Imaging of H2S in Mice

Herein, we report the development of two fluorescent probes for the highly selective and sensitive detection of H2S. The probes take advantage of a Cu(II)-cyclen complex, which acts as a reaction center for H2S and as a quencher of BODIPY (boron-dipyrromethene)-based fluorophores with emissions at 765 and 680 nm, respectively. These non-fluorescent probes could only be turned on by the addition of H2 S, and not by other potentially interfering biomolecules, including reactive oxygen species, cysteine, and glutathione. In a chemical system, both probes detected H2S with a detection limit of 80 nM. The probes were successfully used for the endogenous detection of H2S in HEK 293 cells, for measuring the H2S-release activity of dietary organosulfides in MCF-7 cells, and for the in vivo imaging of H2S in mice.