The reaction of HOCl and cyanocobalamin: corrin destruction and the liberation of cyanogen chloride

Overproduction of hypochlorous acid (HOCl) has been associated with the development of a variety of disorders such as inflammation, heart disease, pulmonary fibrosis, and cancer through its ability to modify various biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl and heme destruction and free iron release from hemoglobin and RBCs. Our current findings extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl), a common pharmacological form of vitamin B12. Cyanocobalamin is a water-soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that although Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC, and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphonucleotide moiety. Additionally, it can liberate free Co, which can perpetuate metal-ion-induced oxidant stress. Taken together, these results are the first report of the generation of toxic molecular products through the interaction of Cobl with HOCl.     

PDCD6 additively cooperates with anti-cancer drugs through activation of NF-κB pathways

The expression of programmed cell death 6 (PDCD6) is known to be down-regulated in cancer cell lines and ovarian cancer tissues compared to normal cells and tissues. In the current study, we characterized the specific function of PDCD6 as a novel pro-apoptotic protein. To define the roles of PDCD6 and cisplatin in tumorigenesis, we either over-expressed PDCD6 or treated it with cisplatin in SKOV-3 ovarian cancer cells. Both PDCD6 and cisplatin respectively inhibited cancer cell proliferation in a dose-dependent manner. The combined treatment of PDCD6 and cisplatin was more effective at suppressing cell growth than with either drug treatment alone, but had no effect with the treatment of caspase-3 and caspase-9 inhibitors. Cleavages of caspase-3, -8, -9, and poly (ADP-ribose) polymerase (PARP) in PDCD6-overexpressing cells were significantly increased after cisplatin treatment. Cell cycle analysis highly correlated with down-regulation of cyclin D1 and CDK4, and the induction of p16 and p27 as a cyclin-dependent kinase inhibitor. Additionally, PDCD6 also suppressed the phosphorylation of signaling regulators downstream of PI3K, including PDK1 and Akt. PDCD6 promotes TNFα-dependent apoptosis through the activation of NF-κB signaling pathways, increasing Bax, p53, and p21 expression, while also down-regulating Bcl-2 and Bcl-xL expression. The p21 and p53 promoter luciferase activities were enhanced by PDCD6, while there was no affect in p53^−/− and p21^−/−. At the same time, p53 activity was confirmed by UV irradiation and siPDCD6. Taken together, these results provide evidence that PDCD6 can mediate the pro-apoptotic activity of cisplatin or TNFα through the down-regulation of NF-κB expression.     

Requirements for mouse mammary tumor virus Rem signal peptide processing and function

Mouse mammary tumor virus (MMTV) encodes a Rev-like protein, Rem, which is involved in the nuclear export and expression of viral RNA. Previous data have shown that all Rev-like functions are localized to the 98-amino-acid signal peptide (SP) at the N terminus of MMTV Rem or envelope proteins. MMTV-SP uses endoplasmic reticulum-associated degradation (ERAD) for protein trafficking. Rem cleavage by signal peptidase in the ER is necessary for MMTV-SP function in a reporter assay, but many requirements for trafficking are not known. To allow detection and localization of both MMTV-SP and the C-terminal cleavage product, we prepared plasmids expressing green fluorescent protein (GFP) tags. N-terminal Rem tagging led to protein accumulation relative to untagged Rem and allowed signal peptidase cleavage but reduced its specific activity. C-terminal tagging also led to Rem accumulation yet dramatically reduced cleavage, GFP fluorescence, and activity relative to N-terminally tagged Rem (GFPRem). Substitutions of an invariant leucine at position 71 between the known RNA-binding and nuclear export sequences interfered with GFPRem accumulation and activity but not cleavage. Similarly, deletion of 100 or 150 C-terminal amino acids from GFPRem dramatically reduced both Rem and MMTV-SP levels and function. Removal of the entire C terminus (203 amino acids) restored both protein levels and activity of MMTV-SP. Only C-terminal GFP tagging, and not other modifications, appeared to trap Rem in the ER membrane. Thus, Rem conformation in both the ER lumen and cytoplasm determines cleavage, retrotranslocation, and MMTV-SP function. These mutants further characterize intermediates in Rem trafficking and have implications for all proteins affected by ERAD.     

Synthesis of novel azo-resveratrol,azo-oxyresveratrol and their derivatives as potent tyrosinase inhibitors

Ten azo compounds including azo-resveratrol (5) and azo-oxyresveratrol (9) were synthesized using a modified Curtius rearrangement and diazotization followed by coupling reactions with various phenolic analogs. All synthesized compounds were evaluated for their mushroom tyrosinase inhibitory activity. Compounds 4 and 5 exhibited high tyrosinase inhibitory activity (56.25% and 72.75% at 50 μM, respectively). The results of mushroom tyrosinase inhibition assays indicate that the 4-hydroxyphenyl moiety is essential for high inhibition and that 3,5-dihydroxyphenyl and 3,5-dimethoxyphenyl derivatives are better for tyrosinase inhibition than 2,5-dimethoxyphenyl derivatives. Particularly, introduction of hydroxyl or methoxy group into the 4-hydroxyphenyl moiety diminished or significantly reduced mushroom tryosinase inhibition. Among the synthesized azo compounds, azo-resveratrol (5) showed the most potent mushroom tyrosinase inhibition with an IC₅₀ value of IC₅₀ = 36.28 ± 0.72 μM, comparable to that of resveratrol, a well-known tyrosinase inhibitor.     

Aqueous two-phase system-derived biofilms for bacterial interaction studies

We describe patterning of bacterial biofilms using polymer-based aqueous two-phase system (ATPS) microprinting protocols. The fully aqueous but selectively bacteria-partitioning nature of the ATPS allows spatially distinct localization of suspensions of bacteria such as Pseudomonas aeruginosa and Escherichia coli with high precision. The ATPS patterned bacterial suspensions form spatially distinct biofilms over time. Due to the fully aqueous and gentle noncontact printing procedures employed, coculture biofilms composed of multiple types of bacteria could be printed not only adjacent to each other but also directly over another layer of existing biofilm. In addition, the ATPS environment also allows free diffusion of small molecules between spatially distinct and localized bacterial suspensions and biofilms. This enables biofilms to chemically affect or be affected by neighboring biofilms or planktonic cells, even if they consist of different strains or species. We show that a β-lactamase producing biofilm confers ampicillin resistance to neighboring nonresistant planktonic cells, as seen by a 3,600-fold increase in survival of the ampicillin-sensitive strain. These examples demonstrate the ability of ATPS-based biofilm patterning methods to enable unique studies on commensalistic effects between bacterial species.     

sMEK1 enhances gemcitabine anti-cancer activity through inhibition of phosphorylation of Akt/mTOR

Recently, we reported that sMEK1 is down-regulated in cancer cells and tissues, and that it enhances the pro-proliferative effect as a novel pro-apoptotic protein. However, the biological mechanism of the sMEK1 tumor suppressor in the cellular signal pathway has not been well understood. In our current work, we examined whether sMEK1 could promote the cytotoxic activity of gemcitabine in the human ovarian carcinoma system. Initially, we attempted to use a treatment of gemcitabine traditional chemotherapeutic agent and over-expression of sMEK1 in OVCAR-3 cancer cells. The combined treatment of sMEK1 and gemcitabine was more effective at inhibiting cell proliferation than either chemotherapeutic agent treatment alone. In addition, sMEK1 actively contributes to cell migration through its ability to promote gemcitabine-inhibited cell migration in tumorigenesis. Cell cycle-related proteins are highly associated with the down-regulation of cyclin D1 and CDK4, and the promotion of p16 and p27 as a cyclin-dependent kinase inhibitor. At the same time, sMEK1 arrests cell cycle progression in the G(1)-G(0) phase, and activates p53 and p21 expression, whereas Bcl-2 and Bcl-xL protein expression is reduced. Additionally, sMEK1 and gemcitabine suppresses the phosphorylation of signaling modulators downstream of PI3K, such as PDK1 and Akt. The p53 and p21 promoter luciferase activities were promoted by either sMEK1 or gemcitabine, and sMEK1 and gemcitabine combined additively activated the promoter further. Furthermore, as expected, sMEK1 plus gemcitabine markedly reduced the phosphorylation of p70S6K and the phosphorylation of 4E-BP1, which is one of the best characterized targets of the mTOR complex cascade. Taken together, these results provide evidence that sMEK1 can effectively regulate the pro-apoptotic activity of gemcitabine through the up-regulation of p53 expression.