Cell-free Conditions Research Articles

Adsorption of lactate dehydrogenase enzyme on carbon nanotubes: how to get accurate results for the cytotoxicity of these nanomaterials.

Carbon nanotube (CNT) cytotoxicity is frequently investigated using in vitro classical toxicology assays. However, these cellular tests, usually based on the use of colorimetric or fluorimetric dyes, were designed for chemicals and may not be suitable for nanosized materials. Indeed, because of their unique physicochemical properties CNT can interfere with the assays and bias the results. To get accurate data and draw reliable conclusions, these artifacts should be carefully taken into account. The aim of this study was to evaluate qualitatively and quantitatively the interferences occurring between CNT and the commonly used lactate dehydrogenase (LDH) assay. Experiments under cell-free conditions were performed, and it was clearly demonstrated that artifacts occurred. They were due to the intrinsic absorbance of CNT on one hand and the adsorption of LDH at the CNT surface on the other hand. The adsorption of LDH on CNT was modeled and was found to fit the Langmuir model. The K(ads) and n(eq) constants were defined, allowing the correction of results obtained from cellular experiments to get more accurate data and lead to proper conclusions on the cytotoxicity of CNT.

Macrocyclic compounds as anti-cancer agents: Design and synthesis of multi-acting inhibitors against HDAC, FLT3 and JAK2

A novel series of macrocyclic compounds were designed and synthesized as multi-target inhibitors targeting HDAC, FLT3 and JAK2. Some of these compounds exhibited potent HDAC inhibition as well as FLT3 and JAK2 inhibition under both cell-free and cellular conditions. In vitro antiproliferative assay indicated that these compounds were interestingly more cytotoxic to MV4-11 cells bearing FLT3-ITD mutation and HEL cells bearing JAK2V617F mutation.

Inactivation of Protein Tyrosine Phosphatases by Peracids Correlates with the Hydrocarbon Chain Length

Background/Aims: Protein tyrosine phosphatases are crucial enzymes controlling numerous physiological and pathophysiological events and can be regulated by oxidation of the catalytic domain cysteine residue. Peracids are highly oxidizing compounds, and thus may induce inactivation of PTPs. The aim of the present study was to evaluate the inhibitory effect of peracids with different length of hydrocarbon chain on the activity of selected PTPs. Methods: The enzymatic activity of human CD45, PTP1B, LAR, bacterial YopH was assayed under the cell-free conditions, and activity of cellular CD45 in human Jurkat cell lysates. The molecular docking and molecular dynamics were performed to evaluate the peracids binding to the CD45 active site. Results: Here we demonstrate that peracids reduce enzymatic activity of recombinant CD45, PTP1B, LAR, YopH and cellular CD45. Our studies indicate that peracids are more potent inhibitors of CD45 than hydrogen peroxide (with an IC50 value equal to 25 nM for peroctanoic acid and 8 µM for hydrogen peroxide). The experimental data show that the inactivation caused by peracids is dependent on hydrocarbon chain length of peracids with maximum inhibitory effect of medium-chain peracids (C8-C12 acyl chain), which correlates with calculated binding affinities to the CD45 active site. Conclusion: Peracids are potent inhibitors of PTPs with the strongest inhibitory effect observed for medium-chain peracids.

Sonic hedgehog processing and release are regulated by glypican heparan sulfate proteoglycans.

All Hedgehog morphogens are released from producing cells, despite being synthesized as N- and C-terminally lipidated molecules, a modification that firmly tethers them to the cell membrane. We have previously shown that proteolytic removal of both lipidated peptides, called shedding, releases bioactive Sonic hedgehog (Shh) morphogens from the surface of transfected Bosc23 cells. Using in vivo knockdown together with in vitro cell culture studies, we now show that glypican heparan sulfate proteoglycans regulate this process, through their heparan sulfate chains, in a cell autonomous manner. Heparan sulfate specifically modifies Shh processing at the cell surface, and purified glycosaminoglycans enhance the proteolytic removal of N- and C-terminal Shh peptides under cell-free conditions. The most likely explanation for these observations is direct Shh processing in the extracellular compartment, suggesting that heparan sulfate acts as a scaffold or activator for Shh ligands and the factors required for their turnover. We also show that purified heparan sulfate isolated from specific cell types and tissues mediates the release of bioactive Shh from pancreatic cancer cells, revealing a previously unknown regulatory role for these versatile molecules in a pathological context.

Cell-free reconstitution of multivesicular body (MVB) cargo sorting.

The signaling activity of cell surface localized membrane proteins occurs primarily while these proteins are located on the plasma membrane but is, in some cases, not terminated until the proteins are degraded. Following internalization and movement through the endocytic pathway en route to lysosomes, membrane proteins transit a late endosomal organelle called the multivesicular body (MVB). MVBs are formed by invagination of the limiting membrane of endosomes, resulting in an organelle possessing a limiting membrane and containing internal vesicles. The fate of an internalized membrane protein depends on whether it buds outwardly from the endosomal membrane, promoting recycling and continued signaling, or is internalized into internal MVB vesicles and is ultimately degraded upon MVB-lysosome fusion. The molecular machinery that regulates the separation of membrane proteins destined for degradation from those resulting in surface expression is not well understood.To elucidate the molecular mechanisms that underlie membrane protein sorting, we have reconstituted an endosomal sorting event under cell-free conditions. We took advantage of the itinerary of a prototypical membrane protein, the epidermal growth factor receptor (EGFR) and designed a biochemical monitor for cargo movement into internal MVB vesicles that is generally modifiable for other membrane proteins. Since is it not known how internal vesicle formation is related to cargo sorting, morphological examination using transmission electron microscopy (TEM) allows separate monitoring of vesicle formation. We have determined that MVB sorting is dependent on cytosolic components, adenosine triphosphate (ATP), time, temperature, and an intact proton gradient. This assay reconstitutes the maturation of late endosomes and allows the morphological and biochemical examination of vesicle formation and membrane protein sorting.

The mycotoxin patulin reacts with DNA bases with and without previous conjugation to GSH: implication for related α,β-unsaturated carbonyl compounds?

The α,β-unsaturated carbonyl group is recognized as alert for mutagenicity, attributed to (1) its direct reaction with DNA, counteractable by glutathione (GSH), and (2) oxidative stress caused indirectly by GSH depletion. Accordingly, the α,β,γ,δ-unsaturated lactone patulin (PAT), a mycotoxin detected in fruits and products derived thereof, is known to induce gene, chromosome, and genome mutations in vitro, its mutagenicity correlating inversely with intracellular GSH levels. Thus, the reactivity of PAT against DNA bases and nucleosides in the absence and presence of GSH and glutathione S-transferases (GSTs) was investigated under cell-free conditions using HPLC mass spectrometry techniques for identification of reaction products. Adduct formation with all four nucleobases as well as with purine base nucleosides occurred even in the presence of GSH, revealing several adducts of PAT, mono- and disubstituted with nucleobases/nucleosides as well as novel GSH-PAT adducts. In addition, novel mixed GSH-PAT-nucleobase adducts were observed. These adducts exhibited a ketohexanoic acid-type structure of the PAT molecule, C6 substituted with GSH and linking C1 of PAT with nitrogens of nucleobases/nucleosides via an amide bond. Formation of GSH-PAT-adenine adducts was not prevented by GSTs, and excess of GSH needed to reduce their formation was higher than for PAT-adenine adducts. The formation of mixed GSH-DNA base adducts has not been described for PAT or any other α,β-unsaturated carbonyl before, although the reaction mechanism seems to be applicable to a variety of α,β-unsaturated carbonyls occurring in food and in the environment.

Thioredoxin-mimetic peptides as catalysts of S-denitrosylation and anti-nitrosative stress agents

S-nitrosylation, the coupling of a nitric oxide moiety to a reactive cysteine residue to form an S-nitrosothiol (SNO), is an important posttranslational mechanism for regulating protein activity. Growing evidence indicates that hyper-S-nitrosylation may contribute to cellular dysfunction associated with various human diseases. It is also increasingly appreciated that thioredoxin and thioredoxin reductase play significant roles in the cellular catabolism of SNO and protection from nitrosative stress. Here, we investigated the SNO reductase activity and protective effects of thioredoxin-mimetic peptides (TXMs), Ac-Cys-Pro-Cys-amide (CB3) and Ac-Cys-Gly-Pro-Cys-amide (CB4), both under cell-free conditions and in nitrosatively stressed cultured cells. In vitro biochemical analyses revealed that the TXM peptides reduced small-molecule SNO compounds, such as S-nitrosoglutathione (GSNO), and acted as general and efficient protein-denitrosylating agents. In particular, CB3 was found to be a highly potent SNO-metabolizing agent. Notably, CB3 mimicked the activity of thioredoxin by coupling with thioredoxin reductase to enhance GSNO reduction. Moreover, in a cell-free lysate system, both CB3 and CB4 synergized with an NADPH-dependent activity to denitrosylate proteins. Further investigation revealed that the TXM peptides protect the peroxiredoxin–thioredoxin system from SNO-dependent inhibition. Indeed, SNO-inhibited Prx1 was efficiently denitrosylated and reactivated by CB3 or CB4. In addition, CB3 protected thioredoxin reductase from SNO-mediated inactivation both in vitro and in intact cells. Finally, CB3 and CB4 partially rescued human neuroblastoma SH-SY5Y cells and rat insulinoma INS-1 832/13 cells from GSNO-induced growth inhibition. Collectively, the present findings indicate the efficient denitrosylation activity and protective effects of TXM peptides and suggest their potential therapeutic value in treating pathological conditions related to nitrosative stress.

Bicyclic tetrapeptide histone deacetylase inhibitors with methoxymethyl ketone and boronic acid zinc-binding groups

Histone deacetylase (HDAC) inhibitors are a class of potential therapeutics for the treatment of cancer. Bicyclic tetrapeptides equipped with methoxymethyl ketone and boronic acid as zinc-binding group were designed and synthesized. The inhibitory activities of these compounds were evaluated against HDAC enzymes. The cell-free and cell-based assay data showed that both potency and selectivity changed with the change in zinc-binding group. Boronic acid-based compound showed poor activity whereas methoxymethyl ketone-based compound displayed impressive activity in both cell-free and cell-based conditions.

Graphene-polydopamine-C60 nanohybrid: an efficient protective agent for NO-induced cytotoxicity in rat pheochromocytoma cells.

Here we have designed and developed a facile and mild synthetic approach to prepare a graphene-polydopamine-C60 nanohybrid for biological applications. Graphene oxide (GO) is surface modified and reduced via polydopamine (PDA) chemistry. Using nucleophilic addition, the rGO-PDA-C60 nanohybrid is then prepared. Benefiting from the unique nanostructure, the versatile hybrid of rGO-PDA-C60 limits the aggregation of C60 and enhances the cell uptake, which displays synergistic protective effects on the NO-induced cytotoxicity. Investigation of the possible NO-scavenging activities of rGO-PDA-C60 demonstrates that it expresses direct scavenging activity toward NO both under cell and cell free conditions. As expected, rGO-PDA-C60 prevents NO-induced cytotoxicity and blocks the following apoptosis events, such as lipid peroxidation, mitochondrial depolarization, and caspase-3 activation. The study presents the potential of nanocarbons for preventing the NO-mediated cell injury.

In Vitro Antitumor Mechanism of (E)-N-(2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)pyridin-3-yl)methanesulfonamide

ON01910.Na [sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate; Rigosertib, Estybon], a styryl benzylsulfone, is a phase III stage anticancer agent. This non-ATP competitive kinase inhibitor has multitargeted activity, promoting mitotic arrest and apoptosis. Extensive phase I/II studies with ON01910.Na, conducted in patients with solid tumors and hematologic cancers, demonstrate excellent efficacy. However, issues remain affecting its development. These include incomplete understanding of antitumor mechanisms, low oral bioavailability, and unpredictable pharmacokinetics. We have identified a novel (E)-styrylsulfonyl methylpyridine [(E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide (TL-77)] which has shown improved oral bioavailability compared with ON01910.Na. Here, we present detailed cellular mechanisms of TL-77 in comparison with ON01910.Na. TL-77 displays potent growth inhibitory activity in vitro (GI50 < 1μM against HCT-116 cells), demonstrating 3- to 10-fold greater potency against tumor cell lines when compared with normal cells. Cell-cycle analyses reveal that TL-77 causes significant G2/M arrest in cancer cells, followed by the onset of apoptosis. In cell-free conditions, TL-77 potently inhibits tubulin polymerization. Mitotically arrested cells display multipolar spindles and misalignment of chromosomes, indicating that TL-77 interferes with mitotic spindle assembly in cancer cells. These effects are accompanied by induction of DNA damage, inhibition of Cdc25C phosphorylation [indicative of Plk1 inhibition], and downstream inhibition of cyclin B1. However, kinase assays failed to confirm inhibition of Plk1. Nonsignificant effects on phosphoinositide 3-kinase/Akt signal transduction were observed after TL-77 treatment. Analysis of apoptotic signaling pathways reveals that TL-77 downregulates expression of B-cell lymphoma 2 family proteins (Bid, Bcl-xl, and Mcl-1) and stimulates caspase activation. Taken together, TL-77 represents a promising anticancer agent worthy of further evaluation.

Polyproline promotes tetramerization of recombinant human butyrylcholinesterase.

Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers and monomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90-98% of purified rBChE (65μM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100μM) for 1.5h at 25°C. However, rBChE tetramerization was inefficient with smaller 8-mer polyproline peptides and larger 300-mer polyproline proteins. Collectively, these studies demonstrated that the eukaryotic cellular machinery is not required for assembly of active BChE into tetramers and that this process can occur invitro with purified rBChE in the presence of peptides containing 15-50 consecutive proline residues.

Excision of Uracil from Transcribed DNA Negatively Affects Gene Expression

Uracil is an unavoidable aberrant base in DNA, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires a succession of intermediate products, including an abasic site and a single strand break, before the original DNA structure can be reconstituted. These repair intermediates are harmful for DNA replication and also interfere with transcription under cell-free conditions. However, their relevance for cellular transcription has not been proved. Here we investigated the influence of uracil incorporated into a reporter vector on gene expression in human cells. The expression constructs contained a single uracil opposite an adenine (to mimic dUTP misincorporation during DNA synthesis) or a guanine (imitating a product of spontaneous cytosine deamination). We found no evidence for a direct transcription arrest by uracil in either of the two settings because the vectors containing the base modification exhibited unaltered levels of enhanced GFP reporter gene expression at early times after delivery to cells. However, the gene expression showed a progressive decline during subsequent hours. In the case of U:A pairs, this effect was retarded significantly by knockdown of UNG1/2 but not by knockdown of SMUG1 or thymine-DNA glycosylase uracil-DNA glycosylases, proving that it is base excision by UNG1/2 that perturbs transcription of the affected gene. By contrast, the decline of expression of the U:G constructs was not influenced by either UNG1/2, SMUG1, or thymine-DNA glycosylase knockdown, strongly suggesting that there are substantial mechanistic or kinetic differences between the processing of U:A and U:G lesions in cells.

Melanogenesis inhibitory effect of aerial part of Pueraria thunbergiana in vitro and in vivo.

Melanin is major factor that determines skin color as well as one of the defense systems that prevent the UV-induced damage. In case of abnormal concentration of melanin, skin diseases or problems occur such as albinism, leukoplakia, melasma, freckles, moles, and lentigo. With the lifespan of humans has been extended, importance of ‘life quality’ has been increased. White and clean skin is very important part of the satisfaction of appearance, especially for Asia women. The aim of this study was to find an anti-melanogenesis activity for which the aerial part of Pueraria thunbergiana can be utilized based on the increase in demands for cosmetics, particularly natural products. We demonstrated anti-pigmentation effects of aerial part of P. thunbergiana by measuring melanin content and through staining in the B16F10 melanoma cell line. The aerial part of P. thunbergiana decreased tyrosinase activity significantly in B16F10 cell cultures, while there is no direct effect on enzyme in cell-free conditions. To define the mechanisms, real-time PCR, western blot, glucosidase activity and antioxidant activity assay were implemented. As results, we demonstrated that aerial part of P. thunbergiana has anti-melanogenesis activity via two mechanisms. One is downgrading microphthalmia-associated transcription factor by activating Akt/GSK-3β. Consequently, transcription of tyrosinase and tyrosinase-related protein 1 is decreased. Another is interrupting maturation of tyrosinase through inhibiting α-glucosidase. Furthermore, aerial part of P. thunbergiana showed great efficacy on pigmentation in vivo. These results suggest that aerial part of P. thunbergiana can be used as an anti-melanogenic agent.

Different effects of two cyclic chalcone analogues on redox status of Jurkat T cells

Chalcones are intermediary compounds of the biosynthetic pathway of the naturally flavonoids. Previous studies have demonstrated that chalcones and their conformationally rigid cyclic analogues have tumour cell cytotoxic and chemopreventive effects. It has been shown that equitoxic doses of the two cyclic chalcone analogues (E)-2-(4′-methoxybenzylidene)-(2) and (E)-2-(4′-methylbenzylidene)-1-benzosuberone (3) have different effect on cell cycle progress of the investigated Jurkat cells. It was also found that the compounds affect the cellular thiol status of the treated cells and show intrinsic (non-enzyme-catalyzed) reactivity towards GSH under cell-free conditions. In order to gain new insights into the cytotoxic mechanism of the compounds, effects on the redox status and glutathione level of Jurkat cells were investigated. Detection of intracellular ROS level in Jurkat cells exposed to 2 and 3 was performed using the dichlorofluorescein-assay. Compound 2 did not influence ROS activity either on 1 or 4h exposure; in contrast, chalcone 3 showed to reduce ROS level at both timepoints. The two compounds had different effects on cellular glutathione status as well. Compound 2 significantly increased the oxidized glutathione (GSSG) level showing an interference with the cellular antioxidant defence. On the contrary, chalcone 3 enhanced the reduced glutathione level, indicating enhanced cellular antioxidant activity. To investigate the chalcone–GSH conjugation reactions under cellular conditions, a combination of a RP-HPLC method with electrospray ionization mass spectrometry (ESI-MS) was performed. Chalcone–GSH adducts could not be observed either in the cell supernatant or the cell sediment after deproteinization. The investigations provide further details of dual – cytotoxic and chemopreventive – effects of the cyclic chalcone analogues.

The radiomimetic enediyne, 20'-deschloro-C-1027 induces inter-strand DNA crosslinks in hypoxic cells and overcomes cytotoxic radioresistance.

The ability of the radiomimetic anti-tumor enediyne C-1027 to induce DNA inter-strand crosslinks (ICLs), in addition to the expected DNA strand breaks, is unique among traditional DNA targeted cancer therapies. Importantly, radiation therapy and most radiomimetic drugs have diminished effect in hypoxic environments due to decreased induction of DNA strand breaks, which is an oxygen requiring process. However, C-1027's induction of ICLs is enhanced under hypoxia and it is actually more potent against hypoxic cells, overcoming this common tumor resistance mechanism. In this study, an analog of C-1027, 20'-deschloro-C-1027 was examined for its ability to induce DNA ICLs under hypoxic conditions. Deschloro-induced ICLs were detected under hypoxic cell-free conditions, with a concomitant reduction in the induction of DNA strand breaks. In cells deschloro behaved similarly, inducing cellular ICLs under hypoxic conditions with a reduction in DNA breaks. The cytotoxicity of deschloro treatment was similar in normoxic and hypoxic cells, suggesting that the ICL induction allows deschloro to retain its cytotoxic activity under hypoxia. It appears that rational engineering of the C-1027 family of radiomimetics holds promise toward overcoming the radioresistance associated with the hypoxic environment associated with solid tumors.

Exposure to inhomogeneous static magnetic field beneficially affects allergic inflammation in a murine model

Previous observations suggest that static magnetic field (SMF)-exposure acts on living organisms partly through reactive oxygen species (ROS) reactions. In this study, we aimed to define the impact of SMF-exposure on ragweed pollen extract (RWPE)-induced allergic inflammation closely associated with oxidative stress. Inhomogeneous SMF was generated with an apparatus validated previously providing a peak-to-peak magnetic induction of the dominant SMF component 389 mT by 39 T m(-1) lateral gradient in the in vivo and in vitro experiments, and 192 mT by 19 T m(-1) in the human study at the 3 mm target distance. Effects of SMF-exposure were studied in a murine model of allergic inflammation and also in human provoked skin allergy. We found that even a single 30-min exposure of mice to SMF immediately following intranasal RWPE challenge significantly lowered the increase in the total antioxidant capacity of the airways and decreased allergic inflammation. Repeated (on 3 consecutive days) or prolonged (60 min) exposure to SMF after RWPE challenge decreased the severity of allergic responses more efficiently than a single 30-min treatment. SMF-exposure did not alter ROS production by RWPE under cell-free conditions, while diminished RWPE-induced increase in the ROS levels in A549 epithelial cells. Results of the human skin prick tests indicated that SMF-exposure had no significant direct effect on provoked mast cell degranulation. The observed beneficial effects of SMF are likely owing to the mobilization of cellular ROS-eliminating mechanisms rather than direct modulation of ROS production by pollen NAD(P)H oxidases.

Extracellular proteolysis of reelin by tissue plasminogen activator following synaptic potentiation

The secreted glycoprotein reelin plays an indispensable role in neuronal migration during development and in regulating adult synaptic functions. The upstream mechanisms responsible for initiating and regulating the duration and magnitude of reelin signaling are largely unknown. Here we report that reelin is cleaved between EGF-like repeats 6–7 (R6–7) by tissue plasminogen activator (tPA) under cell-free conditions. No changes were detected in the level of reelin and its fragments in the brains of tPA knockouts, implying that other unknown proteases are responsible for generating reelin fragments found constitutively in the adult brain. Induction of NMDAR-independent long-term potentiation with the potassium channel blocker tetraethylammonium chloride (TEA-Cl) led to a specific up-regulation of reelin processing at R6–7 in wild-type mice. In contrast, no changes in reelin expression and processing were observed in tPA knockouts following TEA-Cl treatment. These results demonstrate that synaptic potentiation results in tPA-dependent reelin processing and suggest that extracellular proteolysis of reelin may regulate reelin signaling in the adult brain.

The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.

The chemokine domain of fractalkine (FKN-CD) binds to the classical RGD-binding site of αvβ3 and that the resulting ternary complex formation (integrin-FKN-CX3CR1) is critical for CX3CR1 signaling and FKN-induced integrin activation. However, only certain cell types express CX3CR1. Here we studied if FKN-CD can activate integrins in the absence of CX3CR1. We describe that WT FKN-CD activated recombinant soluble αvβ3 in cell-free conditions, but the integrin-binding defective mutant of FKN-CD (K36E/R37E) did not. This suggests that FKN-CD can activate αvβ3 in the absence of CX3CR1 through the direct binding of FKN-CD to αvβ3. WT FKN-CD activated αvβ3 on CX3CR1-negative cells (K562 and CHO) but K36E/R37E did not, suggesting that FKN-CD can activate integrin at the cellular levels in a manner similar to that in cell-free conditions. We hypothesized that FKN-CD enhances ligand binding to the classical RGD-binding site (site 1) through binding to a second binding site (site 2) that is distinct from site 1 in αvβ3. To identify the possible second FKN-CD binding site we performed docking simulation of αvβ3-FKN-CD interaction using αvβ3 with a closed inactive conformation as a target. The simulation predicted a potential FKN-CD-binding site in inactive αvβ3 (site 2), which is located at a crevice between αv and β3 on the opposite side of site 1 in the αvβ3 headpiece. We studied if FKN-CD really binds to site 2 using a peptide that is predicted to interact with FKN-CD in site 2. Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD. This suggests that FKN-CD actually binds to site 2, and this leads to integrin activation. We obtained very similar results in α4β1 and α5β1. The FKN binding to site 2 and resulting integrin activation may be a novel mechanism of integrin activation and of FKN signaling.

Caspase-3 and caspase-6 cleave STAT1 in leukemic cells.

Signal Transducer and Activator of Transcription-1 (STAT1) is phosphorylated upon interferon (IFN) stimulation, which can restrict cell proliferation and survival. Nevertheless, in some cancers STAT1 can act in an anti-apoptotic manner. Moreover, certain malignancies are characterized by the overexpression and constitutive activation of STAT1. Here, we demonstrate that the treatment of transformed hematopoietic cells with epigenetic drugs belonging to the class of histone deacetylase inhibitors (HDACi) leads to the cleavage of STAT1 at multiple sites by caspase-3 and caspase-6. This process does not occur in solid tumor cells, normal hematopoietic cells, and leukemic cells that underwent granulocytic or monocytic differentiation. STAT1 cleavage was studied under cell free conditions with purified STAT1 and a set of candidate caspases as well as with mass spectrometry. These assays indicate that unmodified STAT1 is cleaved at multiple sites by caspase-3 and caspase-6. Our study shows that STAT1 is targeted by caspases in malignant undifferentiated hematopoietic cells. This observation may provide an explanation for the selective toxicity of HDACi against rapidly proliferating leukemic cells.

Template-directed synthesis of a small molecule-antisense conjugate targeting an mRNA structure

The targeting of structural features in mRNA with specificity remains a great chemical challenge. A hairpin structure near exon 10 in the pre-mRNA encoding the tau protein controls its splicing, and dementia-causing mutations that disrupt this structure increase exon 10 splicing. We previously reported the discovery of small molecules, mitoxantrone (MTX) and analogs, which bind to the tau RNA hairpin structure and the design of bipartite antisense oligonucleotides (ASOs) that simultaneously bind to the discontinuous sequences that flank this hairpin. Herein we report the synthesis of a bipartite ASO conjugated to MTX using the tau RNA hairpin and flanking sequences as a template. A set of six MTX analogs, each containing a linker-azide, and a set of ten bipartite ASOs, each containing a branched linker-alkyne, were synthesized and tested in combinatorial fashion for their ability to conjugate in the presence or absence of template RNA. A single template-dependent MTX–ASO conjugate was identified from among the 60 reaction mixtures, demonstrating that the MTX and ASO precursors could simultaneously bind the RNA template and allow proper positioning of azide and alkyne for 1,3-cycloaddition. While the MTX–ASO conjugate proved too cytotoxic for cell-based assays, the conjugate inhibited tau exon 10 splicing under cell-free conditions more effectively than MTX or bipartite ASO alone.