In vitro anticancer studies of α- and β-d-glucopyranose betulin anomers

Four derivatives of betulin containing a d-glucopyranosyl moiety at C3 position were synthesized and characterized by ¹H and ¹³C NMR spectroscopy as well as mass spectrometry. The crystal structure of 28-O-acetylbetulin-3-yl-β-d-(2′,3′,4′,6′-tetra-O-acetyl)glucopyranoside was determined. The compounds were tested against fifteen tumor cell lines of different histogenic origins. The α- and β-anomers of 28-O-acetylbetulin-3-yl-d-glucopyranoside, exerted a dose dependent antiproliferative action towards the tumor cell lines. Treatment of HCT-116 cells for 24 h induced apoptosis, which was confirmed by the appearance of a typical ladder pattern in the DNA fragmentation assay and cell cycle analysis. The α- and β-anomers of 28-O-acetylbetulin-3-yl-d-glucopyranoside seem to induce apoptosis by activation of different upstream caspases on colon cancer HCT-116 cell line.     

Protective effects of a chalcone derivative against Aβ-induced oxidative stress and neuronal damage

Amyloid Β-peptide (AΒ-peptide)-induced oxidative stress is thought to be a critical component of the pathophysiology of Alzheimer's disease (AD). New chalcone derivatives, the Chana series, were recently synthesized from the retrochalcones of licorice. In this study, we investigated the protective effects of the Chana series against neurodegenerative changes in vitro and in vivo. Among the Chana series, Chana 30 showed the highest free radical scavenging activity (90.7%) in the 1,1-diphenyl-2- picrylhydrazyl assay. Chana 30 also protected against AΒ-induced neural cell injury in vitro. Furthermore, Chana 30 reduced the learning and memory deficits of AΒ(1-42)-peptide injected mice. Taken together, these results suggest that Chana 30 may be a promising candidate as a potent therapeutic agent against neurodegenerative diseases. [BMB reports 2011; 44(11): 730-734].     

Subcellular and regional localization ofγ-glutamyl transpeptidase in sheep brain

Studies of the subcellular distribution of-glutamyl transpeptidase from sheep brain by discontinuous sucrose density gradient centrifugation showed that 40% of the transpeptidase activity associated with the mitochondrial-synaptosomal fraction was localized with the synaptosomal-enriched fraction. The microsomal fraction was found to have the highest specific activity when-glutamylp-nitroanalide was used as substrate. This activity, however, represented only 5% of the total-glutamyl transpeptidase activity. Approximately 90% of the total enzyme activity was apparently associated with the fraction containing cell debris and membrane fragments.The 160,000g supernatant fluid (soluble supernatant fraction) represented the least total activity, with only 1.2% recovery; however, this fraction contained two apparent forms of the enzyme. One form had a highK _mand the other a lowK _m for the substrate,-glutamylp-nitroanilide.It was observed that the enzyme-glutamyl transpeptidase was not evenly distributed in all areas of brain when the homogenate was used as the enzyme source. The brain region with the highest enzyme activity was the thalamus, which was able to form 1.10 molp-nitroanaline/min/g wet brain tissue. The cortex was found to have the lowest activity. The 40,000g supernatant fluid from each region, however, exhibited only slight distribution differences.     

Slow accumulation of mutations in Xpc−/− mice upon induction of oxidative stress

XPC is one of the key DNA damage recognition proteins in the global genome repair route of the nucleotide excision repair (NER) pathway. Previously, we demonstrated that NER-deficient mouse models Xpa^?/? and Xpc^?/? exhibit a divergent spontaneous tumor spectrum and proposed that XPC might be functionally involved in the defense against oxidative DNA damage. Others have mechanistically dissected several functionalities of XPC to oxidative DNA damage sensitivity using in vitro studies. XPC has been linked to regulation of base excision repair (BER) activity, redox homeostasis and recruitment of ATM and ATR to damage sites, thereby possibly regulating cell cycle checkpoints and apoptosis. XPC has additionally been implicated in recognition of bulky (e.g. cyclopurines) and non-bulky DNA damage (8-oxodG). However, the ultimate contribution of the XPC functionality in vivo in the oxidative DNA damage response and subsequent mutagenesis process remains unclear. Our study indicates that Xpc^?/? mice, in contrary to Xpa^?/? and wild type mice, have an increased mutational load upon induction of oxidative stress and that mutations arise in a slowly accumulative fashion. The effect of non-functional XPC in vivo upon oxidative stress exposure appears to have implications in mutagenesis, which can contribute to the carcinogenesis process. The levels and rate of mutagenesis upon oxidative stress correlate with previous findings that lung tumors in Xpc^?/? mice overall arise late in the lifespan and that the incidence of internal tumors in XP-C patients is relatively low in comparison to skin cancer incidence.     

In vitro studies of interactions of NO· donor drugs with superoxide and hydroxyl radicals

Nitric oxide (NO·) is a free radical characterized by a high spontaneous chemical reactivity with many other molecules including the superoxide radical (O₂·^–). This complex interaction may generate a peroxynitrite anion (ONOO^–), which behaves as an important mediator of oxidative stress in many pathological states. In the present study, in vitro experiments were performed to assess directly the O₂·^– and hydroxyl (·OH) radical scavenging effects of various NO· donor drugs, i.e. sodium nitroprusside (SNP), sodium nitrite (NaNO₂), molsidomine and SIN 1, at pH 7.4, 7 or 6. Concentrations of NO· in the incubation medium containing the different NO· donor drugs were measured by the assay based on the reaction of Fe-N-methyl-D-glucamine dithiocarbamate (MGD) with NO· that yields a stable spin-adduct measured by electron paramagnetic resonance (EPR). O₂·^– and ·OH generation was characterized by EPR spin trapping techniques, using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). These free radicals were generated from the enzymatic system xanthine-xanthine oxidase, in phosphate buffer adjusted at pH 7.4, 7 and 6. Under these experimental conditions, SNP exhibited the strongest superoxide scavenging properties, characterized by IC₅₀ values expressed in the µmolar range, which decreased at low pH. Addition of SNP (800 µM) to solution containing MGD and Fe²⁺ (5:1) at pH 7 4 produced a three line EPR spectrum which is identified to [(MGD)₂-Fe²⁺-NO]. In control experiments no EPR signal was observed. We obtained the same results with NaNO₂ and an augmentation of the spin-adduct level was noted with the prolongation of the incubation period. In return, molsidomine (2 mM) did not produce, in our conditions, a detectable production of NO·. NaNO₂ displayed a significant superoxide scavenging effect only at pH 6, whilst neither molsidomine nor SIN 1 had any effect. Therefore, the superoxide scavenging properties of SNP, NaNO₂, and molsidomine appeared to be closely related to their potential for NO· release, which partially depends on the pH conditions. The behaviour of SIN 1 is more complicated, the speed of oxygen diffusion probably acting as a limiting factor in NO· formation in our conditions. The production of NO· was detected in presence of SIN 1. The intensity of the complex is comparable with the signal founded with NaNO₂. By contrast, all molecules exhibited hydroxyl radical scavenging properties, highlighting the capacity of ·OH to react with a wide range of molecules. In conclusion, considering the poor chemical reactivity of O₂·^–, the NO· donor drugs/O₂·^– interactions suggest a special relationship between these two radical species, which, in certain pathological states, could lead to the generation of cytotoxic end-products with strong oxidizing properties.     

In vitro culturing of ciliary respiratory cells—a model for studies of genetic diseases

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by the impaired functioning of ciliated cells. Its diagnosis is based on the analysis of the structure and functioning of cilia present in the respiratory epithelium (RE) of the patient. Abnormalities of cilia caused by hereditary mutations closely resemble and often overlap with defects induced by the environmental factors. As a result, proper diagnosis of PCD is difficult and may require repeated sampling of patients’ tissue, which is not always possible. The culturing of differentiated cells and tissues derived from the human RE seems to be the best way to diagnose PCD, to study genotype–phenotype relations of genes involved in ciliary dysfunction, as well as other aspects related to the functioning of the RE. In this review, different methods of culturing differentiated cells and tissues derived from the human RE, along with their potential and limitations, are summarized. Several considerations with respect to the factors influencing the process of in vitro differentiation (cell-to-cell interactions, medium composition, cell-support substrate) are also discussed.     

The induction of oxidative stress in cervix carcinoma cells by levoglucosenone derived 4-S-salicyl derivative and (1–4)-S-thio-disaccharides. Part 4

(1–4)-S-thiodisaccharides were shown to kill various cancer cell lines, including cervix, lung, mammary-gland and colon by unknown mechanisms. Here we identified two actions of levoglucosenone derived (1–4)-S-thiodisaccharides against cervix cancer cells: induction of oxidative stress and DNA damage. In consequence, (1–4)-S-thiodisaccharides lowered the cellular GSH level and changed the expression profile of genes encoding key proteins involved with oxidative stress response. We also observed that (1–4)-S-thiodisaccharides induced DNA damage and interfered with the thioredoxin (Trx) system. Both actions, as induced by FPC6, were stronger when dihedral angles of sulfur bridge were set to 110°, 100° and 109°, clearly indicating differences when compared to FPC8. These findings demonstrate that the 1–4-thio bridge of disaccharide is a powerful anticancer pharmacophore, and its potential use needs further studies.     

In vitro radioprotection studies of organoselenium compounds: differences between mono- and diselenides

Organoselenium compounds belonging to the class of monoselenides, such as selenomethionine (SeM) and methylselenocysteine (MSeCys) and diselenides including selenocystine (SeCys) and selenopropionic acid (SePA), were examined for their comparative radioprotective effects using in vitro models. Effects of these compounds on the inhibition of γ-radiation induced lipid peroxidation in liposomes, protein carbonylation in bovine serum albumin (BSA) and strand breaks in pBR322 plasmid DNA, assessed, respectively, by the formation of thiobarbituric acid reactive substances, formation of 2,2′-dinitrophenyl hydrazine (DNPH) carbonyl complex and horizontal gel electrophoresis, were used to compare their radioprotective ability. The IC₅₀ values for SeCys, SePA, SeM and MSeCys for lipid peroxidation were 27 ± 1, 33 ± 2, 200 ± 8 and 163 ± 4 μM, respectively, and the values for inhibition of protein carbonylation were >200, 300 ± 6, 464 ± 8 and 436 ± 3 μM, respectively. Inhibition of DNA strand break formation was tested at 200 μM for all the compounds and SePA and SeCys exhibited a protective effect on DNA, while SeM and MSeCys did not lead to any protection. The in vitro cytotoxicity studies in normal and tumor cells revealed that MSeCys and SeM were not cytotoxic to lymphocytes and EL4 tumor cells at the concentrations employed. In contrast, SeCys was toxic, with a higher effect on tumor cells than lymphocytes. Our studies suggest that the non-toxic diselenides like SePA should be explored as protective agents against γ-irradiation induced damage.     

Carnosine and its (S)-Trolox? derivative protect animals against oxidative stress

The novel synthetic derivative of carnosine, (S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl-β-alanyl-L-histidine (S-Trolox?-Carnosine, STC) increases the resistance of rats to experimental acute hypobaric hypoxia (AHH) thus protecting brain from the oxidative damage. This effect is accompanied by better preservation of the acquired skills in Morris water maze possibly by increasing efficiency of the brain antioxidant system. In addition, STC caused an increase in life span of both male and female fruit fly Drosophila melanogaster whereas carnosine increased life span only in male fruit flies. The results indicate that development of the drug based on STC could be beneficial in neurology and gerontology.     

Protective effect of tea against lead and cadmium-induced oxidative stress—a review

Exposure to Cd and Pb reduces the activity of antioxidant enzymes, which points to a decrease in the antioxidant potential of the body as a result of supplying factors which enhance cellular oxidation processes. Man is exposed to the effects of toxic metals because they are present in the environment, including in food. Since no effective ways to reduce the concentrations of Cd an Pb in food exist, studies are undertaken to develop methods of reducing their toxic effect on the body through chelating these metals using nutrients (which reduces their absorption by tissues) or increasing the oxidative capacity of the body (which decreases the possibility of inducing oxidative damage to internal organs). Studies performed on laboratory animals have shown that the use of tea infusions fulfil both functions.     

Subcellular localization and possible functions of acid β-glycerophosphatases and naphthol esterases in plant cells

Summary The subcellular localization of esterases and acid phosphatases in described for cells from roots of Vicia faba. The possible mode of transport of the esterases from a cytoplasmic site of synthesis to the cell wall is discussed.     

Synthesis of thiobarbituric acid derivatives: In vitro α-glucosidase inhibition and molecular docking studies

Synthesis, structure, and evaluation of in vitro α-glucosidase enzyme inhibition of a new class of diethylammonium salts of aryl substituted thiobarbituric acid is described. This protocol is straight, environmentally benign and efficient, involving Aldol-Michael addition reaction in one pot fashion. The 3D chemical structures of the synthesized compounds were assigned based on spectroscopic methods and X-ray single crystal diffraction analyses. All synthesized compounds 3a-3n were evaluated for their in vitro α-glucosidase enzyme inhibitory activity, whereas acarbose was used as the standard drug (IC₅₀ = 840 ± 1.73 µM). All tested compounds were found to possess varying degree of α-glucosidase enzyme inhibition activity with (IC₅₀ = 19.46 ± 1.84–415.8 ± 4.0 µM). Compound 3i (IC₅₀ = 19.4 ± 1.84 µM) exhibited the highest activity. To the best of knowledge this is the first report of the in vitro α-glucosidase enzyme inhibition by the diethylamonium salts of aryl substituted thiobarbituric acid. Furthermore, molecular docking studies of selected compounds were also performed to see interactions between active compounds and binding sites.     

In vitro interactions of histones and α-crystallin

The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone–α-crystallin binding with a K_d of 4 × 10^?7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone–α-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone–α-crystallin mixtures showed a decrease in the oligomeric molecular weight of α-crystallin, indicating that histones alter the oligomerization of α-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of α-crystallin, indicating that the interaction between α-crystallin and histones in the lens is functionally important.     

In vivo radioprotection studies of 3,3′-diselenodipropionic acid,a selenocystine derivative

3,3′-Diselenodipropionic acid (DSePA), a diselenide and a derivative of selenocystine, was evaluated for in vivo radioprotective effects in Swiss albino mice, at an intraperitoneal dose of 2 mg/kg body wt, for 5 days before whole-body exposure to γ-radiation. The radioprotective efficacy was evaluated by assessing protection of the hepatic tissue, the spleen, and the gastrointestinal (GI) tract and survival against sub- and supralethal doses of γ-radiation. DSePA inhibited radiation-induced hepatic lipid peroxidation, protein carbonylation, loss of hepatic function, and damage to the hepatic architecture. DSePA also attenuated the depletion of endogenous antioxidants such as glutathione, glutathione peroxidase, superoxide dismutase, and catalase in the livers of irradiated mice. DSePA also restored the radiation-induced reduction in villus height, crypt cell numbers, and spleen cellularity, indicating protective effects on the GI tract and the hematopoietic system. The results from single-cell gel electrophoresis of the peripheral blood leukocytes showed that DSePA can attenuate radiation-induced DNA damage. The mRNA expression analysis of genes revealed that DSePA augmented GADD45α and inhibited p21 in both spleen and liver tissues. DSePA also inhibited radiation-induced apoptosis in the spleen and reversed radiation-induced alterations in the expression of the proapoptotic BAX and the antiapoptotic Bcl-2 genes. In line with these observations, DSePA improved the 30-day survival of irradiated mice by 35.3%. In conclusion, these findings clearly confirm that DSePA exhibits protective effects against whole-body γ-radiation and the probable mechanisms of action involve the maintenance of antioxidant enzymes, prophylactic action through the attenuation of the DNA damage, and inhibition of apoptosis.     

Nrf2-dependent induction of proteasome and Pa28αβ regulator are required for adaptation to oxidative stress

The ability to adapt to acute oxidative stress (e.g. H(2)O(2), peroxynitrite, menadione, and paraquat) through transient alterations in gene expression is an important component of cellular defense mechanisms. We show that such adaptation includes Nrf2-dependent increases in cellular capacity to degrade oxidized proteins that are attributable to increased expression of the 20 S proteasome and the Pa28αβ (11 S) proteasome regulator. Increased cellular levels of Nrf2, translocation of Nrf2 from the cytoplasm to the nucleus, and increased binding of Nrf2 to antioxidant response elements (AREs) or electrophile response elements (EpREs) in the 5'-untranslated region of the proteasome β5 subunit gene (demonstrated by chromatin immunoprecipitation (or ChIP) assay) are shown to be necessary requirements for increased proteasome/Pa28αβ levels, and for maximal increases in proteolytic capacity and stress resistance; Nrf2 siRNA and the Nrf2 inhibitor retinoic acid both block these adaptive changes and the Nrf2 inducers DL-sulforaphane, lipoic acid, and curcumin all replicate them without oxidant exposure. The immunoproteasome is also induced during oxidative stress adaptation, contributing to overall capacity to degrade oxidized proteins and stress resistance. Two of the three immunoproteasome subunit genes, however, contain no ARE/EpRE elements, and Nrf2 inducers, inhibitors, and siRNA all have minimal effects on immunoproteasome expression during adaptation to oxidative stress. Thus, immunoproteasome appears to be (at most) minimally regulated by the Nrf2 signal transduction pathway.     

β-TrCP-dependent degradation of ASK1 suppresses the induction of the apoptotic response by oxidative stress

Apoptosis signal-regulating kinase 1 (ASK1) is a key player in the homeostatic response of many organisms. Of the many functions of ASK1, it is most well-known for its ability to induce canonical caspase 3-dependent apoptosis through the MAPK pathways in response to reactive oxygen species (ROS). As ASK1 is a regulator of apoptosis, its proper regulation is critical for the well-being of an organism. To date, several E3 ubiquitin ligases have been identified that are capable of degrading ASK1, signifying the importance of maintaining ASK1 expression levels during stress responses. ASK1 protein regulation under unstimulated conditions, however, is still largely unknown. Using tandem mass spectrometry, we have identified beta-transducin repeat containing protein (β-TrCP), an E3 ubiquitin ligase, as a novel interacting partner of ASK1 that is capable of ubiquitinating and subsequently degrading ASK1 through the ubiquitin-proteasome system (UPS). This interaction requires the seven WD domains of β-TrCP and the C-terminus of ASK1. By silencing the β-TrCP genes, we observed a significant increase in caspase 3 activity in response to oxidative stress, which could subsequently be suppressed by silencing ASK1. These findings suggest that β-TrCP is capable of suppressing oxidative stress-induced caspase 3-dependent apoptosis through suppression of ASK1, assisting in the organism's ability to maintain homeostasis in an unstable environment.     

Overview — In vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC₅₀=36.4 μM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC₅₀=4.62 μM). We isolate and identify a carbamoylated peptide at Cys₃₀₂ with the sequence FNQGQC₃₀₁C₃₀₂C₃₀₃. Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys₃₀₂, present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.     

In vitro induction and characterization of hexaploid Pennisetum × advena,an ornamental grass

Plant Cell, Tissue and Organ Culture (PCTOC) - Pennisetum × advena (purple fountain grass) is an ornamental grass with considerable commercial value. In vitro induction of...     

Subcellular localization of endo-β-N-acetylglucosaminidase and high-mannose type free N-glycans in plant cell

Subcellular distribution of plant endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase) and high-mannose type free N-glycans produced by the endoglycosidase has been analyzed using cotyledons of pumpkin seedlings as the model plant cells. Each organelle in the cotyledons was fractionated by ultracentrifugation with the sucrose density gradient system and the endo-β-GlcNAc-ase activity in each fraction was assayed with fluorescence labeled N-glycans as substrates. The endoglycosidase activity was exclusively recovered in the soluble fraction (cytosol fraction) but not in other specific organellar fractions, suggesting that the endoglycosidase would reside predominantly in the cytosol. The quantitative analysis of high-mannose type free N-glycans occurring in each fraction showed that more than 70% of the free N-glycans was recovered from the soluble fraction, suggesting the endoglycosidase would work in the cytosol and the resulting free N-glycans would accumulate in the same fraction. The pumpkin endo-β-GlcNAc-ase (endo-CM) partially purified from the cotyledons showed optimum activity around pH 6.5, supporting this enzyme would reside in the cytosol. Furthermore, the detailed analysis of substrate specificity of endo-CM using various high-mannose type N-glycans showed that the pumpkin enzyme, as well as other plant endo-β-N-acetylglucosaminidases, were highly active toward the high-mannose type glycans bearing the Manα1-2Manα1-3Manβ1-structural unit.