Reversal effects of pantoprazole on multidrug resistance in human gastric adenocarcinoma cells by down-regulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway in vitro and in vivo

To investigate reversal effects of pantoprazole (PPZ) on multidrug resistance (MDR) in human gastric adenocarcinoma cells in vivo and in vitro. Human gastric adenocarcinoma cell SGC7901 was cultured in RPMI‐1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5% CO₂ atmosphere at 37°C. Adriamycin (ADR)‐resistant cells were cultured with addition of 0.8 µg/ml of ADR maintaining MDR phenotype. ADR was used to calculate ADR releasing index; CCK‐8 Assay was performed to evaluate the cytotoxicity of anti‐tumor drugs; BCECF‐AM pH‐sensitive fluorescent probe was used to measure intracellular pH (pHi) value of cells, whereas pH value of medium was considered as extracellular pH (pHe) value; Western blotting and immunofluorescent staining analyses were employed to determine protein expressions and intracellular distributions of vacuolar H⁺‐ATPases (V‐ATPases), mTOR, HIF‐1α, P‐glycoprotein (P‐gp), and multidrug resistant protein 1 (MRP1); SGC7901 and SGC7901/ADR cells were inoculated in athymic nude mice. Thereafter, effects of ADR with or without PPZ pretreatment were compared by determining the tumor size and weight, apoptotic cells in tumor tissues were detected by TUNEL assay. At concentrations greater than 20 µg/ml, PPZ pretreatment reduced ADR releasing index and significantly enhanced intracellular ADR concentration of SGC7901 (P < 0.01). Similarly, PPZ pretreatment significantly decreased ADR releasing index of SGC7901/ADR dose‐dependently (P < 0.01). PPZ pretreatment also decreased cell viabilities of SGG7901 and SGC7901/ADR dose‐dependently. After 24‐h PPZ pretreatment, administration of chemotherapeutic agents demonstrated maximal cytotoxic effects on SGC7901 and SGC7901/ADR cells (P < 0.05). The resistance index in PPZ pretreatment group was significantly lower than that in non‐PPZ pretreatment group (3.71 vs. 14.80). PPZ at concentration >10 µg/ml significantly decreased pHi in SGC7901 and SGC7901/ADR cells and diminished or reversed transmembrane pH gradient (P < 0.05). PPZ pretreatment also significantly inhibited protein expressions of V‐ATPases, mTOR, HIF‐1α, P‐gp, and MRP1, and alter intracellular expressions in parent and ADR‐resistant cells (P < 0.05). In vivo experiments further confirmed that PPZ pretreatment could enhance anti‐tumor effects of ADR on xenografted tumor of nude mice and also improve the apoptotic index in xenografted tumor tissues. PPZ pretreatment enhances the cytotoxic effects of anti‐tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR by downregulating the V‐ATPases/mTOR/HIF‐1α/P‐gp and MRP1 signaling pathway. J. Cell. Biochem. 113: 2474–2487, 2012. © 2012 Wiley Periodicals, Inc.     

Effect of Bioregulator Isatin on Protein–Protein Interactions Involving Isatin-Binding Proteins

Isatin (2,3-dioxoindol) is an endogenous low-molecular-weight nonpeptide compound with a wide spectrum of biological and pharmacological activities. It is assumed that isatin acts through isatin-binding proteins. To date, more than a hundred of these proteins are known. Having a different structure and cellular and subcellular localization, they belong to different functional groups. Using the surface plasmon resonance technology, we found earlier that isatin affected the profile of intracellular amyloid-binding proteins and changed the stability of protein complexes in the model system. In fact, this indicates the selective effect of isatin on certain protein–protein interactions (PPI) that occur primarily with the participation of isatinbinding proteins. Therefore, we had formulated the hypothesis that isatin could be a regulator of a protein interactome. This study focuses on the verification of this assumption. Size-exclusion chromatography (SEC) profile of the rat liver tissue lysate along with mass-spectrometric protein identification has revealed 20 isatinbinding proteins that participate in the formation of the protein interactome. About 65 and 25% of them are involved in the formation of multimeric protein complexes and homo/heterodimers, respectively, and only 10% are detected as single molecules. The addition of isatin had a multidirectional effect on the profile of about half of the identified isatin-binding proteins. In some cases, the formation of protein complexes was induced, while in other cases the protein complexes were dissociated. This result confirms the hypothesis of the regulatory effect of isatin on certain PPIs. The data of this work in combination with our previous results allowed us to formulate an “interactomics image” of isatin as a bioregulator, which selectively controls both the formation and dissociation of a number of protein complexes. Two new isatin-dependent proteins were found in the work. This indicates that not all potential target proteins of the regulatory effect of isatin had been previously detected. The study of the molecular mechanisms of isatin action on PPI remains a difficult but priority task for future research.     

Isatin‐binding proteins of rat and mouse brain: Proteomic identification and optical biosensor validation

Isatin (indole‐2,3‐dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Isatin itself and its analogues exhibit a wide range of pharmacological activities but its specific biological targets still are not well characterized. Affinity chromatography of Triton X‐100 lysates of soluble and particulate fractions of mouse and rat whole brain homogenates on 5‐aminocaproyl‐isatin‐Sepharose followed by subsequent proteomic analysis resulted in identification of 65 and 64 individual proteins, respectively. Isatin‐binding capacity of some of the identified proteins has been validated in an optical biosensor study using a Biacore 3000 optical biosensor, 5‐aminocarproyl‐isatin, and 5‐aminoisatin as the affinity ligands. The K_d values (of 0.1–20 μM) obtained during the optical biosensor experiments were consistent with the range of K_d values recently reported for [³H]isatin binding to brain sections. Although the number of isatin‐binding proteins identified in the mouse and rat brain was similar, only 21 proteins (about one‐third) were identical in the two species. This may be one reason for the differences in isatin effects in rats and mice reported in the literature.     

αv integrin processing interferes with the cross-talk between αvβ5/β6 and α2β1 integrins

Background information. Previous studies have reported that cross‐talk between integrins may be an important regulator of integrin—ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvβ5/β6 integrin represses α2β1‐dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial. Results. Inhibition of convertases by the convertase inhibitor α1‐PDX (α1‐antitrypsin Portland variant), leading to the cell‐surface expression of an uncleaved form of the αv integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, α2β1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen‐activated protein kinase). This outside‐in signalling stimulation was associated with increased levels of activated β1 integrin located in larger than usual focal‐adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3‐kinase)/Akt (also called protein kinase B) pathway. Conclusions. The increase in cell migration observed upon convertases inhibition appears to be due to the up‐regulation of β1 integrins and to their location in larger focal‐adhesion structures. The endoproteolytic cleavage of αv subunits is necessary for αvβ5/β6 integrin to control α2β1 function and could thus play an essential role in colon cancer cell migration.     

l‐NAME‐Induced Dispersion of Melanosomes in Melanophores Activates PKC,MEK and ERK1

Melanosome movement represents a good model of cytoskeleton‐mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nω‐nitro‐l ‐arginine methyl ester (l ‐NAME) induced dispersion in melanophores pre‐aggregated with melatonin. Activation of cyclic adenosine 3′,5′‐monophosphate (cAMP)‐dependent protein kinase (PKA) or calcium‐dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal‐regulated kinase (MEK)‐ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of l ‐NAME‐induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in l ‐NAME‐dispersed melanophores. l ‐NAME also caused dispersion in latrunculin‐B‐treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the l ‐NAME‐induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.     

The endogenous oxindoles 5-hydroxyoxindole and isatin are antiproliferative and proapoptotic

Oxindole-core synthetic molecules are currently being developed as anticancer drugs that target protein tyrosine kinases associated with growth factor receptors. Oxindole, 5-Hydroxyoxindole, and 2, 3-dioxindole [isatin] are natural molecules found in mammalian body fluids and tissues and we addressed the question of similar properties of endogenous oxindoles. 5-Hydroxyoxindole and isatin, but not oxindole, inhibited N1E-115, BALB/c3T3, BBC, PC12, and HL60 proliferation at submicromolar concentrations. Acute treatment with 5-hydroxyoxindole and isatin reduced the activity of extracellular signal regulated protein kinases (ERKs) by 35% at 100 microM and ERK1 activity was strongly inhibited by 5-Hydroxyoxindole at 10 microM. Survival of PMA-differentiated HL60 and FGF(2)-differentiated PC12 cells was not affected by 5-Hydroxyoxindole and isatin treatment, suggesting that endogenous oxindoles interact with growth factors signaling. The physiological implications of these data and the potential utility of 5-Hydroxyoxindole and isatin as antitumor agents are discussed.     

Effect of green microalgal extracts exhibiting antibacterial activity on viability of human malignant and non‐malignant cells

Microalgae have been investigated for their ability to produce metabolites that exhibit antibacterial activity. However, as research on antibacterial activity progresses, the effect of microalgal extracts on mammalian cells needs to be also assessed. The in vitro effect of microalgal extracts with demonstrated antibacterial activity against the human opportunistic pathogen Staphylococcus aureus was examined on the viability of non‐malignant (MCF10A and 184B5 cells) and malignant human cell lines (A2780 and MCF7). Direct cell counts indicated that the MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) proliferation assay was found to under/overestimate cell viability when specific microalgal extracts and/or concentrations were tested. From direct cell counts, the viability of non‐malignant cells was not reduced after exposure to the extracts, whereas the viability of malignant cells was affected by specific microalgal extracts and concentrations. The results suggest that green microalgae are worthy of further investigation as potential sources of antibiotics, since they did not show a negative effect on the non‐malignant cell lines, MCF10A and 184B5. Additional studies should evaluate the compounds responsible for the anti‐proliferative activity of specific microalgal extracts observed on the malignant cells A2780 and MCF7.     

Overexpression of the <Emphasis Type="Italic">Activated Disease Resistance 1-like1</Emphasis> (<Emphasis Type="Italic">ADR1-L1</Emphasis>) Gene Results in a Dwarf Phenotype and Activation of Defense-Related Gene Expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The protein encoded by the activated disease resistance 1-like1 (ADR1-L1) gene (locus name, At4g33300) belongs to the activated disease resistance 1 (ADR1) family of coiled-coil nucleotide-binding site leucine-rich repeat-type disease resistance proteins. This family contains four proteins and they have specific features in their amino acid sequences. It has been reported that ADR1 protein belongs to the ADR1 family, which is related to not only defense response but also drought tolerance. We found that transgenic plants overexpressing the ADR1-L1 gene showed a dwarf phenotype and morphological change in leaves. The expression levels of defense-related genes and the resistance to Pseudomonas syringae pv. tomato DC3000 were increased in transgenic plants. However, enhancement of drought tolerance and activation of abiotic response genes were not observed. When the growth temperature was changed from 22°C to 28°C, the expression of defense-related genes and the enhancement of resistance to a bacterial pathogen were suppressed and the dwarf phenotype and morphological change of leaves recovered.     

Interactome analysis of the EV71 5′ untranslated region in differentiated neuronal cells SH‐SY5Y and regulatory role of FBP3 in viral replication

Enterovirus 71 (EV71), a single‐stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia‐Pacific region. Through interactions with host proteins, the 5′ untranslated region (5′UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5′UTR in neuronal cells, we performed a biotinylated RNA‐protein pull‐down assay in conjunction with LC–MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein–protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far‐upstream element binding protein 3 (FBP3) was able to bind to the EV71 5′UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5′UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells.     

Amelioration of adriamycin-induced cardiotoxicity by Salsola kali aqueous extract is mediated by lowering oxidative stress

Abstract

Objectives

To assess the cardioprotective effect of the Salsola kali aqueous extract against adriamycin (ADR)-induced cardiotoxicity in male Swiss albino mice.

Methods

The aqueous extract of S. kali was phytochemically screened by traditional methods for different classes and further evaluated for antioxidant activity in vitro. In vivo, cardioprotective evaluation of the extract was designed to have four groups of mice: (1) control group (distilled water, orally; normal saline, intraperitoneally (i.p.)); (2) ADR group (15 mg/kg, i.p.); (3) aqueous S. kali extract (200 mg/kg, orally); and (4) ADR + S. kali group. ADR (5 mg/kg) was injected three times over 2 weeks while S. kali was orally administered daily for 3 weeks (1 week before and 2 weeks during ADR treatment). Cardioprotective properties were assessed using biochemical and histopathological approaches.

Results

ADR caused a significant increase in serum enzymes (lactate dehydrogenase, creatine phosphokinase, aspartate aminotransferase, and alanine aminotransferase). Myocardial levels of malondialdehyde, nitric oxide, and reduced glutathione, as well as the activities of superoxide dismutase and catalase increased while the activities of glutathione peroxidase and glutathione S-transferase declined. Histopathological examination of heart sections revealed that ADR caused myofibrils loss, necrosis and cytoplasmic vacuolization.

Discussion

Pretreatment with S. kali aqueous extract normalized serum and antioxidant enzymes minimized lipid peroxidation and cardiac damage. These results have suggested that the extract has antioxidant activity, indicating that the mechanism of cardioprotection during ADR treatment is mediated by lowering oxidative stress.     

The effect of new anthracycline derivatives on the induction of apoptotic processes in human neoplastic cells

This study was an attempt to determine the effect of a selected anthracycline derivative, WP903, on apoptotic processes in human melanoma cells depending on intracellular concentrations of the compound, and to evaluate the significance of apoptosis induction for the cytotoxic effect of anthracycline antibiotics. It was found that the WP903, contrary to ADR (adriamycin) is a strong inducer of apoptotic processes in ME18 human melanoma cells regardless of their susceptibility to adriamycin and WP903. The cells were treated for 24 h with ADR (1 and 5 microg/ml) or WP903 (0.2 and 2 microg/ml). Apoptosis was detected with the use of annexin V-FITC and PI (propidium iodide) and with TUNEL assay. WP903 used at 0.2 microg/ml induced early apoptosis in 23% of ME18 cells and in 60% of ME18/R cells; at 2 microg/ml in 70% of each of cell line tested. Significant late apoptotic effect was observed in ME18 cells. In contrast, ADR was found to be a weak inducer of apoptotic events. The results suggest that apoptosis is not a mechanism directly related to the cytotoxic effect of anthracycline antibiotics.     

Autophosphorylation of αCaMKII affects social interactions in mice

The α‐Ca²⁺/calmodulin‐dependent protein kinase II (αCaMKII), a key regulator of the glutamatergic synapse, has been implicated in many psychiatric disorders characterized by social impairments. Here we tested whether autophosphorylation of αCaMKII at threonine 286, which prolongs the activity of the enzyme, affects social behaviors in mice. We observed that autophosphorylation‐deficient (αCaMKII‐T286A) mutant female mice showed abnormal social behaviors characterized by decreased social preference and interest in conspecifics of the same sex, as compared to their wild‐type littermates. Moreover, we developed a mathematical approach to analyze social interactions in group‐housed mice in the automated IntelliCages. Using this approach we observed that αCaMKII‐T286A mutants show decreased levels of social interactions in a social group, as compared with WT mice. WT mice increased the frequency of close social interactions when learning about the location of the food reward. This phenomenon was absent in the mutants. Overall, our data indicates that autophosphorylation of αCaMKII affects social interactions.     

The Influence of Higher Protein Intake and Greater Eating Frequency on Appetite Control in Overweight and Obese Men

The purpose of this study was to determine the effects of dietary protein intake and eating frequency on perceived appetite, satiety, and hormonal responses in overweight/obese men. Thirteen men (age 51 ± 4 years; BMI 31.3 ± 0.8 kg/m²) consumed eucaloric diets containing normal protein (79 ± 2 g protein/day; 14% of energy intake as protein) or higher protein (138 ± 3 g protein/day; 25% of energy intake as protein) equally divided among three eating occasions (3‐EO; every 4 h) or six eating occasions (6‐EO; every 2 h) on four separate days in randomized order. Hunger, fullness, plasma glucose, and hormonal responses were assessed throughout 11 h. No protein × eating frequency interactions were observed for any of the outcomes. Independent of eating frequency, higher protein led to greater daily fullness (P < 0.05) and peptide YY (PYY) concentrations (P < 0.05). In contrast, higher protein led to greater daily ghrelin concentrations (P < 0.05) vs. normal protein. Protein quantity did not influence daily hunger, glucose, or insulin concentrations. Independent of dietary protein, 6‐EO led to lower daily fullness (P < 0.05) and PYY concentrations (P < 0.05). The 6‐EO also led to lower glucose (P < 0.05) and insulin concentrations (P < 0.05) vs. 3‐EO. Although the hunger‐related perceived sensations and hormonal responses were conflicting, the fullness‐related responses were consistently greater with higher protein intake but lower with increased eating frequency. Collectively, these data suggest that higher protein intake promotes satiety and challenge the concept that increasing the number of eating occasions enhances satiety in overweight and obese men.     

Shotgun proteomic profiling of five species of New Zealand Pachycladon

The genus Pachycladon consists of ten species of alpine plants, nine of which are endemic to New Zealand. The species are closely related to the model plant Arabidopsis thaliana with respect to their sequence divergence and chromosome synteny, occupy distinct geographical habitats in terms of both latitude and altitude, and display a range of morphologies. We have performed label‐free quantitative shotgun proteomic analysis of five different species of Pachycladon, namely P. cheesemanii (CH), P. exile (EX), P. fastigiatum (FA), P. enysii (EN) and P. novae‐zelandiae (NZ). The total non‐redundant data set for all five species contained 1489 proteins. The numbers of proteins identified reproducibly in each species ranged from 629 for CH to 987 for NZ, with 681 for EN, 741 for EX and 934 for FA. Previous metabolite‐based studies have shown that FA hydrolyzes glucosinolates completely to isothiocyanates while EN converts glucosinolates to nitriles. In this study, we observed high expression of ESP (At1g54040, epithiospecifying senescence regulator protein) and myrosinase 2 (At5g25980, glycosyl hydrolase family protein), which result in production of nitriles and epithionitriles, in EN and NZ, and we also observed higher expression of ESM1 (At3g14210, GDSL esterase/lipase), which mediates the formation of isothiocyanate, in FA.     

Opening the Black Box of Developmental Experiments: Behavioural Mechanisms Underlying Long‐Term Effects of Early Social Experience

Typically, animals spend a considerable portion of their time with social interactions involving mates, offspring, competitors and group members. The social performance during these interactions can strongly depend on the social environment individuals have experienced early in life. Despite a considerable number of experiments investigating long‐term effects of the early social environment, our understanding of the behavioural mechanisms mediating these effects is still limited, mainly for two reasons. (1) Only in few experimental studies have researchers actually observed and quantified the behaviour of their study animals during the social treatment. (2) Even if differences in social interactions between social rearing treatments are reported, these differences might not be causally linked to any observed long‐term effects later in life. The aim of this review was to investigate whether behavioural records of animals during the experimental manipulation of their social environment can help (1) identifying behavioural mechanisms involved in a long‐term effect and (2) obtaining a better understanding of the long‐term consequences of early manipulations. First, I review studies that manipulated the social environment at an early stage of the ontogeny, observed the social interactions and behaviour during the social experience phase and subsequently tested the performance in social and non‐social behavioural tasks at a later life stage. In all reviewed studies, treatment differences were reported both in social interactions during the social experience phase and in social and/or non‐social behaviours later in life. Second, I discuss four classes of behavioural mechanisms that can cause the reported long‐term effects of social experience, namely learning by experience, social learning, sensory stimulation and social cueing. I conclude that social interactions during the social experience phase should always be recorded for at least two reasons. Knowledge about how the social interactions differ between rearing treatments (1) permits researchers to formulate hypotheses about candidate mechanisms causing long‐term effects on behaviour and (2) can help to interpret unexpected outcomes of developmental experiments. Finally, I propose that as a crucial ultimate step towards understanding effects of the early social environment, we should develop targeted experiments testing for the causality of identified candidate mechanism.     

Stereoselective Accumulation of Propranolol Enantiomers in K562 and K562/ADR Cells

The stereoselective uptake of propranolol enantiomers was investigated by using the K562 and K562 adriamycin‐resistant cell line (K562/ADR) as a model. An enantioselective RP‐HPLC method was applied to determine the accumulation of propranolol (PPL) stereoisomers in K562 and K562/ADR cells. The concentration, time and temperature dependent studies showed that the accumulation of S‐(?)‐PPL was higher than R‐(+)‐PPL in K562 cells and uptake of R‐(+)‐PPL was significantly higher than that of S‐(?)‐PPL in K562/ADR cells. The results indicate the enantioselective accumulation of propranolol enantiomers in K562 and K562 / ADR cells. Chirality 25:361–364, 2013. © 2013 Wiley Periodicals, Inc.