6‐Methyl 3‐chromonyl 2,4‐thiazolidinedione/2,4‐imidazolidinedione/2‐thioxo‐imidazolidine‐4‐one compounds: novel scavengers of reactive oxygen species

The benefits of antioxidants on human health are usually ascribed to their potential ability to remove reactive oxygen species providing protection against oxidative stress. In this paper the free radicals scavenging activities of nine 6‐methyl 3‐chromonyl derivatives (CMs) were evaluated for the first time by the chemiluminescence, electron paramagnetic resonance, spin trapping and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) methods. The total antioxidant capacity was also measured using a ferric‐ferrozine reagent. Compounds having a hydrogen atom at the N3‐position of the β‐ring were effective in quenching CL resulted from the KO₂/18‐crown‐6‐ether system (a source of superoxide anion radical, ) in a dose‐dependent manner over the range of 0.05–1 mmol/L [IC₅₀ ranged from 0.353 (0.04) to 0.668 (0.05) mmol/L]. The examined compounds exhibited a significant scavenging effect towards hydroxyl radicals (HO^? HO^?), produced by the Fenton reaction, and this ranged from 24.0% to 61.0%, at the concentration of 2.5 mmol/L. Furthermore, the compounds examined were also found to inhibit DPPH^? and this ranged from 51.9% to 97.4% at the same concentration. In addition, the use of the total antioxidant capacity assay confirmed that CM compounds are able to act as reductants. According to the present study, CM compounds showed effective in vitro free radical scavenging activity and may be considered as potential therapeutics to control diseases of oxidative stress‐related etiology. Copyright © 2013 John Wiley & Sons, Ltd.     

Neuroprotective effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on ischaemia/reperfusion‐induced neuronal injury by activating the Nrf2/HO‐1 pathway

1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a lipophilic phenolic agent, has an antioxidant activity and reactive oxygen species (ROS) scavenging property. However, the role of HTHQ on cerebral ischaemic/reperfusion (I/R) injury and the underlying mechanisms remain poorly understood. In the present study, we demonstrated that HTHQ treatment ameliorated cerebral I/R injury in vivo, as demonstrated by the decreased infarct volume ration, neurological deficits, oxidative stress and neuronal apoptosis. HTHQ treatment increased the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream antioxidant protein, haeme oxygenase‐1 (HO‐1). In addition, HTHQ treatment decreases oxidative stress and neuronal apoptosis of PC12 cells following hypoxia and reperfusion (H/R) in vitro. Moreover, we provided evidence that PC12 cells were more vulnerable to H/R‐induced oxidative stress after si‐Nrf2 transfection, and the HTHQ‐mediated protection was lost in PC12 cells transfected with siNrf2. In conclusion, these results suggested that HTHQ possesses neuroprotective effects against oxidative stress and apoptosis after cerebral I/R injury via activation of the Nrf2/HO‐1 pathway.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

Crystal structure of heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding

The heart‐specific isoform of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well‐known inhibitor of PFKFB2, co‐crystallized in the 2‐kinase domains of both orthologues, occupying the fructose‐6‐phosphate binding‐site and extending into the γ‐phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ‐phosphate site by citrate proved highly consequential to the binding of co‐complexed ATP analogues. The bovine structure, which co‐crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co‐complexed with AMPPNP, which, unlike ADP, contains a γ‐phosphate. The presence of this γ‐phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding‐pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed‐back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117–124. © 2016 Wiley Periodicals, Inc.     

CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose

Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N‐linked glycans, including the presence of β‐1,2‐xylose and core α‐1,3‐fucose residues in plants, can affect the activity, potency and immunogenicity of plant‐derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N‐glycosylation machinery to allow the synthesis of complex N‐glycans lacking β‐1,2‐xylose and core α‐1,3‐fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant‐specific α‐1,3‐fucosyltransferase and β‐1,2‐xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry‐based N‐glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64‐binding affinity of 2G12 glycovariants produced in wild‐type N. benthamiana, the newly generated FX‐KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco‐engineered antibody performed as well as its CHO‐produced counterpart.     

Cell‐cycle‐dependent PC‐PLC regulation by APC/CCdc20‐mediated ubiquitin‐proteasome pathway

Phosphatidylcholine‐specific phospholipase C (PC‐PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC‐PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC‐PLC were co‐immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH‐7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC‐PLC and Cdc20 were co‐localized in the perinuclear endoplasmic reticulum region (the “juxtanuclear quality control” compartment, JUNQ). The expression level and activities of PC‐PLC changed in a cell‐cycle‐dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC‐PLC, and caused PC‐PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC‐PLC regulation in cell cycles is controlled by APC/C^Cdc20‐mediated UPP. J. Cell. Biochem. 107: 686–696, 2009. © 2009 Wiley‐Liss, Inc.     

Targeting the ROS/PI3K/AKT/HIF‐1α/HK2 axis of breast cancer cells: Combined administration of Polydatin and 2‐Deoxy‐d‐glucose

It is well established that cancer cells depend upon aerobic glycolysis to provide the energy they need to survive and proliferate. However, anti‐glycolytic agents have yielded few positive results in human patients, in part due to dose‐limiting side effects. Here, we discovered the unexpected anti‐cancer efficacy of Polydatin (PD) combined with 2‐deoxy‐D‐glucose (2‐DG), which is a compound that inhibits glycolysis. We demonstrated in two breast cell lines (MCF‐7 and 4T1) that combination treatment with PD and 2‐DG induced cell apoptosis and inhibited cell proliferation, migration and invasion. Furthermore, we determined the mechanism of PD in synergy with 2‐DG, which decreased the intracellular reactive oxygen (ROS) levels and suppressed the PI3K/AKT pathway. In addition, the combined treatment inhibited the glycolytic phenotype through reducing the expression of HK2. HK2 deletion in breast cancer cells thus improved the anti‐cancer activity of 2‐DG. The combination treatment also resulted in significant tumour regression in the absence of significant morphologic changes in the heart, liver or kidney in vivo. In summary, our study demonstrates that PD synergised with 2‐DG to enhance its anti‐cancer efficacy by inhibiting the ROS/PI3K/AKT/HIF‐1α/HK2 signalling axis, providing a potential anti‐cancer strategy.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.     

MiR‐30‐5p suppresses cell chemoresistance and stemness in colorectal cancer through USP22/Wnt/β‐catenin signaling axis

Colorectal cancer (CRC) remains both common and fatal, and its successful treatment is greatly limited by the development of stem cell‐like characteristics (stemness) and chemoresistance. MiR‐30‐5p has been shown to function as a tumor suppressor by targeting the Wnt/β‐catenin signaling pathway, but its activity in CRC has never been assessed. We hypothesized that miR‐30‐5p exerts anti‐oncogenic effects in CRC by regulating the USP22/Wnt/β‐catenin signaling axis. In the present study, we demonstrate that tissues from CRC patients and human CRC cell lines show significantly decreased miR‐30‐5p family expression. After identifying the 3’UTR of USP22 as a potential binding site of miR‐30‐5p, we constructed a luciferase reporter containing the potential miR‐30‐5p binding site and measured the effects on USP22 expression. Western blot assays showed that miR‐30‐5p decreased USP22 protein expression in HEK293 and Caco2 CRC cells. To evaluate the effects of miR‐30‐5p on CRC cell stemness, we isolated CD133 + CRC cells (Caco2 and HCT15). We then determined that, while miR‐30‐5p is normally decreased in CD133 + CRC cells, miR‐30‐5p overexpression significantly reduces expression of stem cell markers CD133 and Sox2, sphere formation, and cell proliferation. Similarly, we found that miR‐30‐5p expression is normally reduced in 5‐fluorouracil (5‐FU) resistant CRC cells, whereas miR‐30‐5p overexpression in 5‐FU resistant cells reduces sphere formation and cell viability. Inhibition of miR‐30‐5p reversed the process. Finally, we determined that miR‐30‐5p attenuates the expression of Wnt/β‐catenin signaling target genes (Axin2 and MYC), Wnt luciferase activity, and β‐catenin protein levels in CRC stem cells.     

Light modulated activity of root alkaline/neutral invertase involves the interaction with 14‐3‐3 proteins

Alkaline/neutral invertases (A/N‐Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At‐A/N‐InvG or AT1G35580). We demonstrate that Ser547 at the extreme C‐terminus of the AtCINV1 protein is a substrate of calcium‐dependent kinases (CPK3 and 21) and that phosphorylation creates a high‐affinity binding site for 14‐3‐3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14‐3‐3 proteins enhances its activity. The analysis of three quadruple 14‐3‐3 mutants generated from six T‐DNA insertion mutants of the non‐epsilon family shows both specificity as well as redundancy for this function of 14‐3‐3 proteins. The strong reduction in hexose levels in the roots of one 14‐3‐3 quadruple mutant plant is in line with the activating function of 14‐3‐3 proteins. The physiological relevance of this mechanism that affects A/N‐invertase activity is underscored by the light‐induced activation and is another example of the central role of 14‐3‐3 proteins in mediating dark/light signaling. The nature of the light‐induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca⁺⁺ changes that activate calcium‐dependent kinases, await further study.     

Mir‐29b promotes human aortic valve interstitial cell calcification via inhibiting TGF‐β3 through activation of wnt3/β‐catenin/Smad3 signaling

Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.     

Application of NMR spectroscopy for assignment of the absolute configuration of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one

In order to assign the absolute configurations of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one ( 2a , 2b ), their esters ( 5a , 5b , 5c , 5d ) with (R)‐ or (S)‐2‐methoxyphenylacetic acid ( 4a , 4b ) have been synthesized. The absolute configurations of these compounds have been determined on the basis of NOESY correlations between the protons of the tert‐butyl group and the cyclopentane fragment of the molecules. The crucial part of this analysis was assignment of the absolute configuration at C‐5. Additionally, by calculation of the chemical shift anisotropy, δ^RS, for the relevant protons, it was also possible to confirm the absolute configurations at the C‐2 centres of compounds 2a , 2b and 5a , 5b , 5c , 5d . Chirality, 25:422–426, 2013.© 2013 Wiley Periodicals, Inc.     

Suppression of microRNA‐144‐3p attenuates oxygen–glucose deprivation/reoxygenation‐induced neuronal injury by promoting Brg1/Nrf2/ARE signaling

Accumulating evidence has reported that microRNA‐144‐3p (miR‐144‐3p) is highly related to oxidative stress and apoptosis. However, little is known regarding its role in cerebral ischemia/reperfusion‐induced neuronal injury. Herein, our results showed that miR‐144‐3p expression was significantly downregulated in neurons following oxygen–glucose deprivation and reoxygenation (OGD/R) treatment. Overexpression of miR‐144‐3p markedly reduced cell viability, promoted cell apoptosis, and increased oxidative stress in neurons with OGD/R treatment, whereas downregulation of miR‐144‐3p protected neurons against OGD/R‐induced injury. Brahma‐related gene 1 (Brg1) was identified as a potential target gene of miR‐144‐3p. Moreover, downregulation of miR‐144‐3p promoted the nuclear translocation of nuclear factor erythroid 2‐related factor 2 (Nrf2) and increased antioxidant response element (ARE) activity. However, knockdown of Brg1 significantly abrogated the neuroprotective effects of miR‐144‐3p downregulation. Overall, our results suggest that miR‐144‐3p contributes to OGD/R‐induced neuronal injury in vitro through negatively regulating Brg1/Nrf2/ARE signaling.     

MicroRNA‐1275 induces radiosensitization in oesophageal cancer by regulating epithelial‐to‐mesenchymal transition via Wnt/β‐catenin pathway

Acquired radioresistance is one of the main obstacles for the anti‐tumour efficacy of radiotherapy in oesophageal cancer (EC). Recent studies have proposed microRNAs (miRNAs) as important participators in the development of radioresistance in various cancers. Here, we investigated the role of miR‐1275 in acquired radioresistance and epithelial‐mesenchymal transition (EMT) in EC. Firstly, a radioresistant cell line KYSE‐150R was established, with an interesting discovery was observed that miR‐1275 was down‐regulated in KYSE‐150R cells compared to the parental cells. Functionally, miR‐1275 inhibition elevated radioresistance in KYSE‐150 cells via promoting EMT, whereas enforced expression of miR‐1275 increased radiosensitivity in KYSE‐150R cells by inhibiting EMT. Mechanically, we demonstrated that miR‐1275 directly targeted WNT1 and therefore inactivated Wnt/β‐catenin signalling pathway in EC cells. Furthermore, WNT1 depletion countervailed the promoting effect of miR‐1275 suppression on KYSE‐150 cell radioresistance through hampering EMT, whereas WNT1 overexpression rescued miR‐1275 up‐regulation‐impaired EMT to reduce the sensitivity of KYSE‐150R cells to radiation. Collectively, our findings suggested that miR‐1275 suppressed EMT to encourage radiosensitivity in EC cells via targeting WNT1‐activated Wnt/β‐catenin signalling, providing a new therapeutic outlet for overcoming radioresistance of patients with EC.