HIF-1α is necessary for activation and tumour-promotion effect of cancer-associated fibroblasts in lung cancer

Cancer-associated fibroblasts (CAFs) activation is crucial for the establishment of a tumour promoting microenvironment, but our understanding of CAFs activation is still limited. In this study, we found that hypoxia-inducible factor-1α (HIF-1α) was highly expressed in CAFs of human lung cancer tissues and mouse spontaneous lung tumour. Accordingly, enhancing the expression of HIF-1α in fibroblasts via hypoxia induced the conversion of normal fibroblasts into CAFs. HIF-1α-specific inhibitor or HIF-1α knockout (KO) significantly attenuated CAFs activation, which was manifested by the decreased expression of COL1A2 and α-SMA. In vivo, during tumour formation, the expression of Ki-67 and proliferating cell nuclear antigen (PCNA) in the tumour tissue with HIF-1α KO fibroblasts was significantly lower than that of normal fibroblasts. Moreover, HIF-1α in fibroblasts could activate the NF-κB signalling pathway and enhance a subsequent secretion of CCL5, thus promoting the tumour growth. In conclusion, our results suggest that HIF-1α is essential for the activation and tumour-promotion function of CAFs in lung cancer (LC). And targeting HIF-1α expression on CAFs may be a promising strategy for LC therapy.     

p-Nitrophenyl-β-d-xyloside modulates proteoglycan synthesis and secretory differentiation in mouse mammary epithelial cell cultures

Summary Primary cultures of mouse mammary epithelial cells synthesize significant quantities of chondroitin and heparan sulfate proteoglycans (16). Long term treatment of such cultures with p-nitrophenyl-β-D-xylopyranoside leads to a 10–20 fold increase in the synthesis and secretion of free chondroitin sulfate glycosaminoglycan (GAG) chains and assembly of a cell-associated matrix that is relatively enriched in heparan sulfate proteoglycan. This modulation of cell-synthesized proteoglycans leads to significant changes in cell morphology and cellular differentiation. Notably cells cultured on plastic culture dishes change from being flattened to cuboidal. The synthesis of the milk proteins α1, α2, and β-casein is also increases as is the formation of fat droplets and fat droplet membrane components. Promotion of differentiation increases with increasing xyloside concentration in the range 0–1.5 mM, but there may be a block in secretion at higher xyloside concentrations. While the detailed mechanisms remain to be elucidated, we conclude that the composition of proteoglycans incorporated into the matrix (and possibly the glycosaminoglycans secreted into the medium), may play a significant role in maintaining the phenotypic characteristics of terminally differentiated mammary epithelial cells. This research was supported by the Office of Health and Environmental Research, Office of Energy Research, U.S. Dept. of Energy under contract No. DEAC-03-76SF00098 and by National Institutes of Health Grant CA44398-01 (G. Parry) Editor's Statement Exogenous elements of extracellular matrix affect expression of cultured mammary cell function. This work reports manipulation of cell-derived endogenous matrix elements and shows correlative changes in cell functions.     

Facile one-pot synthesis of fluorescent aminoclay and the applications for fluorescence sensing Cr2O72−

Here, we developed a facile one-pot strategy for the fabrication of fluorescent aminoclay (F-AC) through in situ solvothermal treatment of 3-aminopropyltrimethoxysilane, MgCl₂, and sodium ascorbate at 180°C for 6 h. The obtained F-AC exhibited blue emission, good water solubility, and satisfactory photostability. It was observed that Cr₂O₇²⁻ could selectively quench the fluorescence of F-AC through the inner filter effect and static quenching process. As a result, a novel fluorescent F-AC-based nanosensor was constructed with good linearity in the range 0.1–75 μM. The nanosensor was successfully applied in real water samples with satisfactory results. This work not only provides a novel nanosensor for Cr₂O₇²⁻, but also highlights the F-AC's promising applications in wider fields due to the versatility and simplicity of the preparation strategy.     

Cardioprotection from oxidative stress in the newborn heart by activation of PPARγ is mediated by catalase

Regulation of catalase (CAT) by peroxisome proliferator-activated receptor-γ (PPARγ) was investigated to determine if PPARγ activation provides cardioprotection from oxidative stress caused by hydrogen peroxide (H(2)O(2)) in an age-dependent manner. Left ventricular developed pressure (LVDP) was measured in Langendorff perfused newborn or adult rabbit hearts, exposed to 200μM H(2)O(2), with perfusion of rosiglitazone (RGZ) or pioglitazone (PGZ), PPARγ agonists. We found: (1) H(2)O(2) significantly decreased sarcomere shortening in newborn ventricular cells but not in adult cells. Lactate dehydrogenase (LDH) release occurred earlier in newborn than in adult heart, which may be due, in part, to the lower expression of CAT in newborn heart. (2) RGZ increased CAT mRNA and protein as well as activity in newborn but not in adult heart. GW9662 (PPARγ blocker) eliminated the increased CAT mRNA by RGZ. (3) In newborn heart, RGZ and PGZ treatment inhibited release of LDH in response to H(2)O(2) compared to H(2)O(2) alone. GW9662 decreased this inhibition. (4) LVDP was significantly higher in both RGZ+H(2)O(2) and PGZ+H(2)O(2) groups than in the H(2)O(2) group. Block of PPARγ abolished this effect. In contrast, there was no effect of RGZ in adult. (5) The cardioprotective effects of RGZ were abolished by inhibition of CAT. In conclusion, PPARγ activation is cardioprotective to H(2)O(2)-induced stress in the newborn heart by upregulation of catalase. These data suggest that PPARγ activation may be an effective therapy for the young cardiac patient.     

Endothelial NO and O2− production rates differentially regulate oxidative,nitroxidative, and nitrosative stress in the microcirculation

Endothelial dysfunction causes an imbalance in endothelial NO and O₂⁻ production rates and increased peroxynitrite formation. Peroxynitrite and its decomposition products cause multiple deleterious effects including tyrosine nitration of proteins, superoxide dismutase (SOD) inactivation, and tissue damage. Studies have shown that peroxynitrite formation during endothelial dysfunction is strongly dependent on the NO and O₂⁻ production rates. Previous experimental and modeling studies examining the role of NO and O₂⁻ production imbalance on peroxynitrite formation showed different results in biological and synthetic systems. However, there is a lack of quantitative information about the formation and biological relevance of peroxynitrite under oxidative, nitroxidative, and nitrosative stress conditions in the microcirculation. We developed a computational biotransport model to examine the role of endothelial NO and O₂⁻ production on the complex biochemical NO and O₂⁻ interactions in the microcirculation. We also modeled the effect of variability in SOD expression and activity during oxidative stress. The results showed that peroxynitrite concentration increased with increase in either O₂⁻ to NO or NO to O₂⁻ production rate ratio (Q_O2⁻/Q_NO or Q_NO/Q_O2⁻, respectively). The peroxynitrite concentrations were similar for both production rate ratios, indicating that peroxynitrite-related nitroxidative and nitrosative stresses may be similar in endothelial dysfunction or inducible NO synthase (iNOS)-induced NO production. The endothelial peroxynitrite concentration increased with increase in both Q_O2⁻/Q_NO and Q_NO/Q_O2⁻ ratios at SOD concentrations of 0.1–100 μM. The absence of SOD may not mitigate the extent of peroxynitrite-mediated toxicity, as we predicted an insignificant increase in peroxynitrite levels beyond Q_O2⁻/Q_NO and Q_NO/Q_O2⁻ ratios of 1. The results support the experimental observations of biological systems and show that peroxynitrite formation increases with increase in either NO or O₂⁻ production, and excess NO production from iNOS or from NO donors during oxidative stress conditions does not reduce the extent of peroxynitrite mediated toxicity.     

When it is too hot for photosynthesis: heat-induced instability of photosynthesis in relation to respiratory burst, cell permeability changes and H₂O₂ formation

Photosynthesis rate (A_n) becomes unstable above a threshold temperature, and the recovery upon return to low temperature varies because of reasons not fully understood. We investigated responses of A_n, dark respiration and chlorophyll fluorescence to supraoptimal temperatures of varying duration and kinetics in Phaseolus vulgaris asking whether the instability of photosynthesis under severe heat stress is associated with cellular damage. Cellular damage was assessed by Evans blue penetration (enhanced membrane permeability) and by H₂O₂ generation [3,3′‐diaminobenzidine 4HCl (DAB)‐staining]. Critical temperature for dark fluorescence (F₀) rise (T_F) was at 46–48 °C, and a burst of respiration was observed near T_F. However, A_n was strongly inhibited already before T_F was reached. Membrane permeability increased with temperature according to a switch‐type response, with enhanced permeability observed above 48 °C. Experiments with varying heat pulse lengths and intensities underscored the threshold‐type loss of photosynthetic function, and indicated that the degree of photosynthetic deterioration and cellular damage depended on accumulated heat‐dose. Beyond the ‘point of no return’, propagation of cellular damage and reduction of photosynthesis continued upon transfer to lower temperatures and photosynthetic recovery was slow or absent. We conclude that instability of photosynthesis under severe heat stress is associated with time‐dependent propagation of cellular lesions.     

Indoleamine 2, 3-dioxygenase （IDO） is essential for dendritic cell activation and chemotactic responsiveness to chemokines

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs),and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC functions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflammatory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regulated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.     

Tat-DJ-1 enhances cell survival by inhibition of oxidative stress,NF-κB and MAPK activation in HepG2 cells

Objectives

To identify the protective effect of DJ-1 protein against oxidative stress-induced HepG2 cell death, we used cell-permeable wild type (WT) and a mutant (C106A Tat-DJ-1) protein.

Results

By using western blotting and fluorescence microscopy, we observed WT and C106A Tat-DJ-1 proteins were efficiently transduced into HepG2 cells. Transduced WT Tat-DJ-1 proteins increased cell survival and protected against DNA fragmentation and intracellular ROS generation levels in H₂O₂-exposed HepG2 cells. At the same time, transduced WT Tat-DJ-1 protein significantly inhibited NF-κB and MAPK (JNK and p38) activation as well as regulated the Bcl-2 and Bax expression levels. However, C106A Tat-DJ-1 protein did not show any protective effect against cell death responses in H₂O₂-exposed HepG2 cells.

Conclusions

Oxidative stress-induced HepG2 cell death was significantly reduced by transduced WT Tat-DJ-1 protein, not by C106A Tat-DJ-1 protein. Thus, transduction of WT Tat-DJ-1 protein could be a novel strategy for promoting cell survival in situations of oxidative stress-induced HepG2 cell death.

     

Dephosphorylation of eIF2α is essential for protein synthesis increase and cell cycle progression after sea urchin fertilization

The eukaryotic Initiation Factor 2 (eIF2) is a key regulator of protein synthesis in eukaryotic cells, implicated in the initiation step of translation. Fertilization of the sea urchin eggs triggers a rapid increase in protein synthesis activity, which is necessary for the progress into embryonic cell cycles. Here we demonstrate that fertilization triggers eIF2α dephosphorylation, concomitant with an increase in protein synthesis and that induction of the eIF2α phosphorylation is intimately linked with an inhibition of protein synthesis and cell cycle arrest. Using a phospho-mimetic protein microinjected into sea urchin eggs, we showed that dephosphorylation of eIF2α is necessary for protein synthesis activity and cell division progression following fertilization. Our results demonstrate that regulation of eIF2α plays an important role in the protein synthesis rise that occurs during early development following fertilization.     

Dexmedetomidine alleviates H2O2-induced oxidative stress and cell necroptosis through activating of α2-adrenoceptor in H9C2 cells

Molecular Biology Reports - Oxidative stress induced necroptosis is important in myocardial ischemia/reperfusion injury. Dexmedetomidine (Dex), an α2-adrenoceptor (α2-AR) agonist, has...     

Bioprocess parameters of cell growth and human μ opioid receptor expression in recombinant Drosophila S2 cell cultures in a bioreactor

The paper describes a recombinant Schneider 2 (rS2) cell culture and protein expression in a bioreactor. S2 cells were transfected with a plasmid containing a fusion protein (human μ opioid receptor, hMOR, and green fluorescent protein, EGFP) under the control of inducible metallothionein promoter. A bioprocess in a bioreactor with 5% dissolved oxygen, 27°C and 120 rpm enabled the cell culture to attain 5.3×10⁷ viable cells/mL at 96 h. The induction decreased the cell multiplication (2.5×10⁷ viable cells/mL at 72 h). Glutamine and glucose and low levels of lactate were consumed. A fast recombinant protein synthesis took place and, at 6 h of induction, 2×10⁴ receptors/cell could be detected by a functional binding assay. Fluorescence measurements showed a progressive increase of recombinant protein expression with a maximal value of 1.26×10⁵ fluo counts/s at 24 h of induction. The data shown in this paper indicate a practical and scaleable cell culture bioprocess procedure for the preparation of recombinant proteins expressed in S2 cells.     

Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth

Casein kinase 1δ/ε (CK1δ/ε) and their yeast homologue Hrr25 are essential for cell growth. Further, CK1δ is overexpressed in several malignancies, and CK1δ inhibitors have shown promise in several preclinical animal studies. However, the substrates of Hrr25 and CK1δ/ε that are necessary for cell growth and survival are unknown. We show that Hrr25 is essential for ribosome assembly, where it phosphorylates the assembly factor Ltv1, which causes its release from nascent 40S subunits and allows subunit maturation. Hrr25 inactivation or expression of a nonphosphorylatable Ltv1 variant blocked Ltv1 release in vitro and in vivo, and prevented entry into the translation-like quality control cycle. Conversely, phosphomimetic Ltv1 variants rescued viability after Hrr25 depletion. Finally, Ltv1 knockdown in human breast cancer cells impaired apoptosis induced by CK1δ/ε inhibitors, establishing that the antiproliferative activity of these inhibitors is due, at least in part, to disruption of ribosome assembly. These findings validate the ribosome assembly pathway as a novel target for the development of anticancer therapeutics.     

Peroxiredoxin 2 is upregulated in colorectal cancer and contributes to colorectal cancer cells’ survival by protecting cells from oxidative stress

Peroxiredoxin 2 (Prdx2) is a member of the peroxiredoxin family, which is responsible for neutralizing reactive oxygen species. Prdx2 has been found to be elevated in several human cancer cells and tissues, including colorectal cancer (CRC), and it influences diverse cellular processes involving cells’ survival, proliferation, and apoptosis, which suggests a possible role for Prdx2 in the maintenance of cancer cell. However, the mechanism by which Prdx2 modulates CRC cells’ survival is unknown. The current study aimed to determine the effect of elevated Prdx2 on CRC cells and to further understand the underlying mechanisms. The results of this study showed that Prdx2 was upregulated in CRC tissues compared with the matched noncancer colorectal mucosa tissues and that Prdx2 expression was positively associated with tumor metastasis and the TNM stage. In the LoVo CRC cell line, Prdx2 was upregulated at both the RNA and protein levels compared with the normal FHC colorectal mucosa cell line. In addition, the LoVo CRC cell line was significantly more resistant to hydrogen peroxide (H₂O₂)-induced apoptosis because of a failure to activate pro-apoptotic pathways in contrast to Prdx2 knockdown cells. Suppression of Prdx2 using a lentiviral vector-mediated Prdx2-specific shRNA in the LoVo cell line restored H₂O₂ sensitivity. Our results suggested that Prdx2 has an essential role in regulating oxidation-induced apoptosis in CRC cells. Prdx2 may have potential as a therapeutic target in CRC.     

The role and mechanism of metal ions and their complexes in enhancing damage in biological systems or in protecting these systems from these systems from the toxicity of O2−

Cooper complexes of 1,10-phenanthroline and some substituted 1,10-phenanthroline cleave DNA in the presence of a reducing agent and molecular oxygen. Generally, the damage is attributed to hydroxyl radicals which are formed through the Haber-Weiss reaction. It is assumed that this reaction occurs with the ternary metal complexes with the biological target and the mechanism is defined as the “site specific mechanism.” In these systems, O₂⁻ drives the cycle through the reduction of copper(II). On the other hand, these same copper complexes catalyze the dismutation of O₂⁻ and thus should protect the systems from O₂⁻ toxicity. In this article, the toxicity of these complexes is explained on kinetic grounds. A general discussion on the various factors which could cause the metal ions or their complexes to act either as protectors from O₂⁻ toxicity or as sensitizers of toxic effects of O₂⁻ is given.     

Effect of serum from chickens treated with clenbuterol on myosin accumulation, β-adrenergic receptor population, and cyclic AMP synthesis in embryonic chicken skeletal muscle cell cultures

Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 microM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The beta-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25-30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 microM isoproterenol, limited increases of 12-20% in cAMP concentration above the basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 microM isoproterenol, cAMP concentration was stimulated 5- to 9-fold above the basal levels. Thus, not only did cells grown in horse serum have a higher betaAR population, but also each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration was tested. No correlation was apparent between basal cAMP concentration and MHC content.