Quercetin Inhibits the Proliferation of Glycolysis-Addicted HCC Cells by Reducing Hexokinase 2 and Akt-mTOR Pathway.

Increased glycolysis in tumor cells is associated with increased risk of tumor progression and mortality. Therefore, disruption of glycolysis, one of the main sources of cellular energy supply, can serve as a target for suppressing tumor growth and progression. Of note, hexokinase-2 (HK2) plays vital roles in glucose metabolism. Moreover, the expression of HK2 alters the metabolic phenotype and supports the continuous growth of tumor cells, making it an attractive target for cancer therapy. Quercetin (QUE), a bioactive flavonoid, has a profound anti-tumor effect on hepatocellular carcinoma (HCC), but the precise underlying mechanism of this effect is unclear. In the present study, we reported that QUE inhibited the proliferation of HCC cells that relied on aerobic glycolysis. We further found that QUE could decrease the protein levels of HK2 and suppress the AKT/mTOR pathway in HCC cells. In addition, QUE significantly restrained the growth of HCC xenografts and decreased HK-2 expression in vivo. Taken together, we have revealed that QUE suppresses the progression of HCC by inhibiting HK2-dependentglycolysis, which may have a promising potential to be an effective treatments for HCC, especially for those patients with high HK2 expression.

Resveratrol suppresses pulmonary tumor metastasis by inhibiting platelet-mediated angiogenic responses.

BACKGROUND: To explore the impact of Resveratrol (RSV) on the angiogenic potential of activated platelets and to elucidate the underlying mechanism. METHODS: Vascular endothelial growth factor concentrations were measured by enzyme-linked immunosorbent assay. Capillary tube formation assay was used to examine the impact of RSV on the angiogenic potential of activated platelets. The levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate (cGMP) in the supernatant were evaluated using corresponding enzyme-linked immunosorbent assay kits. Immunoblotting assays were used to determine the expression of vasodilator-stimulated phosphoprotein and Akt phosphorylation. A pulmonary metastasis experiment with male nude mice model was performed to test the effect of RSV on pulmonary metastasis and angiogenesis in vivo. RESULTS: RSV inhibited platelets-mediated angiogenic responses induced by adenosine diphosphate (ADP)ADP through increased cGMP generation and cGMP-mediated vasodilator-stimulated phosphoprotein phosphorylation along with reduced intracellular Ca2+ mobilization. In addition, RSV attenuated the platelet secretion and angiogenic responses induced by A549 cells in vitro and suppressed A549 lung cancer metastasis and angiogenesis in nude mice. CONCLUSIONS: RSV is a potential therapeutic drug for the prevention of tumor metastasis by interrupting the platelet-tumor cell amplification loop.

Molecular cloning, expression and functional characterization of interferon-γ-inducible lysosomal thiol reductase (GILT) gene from mandarin fish (Siniperca chuatsi).

Interferon-γ-inducible lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. For this important function in the immune system, we cloned a GILT gene homologue from mandarin fish (designated mGILT), a kind of precious freshwater fish with high market value. Through reverse transcription PCR and rapid amplification of cDNA ends (RACE) strategies, we obtained the full-length cDNA of mGILT, which consists of 1008 bp with a 771 bp open reading frame, encoding a protein of 256 amino acids, with a putative molecular weight of 28.47 kDa. The deduced protein possesses the typical structural features of known GILT proteins, including an active-site motif, a GILT signature sequence, and 6 conserved cysteines. The result of real-time quantitative PCR showed that mGILT mRNA was expressed in a tissue-specific manner. In addition, the expression of mGILT mRNA was obviously up-regulated in splenocytes and kidney after induction with lipopolysaccharide (LPS). Recombinant mGILT fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified using Ni-nitrilotriacetic acid resin. Further study revealed that mGILT exhibit thiol reductase activity on IgG substrate. These results suggest mGILT is highly likely to play a role in the immune responses in mandarin fish.

Exenatide improves hypogonadism and attenuates inflammation in diabetic mice by modulating gut microbiota.

With the rising incidence of diabetes and its onset at a younger age, the impact on the male reproductive system has gradually gained attention. Exenatide is a glucagon-like peptide-1 receptor agonist effective in the treatment of diabetes. However, its role in diabetes-induced reproductive complications has rarely been reported. The study aimed to investigate the mechanism by which exenatide improved diabetic hypogonadism by regulating gut microbiota (GM) mediated inflammation. C57BL/6J mice were equally divided into normal control (NC), diabetic model control (DM) and exenatide-treated (Exe) groups. Testicular, pancreatic, colonic, and fecal samples were collected to assess microbiota, morphologic damage, and inflammation. Exenatide significantly reduced the fasting blood glucose (FBG) level in diabetic mice, increased the testosterone level, ameliorated the pathological morphological damage of islet, colon, and testes, and reduced the expression of pro-inflammatory factors, tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 in colon and testis. Furthermore, exenatide significantly reduced the abundance of some pathogenic bacteria, such as Streptococcaceae and Erysipelotrichaceae, and increased that of beneficial bacteria Akkermansia. Probiotics, such as Lactobacillus were negatively correlated with TNF-α, nuclear factor-kappa-B (NF-κB), IL-6, and FBG. Conditional pathogenic bacteria such as Escherichia/Shigella Streptococcus were positively correlated with TNF-α, NF-κB, IL-6, and FBG. The fecal bacteria transplantation experiment revealed that the abundance of pathogenic bacteria, Peptostreptococcaceae, significantly decreased from Exe group mice to pseudo-sterile diabetic mice, and the pathological damage to testes was also alleviated. These data suggested the protective effects of exenatide on male reproductive damage induced by diabetes by regulating GM.

Cloning, expression, and characterization of Der f 7, an allergen of Dermatophagoides farinae from China.

A full-length cDNA encoding house dust mite allergen Der f 7 from Dermatophagoides farina (Acari: Pyroglyphidae) from China was cloned, sequenced, and successfully expressed. A reference sequence (GenBank accession AY283292) was used to design polymerase chain reaction primers. Analysis revealed eight mismatched nucleotides in five Der f 7 cDNA clones, and the projected amino acid sequence contained six incompatible residues. These results suggest that the sequence of Der f 7 may be polymorphic. Further bioinformatic analysis revealed that the mature Der f 7 allergen had a molecular mass of approximately 21.88 kDa and a theoretical isoelectric point of 4.90. Der f 7 protein secondary structure was composed of a helix (56.63%), extended strand (5.10%), and random coil (38.27%). Group 7 allergens are present in Pyroglyphidae, Acaridae, and Glycyphagidae families, and homology analysis revealed a 86% similarity between Der f 7 and Der p 7. Furthermore, a phylogenetic tree constructed of group 7 allergens from different mite species revealed that Der f 7 and Der p 7 clustered with 100% bootstrap support. Bioinformatics-driven characterization of Der f 7 allergen as conducted in this study may contribute to diagnostic and therapeutic applications for dust mite allergies.

[Preliminary investigation on phylogenetic relationship among three common species of house dust mites].

Dermatophagoides farinae, D. pteronyssinus and Euroglyphus maynei were used for the investigation. The cDNA fragment coding for Der f1 and Der f2 were amplified by PCR, cloned and sequenced. By bioinformatics softwares, the amino acid sequences for Der f1 and Der f2 were deduced and compared with those for the groups 1 and 2 allergens of D. pteronyssinus and E. maynei available in GenBank. Amino acid sequence similarity analysis showed that Der p1 shared 84% identical residues with Eur m1 and 83% with Der f1. Similarly, Der p2 shared 87% identical residues with Eur m2 and 68% with Der f2. In the two phylogenetic trees constructed with group 1 and 2 allergens, D. pteronyssinus was clustered with E. maynei but not with D. farinae, although D. pteronyssinus and D. farinae belong to the same genus. D. pteronyssinus should be more similar to E. maynei than to D. farinae at evolutional level, which was not consistent with the conventional taxonomical relationship based on their morphological characteristics.

SNORA74B gene silencing inhibits gallbladder cancer cells by inducing PHLPP and suppressing Akt/mTOR signaling.

Small nucleolar RNAs (snoRNAs) have been implicated in the development of many cancers. We therefore examined the differential expression of snoRNAs between gallbladder cancer (GBC) tissues and matched adjacent non-tumor tissues using expression microarray analysis with confirmation by quantitative real-time PCR (qRT-PCR). Western blot analysis showed that SNORA74B levels were higher in GBC than non-tumor tissues. SNORA74B expression was positively associated with local invasion, advanced TNM stage, CA19-9 level, and Ki67 expression in patients with GBC, while it was negatively associated with expression of PHLPP, an endogenous Akt inhibitor. Moreover, SNORA74B expression was prognostic for overall survival (OS) and disease-free survival (DFS). Functional studies revealed that silencing SNORA74B in GBC cells using sh-SNORA74B suppressed cell proliferation, induced G1 arrest, and promoted apoptosis. Preliminary molecular investigation revealed that SNORA74B silencing inhibited activation of the AKT/mTOR signaling pathway, while increasing PHLPP expression. PHLPP depletion using shRNA abrogated sh-SNORA74B suppression of GBC cell proliferation, indicating that the antitumor effects of SNORA74B silencing were mediated by PHLPP. These findings define the important role of SNORA74B in cell proliferation, cell cycle, and apoptosis of GBC, and suggest that it may serve as a novel target for GBC treatment.

Blocking beta 2-adrenergic receptor inhibits dendrite ramification in a mouse model of Alzheimer's disease.

Dendrite ramification affects synaptic strength and plays a crucial role in memory. Previous studies revealed a correlation between beta 2-adrenergic receptor dysfunction and Alzheimer's disease (AD), although the mechanism involved is still poorly understood. The current study investigated the potential effect of the selective ß2-adrenergic receptor antagonist, ICI 118551 (ICI), on Aß deposits and AD-related cognitive impairment. Morris water maze test results demonstrated that the performance of AD-transgenic (TG) mice treated with ICI (AD-TG/ICI) was significantly poorer compared with NaCl-treated AD-TG mice (AD-TG/NaCl), suggesting that ß2-adrenergic receptor blockage by ICI might reduce the learning and memory abilities of mice. Golgi staining and immunohistochemical staining revealed that blockage of the ß2-adrenergic receptor by ICI treatment decreased the number of dendritic branches, and ICI treatment in AD-TG mice decreased the expression of hippocampal synaptophysin and synapsin 1. Western blot assay results showed that the blockage of ß2-adrenergic receptor increased amyloid-ß accumulation by downregulating hippocampal α-secretase activity and increasing the phosphorylation of amyloid precursor protein. These findings suggest that blocking the ß2-adrenergic receptor inhibits dendrite ramification of hippocampal neurons in a mouse model of AD.

Cloning, sequence analysis and expression in E. coli of the group 3 allergen of Dermatophagoides farinae.

BACKGROUND: The dust mites, which are mostly represented by Dermatophagoides spp. (Acari: Pyroglyphidae), are the major sources of indoor allergens. Identification and characterization of these mite allergen molecules are an important step in the development of new effective diagnostic procedures and possible therapeutic strategies for allergic disorders associated with dust mites. METHODS: Total RNA was extracted from Dermatophagoides farinae. The gene coding for Der f 3 was amplified by RT-PCR with the primers designed based on previous sequence published in GenBank. The target gene was cloned intermediately into pMD19-T plasmid and finally into plasmid pET28a (+), expressed in E. coli BL21 at the aid of the inducer isopropyl-D-thiogalactopyranoside (IPTG). The physicochemical properties, spatial structure of the allergen were analyzed with bioinformatics software. RESULTS: The cDNA coding for group 3 allergen of Dermatophagoides farinae from China was cloned and expressed successfully. Sequencing analysis showed that there were nineteen mismatched nucleotides in five Der f 3 cDNA clones in comparison with the reference (GenBank Accession No. AY283291), which resulted in deduced amino acid sequence incompatibility in eleven residues. Bioinformatics analysis revealed that the Der f 3 pro-protein was an extracellular hydrophobic protein, consisting of 259 amino acids with a 16 amino acid signal peptide. The protein was deduced to have three chymotrypsin active sites (53-68 AA, 108-122 AA and 205-217 AA), one N-glycosylation site, one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, and five N-myristoylation sites. CONCLUSIONS: Der f 3 is an extracellular hydrophobic protein which possesses multiple activation and phosphorylation sites. Polymorphism may exist in the Der f 3 gene but this needs to be further confirmed in the future.