Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E₂ without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.     

Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps

Gaussia luciferase secreted by the copepod Gaussia princeps catalyzes the oxidation of coelenterazine to produce blue light. The primary structure of Gaussia luciferase deduced from the cDNA sequence shows two repeat sequences of 71 amino acid residues, suggesting the luciferase consists of two structural domains. Two domains in Gaussia luciferase were expressed independently in Escherichia coli cells, purified and characterized. We found that both domains have luminescence activity with coelenterazine, and the catalytic properties including luminescence spectrum, optimal pH, substrate specificity and luminescence stimulation by halogen ions (Cl⁻, Br⁻ and I⁻) are identical to intact Gaussia luciferase. Thus, Gaussia luciferase has two catalytic domains for the luminescence reaction.     

Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes. Combined treatment with B:9-23 peptide and polyinosinic-polycytidylic acid (poly I:C), but neither alone, induce insulitis in normal BALB/c mice. In contrast, the combined treatment accelerated insulitis, but prevented diabetes in NOD mice. Our immunofluorescence study with anti-CD4/anti-Foxp3 revealed that the proportion of Foxp3 positive CD4⁺CD25⁺ regulatory T cells (Tregs) was elevated in the islets of NOD mice treated with B:9-23 peptide and poly I:C, as compared to non-treated mice. Depletion of Tregs by anti-CD25 antibody hastened spontaneous development of diabetes in non-treated NOD mice, and abolished the protective effect of the combined treatment and conversely accelerated the onset of diabetes in the treated mice. These results indicate that poly I:C combined with B:9-23 peptide promotes infiltration of both pathogenic T cells and predominantly Tregs into the islets, thereby inhibiting progression from insulitis to overt diabetes in NOD mice.     

Expression of highly sulfated keratan sulfate synthesized in human glioblastoma cells

Keratan sulfate (KS) proteoglycan is expressed in the extracellular matrix or cell surface in numerous tissues, predominantly in those of the cornea, cartilage, and brain. However, its structure, function, and regulation remain poorly understood. Our investigation of KS expression in glioblastoma cell lines using Western-blot and flow cytometry with anti-KS antibody (5D4) revealed that LN229 glioblastoma cell highly expresses KS on a cell surface. Real-time PCR analysis showed that LN229 expresses a high level of keratan sulfate Gal-6-sulfotransferase. Results of this study also demonstrate that recombinant 5D4-reactive aggrecan is produced in LN229. Taken together, these results suggest that LN229 produces 5D4-reactive highly sulfated KS and is useful to investigate the KS structure and function in glioblastoma.     

Validation of HB-EGF and amphiregulin as targets for human cancer therapy

Aberrant expression levels of epidermal growth factor receptor (EGFR) and its cognate ligands have been recognized as one of the causes of cancer progression. To investigate the validity of EGFR ligands as targets for cancer therapy, we examined the expression of EGFR ligands and in vitro anti-tumor effects of small interference RNA (siRNA) for EGFR ligands in various cancer cells. HB-EGF expression was dominantly elevated in ovarian, gastric, and breast cancer, melanoma and glioblastoma cells, whereas amphiregulin was primarily expressed in pancreatic, colon, and prostate cancer, renal cell carcinoma and cholangiocarcinoma cells. Transfection of siRNAs for HB-EGF or amphiregulin into these cells significantly increased the numbers of apoptotic cells with attenuation of EGFR and ERK activation. In lung cancer cells, any EGFR ligand was not recognized as a validated target for cancer therapy. These results suggest that HB-EGF and amphiregulin are promising targets for cancer therapy.     

Spectroscopic studies of perturbed T1 Cu sites in the multicopper oxidases Saccharomyces cerevisiae Fet3p and Rhus vernicifera laccase: allosteric coupling between the T1 and trinuclear Cu sites

The multicopper oxidases catalyze the 4e- reduction of O2 to H2O coupled to the 1e- oxidation of 4 equiv of substrate. This activity requires four Cu atoms, including T1, T2, and coupled binuclear T3 sites. The T2 and T3 sites form a trinuclear cluster (TNC) where O2 is reduced. The T1 is coupled to the TNC through a T1-Cys-His-T3 electron transfer (ET) pathway. In this study the two T3 Cu coordinating His residues which lie in this pathway in Fet3 have been mutated, H483Q, H483C, H485Q, and H485C, to study how perturbation at the TNC impacts the T1 Cu site. Spectroscopic methods, in particular resonance Raman (rR), show that the change from His to Gln to Cys increases the covalency of the T1 Cu-S Cys bond and decreases its redox potential. This study of T1-TNC interactions is then extended to Rhus vernicifera laccase where a number of well-defined species including the catalytically relevant native intermediate (NI) can be trapped for spectroscopic study. The T1 Cu-S covalency and potential do not change in these species relative to resting oxidized enzyme, but interestingly the differences in the structure of the TNC in these species do lead to changes in the T1 Cu rR spectrum. This helps to confirm that vibrations in the cysteine side chain of the T1 Cu site and the protein backbone couple to the Cu-S vibration. These changes in the side chain and backbone provide a possible mechanism for regulating intramolecular T1 to TNC ET in NI and partially reduced enzyme forms for efficient turnover.     

Characterization of microsatellite loci from the Japanese eel Anguilla japonica

The Japenese eel, Anguilla japonica, is generally assumed to be composed of a single population with wide distribution range, and some genetic studies using allozyme or mitochondrial DNA methods supported this population model. However, one genetic study suggested the existence of multiple populations in this species, and thus, more detailed studies on the population structure is needed. Here we characterized a total of 11 microsatellite markers of the Japanese eel. These will serve as powerful tools for detailed population study for the Japanese eel, though two of them showed the significant departure from the Hardy–Weinberg expectations.     

Does Waist Circumference Add to the Predictive Power of the Body Mass Index for Coronary Risk?

Objectives: To examine the power of the combined measurements of body mass index (BMI) and waist circumference (WC) for the prediction of abnormality in coronary heart disease risk factors and to determine whether the additional measurement of WC is predictive in older men and women. Research Methods and Procedures: 1190 men and 751 women of the Baltimore Longitudinal Study of Aging were dichotomized into younger (<65 years) and older (65+ years) age groups. Coronary risk factors in the realms of glucose/insulin metabolism, blood pressure, and plasma lipids were assessed. The relationship of BMI and WC, singly and combined, to 10 risk factors for coronary heart disease was examined. Results: In younger and older men and women, BMI and WC are highly correlated (0.84 to 0.88). BMI and WC are also significantly correlated to all 10 coronary risk factors in younger men and women and to 8 of the 10 in the older men and women. Both partial correlation and logistic regression analyses revealed a modest but significant improvement in the prediction of coronary risk in younger men and women by WC after controlling for the level of BMI. There was no improvement in the older subjects. Discussion: WC adds only modestly to the prediction of coronary risk in younger subjects once BMI is known, and adds nothing to the production of risk in older subjects.