Deregulation of protein phosphatase 2A inhibitor SET is associated with malignant progression in breast cancer.

To understand the mechanism underlying metastasis, identification of a mechanism-based and common biomarker for circulating tumour cells (CTCs) in heterogenous breast cancer is needed. SET, an endogenous inhibitor of protein phosphatase 2A, was overexpressed in all subtypes of invasive breast carcinoma tissues. Treatment with SET-targeted siRNAs reduced the motility of MCF-7 and MDA-MB-231 cells in transwell assay. SET knockdown reduced the number of mammospheres by 60-70% in MCF-7 and MDA-MB-231 cells, which was associated with the downregulation of OCT4 and SLUG. Hence, we analysed the presence of SET-expressing CTCs (SET-CTCs) in 24 breast cancer patients. CTCs were enriched using a size-based method and then immunocytochemically analysed using an anti-SET antibody. SET-CTCs were detected in 6/6 (100%) patients with recurrent breast cancer with a median value of 12 (12 cells/3 mL blood), and in 13/18 (72.2%) patients with stage I-III breast cancer with a median value of 2.5, while the median value of healthy controls was 0. Importantly, high numbers of SET-CTCs were correlated with lymph node metastasis in patients with stage I-III disease. Our results indicate that SET contributes to breast cancer progression and can act as a potential biomarker of CTCs for the detection of metastasis.

Gαq protein signaling in the bed nucleus of the stria terminalis regulate the lipopolysaccharide-induced despair-like behavior in mice.

Major depressive disorder (MDD) is highly comorbid with anxiety disorders. It has been reported that the bed nucleus of the stria terminalis (BNST) is important for the induction of anxiety and MDD. Recently, the Gαq protein signaling within the BNST is involved in the induction of anxiety through Gαq protein signaling-mediated RNA-editing of GluR2 subunit, which produces the calcium (Ca2+)-impermeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. On the other hand, the role of Gαq protein signaling within the BNST on the induction of MDD has never been reported yet. Therefore, we investigated whether Gαq protein signaling-producing the Ca2+-impermeable AMPA receptors in the BNST is involved in the lipopolysaccharide (LPS)-induced depressive-like behavior, particularly, despair-like behavior. When mice were systemically challenged with a single dose of LPS (1.2 mg/kg, i.p.), the immobility time during tail suspension test (TST) was increased 24 h after LPS injection. However, pretreatment with bilateral intra-BNST injection of neomycin (6.5 mM, 0.125 µL/side), an inhibitor of phospholipase C that is activated by Gαq protein-coupled receptor stimulation, extended the LPS-induced increase in the immobility time of TST. Furthermore, the co-pretreatment with bilateral intra-BNST injection of neomycin with 1-naphthylacetyl spermine (3 mM, 0.125 µL/side), an antagonist of Ca2+-permeable AMPA receptor, to mimic one of the final forms of Gαq protein activation, abolished the aggravated effect of neomycin and significantly shortened the immobility time compared with the control mice with an intra-BNST injection of artificial cerebrospinal fluid before LPS injection. However, pretreatment with bilateral intra-BNST injection of MDL-12,330A (10 µM, 0.125 µL/side), an inhibitor of adenylyl cyclase that is activated by Gαs protein-coupled receptor stimulation, did not affect the LPS-induced increase in the immobility time of TST. These results indicate that the Gαq protein signaling-mediated RNA-editing of GluR2, which produces the Ca2+-impermeable AMPA receptors within the BNST, regulates the LPS-induced despair-like behavior.

Diagnostic value of a nested polymerase chain reaction for diagnosing cutaneous sporotrichosis from paraffin-embedded skin tissue.

BACKGROUND: The gold standard for diagnosis of cutaneous sporotrichosis involves the isolation of the fungus, Sporothrix, by a culture test. Generally, the sampling for the culture test is performed at the same time as skin biopsy under local anaesthesia. However, the culture test may occasionally return a false negative result. OBJECTIVE: The aim of our study was to investigate the diagnostic value of a molecular method for diagnosing cutaneous sporotrichosis from formalin-fixed and paraffin-embedded (FFPE) tissues. METHODS: Over a 30-year period, we collected 52 cases of cutaneous sporotrichosis from biopsied specimens that had been positively diagnosed by a culture test. A nested PCR specific for Sporothrix detection was applied using FFPE tissue as template. The results were compared with control samples from 79 patients diagnosed with other cutaneous diseases according to histopathological, clinical findings and a cutler test. RESULTS: Of the 52 patients who were tested positive on the culture test, all cutaneous diseases were detected by PCR. Of the 59 patients in the control group, 58 tested negative by PCR. Under our conditions, the calculated sensitivity of this method was 100%, the specificity was 98.7% and the kappa coefficient was 0.984 (95% CI: 0.953-1.000). CONCLUSIONS: The specific PCR assay used appears to be a useful tool for the prompt and accurate diagnosis of sporotrichosis. Using this method, it would be possible to diagnose cutaneous sporotrichosis for patients who were suspected of cutaneous sporotrichosis but tested negative on culturing, and for pathologically suspected cutaneous sporotrichosis patients for whom the culture test was not undertaken.

Down-regulation of histone deacetylase 4, -5 and -6 as a mechanism of synergistic enhancement of apoptosis in human lung cancer cells treated with the combination of a synthetic retinoid, Am80 and green tea catechin.

(-)-Epigallocatechin gallate (EGCG), a green tea catechin, acts as a synergist with various anticancer drugs, including retinoids. Am80 is a synthetic retinoid with a different structure from all-trans-retinoic acid: Am80 is now clinically utilized as a new drug for relapsed and intractable acute promyelocytic leukemia patients. Our experiments showed that the combination of EGCG and Am80 synergistically induced both apoptosis in human lung cancer cell line PC-9 and up-regulated expressions of growth arrest and DNA damage-inducible gene 153 (GADD153), death receptor 5, and p21waf1 genes in the cells. To understand the mechanisms of synergistic anticancer activity of the combination, we gave special attention to the lysine acetylation of proteins. Proteomic analysis using nanoLC-ESI-MS/MS revealed that PC-9 cells treated with the combination contained 331 acetylated proteins, while nontreated cells contained 553 acetylated proteins, and 59 acetylated proteins were found in both groups. Among them, the combination increased acetylated-p53 and acetylated-α-tubulin through reduction of histone deacetylase (HDAC) activity in cytosol fraction, although the levels of acetylation in histones H3 or H4 did not change, and the combination reduced protein levels of HDAC4, -5 and -6 by 20% to 80%. Moreover, we found that a specific inhibitor of HDAC4 and -5 strongly induced p21waf1 gene expression, and that of HDAC6 induced both GADD153 and p21waf1 gene expression, which resulted in apoptosis. All results demonstrate that EGCG in combination with Am80 changes levels of acetylation in nonhistone proteins via down-regulation of HDAC4, -5 and -6 and stimulates apoptotic induction.

Mechanism-based inhibition of cancer metastasis with (-)-epigallocatechin gallate.

Cell motility and cell stiffness are closely related to metastatic activity of cancer cells. (-)-Epigallocatechin gallate (EGCG) has been shown to inhibit spontaneous metastasis of melanoma cell line into the lungs of mice, so we studied the effects of EGCG on cell motility, cell stiffness, and expression of vimentin and Slug, which are molecular phenotypes of epithelial-mesenchymal transition (EMT). Treatments of human non-small cell lung cancer cell lines H1299 and Lu99 with 50 and 100 µM EGCG reduced cell motility to 67.5% and 43.7% in H1299, and 71.7% and 31.5% in Lu99, respectively in in vitro wound healing assay. Studies on cell stiffness using atomic force microscope (AFM) revealed that treatment with 50 µM EGCG increased Young's modulus of H1299 from 1.24 to 2.25 kPa and that of Lu99 from 1.29 to 2.28 kPa, showing a 2-fold increase in cell stiffness, i.e. rigid elasticity of cell membrane. Furthermore, treatment with 50 µM EGCG inhibited high expression of vimentin and Slug in the cells at a leading edge of scratch. Methyl-ß-cyclodextrin, a reagent to deplete cholesterol in plasma membrane, showed inhibition of EMT phenotypes similar that by EGCG, suggesting that EGCG induces inhibition of EMT phenotypes by alteration of membrane organization.

Epithelial-mesenchymal transition in human gastric cancer cell lines induced by TNF-α-inducing protein of Helicobacter pylori.

Helicobacter pylori strains produce tumor necrosis factor-α (TNF-α)-inducing protein, Tipα as a carcinogenic factor in the gastric epithelium. Tipα acts as a homodimer with 38-kDa protein, whereas del-Tipα is an inactive monomer. H. pylori isolated from gastric cancer patients secreted large amounts of Tipα, which are incorporated into gastric cancer cells by directly binding to nucleolin on the cell surface, which is a receptor of Tipα. The binding complex induces expression of TNF-α and chemokine genes, and activates NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells). To understand the mechanisms of Tipα in tumor progression, we looked at numerous effects of Tipα on human gastric cancer cell lines. Induction of cell migration and elongation was found to be mediated through the binding to surface nucleolin, which was inhibited by the nucleolin-targeted siRNAs. Tipα induced formation of filopodia in MKN-1 cells, suggesting invasive morphological changes. Tipα enhanced the phosphorylation of 11 cancer-related proteins in serine, threonine and tyrosine, indicating activation of MEK-ERK signal cascade. Although the downregulation of E-cadherin was not shown in MKN-1 cells, Tipα induced the expression of vimentin, a significant marker of the epithelial-mesenchymal transition (EMT). It is of great importance to note that Tipα reduced the Young's modulus of MKN-1 cells determined by atomic force microscopy: This shows lower cell stiffness and increased cell motility. The morphological changes induced in human gastric cancer cells by Tipα are significant phenotypes of EMT. This is the first report that Tipα is a new inducer of EMT, probably associated with tumor progression in human gastric carcinogenesis.