Mutually exclusive antiproliferative effect of cell line-specific HOX inhibition in epithelial ovarian cancer cell lines: SKOV-3 vs RMUG-S

We aimed to discover cell line-specific overexpressed HOX genes responsible for chemoresistance and to identify the mechanisms behind HOX-induced cell line-specific chemoresistance in EOC. Ten HOX genes and eight EOC cell lines were tested for any cell line-specific overexpression that presents a mutually exclusive pattern. Cell viability was evaluated after treatment with cisplatin and/or siRNA for cell line-specific overexpressed HOX genes. Immunohistochemical (IHC) staining for HOXB9 was performed in 84 human EOC tissues. HOXA10 and HOXB9 were identified as cell line-specific overexpressed HOX genes for SKOV-3 and RMUG-S, respectively. Inhibiting the expression of cell line-specific HOX genes, but not of other HOX genes, significantly decreased cell viability. In SKOV-3 cells, cell viability decreased to 46.5% after initial 10 µM cisplatin treatment; however, there was no further decrease upon additional treatment with HOXA10 siRNA. In contrast, cell viability did not significantly decrease upon cisplatin treatment in RMUG-S cells, but decreased to 65.5% after additional treatment with HOXB9 siRNA. In both cell lines, inhibiting cell line-specific HOX expression enhanced apoptosis but suppressed the expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, MMP9, and Oct4. IHC analysis showed that platinum-resistant cancer tissues more frequently had high HOXB9 expression than platinum-sensitive cancer tissues. HOXB9, which is overexpressed in RMUG-S but not in SKOV-3 cells, appeared to be associated with cell line-specific platinum resistance in RMUG-S. Inhibiting HOXB9 overexpression in RMUG-S cells may effectively eliminate platinum-resistant ovarian cancer cells by facilitating apoptosis and inhibiting EMT.     

Feedback regulation of phospholipase C-beta by protein kinase C

Treatment of a variety of cells and tissues with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC) results in the inhibition of receptor-coupled inositol phospholipid-specific phospholipase C (PLC) activity. To determine whether or not the targets of TPA-activated PKC include one or more isozymes of PLC, studies were carried out with PC12, C6Bu1, and NIH 3T3 cells, which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of the cells with TPA stimulated the phosphorylation of serine residues in PLC-beta, but the phosphorylation state of PLC-gamma and PLC-delta was not changed significantly. Phosphorylation of bovine brain PLC-beta by PKC in vitro resulted in a stoichiometric incorporation of phosphate at serine 887, without any concomitant effect on PLC-beta activity. We propose, therefore, that rather than having a direct effect on enzyme activity, the phosphorylation of PLC-beta by PKC may alter its interaction with a putative guanine nucleotide-binding regulatory protein and thereby prevent its activation.     

Susceptibilities of bacterial cellulose containing N-acetylglucosamine residues for cellulolytic and chitinolytic enzymes.

Detailed characterization of enzyme susceptibility of bacterial cellulose containing N-acetylglucosamine (GlcNAc) residues (N-AcGBC) which possess high susceptibility for cellulase and lysozyme and slight susceptibility for chitinase was studied. Turbidimetric lysozyme assay of N-AcGBC showed that (i) the susceptibilities of various N-AcGBCs for lysozyme were proportional to GlcNAc content, and (ii) N-AcGBC homogenates were divided into two groups based on the rate of turbidity reduction (not dependent on GlcNAc content). High reactivity of N-AcGBC for lysozyme would arise from fine microfibrils characteristic of bacterial cellulose (BC) and random distribution of GlcNAc residues in N-AcGBC because water soluble oligomers of N-AcGBC produced by lysozymic hydrolysis did not inhibit lysozyme activity; however, the random distribution of GlcNAc seemed to result in the slight susceptibility of N-AcGBC for chitinase. The rate of cellulolytic turbidity reduction of N-AcGBC was slower than that of BC, which arose from the inhibition for binding of cellulase by GlcNAc residues.     

Chemoimmunotherapeutic effect of cyclophosphamide on the highly metastatic MAT 13762 tumor

Summary We have observed that cyclophosphamide (CY) treatment of 13762 mammary adenocarcinoma tumor-bearing rats was found to cause tumor regression. Tumor-bearing animals cured with three low doses of CY were partially immune against IV and SC challenge with a high dose of 13762 cells. This immune protection mechanism in CY-cured animals appears to be a T (Ig^–) cell-mediated response. Irradiated rats reconstituted with CY-cured animal spleen cells were also partially protected against IV and SC challenge with 13762 cells, whereas irradiated rats reconstituted with CY-control animal spleen cells were not. In vitro primary and secondary cell-mediated cytotoxic activity of CY-cured spleen cells against target 13762 cells was low. The possible relevance of this tumor-model study is in the understanding of CY-induced tumor immune response and its role in preventing metastases or perhaps recurrent tumor growth.     

Kushenol A and 8-prenylkaempferol,tyrosinase inhibitors,derived from Sophora flavescens

Tyrosinase is known for an enzyme that plays a key role in producing the initial precursor of melanin biosynthesis. Inhibition of the catalytic reaction of this enzyme led to some advantage such as skin-whitening and anti-insect agents. To find a natural compound with inhibitory activity towards tyrosinase, the five flavonoids of kushenol A (1), 8-prenylkaempferol (2), kushenol C (3), formononetin (4) and 8-prenylnaringenin (5) were isolated by column chromatography from a 95% methanol extract of Sophora flavescens. The ability of these flavonoids to block the conversion of L-tyrosine to L-DOPA by tyrosinase was tested in vitro. Compounds 1 and 2 exhibited potent inhibitory activity, with IC50 values less than 10?µM. Furthermore, enzyme kinetics and molecular docking analysis revealed the formation of a binary encounter complex between compounds 1–4 and the enzyme. Also, all of the isolated compounds (1–5) were confirmed to possess antioxidant activity.     

Gallic acid attenuates calcium calmodulin‐dependent kinase II‐induced apoptosis in spontaneously hypertensive rats

Hypertension causes cardiac hypertrophy and leads to heart failure. Apoptotic cells are common in hypertensive hearts. Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is associated with apoptosis. We recently demonstrated that gallic acid reduces nitric oxide synthase inhibition‐induced hypertension. Gallic acid is a trihydroxybenzoic acid and has been shown to have beneficial effects, such as anti‐cancer, anti‐calcification and anti‐oxidant activity. The purpose of this study was to determine whether gallic acid regulates cardiac hypertrophy and apoptosis in essential hypertension. Gallic acid significantly lowered systolic and diastolic blood pressure in spontaneously hypertensive rats (SHRs). Wheat germ agglutinin (WGA) and H&E staining revealed that gallic acid reduced cardiac enlargement in SHRs. Gallic acid treatment decreased cardiac hypertrophy marker genes, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), in SHRs. The four isoforms, α, β, δ and γ, of CaMKII were increased in SHRs and were significantly reduced by gallic acid administration. Gallic acid reduced cleaved caspase‐3 protein as well as bax, p53 and p300 mRNA levels in SHRs. CaMKII δ overexpression induced bax and p53 expression, which was attenuated by gallic acid treatment in H9c2 cells. Gallic acid treatment reduced DNA fragmentation and the TUNEL positive cells induced by angiotensin II. Taken together, gallic acid could be a novel therapeutic for the treatment of hypertension through suppression of CaMKII δ‐induced apoptosis.     

Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus,Epstein-Barr virus,and polyomavirus BK in whole blood from transplant candidates

Transplant recipients are more susceptible to bacterial and viral infections. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyomavirus BK (BK) are risk factors for graft dysfunction. All three of them are latent viruses that can cause serious disease in immunocompromised patients. Mainly qualitative PCR tests are required for diagnosis and quantitative monitoring, which are used to follow the response to transplantation. We developed a multiplex real-time PCR (qPCR) method to detect these viruses during blood screenings of transplant recipients. We also validated analytical and clinical performance tests using the developed multiplex qPCR. The limit of detection (LOD) was 100, 125, and 183 copies/ml for CMV, EBV, and BK, respectively. These results had high linearity (R² = 0.997) and reproducibility (CV range, 0.95–2.38%, 0.52–3.32%, and 0.31–2.45%, respectively). Among 183 samples, we detected 8 samples that were positive for CMV, while only 6 were positive for EBV, and 3 were positive for BK. Therefore, the viral infection prevalence in transplant candidates was 4.40% for CMV, 3.29% for EBV, and 1.64% for BK. This multiplex qPCR method should be used widely for diagnosing and monitoring latent viral infections in transplant recipients.     

Resistance of transgenic rice events (rbcS:cry1Ac) against three lepidopteran rice pests

The transgenic rice expressing cry1Ac gene, which is linked to the rice rbcS promoter and its transit peptide sequence (tp), was highly resistant against all instars of Cnaphalocrocis medinalis (Guenetée) (Lepidoptera: Crambidae). In this study, we evaluated the larval mortality, behavior change, and field occurrence of three main rice pests, C. medinalis, Naranga aenescens (Moore) (Lepidoptera: Noctuidae), and Parnara guttata (Bremer & Grey) (Lepidoptera: Hesperiidae) in T4 generations of three Bt rice events (rbcS3:cry1Ac; 608102, 608104 and 608107) and non-Bt rice. All of the three Bt rice events were resistant to C. medinalis which showed significantly higher mortality for all instars compared to non-Bt rice. The resistance of Bt rice events against the larvae decreased gradually as the larvae developed. However, the survived larvae which ingested Bt rice events died eventually without further development. The resistance of three Bt rice events was investigated in the pot test, which was conducted with 3rd instars of C. medinalis, N. aenescens, and P. guttata, showed mortalities of over 70%. In behavioral assay, C. medinalis fed on the Bt rice events showed feeding avoidance and less leaf rolling behavior compared to that of the larvae fed on non-Bt rice. A 2-yr field survey conducted with larvae of C. medinalis and P. guttata also showed that the three Bt rice events significantly had lower damaged on leaves compared to that of non-Bt rice. Overall, the three Bt rice events were highly resistant to the larvae of lepidopteran target rice pests.