Hedgehog signaling in skin cancers

An increasing progress on the role of Hedgehog (Hh) signaling for carcinogenesis has been achieved since the link of Hh pathway to human cancer was firstly established. In particular, the critical role of Hh signaling in the development of Basal cell carcinoma (BCC) has been convincingly demonstrated by genetic mutation analyses, mouse models of BCCs, and successful clinical trials of BCCs using Hh signaling inhibitors. In addition, the Hh pathway activity is also reported to be involved in the pathogenesis of Squamous Cell Carcinoma (SCC), melanoma and Merkel Cell Carcinoma. These findings have significant new paradigm on Hh signaling transduction, its mechanisms in skin cancer and even therapeutic approaches for BCC. In this review, we will summarize the major advances in the understanding of Hh signaling transduction, the roles of Hh signaling in skin cancer development, and the current implications of “mechanism-based” therapeutic strategies.     

Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells

Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H₂O₂ to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H₂O₂ was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.     

Synthesis and biological evaluation of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors

A series of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles 14a-ae, 16a, 16b, and 21a-c has been prepared and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-(4-methoxyphenyl)-3-(6-methylpyridin-2-yl)-1H-pyrazole-1-carbothioamide (14n) inhibited ALK5 phosphorylation with IC(50) value of 0.57 nM and showed 94% inhibition at 100 nM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.     

Classification of viral zoonosis through receptor pattern analysis

Background  

Viral zoonosis, the transmission of a virus from its primary vertebrate reservoir species to humans, requires ubiquitous cellular proteins known as receptor proteins. Zoonosis can occur not only through direct transmission from vertebrates to humans, but also through intermediate reservoirs or other environmental factors. Viruses can be categorized according to genotype (ssDNA, dsDNA, ssRNA and dsRNA viruses). Among them, the RNA viruses exhibit particularly high mutation rates and are especially problematic for this reason. Most zoonotic viruses are RNA viruses that change their envelope proteins to facilitate binding to various receptors of host species. In this study, we sought to predict zoonotic propensity through the analysis of receptor characteristics. We hypothesized that the major barrier to interspecies virus transmission is that receptor sequences vary among species--in other words, that the specific amino acid sequence of the receptor determines the ability of the viral envelope protein to attach to the cell.     

One-step knockin for inducible expression in mouse embryonic stem cells

Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline (dox)-controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units, and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor, and money. Here, we describe a one-step Cre-mediated knockin system in mouse embryonic stem cells that positions the TA and inducible expression cassette to a single location. Using this system, we show dox-dependent regulation of eGFP at the DNA topoisomerase 3β promoter. Because Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.     

Serum 27-nor-5β-cholestane-3,7,12,24,25 pentol glucuronide discovered by metabolomics as potential diagnostic biomarker for epithelium ovarian cancer

The aim of this study was to use a two steps strategy metabolomics to screen/identify and validate novel metabolic biomarker(s) for epithelial ovarian cancer (EOC). In the screening step, serum samples from 27 healthy women, 28 benign ovarian tumors, and 29 EOCs were analyzed by using LC-MS based nontargeted metabolomics. The three groups were separated with OSC filtered PLS-DA model, and six metabolites (27-nor-5β-cholestane-3,7,12,24,25 pentol glucuronide (CPG), phenylalanine, glycocholic acid, propionylcarnitine, Phe-Phe and Lyso PC (18:2)) were considered as potential biomarker candidates. In the validation step, the six metabolites were analyzed in targeted metabolomics by LC-selective ion monitoring mass spectrometry in another 685 serum samples with various clinical backgrounds. As a result, CPG was evaluated to be a potential biomarker and its content was elevated in EOC tissues compared with benign ovarian tumor tissues (p = 0.0005). Besides, CPG levels were found to be up-regulated in early stage EOC and in the three types of EOC histological types. Other variables such as nonovarian diseases, medicine consumption, gynecological inflammations, and menopausal state did not interfere in using CPG as diagnosis marker. CPG was found to be complementary to CA125. Our findings suggest that CPG can be considered a statistical relevant biomarker of EOC, ready for early stage detection.     

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.     

Statistical optimization of fermentation conditions and comparison of their influences on production of cellulases by a psychrophilic marine bacterium, <Emphasis Type="Italic">Psychrobacter aquimaris</Emphasis> LBH-10 using orthogonal array method

The optimal conditions for the production of cellulases by a marine bacterium, Psychrobacter aquimaris LBH-10, were established and their effects were compared using orthogonal array experiments based on the Taguchi method. The optimal conditions of rice bran, peptone and initial pH for the production of avicelase and CMCase by P. aquimaris LBH-10 were 50.0, 3.0, and 8.0 g/L, respectively, whereas those for filter paperase (FPase) were 100.0, 3.0, and 8.0 g/L, respectively. Rice bran was found to be the most important factor for the production of cellulases based on the calculated percentage of participation P (%) from an analysis of the variance (ANOVA). The optimal temperature for the cell growth of P. aquimaris LBH-10 was 25°C, whereas that for the production of avicelase, CMCase and FPase was 30°C. The optimal agitation speed and aeration rate for cell growth was 400 rpm and 1.5 vvm, respectively, whereas those for the production of CMCase were 300 rpm and 1.0 vvm, respectively. Aeration was found to be more important for cell growth and CMCase production than agitation. The maximum production of avicelase, CMCase and FPase in a 100 L bioreactor for 72 h under optimized conditions was 83.2, 388.7, and 75.4 U/mL, respectively.     

Biomass and Carbon Storage in an Age-Sequence of Korean Pine (<Emphasis Type="Italic">Pinus koraiensis</Emphasis>) Plantation Forests in Central Korea

This study examined the biomass and carbon pools of the main ecosystem components in an age sequence of five Korean pine plantation forest stands in central Korea. The C contents in the tree and ground vegetation biomass, coarse woody debris, forest floor, and mineral soil were estimated by analyzing the C concentration of each component. The aboveground and total tree biomass increased with increasing stand age. The highest C concentration across this chronosequence was found in the tree branch while the lowest C concentration was found in the ground vegetation. The observed C contents for tree components, ground vegetation, and coarse woody debris were generally lower than the predicted C contents estimated from a biomass C factor of 0.5. Forest floor C content was age-independent. Total mineral soil C content appeared to decline initially after establishing Korean pine plantations and recover by the stand age of 35 years. Although aboveground tree biomass C content showed considerable accumulation with increasing age, the relative contribution of below ground C to total ecosystem C content varied substantially. These results suggest that successional development as temporal factor has a key role in estimating the C storage in Korean pine plantation forests.