The Chemotherapeutic Agents XK469 (2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid) and SH80 (2-{4-[(7-Bromo-2-quinolinyl)oxy]phenoxy}propionic acid) Inhibit Cytokinesis and Promote Polyploidy and Induce Senescence

The therapeutic usefulness of the quinoxaline derivatives XK469 (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid) and SH80 (2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid) has been attributed to their abilities to induce G2/M arrest and apoptotic or autophagic cell death. Concentrations of XK469 or SH80 ≥ 5 μM were cytostatic to cultures of the normal murine melanocyte cell line Melan-a. Higher concentrations caused dose-dependent cytotoxicity. Concentrations ≥10 μM provoked dramatic morphological changes typified by marked increases in cell size and granularity. XK469/SH80-treated cultures accumulated tetraploid (4N) DNA-containing cells within 24 h of treatment, an 8N population within 3 days, and a 16N population within 5 days. Increases in ploidy correlated with the appearance of multinucleated cells. Under no circumstances did cells exhibit evidence of furrow formation. Both drugs suppressed cytokinesis in additional mammalian cell lines. Cytotoxic concentrations of XK469 elevated DEVDase activities (a measure of procaspase-3/7 activation) and enhanced cellular staining by a fluorescent analog of the pan caspase inhibitor valine-alanine-aspartic acid-fluoromethyl ketone within 48 to 96 h of treatment. Within 48 h of treatment, cytostatic and cytotoxic concentrations of XK469 elevated p21 contents, reduced Bcl-2 and Bcl-XL contents, and induced autophagy, as monitored by the accumulaton of phosphatidylethanolamine-modified cleavage product of microtubule-associated protein light chain 3 (LC3-II). Cultures treated with ≥10 μM XK469 or SH80 for 5 days could not be induced to divide upon removal of drugs. Such cultures maintained high LC3-II contents, exhibited reduced cyclin E and D1 contents, and extensively expressed senescence-associated β-galactosidase within 14 to 17 days of cessation of drug treatment. Hence, XK469 and SH80 inhibit cytokinesis, promote polyploidy, and induce senescence in Melan-a cells

p-Anilinoaniline Enhancement of Dioxin-Induced CYP1A1 Transcription and Aryl Hydrocarbon Receptor Occupancy of CYP1A1 Promoter: Role of the Cell Cycle □ S

ABSTRACT: The aryl hydrocarbon receptor (AhR) is targeted by ubiquitination for degradation by the proteasome shortly after its activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In silico screening identified p-anilinoaniline (pAA) as a putative inhibitor of an E2 ligase that partners with an E3 ligase implicated in AhR ubiquitination. We investigated whether pAA could modify AhR-dependent activation of its target gene CYP1A1. pA

Improvement of prostate cancer diagnosis using a multiplex test of PSA, GDF-15 (NAG-1) and glycan-binding auto-IgG in plasma

The blood prostate-specific antigen (PSA) assay is used for prostate cancer diagnosis but specificity of the assay is not satisfactory. Previously a combined PSA and GDF-15 (NAG-1) score was shown to improve specificity of prostate cancer detection. Our hypothesis was that, in prostate cancer, glycoproteins were secreted from tumor tissues into blood and induce autoimmune immunoglobulin G (auto-IgG) production and, thus, measurement of the glycan-auto-IgG in blood would improve prostate cancer diagnosis. The 24 glycan-containing microarray analyses have been carried out with plasma samples obtained from 35 prostate cancer patients and 46 healthy subjects to identify glycan-binding auto-IgG biomarker candidates by incubating the auto-IgG captured by glycans spotted on the slide with biotinylated anti-human secondary IgG/streptavidin-Alexa647. Among the 24 glycans, GlcNAc-polyacrylamide (PAA) (G09) and Fucα1-3GlcNAcβ-PAA (G24) glycans showed lower signals of the auto-IgG in the prostate cancer after quantile normalization. β-D-Galactose-PAA (G04) and L-rhamnose-PAA (G08) glycans showed higher signals of the auto-IgG in prostate cancer. No auto-IgM signal was detected. Subsequently, a 5-glycan subarray analysis was developed and lower signals of G09 and G24 glycan-binding auto-IgG were verified by the subarray analysis using 35 prostate cancer plasma samples compared with 54 controls. A higher signal of Neu5Acα2-8Neu5Acα2-8Neu5Acα-sp-PAA (G81)-binding auto-IgG in prostate cancer was detected by the subarray analysis. When the result obtained with the G81-binding auto-IgG was combined with levels of PSA and NAG-1, the prediction rate of prostate cancer increased to 86.2% from 78.2% with PSA levels alone, improving diagnostic accuracy of prostate cancer. The G81 glycan-binding auto-IgG was isolated from prostate cancer and control plasma samples using G81 glycan-affinity chromatography. Western blot analysis of the auto-IgG eluate with a secondary IgG antibody revealed that the level of the 50 kDa heavy chain of the auto-IgG obtained from the prostate cancer patient was ~3-fold higher than the control sample. The 50 kDa fragment was identified as an IgG heavy chain variable region by N-terminal sequencing (Edman\u27s degradation). Our result demonstrated that a multiplex biomarker diagnostic consisting of glycan-binding auto-IgG, PSA and NAG-1 increased specificity and sensitivity of prostate cancer diagnosis. Supported by NCI SBIR Phase I, CA159721

Bisphenol A (BPA) in liquid portions of canned foods obtained from domestic and Asian markets in the United States

Bisphenol A (BPA) is a phenolic environmental estrogen that disrupts endocrine activity thereby increasing the risk of hormone-related health problems. The human population is highly exposed to BPA and food is believed to be a primary source of BPA exposure. The aim of this study was to test the sensitivity and specificity of a BPA enzyme-linked immunosorbent assay (ELISA) and to measure levels of BPA in supernatants obtained from various canned foods from different countries. The concentration of BPA was measured in supernatant from different types of canned soup and vegetable mixes produced by US companies and two companies each from three different Asian countries (Korea, Japan and China), which are available at markets in the USA. ELISA results were confirmed by LC/MS/MS and shown to be in agreement. Cross-reactivity tests demonstrated that BPA ELISA kit does not cross-react with other tested phenolic compounds. There was no significant difference of BPA levels in different types of soups from different US companies. However, levels of BPA in supernatants of canned vegetable mixes of a company in the USA were 200-fold lower than the levels in canned vegetable soups of the US companies. BPA levels varied greatly among canned foods among companies in various countries. Thus, this study validated the use of a simple ELISA assay to measure levels of BPA in supernatants of canned food, which would facilitate the routine monitoring of dietary exposure to BPA. Decreasing the consumption of BPA will lead to a reduction in the risk of adverse health effects

Levels of plasma glycan-binding auto-IgG biomarkers improve the accuracy of prostate cancer diagnosis

Strategies to improve the early diagnosis of prostate cancer will provide opportunities for earlier intervention. The blood-based prostate-specific antigen (PSA) assay is widely used for prostate cancer diagnosis but specificity of the assay is not satisfactory. An algorithm based on serum levels of PSA combined with other serum biomarkers may significantly improve prostate cancer diagnosis. Plasma glycan-binding IgG/IgM studies suggested that glycan patterns differ between normal and tumor cells. We hypothesize that in prostate cancer glycoproteins or glycolipids are secreted from tumor tissues into the blood and induce auto-immunoglobulin (Ig) production. A 24-glycan microarray and a 5-glycan subarray were developed using plasma samples obtained from 35 prostate cancer patients and 54 healthy subjects to identify glycan-binding auto-IgGs. Neu5Acα2-8Neu5Acα2-8Neu5Acα (G81)-binding auto-IgG was higher in prostate cancer samples and, when levels of G81-binding auto-IgG and growth differentiation factor-15 (GDF-15 or NAG-1) were combined with levels of PSA, the prediction rate of prostate cancer increased from 78.2% to 86.2% than with PSA levels alone. The G81 glycan-binding auto-IgG fraction was isolated from plasma samples using G81 glycan-affinity chromatography and identified by N-terminal sequencing of the 50 kDa heavy chain variable region of the IgG. G81 glycan-binding 25 kDa fibroblast growth factor-1 (FGF1) fragment was also identified by N-terminal sequencing. Our results demonstrated that a multiplex diagnostic combining G81 glycan-binding auto-IgG, GDF-15/NAG-1 and PSA (≥ 2.1 ng PSA/ml for cancer) increased the specificity of prostate cancer diagnosis by 8%. The multiplex assessment could improve the early diagnosis of prostate cancer thereby allowing the prompt delivery of prostate cancer treatment

RNA-binding protein HuR regulates RGS4 mRNA stability in rabbit colonic smooth muscle cells

Regulator of G protein signaling 4 (RGS4) regulates the strength and duration of G protein signaling and plays an important role in smooth muscle contraction, cardiac development, and psychiatric disorders. Little is known about the posttranscriptional regulation of RGS4 expression. We cloned the full-length cDNA of rabbit RGS4, which contains a long 3′-untranslated region (UTR) with several AU-rich elements (AREs). We determined whether RGS4 mRNA stability is mediated by the RNA-binding protein human antigen R (HuR) and contributes to IL-1β-induced upregulation of RGS4 expression. We show that IL-1β treatment in colonic smooth muscle cells doubled the half-life of RGS4 mRNA. Addition of RGS4 3′-UTR to the downstream of Renilla luciferase reporter induced dramatic reduction in the enzyme activity and mRNA expression of luciferase, which was attenuated by the site-directed mutation of the two 3′-most ARE sites. IL-1β increased luciferase mRNA stability in a UTR-dependent manner. Knockdown of HuR significantly aggravated UTR-mediated instability of luciferase and inhibited IL-1β-induced upregulation of RGS4 mRNA. In addition, IL-1β increased cytosolic translocation and RGS4 mRNA binding of HuR. These findings suggest that 3′-most ARE sites within RGS4 3′-UTR are essential for the instability of RGS4 mRNA and IL-1β promotes the stability of RGS4 mRNA through HuR