Antiviral activity of 2,3-Dihydro-1H-imidazo[1,2-b]pyrazole in herpes simplex virus type 1-infected mammalian cells

2,3-Dihydro-1H-imidazo[1,2-b]pyrazole (IMPY), a known inhibitor of DNA synthesis, has been shown to be a useful drug for the synchronization of mammalian cells in culture. Recent studies in our laboratory indicate that IMPY may possess significant antiviral activity against herpes simplex virus (HSV) type 1. IMPY. at a concentration of 80 [mu]g/ml or 0.73 mM, reduced syncytia formation approximately 80 per cent. A 50 per cent inhibitory dose was calculated for each drug in order to compare potency in syncytia reduction of IMPY with that of arabinosyladenine (ara-A) and arabinosyl-hypoxanthine (ara-H). Our results indicated that the antiviral potencies of the three drugs were ranked in the order ara-A > ara-H > IMPY, the 50 per cent inhibitory doses being 22, 195 and 309 [mu]M respectively. Utilizing the microplate procedure of Sidwell and Huffman [Appl. Microbiol. 22, 797 (1971)], inhibition of viral cytopathic effect was rated against drug cytotoxicity and a virus rating (VR) established. A virus rating of 0.68 was calculated for IMPY. In comparison, VR values of 0.84 and 0.66 were obtained for ara-A and ara-H respectively. In contrast to the syncytia reduction studies, IMPY appeared to possess antiviral activity equivalent to that of ara-H according to the criteria of the virus rating assay. A technique was developed for evaluating the degree of selectivity ([deg]S) of a drug with respect to its differential effect on viral and cellular DNA synthesis. IMPY was found to possess a negative selectivity at all concentrations studied, reflecting the fact that it inhibited cellular DNA synthesis more than viral DNA synthesis. In contrast, ara-A and ara-H both expressed positive degrees of selectivity in that they inhibited viral DNA synthesis more extensively than cellular DNA synthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21651/1/0000035.pd

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys.BackgroundThe proliferation of mural epithelial cells is a major cause of progressive cyst enlargement in autosomal-dominant polycystic kidney disease (ADPKD). Adenosine 3′, 5′ cyclic monophosphate (cAMP) stimulates the proliferation of cells from ADPKD cysts, but not cells from normal human kidney cortex (HKC), through the activation of protein kinase A (PKA), mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK/MAPK). In the current study, we examined the signaling pathway between PKA and MEK in ADPKD and HKC cells.MethodsPrimary cultures of human ADPKD and HKC cells were prepared from nephrectomy specimens. We determined the effects of cAMP and epidermal growth factor (EGF) on the activation of ERK, B-Raf and Raf-1 in ADPKD and HKC cells by immune kinase assay and Western blot.Results8-Br-cAMP increased phosphorylated ERK (2.7- ± 0.6-fold, N = 7), and B-Raf kinase activity (3.6- ± 1.1-fold, N = 5) in cells from ADPKD kidneys; levels of phosphorylated Raf-1 were not changed. Inhibition of PKA by H89 strikingly decreased cAMP-stimulated phosphorylation of ERK and B-Raf, and MAPK inhibition by PD98059 blocked the effect of the nucleotide to activate ERK. By contrast, in HKC cells 8-Br-cAMP did not activate B-Raf and ERK. EGF stimulated the phosphorylation of ERK and Raf-1 in both ADPKD and HKC cells, but had no effect on B-Raf. 8-Br-cAMP and EGF conjointly increased ERK activation above that of either agonist alone in ADPKD cells, and this combined effect was abolished by PD98059, indicating that ERK was activated by EGF- and cAMP-responsive cascades that converge at MAPK.ConclusioncAMP activates ERK and increases proliferation of ADPKD epithelial cells, but not cells from normal human kidney cortex, through the sequential phosphorylation of PKA, B-Raf and MAPK in a pathway separate from, but complementary to, the classical receptor tyrosine kinase cascade. Consequently, cAMP and EGF have great potential to accelerate the progressive enlargement of renal cysts

Apigenin Prevents UVB-Induced Cyclooxygenase 2 Expression: Coupled mRNA Stabilization and Translational Inhibition

Cyclooxygenase 2 (COX-2) is a key enzyme in the conversion of arachidonic acid to prostaglandins, and COX-2 overexpression plays an important role in carcinogenesis. Exposure to UVB strongly increased COX-2 protein expression in mouse 308 keratinocytes, and this induction was inhibited by apigenin, a nonmutagenic bioflavonoid that has been shown to prevent mouse skin carcinogenesis induced by both chemical carcinogens and UV exposure. Our previous study suggested that one pathway by which apigenin inhibits UV-induced and basal COX-2 expression is through modulation of USF transcriptional activity in the 5′ upstream region of the COX-2 gene. Here, we found that apigenin treatment also increased COX-2 mRNA stability, and the inhibitory effect of apigenin on UVB-induced luciferase reporter gene activity was dependent on the AU-rich element of the COX-2 3′-untranslated region. Furthermore, we identified two RNA-binding proteins, HuR and the T-cell-restricted intracellular antigen 1-related protein (TIAR), which were associated with endogenous COX-2 mRNA in 308 keratinocytes, and apigenin treatment increased their localization to cell cytoplasm. More importantly, reduction of HuR levels by small interfering RNA inhibited apigenin-mediated stabilization of COX-2 mRNA. Cells expressing reduced TIAR showed marked resistance to apigenin's ability to inhibit UVB-induced COX-2 expression. Taken together, these results indicate that in addition to transcriptional regulation, another mechanism by which apigenin prevents COX-2 expression is through mediating TIAR suppression of translation

Apigenin Prevents UVB-Induced Cyclooxygenase 2 Expression: Coupled mRNA Stabilization and Translational Inhibition

Cyclooxygenase 2 (COX-2) is a key enzyme in the conversion of arachidonic acid to prostaglandins and COX-2 overexpression plays an important role in carcinogenesis. Exposure to UVB strongly increased COX-2 protein expression in mouse 308 keratinocytes and this induction was inhibited by apigenin a nonmutagenic bioflavonoid that has been shown to prevent mouse skin carcinogenesis induced by both chemical carcinogens and UV exposure. Our previous study suggested that one pathway by which apigenin inhibits UV-induced and asal COX-2 expression is through modulation of USF transcriptional activity in the 5 upstream region of the COX-2 gene. Here we found that apigenin treatment also increased COX-2 mRNA stability and the inhibitory effect of apigenin on UVB-induced luciferase reporter gene activity was dependent on the AU-rich element of the COX-2 3 -untranslated region. Furthermore we identified two RNA-binding proteins HuR and the T-cell-restricted intracellular antigen 1-related protein (TIAR) which were associated with endogenous COX-2 RNA in 308 keratinocytes and apigenin treatment increased their localization to cell cytoplasm. More importantly reduction of HuR levels by small interfering RNA inhibited apigenin-mediated stabilization of COX-2 mRNA. Cells expressing reduced TIAR showed marked resistance to apigenin’s ability to inhibit UVB-induced COX-2 expression. Taken together these results indicate that in addition to transcriptional regulation another mechanism by which apigenin prevents COX-2 expression is through mediating TIAR suppression of translation. Originally published Molecular and Cellular Biology Vol. 27 No. 1 Jan 200

The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

<div><p>Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG’s effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.</p> </div

The Chemopreventive Bioflavonoid Apigenin Inhibits Prostate Cancer Cell Motility through the Focal Adhesion Kinase/Src Signaling Mechanism

Prostate cancer mortality is primarily attributed to metastatic rather than primary, organ-confined disease. Acquiring a motile and invasive phenotype is an important step in development of tumors and ultimately metastasis. This step involves remodeling of the extracellular matrix and of cell-matrix interactions, cell movement mediated by the actin cytoskeleton, and activation of focal adhesion kinase (FAK)/Src signaling. Epidemiologic studies suggest that the metastatic behavior of prostate cancer may be an ideal target for chemoprevention. The natural flavone apigenin is known to have chemopreventive properties against many cancers, including prostate cancer. Here, we study the effect of apigenin on motility, invasion, and its mechanism of action in metastatic prostate carcinoma cells (PC3-M). We found that apigenin inhibits PC3-M cell motility in a scratch-wound assay. Live cell imaging studies show that apigenin diminishes the speed and affects directionality of cell motion. Alterations in the cytoskeleton are consistent with impaired cell movement in apigenin-treated cells. Apigenin treatment leads to formation of &quot;exaggerated filopodia,&quot; which show accumulation of focal adhesion proteins at their tips. Furthermore, apigenin-treated cells adhere more strongly to the extracellular matrix. Additionally, apigenin decreases activation of FAK and Src, and phosphorylation of Src substrates FAK Y576/577 and Y925. Expression of constitutively active Src blunts the effect of apigenin on cell motility and cytoskeleton remodeling. These results show that apigenin inhibits motility and invasion of prostate carcinoma cells, disrupts actin cytoskeleton organization, and inhibits FAK/Src signaling. These studies provide mechanistic insight into developing novel strategies for inhibiting prostate cancer cell motility and invasiveness

PG promotes cell-cell adhesion in prostate cancer cell lines.

<p>A. Representative image of a dispase assay in PC3-M cells after transduction with PG OE adenovirus or control GFP adenovirus. B. Quantitations of dispase assays performed in triplicate; LNCaP, C4-2B, or PC3-M cells were plated in 6-well plates, and allowed to attach and spread. Cultures were transduced with GFP-containing adenovirus (GFP) or PG-containing adenovirus (PG OE), and after 24 hours the dispase assay was performed. Overexpression of PG in all 3 cell lines strengthens cell-cell adhesion. C. LNCaP cells were plated in 6-well plates and allowed to attach and spread. The cells were then transfected with control siRNA or PG siRNA pool. The dispase assay was performed 96 hours after transfection. Suppression of PG expression results in a weakening of cell-cell adhesion in LNCaP cells. D. Representative image of a hanging drop assay in DU145 cells after transfection with control or PG siRNA pool. E–F. Quantitation of hanging drop assays performed in triplicate; C4-2B and PC3-M cells were transduced with GFP- or PG-containing adenoviruses (E), ARCaP E, LNCaP, DU145 and PC3 cells were transfected with control siRNA or PG siRNA pool (F). Aggregation assays were done 24 h (E), or 96 h (F) after the treatment. Graphs represent averages +/− SEM. *P<0.06; **P<0.005; ***P<0.0007, by paired Student t test.</p