Functional Effects of let-7g Expression in Colon Cancer Metastasis.

MicroRNA regulation is crucial for gene expression and cell functions. It has been linked to tumorigenesis, development and metastasis in colorectal cancer (CRC). Recently, the let-7 family has been identified as a tumor suppressor in different types of cancers. However, the function of the let-7 family in CRC metastasis has not been fully investigated. Here, we focused on analyzing the role of let-7g in CRC. The Cancer Genome Atlas (TCGA) genomic datasets of CRC and detailed data from a Taiwanese CRC cohort were applied to study the expression pattern of let-7g. In addition, in vitro as well as in vivo studies have been performed to uncover the effects of let-7g on CRC. We found that the expression of let-7g was significantly lower in CRC specimens. Our results further supported the inhibitory effects of let-7g on CRC cell migration, invasion and extracellular calcium influx through store-operated calcium channels. We report a critical role for let-7g in the pathogenesis of CRC and suggest let-7g as a potential therapeutic target for CRC treatment.

Involvement of intracellular calcium mobilization in IL-8 activation in human retinal pigment epithelial cells.

PURPOSE: Calcium signaling is an important intracellular pathway. Increased intracellular calcium is associated with cytokine regulation and inflammatory signals secretion. The purpose of this study is to understand the molecular mechanisms by which calcium signaling controls IL-8 activation in human RPE cells. METHODS: Fluorescence-based calcium imaging and different mutants of IL-8 plasmids were used in this study. The IL-8 promoter activation, gene expression, and secretion were detected by using luciferase reporter assay, quantitative real-time PCR (Q-PCR), and ELISA, respectively. In addition, pharmacological inhibitors and small interfering RNA (siRNA) were applied to clarify the mechanisms of IL-8 activation. RESULTS: Our study reported that intracellular calcium mobilization activated IL-8 gene expression and secretion. Application of pharmacological inhibitor BAY 11-7082, siRNA, and plasmids of the nuclear factor κ light chain enhancer of activated B cells (NF-κB) binding site, we identified that NF-κB is the main transcription factor involved in intracellular calcium mobilization-mediated IL-8 activation in human RPE cells. CONCLUSIONS: Collectively, our findings highlight the important role of intracellular calcium mobilization in the activation of IL-8. These findings may be helpful for the clinical applications in the age-related macular degeneration (AMD) prevention and treatment.

Pb²âº induced IL-8 gene expression by extracellular signal-regulated kinases and the transcription factor, activator protein 1, in human gastric carcinoma cells.

Divalent lead (Pb(2+) ) is a common industrial pollutant epidemiologically associated with gastric cancers. Pb(2+) was found to promote tumorigenesis, which may include interleukin (IL)-8, a pro-inflammatory chemokine that promotes angiogenesis and tumor metastasis. Given that the gastrointestinal tract is a major route of Pb(2+) exposure, we investigated the ability of Pb(2+) to induce IL-8 expression in gastric carcinoma cells and its underlying mechanism. At a concentration of 0.1 µM, Pb(2+) induced IL-8 gene activation in gastric carcinoma AGS cells. Using a IL-8 promoter-deletion analysis, transcription factor activator protein 1 (AP-1) was identified as a necessary component of Pb(2+) -induced IL-8 gene activation. Upregulation of the IL-8 gene was abrogated by the MEK inhibitor, PD98059, and partially suppressed by the epidermal growth factor receptor inhibitors, AG1478 and PD153035. Furthermore, c-Jun protein expression was induced in cells treated with Pb(2+) , and overexpression of c-Jun enhanced Pb(2+) -induced IL-8 activation. Collectively, our findings highlight the pivotal roles of AP-1 and extracellular signal-regulated kinase in signal transduction of Pb(2+) -induced IL-8 gene activation. These molecules may be potential therapeutic targets for Pb(2+) -related inflammation leading to stomach carcinogenesis. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 315-322, 2015.

Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 µM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture.

Function of DNA methyltransferase 3a in lead (Pb(2+) )-Induced Cyclooxygenase-2 gene.

Lead ions (Pb(2+) ) are toxic industrial pollutants associated with chronic inflammatory diseases in humans and animals. Previously, we found that Pb(2+) ions induce COX-2 gene expression via the EGF receptor/nuclear factor-κB signal transduction pathway in epidermoid carcinoma cell line A431. In this study, to see whether Pb(2+) ions affect COX-2 expression by epigenetic mechanisms, we looked at the mRNAs of DNA methyltransferases (DNMTs) using real-time PCR of total RNA from these cells. Cells exposed to Pb(2+) had low levels of DNMT3a mRNA, whereas the levels of DNMT1 and DNMT3b mRNAs remained unchanged. Pretreatment of cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5 µM) followed by Pb(2+) (1 µM) significantly increased levels of COX-2 mRNA compared with cells treated with Pb(2+) alone. Overexpression of tumor suppressor gene Rb correlated with an increase in COX-2 mRNA and a decrease in DNMT3a mRNA. Conversely, overexpression of transcription factor E2F1 correlated with a decrease in COX-2 mRNA and an increase in DMNT3a mRNA. Pretreatment with EGFR inhibitors AG1478 and PD153035 significantly limited Pb(2+) -induced reduction in DNMT3a mRNA. In addition, gene knockdown of DNMT3a with short hairpin RNA correlated with increased COX-2 mRNA induced by Pb(2+) . Our findings suggest Pb(2+) ions induce COX-2 expression indirectly by reducing DNMT3a methylation of the COX-2 promoter via transcription factors Rb and E2F1.

A soft coral natural product, 11-episinulariolide acetate, inhibits gene expression of cyclooxygenase-2 and interleukin-8 through attenuation of calcium signaling.

Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancer cells. EGFR-mediated signaling involves inflammatory gene expression including cyclooxygenase (COX)-2 and interleukin (IL)-8, and is associated with cancer pathogenesis. In a search of phytochemicals with anti-inflammatory activity, the COX-2 and IL-8 inhibitory activities of some marine compounds were examined. After screening these compounds 11-episinulariolide acetate (1) from soft coral exhibited the most potent activity. Reverse-transcription PCR; western blotting; ELISA and luciferase assays were used to test the effect of compound 1 on EGF-stimulated expressions of COX-2 and IL-8 in A431 human epidermoid carcinoma cells. After exposure to 10 µM of compound 1, expression levels of COX-2 and IL-8 were reduced. In addition; intracellular Ca²âº increase and Ca²âº-dependent transcription factor activation were blocked by compound 1. Thus, compound 1 can potentially serve as a lead compound for targeting Ca²âº signaling-dependent inflammatory diseases.

Indomethacin inhibits cancer cell migration via attenuation of cellular calcium mobilization.

Non-steroidal anti-inflammatory drugs (NSAIDs) were shown to reduce the risk of colorectal cancer recurrence and are widely used to modulate inflammatory responses. Indomethacin is an NSAID. Herein, we reported that indomethacin can suppress cancer cell migration through its influence on the focal complexes formation. Furthermore, endothelial growth factor (EGF)-mediated Ca2+ influx was attenuated by indomethacin in a dose dependent manner. Our results identified a new mechanism of action for indomethacin: inhibition of calcium influx that is a key determinant of cancer cell migration.

Involvement of STIM1 and Orai1 in EGF-mediated cell growth in retinal pigment epithelial cells.

BACKGROUND: In non-excitable cells, one major route for calcium entry is through store-operated calcium (SOC) channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca²âº store. STIM1 and Orai1 are major regulators of SOC channels. In this study, we explored the functions of STIM1 and Orai1 in epidermal growth factor (EGF)-induced cell proliferation and migration in retinal pigment epithelial cells (ARPE-19 cell line). RESULTS: EGF triggers cell proliferation and migration in ARPE-19 cells. Cell proliferation and migration involve STIM1 and Orai1, as well as phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, and Akt. Pharmacological inhibitors of SOC channels and siRNA of Orai1 and STIM1 suppress cell proliferation and migration. Pre-treatment of mitogen-activated protein kinase kinase (MEK) inhibitors and a phosphatidylinositol 3 kinases (PI3K) inhibitor attenuated cell proliferation and migration. However, inhibition of the SOC channels failed to prevent EGF-mediated ERK 1/2 and Akt phosphorylation. CONCLUSIONS: Our results showed that STIM1, Orai1, ERK 1/2, and Akt are key determinants of EGF-mediated cell growth in ARPE-19 cells. EGF is a potent growth molecule that has been linked to the development of PVR, and therefore, STIM1, Orai1, as well as the MEK/ERK 1/2 and PI3K/Akt pathways, might be potential therapeutic targets for drugs aimed at treating such disorders.

Lack of association between CLEC5A gene single-nucleotide polymorphisms and Kawasaki disease in Taiwanese children.

BACKGROUND: Kawasaki disease is characterized by systemic vasculitis of unknown etiology. Previous genetic studies have identified certain candidate genes associated with susceptibility to KD and coronary artery lesions. Host innate immune response factors are involved in modulating the disease outcome. The aim of this study was to investigate CLEC5A (C-type lectin domain family 5) genetic polymorphisms with regards to the susceptibility and outcome of KD. METHODS: A total of 1045 subjects (381 KD patients and 664 controls) were enrolled to identify 4 tagging single-nucleotide polymorphisms (tSNPs) of CLEC5A (rs1285968, rs11770855, rs1285935, rs1285933) by using the TaqMan Allelic Discrimination Assay. The Hardy-Weinberg equilibrium was assessed in cases and controls, and genetic effects were evaluated by the chi-square test. RESULTS: No significant associations were noted between the genotypes and allele frequency of the 4 CLEC5A tSNPs between controls and patients. In the patients, polymorphisms of CLEC5A showed no significant association with coronary artery lesion formation and intravenous immunoglobulin treatment response. CONCLUSIONS: This study showed for the first time that polymorphisms of CLEC5A are not associated with susceptibility to KD, coronary artery lesion formation, and intravenous immunoglobulin treatment response in a Taiwanese population.

Isodesacetyluvaricin, an Annonaceous acetogenin, specifically inhibits gene expression of cyclooxygenase-2.

Cyclooxygenase-2 (COX-2) is an inducible isoform of the enzyme responsible for the synthesis of several inflammatory mediators. In a search for phytochemicals with anti-inflammatory activity, the COX-2 inhibitory activity of 15 typical Annonaceous acetogenins was examined. Isodesacetyluvaricin (1), from the Formosan tropical fruit tree Annona glabra, exhibited the most potent activity. Reverse transcription PCR was used to test the effect of 1 on epidermal growth factor-stimulated expression of COX-2 in cultures of A431 human epidermoid carcinoma cells. Three hours after exposure to 1 (5 µM), A431 cells had barely detectable levels of COX-2 mRNA. A corresponding but smaller decline in the COX-2 protein appeared on using Western blots. Lipopolysaccharide-stimulated expression of COX-2 in Raw 264.7 mouse leukemic monocyte-macrophages showed a similar decrease. Luciferase assays revealed that cells exposed to 1 had reduced activities of two COX-2 promoter-transcription factors: cAMP response element-binding factor and nuclear factor of activated T-cells. Compound 1 did not affect cell proliferation, as measured by a colorimetric assay, or intracellular store-operated calcium influx, as determined by fluorescence imaging. Thus, 1 may serve as a lead compound for targeting inflammatory diseases as well as angiogenesis and cancer metastasis.

Involvement of store-operated calcium signaling in EGF-mediated COX-2 gene activation in cancer cells.

Growing evidence shows that chronic inflammation drives the progression of colorectal cancer (CRC). Cyclooxygenase-2 (COX-2) is one of the most important inflammatory genes involved in solid tumor metastasis. Epidermal growth factor receptor (EGFR) also plays a key role in cancer cell development. We compared the expression levels of EGFR and COX-2 between tumor and normal tissues from 20 CRC patients and studied the molecular mechanism of EGFR-mediated COX-2 gene expression in cancer cells. Our results indicated that COX-2 expression was markedly increased after EGF stimulation. COX-2 promoter analysis indicated the involvement of cyclic AMP-responsive element (CRE) and nuclear factor of activated T cells/nuclear factor interleukin-6 (NFAT/NF-IL6)-binding sites in EGF-mediated signaling pathways. Furthermore, EGF-mediated COX-2 activation was prevented by 2-aminoethoxydiphenyl borate (2-APB), a store-operated Ca(2+) channel inhibitor. Transfection of siRNA against ORAI1 or STIM1, the key regulators of store-operated Ca(2+) channels, showed significant inhibitory effects on EGF-mediated COX-2 expression. In conclusion, store-operated Ca(2+) entry is involved in the activation of transcription factors (CREB/NFAT) that are responsible for delivering EGF-mediated signals to evoke inflammatory cascades and is eventually related to CRC tumorigenesis.

Divalent lead cations induce cyclooxygenase-2 gene expression by epidermal growth factor receptor/nuclear factor-kappa B signaling in A431carcinoma cells.

Divalent lead cations (Pb²+) are toxic metal pollutants that may contribute to inflammatory diseases in people and animals. Human vascular smooth muscle cells in culture respond to low concentrations of Pb²+ ions by activating mediators of inflammation via the plasma membrane epidermal growth factor receptor (EGFR). These include cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 as well as the hormone-like lipid compound prostaglandin E2. To further clarify the mechanism by which Pb²+ induces such mediators of inflammation, we tested human epidermoid carcinoma cell line A431 that expresses high levels of EGFR. Reverse transcription PCR and western blots confirmed A431 cells treated with a low concentration (1 µM) of Pb²+ in the form of lead (II) nitrate increased expression of COX-2 mRNA and its encoded protein in a time-dependent manner. Promoter deletion analysis revealed the transcription factor known as nuclear factor-kappa B (NF-κB) was a necessary component of the COX-2 gene response. NF-κB inhibitor BAY 11-7082 suppressed Pb²+-induced COX-2 mRNA expression, and EGFR inhibitors AG1478 and PD153035 as well as EGFR small interfering RNA reduced the coincident nuclear translocation of NF-κB. Our findings support the hypothesis that low concentrations of Pb²+ ions incite inflammation by inducing COX-2 gene expression via the EGFR/NF-κB signal transduction pathway.