Cell toxicity of compound C2, C6, and their 2D analogs tested in a cancer cell line and normal fibroblasts.

<p><b>A)</b> SCC 22B cells were treated with increasing concentrations of the indicated compound and 48 hours later cell numbers were determined (n = 3). <b>B)</b> MRC5 cells (fibroblast) were treated with increasing concentrations of the indicated compounds and 48 hours later cell numbers were determined using the CyQuant kit (n = 3).</p

Compound C6 and C2 impair La binding to different RNA substrates.

<p>Constant concentration of recombinant La as indicated and fluorescence labeled RNA oligonucleotides (50 nM) were used with increasing concentrations of compound C6 or C2, respectively. Lane 1 and 2 contained the highest vehicle (DMSO) concentration used with the compounds. The compound was first incubated with La and RNA oligoribonucleotides were quickly added thereafter. Lane 10 always contained the fluorescence labeled RNA oligoribonucleotides and the highest concentration of compound tested. Reactions were analyzed on a 10% native polyacrylamide gel and representative gels of independent experiments are shown. <b>A)</b> fD1-RNA, La-WT (60 nM) and decreasing concentrations of C6, <b>B)</b> fD1-RNA, La-RRM1+2 (60 nM) and decreasing concentrations of C6, <b>C)</b> fPolyU-RNA, La-WT (200 nM) and decreasing concentrations of C6, <b>D)</b> TOPf-RNA, La-WT (150 nM) and decreasing concentrations of C6, <b>E)</b> fD1-RNA, La-WT (60 nM) and decreasing concentrations of C2, <b>F)</b> fD1-RNA, La-RRM1+2 (60 nM) and decreasing concentrations of C2., <b>G)</b> fPolyU-RNA, La-WT (200 nM) and decreasing concentrations of C2, <b>H)</b> TOPf-RNA, La-WT (200 nM) and decreasing concentrations of C2. All fluorescence labeled RNA oligoribonucleotides were used at a concentration of 50 nM. All experiments were performed at least twice.</p

The La:RNA fluorescence polarization assay (La-FP assay).

<p><b>A)</b> Domain structure of La wildtype (La-WT). RRM = RNA recognition motif with RNP1 and RNP2 consensus sequences. Lines indicate which domains are required for binding to different RNA substrates. NRE = nuclear retention element; NoLS = nucleolar localization signal; RCD = RNA chaperone domain; C-terminal dark bar = nuclear localization signal. <b>B)</b> Fluorescence polarization (FP) of the fluorescence-labeled cyclin D1-RNA oligonucleotide (fD1-RNA) in the absence and presence of recombinant La protein. <b>C)</b> Quality statistic for the La-FP assay. Z’ factor, signal to noise ratio and the coefficient of variation (CV) were determined by recording fluorescence polarization signals of fD1-RNA alone and in the presence of recombinant La. Measurements from seven microtiter plates performed at different days were combined to ensure proper statistics. <b>D)</b> La-FP on fD1-RNA in the presence of compound C1 to C6 at increasing concentrations. La protein (1.35 μM) was incubated with each compound at 25, 50, 100, 1000 μM for 30 min, followed by additional 15 min incubation with 0.8 μM fD1-RNA to detect the residual activity of the labeled fD1-RNA binding. Mean values of B/B_max (B_max = maximal binding in absence of compound) were plotted against the compound concentrations (μM). B_max = ΔmP_max = mPs−mP_free is the maximum specific binding, which defines complete saturation of La protein with fD1-RNA in the absence of a compound. B was determined as the difference between fluorescence polarization of fD1-RNA plus La plus compound and fluorescence polarization of fD1-RNA plus compound. IC₅₀ values were determined using mean values of B/ B_max in a non-linear regression analysis by GraphPad Prism version 4.00 for Windows (GraphPad Software, Inc, San Diego, CA). <b>E)</b> The structures of the best two hits, C2 and C6, are shown.</p

Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions <i>in vitro</i> and in cells

<div><p>The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. Hence, small molecules able to block the binding of La to specific RNAs could have a therapeutic impact by reducing the expression of tumor-promoting and anti-apoptotic factors. Toward this novel therapeutic strategy, we aimed to develop a high-throughput fluorescence polarization assay to screen small compound libraries for molecules blocking the binding of La to an RNA element derived from cyclin D1 mRNA. Herein, we make use of a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. We showed recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here, we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA in cells and sensitizes cells for cipslatin-induced cell death. In summary, we report the application of a high-throughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs <i>in vitro</i> and in cells.</p></div

The 2D analogs C6.01, C6.02 and C2.01, C2.02 compete for La binding to fD1-RNA.

<p>Chemical structure of 2D analogs of C6 and C2, respectively, are shown: C6.01 <b>(A)</b>, C6.02 <b>(B)</b>, and C2.01 <b>(C)</b>, C2.02 <b>(D)</b>. Constant concentration of recombinant La protein (60 nM) and fluorescence labeled RNA oligonucleotides (50 nM) was used with increasing concentrations of compound C6.01, C6.02, C2.01 or C2.02. Lanes 1 and 2 contained the highest vehicle (DMSO) concentration used with the compounds. The compound was first incubated with La and RNA oligonucleotides were quickly added thereafter. Lane 10 contains the fluorescence labeled RNA oligonucleotides and the highest concentration of compound tested. Reactions were analyzed on a 10% native polyacrylamide gel and representative gels of independent experiments are shown. <b>E)</b> fD1-RNA, La-WT and decreasing concentrations of C6.01, <b>F)</b> fD1-RNA, La-WT and decreasing concentrations of C6.02, <b>G)</b> fD1-RNA, La-WT and decreasing concentrations of C2.01, and <b>H)</b> fD1-RNA, La-WT and decreasing concentrations of C2.02. All experiments were performed at least twice.</p

Binding of La wilstype and the minimal RNA-binding competent La-RRM1+2 mutant to different RNA substrates.

<p>Domain structure of La wildtype (La-WT) <b>(A)</b> and La-RRM1+2 mutant <b>(B).</b> Either recombinant La-WT or La-RRM1+2 protein was titrated into binding reactions containing 50 nM fluorescence labeled fD1-RNA <b>(C, F)</b>, fPolyU-RNA <b>(D, G)</b>, or TOPf-RNA oligoribonucleotides <b>(E, H)</b>. The EMSA reactions were loaded without dye and separated on 10% native polyacrylamide gels. Representative gels of independent experiments are shown. All experiments were performed at least three times.</p

Characterization of La binding to the TOP element of RPL5 mRNA.

<p><b>A)</b> and <b>B)</b> Comparison of La binding to TOPf- or 5’ capped TOPf-RNA (capTOPf) oligonucleotide (50 nM). Recombinant La-WT was titrated into binding reactions contain fluorescence labeled TOPf-RNA <b>(A)</b>, or fluorescence labeled capTOPf-RNA <b>(B)</b> oligoribonucleotides (both at 50 nM) and analyzed on a 10% native polyacrylamide gel. Representative gels of independent experiments are shown. <b>C)</b> and <b>D)</b> Competition experiments using three different RNA oligonucleotides <b>(E)</b>. Recombinant La-WT (100 nM) was used in binding reactions contain TOPf-RNA (50 nM) and increasing concentrations of unlabeled competitor RNA oligonucleotides: TOP-WT, TOP-mu1 or TOP-mu2. Reactions were analyzed on a 10% native polyacrylamide gel. <b>E)</b> Sequence of unlabeled competitor RNA oligonucleotides used in this study. All experiments were at least performed twice.</p

Modulation of SOCS3 expression in gain and loss of function experiments in HNSCC only reduces proliferation of cells without (UM-SCC-22B) detectable levels of endogenous SOCS3.

<p>(A) Immunoblot analysis of whole cell extracts shows that transfection of the SOCS3 expression plasmid resulted in increased levels of SOCS3 after 24 and 48 h in OSCC3 and UM-SCC-22B cells. Pre-treatment of both cell lines with the indicated concentrations of the biochemical inhibitor effectively decreased phosphorylation of STAT3 30 min after stimulation with IL-6 (25 ng/mL). (B) Subcellular localization of SOCS3 in the overexpression experiments was confirmed by immunofluorescence analysis. Images are representative of three independent experiments assessing SOCS3 transgene expression 48 h after plasmid transfection. (C) Cell proliferation was determined by direct counting the cells and the trypan blue dye exclusion test 24 and 48 h after transfection. (D) Cell proliferation was also determined in loss of function experiments by direct counting of cells using trypan blue exclusion test 24 and 48 h after siRNA transfection in OSCC3 cells. Graphs (C and D) represent growth curves (number of viable cells) over time, according to the experimental condition (in C: transfection of empty vector, SOCS3 expressing vector, or treatment with STAT3 inhibitor; in D: reagent control, SOCS3 siRNA and non-target control siRNA). Vertical lines standard deviations of three independent experiments and * indicates p<0.05 for comparison with vehicle control (empty vector or non-targeting siRNA in gain and loss of function experiments, respectively). (E) Immunoblot analysis verifying the efficiency of siRNA-mediated inhibition of endogenous SOCS3 expression 24, 48 and 72 h after transfection. Images are representative of three to five independent experiments.</p

Decrease of SOCS3 expression is an early event in head and neck cancer.

<p>Immunohistochemical staining for SOCS3 in four different tissue microarrays was analyzed with a digital slide scanner system. The intensity of positive staining was normalized to the area of positive staining to account for the larger area occupied by epithelial cells in the tumor samples. (A) Representative images of the results for immunohistochemical staining of SOCS3 are shown for 4 of the squamous cell carcinoma (SCC) cases that had both neoplastic and non-neoplastic samples with the indicated pathological information (location, TNM staging) (400X). Below are representative images of the classification system with the slide scanner. The protocol designed recognizes positive staining (in red), non-stained tissue (in yellow), cell nuclei (in blue) and empty spaces (in gray) (100X). (B) Results for the normalized intensity of SOCS3 expression according to tumor staging classification. The number of cases analyzed in each condition is indicated above the graphs (‘n’). This number varies because some TMAs did not have all the staging information and because some spots were lost during the staining process. Bars represent averages and vertical lines the standard deviations. Different letters above the columns indicate a statistically significant difference (p<0.05).</p

Differential effects of modulation of SOCS3 expression and STAT3 inhibition on HNSCC migration.

<p>(A) Representative phase-contrast images of the in vitro wound area in the different cell cultures (100×magnification) for the gain of function experiments by transfection of a CMV-driven SOCS3 expression plasmid or the empty vector control. STAT3 biochemical inhibitor was used to assess possible effects of SOCS3 that are independent of modulation of STAT3 activity. The distance between the edges of the wound was measured on digital images captured adjacent to the reference notch made in the cell culture plastic (black area on the right of the images) Non-neoplastic HaCAT cells were transfected with the empty vector to control for non-specific effects of the transfection procedure on cell migration. Bars represent averages and vertical lines standard deviation of three independent wounding experiments, measured in triplicate (*p<0.05 indicates significant difference in comparison to the distance between edges of the wound at the same time period in empty-vector transfected cells). (B) Representative phase-contrast images of the in vitro wound area in the loss of function experiments by transfection of SOCS3 siRNA or non-targeting siRNA in OSCC3 cells. Quantitation of cell migration was performed as described in (A) and the graph represent average and standard deviations of three independent wounding experiments measured in triplicate.</p