R-Ras binding to FLNa enhances fibronectin matrix assembly.

<p>Fibronectin matrix assembly in cultures expressing R-Ras G38V and either wild type FLNa (<b>A</b>), FLNaΔ3 (deletion of FLNa repeat 3; <b>B</b>), or cell cultures that do not express FLNa (FLNa-; <b>C</b>) were assessed. FLNa cultures expressing PcDNA3 vector and control scrambled siRNA (<b>D</b>) or knockdown of endogenous R-Ras using R-Ras siRNA (<b>E</b>) or control cultures not expressing FLNa (<b>F</b>) were also assessed. 24 hr after plating the culture media was replaced with media containing human FN (100 µg/ml) and cells were cultured for a further 48 hr before fibronectin matrix was assessed by immunostaining for FN. Each experiment was repeated a minimum of 3X.</p

The association of R-Ras with FLNa enhances melanoma cell migration.

<p><b>A</b>) Transwell migration assay was used to measure the migration of M2 melanoma cells in which dominant active R-Ras G38V was expressed in stable clones expressing wild type FLNa, FLNaΔ3, or vector control with no FLNa expression. The bottom side of filters were coated with 15 µg/ml FN. Migration was expressed as the number of migrated β-Gal expressing cells. Data represent the mean ± S.D. of three independent experiments. <b>Insert</b>: Western blot of stable M2 cell clones expressing wild type FLNa, FLNaΔ3 (deletion of FLNa repeat 3), or vector control with no FLNa expression and R-Ras G38V. Tubulin was used as a loading control. Data represent at least 3 independent experiments. Statistically significant differences are reported in the graph as <i>P</i> values (Student's <i>t</i>-test), **, <i>P</i><0.01 <b>B</b>) Transwell migration assay was used to measure the migration of M2 (FLNa-) melanoma parental cell line in which FLNa, FLNaΔ3, or vector control and active R-Ras G38V and the transfection marker β-Gal were transiently transfected. Migration was expressed as the number of migrated β-Gal expressing cells. Data represent the mean ± S.D. of three independent experiments. Statistically significant differences are reported in the graph as <i>P</i> values (Student's <i>t</i>-test), **, <i>P</i><0.01; *, <i>P</i><0.04. <b>Insert</b>: Western blot of FLNa+, FLNaΔ3, or FLNa- with active R-Ras G38V expression in parental M2 (FLNa-) cell line or FLNa+, FLNaΔ3, or FLNa- cells with no active R-Ras G38V. Tubulin was used as a loading control. Data represent at least 3 independent experiments. <b>C</b>) Adhesion assay of cells expressing FLNa, FLNaΔ3, or no FLNa ± active R-Ras. Data represent at least 3 independent experiments. Top Insert: protein expression levels shown are for both A and C.</p

R-Ras and FLNa co-localize in M2 melanoma cells.

<p>FLNa and active R-Ras G38V partially co-localize in cells as examined by immunofluorescence confocal microscopy at 63X. Co-localization of FLNa and active R-Ras is displayed in yellow in the merged image. Arrows indicate co-localization. M2 (FLNa-) cells were transiently transfected with either wild type FLNa, dominant active R-Ras G38V (DA R-Ras), FLNa + DA R-Ras, FLNa + dominant negative R-Ras T43N (DN R-Ras), FLNaΔ3 (FLNa repeat 3 deleted) ± R-Ras, FLNa+R-Ras siRNA or FLNa + control scrambled siRNA and plated on FN coated glass slides. R-Ras was detected by immunostaining with an anti-R-Ras antibody followed by a Rhodamine conjugated secondary. FLNa and FLNaΔ3 were detected by immunostaining with an anti-FLNa antibody followed by Alexa Fluor 488 conjugated secondary. 63X. Data shown is representative of 3 independent experiments.</p

R-Ras binding to FLNa enhances deoxycholate-insoluble fibronectin matrix deposition.

<p><b>A</b>) Deoxycholate (DOC)-insoluble fibronectin matrix formation was examined in M2 cultures expressing active R-Ras and either wild type FLNa, FLNaΔ3 (deletion of FLNa repeat 3), or M2 (FLNa-) control cell cultures. FLNa cultures with knockdown of endogenous R-Ras using R-Ras siRNA were assessed. Control scrambled siRNA and cultures not expressing FLNa (FLNa-) were also assessed. 48 hr after the addition of human FN to the wells, the DOC-insoluble material was subjected to SDS-PAGE analysis under reducing conditions and FN was detected by immunostaining. FN = Fibronectin. Vimentin was used as a loading control. <b>B</b>) Fold induction of matrix was normalized to M2 (FLNa-) control cultures. Graph shown represents pooled data from three independent experiments. Error bars represent S.E.M. Statistically significant differences are reported in the graph as <i>P</i> values (Student's <i>t</i>-test), **, <i>P</i><0.05. <b>C</b>) Knockdown of endogenous R-Ras using siRNA or scrambled control siRNA is demonstrated via Western blot. 60 hr post transfection cells were lysed, run on SDS-PAGE gel and immunoblotted for R-Ras using a polyclonal anti-R-Ras antibody. Tubulin was used as a loading control. Results are representative of 3 independent experiments.</p

R-Ras associates with FLNa at repeat 3.

<p><b>A</b>) Yeast two hybrid screen of R-Ras binding to FLNa. Colonies from + histidine (+his) plates obtained by co-transformation of the L40 yeast strain with BTM116R-Ras-CAAX^- or negative control BTM116-lamin C and VP16-FLNa (nucleotides 1699–1995). The plate was photographed after 3 days of growth. <b>B</b>) R-Ras binds FLNa. GST fusion proteins derived from indicated FLNa fragments were incubated with M2 (FLNa-) cell total lysates. Bound R-Ras was detected by Western blotting for R-Ras. Results are representative of 3 independent experiments. Coomassie stained gel containing purified FLNa fragments is shown to confirm FLNa fragment protein expression. <b>C</b>) R-Ras and FLNa co-immunoprecipitate in M2 melanoma cells. M2 cells were co-transfected with expression vectors for active R-Ras G38V, His-tagged wild type FLNa, His-tagged FLNaΔ3 (deletion of FLNa repeat 3) or vector alone. 48 h post transfection cells were lysed, immunoprecipitated (IP) with anti-His conjugated IgG beads or IgG beads alone and immunoprecipitates were analyzed by immunoblotting with an anti-R-Ras antibody. Input R-Ras and FLNa levels are shown (Bottom). Results are representative of 3 independent experiments. <b>D</b>) The domain structure of FLNa and the location of R-Ras binding site as determined by yeast two hybrid screen. The β-integrin subunits, the actin-binding domain, and the dimerization domain at the C-terminus have been established previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011269#pone.0011269-Pfaff1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011269#pone.0011269-Gorlin1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011269#pone.0011269-Loo1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011269#pone.0011269-Sharma1" target="_blank">[51]</a>. FLNa fragments repeats 1–10, 11–16, and 17–23 + dimerization domain are shown on the left.</p

The association between R-Ras and FLNa enhances integrin activation.

<p><b>A</b>) M2 cells express a subset of integrins on their surface. FACS analysis of a subset of integrins expressed on the surface of M2 cells. M2 cells express α5 and β1 subunits on their surface but not αvβ3. <b>B</b>) Forced expression of activated R-Ras promotes integrin activation in M2 cells as determined by the 3Fn-(9–11) activation assay. M2 (FLNa-) cells were transiently transfected with expression vectors encoding GFP as transfection reporter and FLNa, constitutively active R-Ras G38V alone or ±FLNa or FLNaΔ3. Cells were harvested and analyzed by two-color FACS for transfected cells and 3FN-(9–11) binding. Shown on the <i>Y-Axis</i> is mean activation index ±S.E. of three independent experiments. Constitutively expressed active R-Ras G38V and FLNa increased the activation index more than R-Ras alone. FLNaΔ3 did not block R-Ras-mediated activation compared with controls. Statistically significant differences are reported in the graph as <i>P</i> values (Student's <i>t</i>-test), *, <i>P</i><0.05 **, <i>P</i><0.04 <b>C</b>) Western blot of R-Ras levels when transiently transfected with R-Ras G38V. Tubulin was used as a loading control. Data represent at least 3 independent experiments.</p

Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

Early–late transition metal TiAu₂ compounds [(η-C₅H₅)₂Ti­{OC­(O)­CH₂PPh₂AuCl}₂] (<b>3</b>) and new [(η-C₅H₅)₂Ti­{OC­(O)-4-C₆H₄­PPh₂AuCl}₂] (<b>5</b>) were evaluated as potential anticancer agents <i>in vitro</i> against renal and prostate cancer cell lines. The compounds were significantly more effective than monometallic titanocene dichloride and gold­(I) [{HOC­(O)­RPPh₂}­AuCl] (R = −CH₂– <b>6</b>, −4-C₆H₄– <b>7</b>) derivatives in renal cancer cell lines, indicating a synergistic effect of the resulting heterometallic species. The activity on renal cancer cell lines (for <b>5</b> in the nanomolar range) was considerably higher than that of cisplatin and highly active titanocene Y. Initial mechanistic studies in Caki-1 cells <i>in vitro</i> coupled with studies of their inhibitory properties on a panel of 35 kinases of oncological interest indicate that these compounds inhibit protein kinases of the AKT and MAPKAPK families with a higher selectivity toward MAPKAPK3 (IC₅₀ <b>3</b> = 91 nM, IC₅₀ <b>5</b> = 117 nM). The selectivity of the compounds <i>in vitro</i> against renal cancer cell lines when compared to a nontumorigenic human embryonic kidney cell line (HEK-293T) and the favorable preliminary toxicity profile on C57black6 mice indicate that these compounds (especially <b>5</b>) are excellent candidates for further development as potential renal cancer chemotherapeutics

Englerin A Selectively Induces Necrosis in Human Renal Cancer Cells

<div><p>The number of renal cancers has increased over the last ten years and patient survival in advanced stages remains very poor. Therefore, new therapeutic approaches for renal cancer are essential. Englerin A is a natural product with a very potent and selective cytotoxicity against renal cancer cells. This makes it a promising drug candidate that may improve current treatment standards for patients with renal cancers in all stages. However, little is known about englerin A's mode of action in targeting specifically renal cancer cells. Our study is the first to investigate the biological mechanism of englerin A action in detail. We report that englerin A is specific for renal tumor cells and does not affect normal kidney cells. We find that englerin A treatment induces necrotic cell death in renal cancer cells but not in normal kidney cells. We further show that autophagic and pyroptotic proteins are unaffected by the compound and that necrotic signaling in these cells coincided with production of reactive oxygen species and calcium influx into the cytoplasm. As the first study to analyze the biological effects of englerin A, our work provides an important basis for the evaluation and validation of the compound's use as an anti-tumor drug. It also provides a context in which to identify the specific target or targets of englerin A in renal cancer cells.</p> </div

Neopetrocyclamines A and B, Polycyclic Diamine Alkaloids from the Sponge <i>Neopetrosia</i> cf <i>exigua</i>

Two new polycyclic alkaloids, neopetrocyclamines A and B (<b>1</b> and <b>2</b>), along with the known metabolites papuamine (<b>3</b>) and haliclonadiamine (<b>4</b>), were isolated from the Indonesian sponge <i>Neopetrosia</i> cf <i>exigua</i>. Neopetrocyclamine A contains a formamidinium moiety, a rare functional group. While these compounds share the same basic biosynthetic building blocks, the size of the ring system differs in <b>1</b> and <b>2</b> because of the formamidinium moiety. Biological evaluations of <b>1</b>–<b>4</b> revealed that papuamine is cytotoxic against glioblastoma SF-295 cells (GI₅₀ = 0.8 μM)

Englerin A does not lead to up-regulation of extracellular phosphatidyl serine.

<p>Cells were treated with either 1 μM englerin A or carrier DMSO for 60 min, or 5 μM staurosporine for 3 h. After incubation, cells were trypsinized and stained for extracellular phosphatidyl serine expression using FITC-tagged Annexin V and propidium iodide (PI) as co-stain to test cell membrane integrity. Shown is a result representative of three independent experimental repeats. Quantifications and statistics of all data are depicted as bar graphs and show the distribution of cells testing positive for Annexin V binding (early apoptotic stages) or Annexin V binding and propidium iodide uptake (late apoptotic stages/necrotic death). Values shown are mean ± SEM (n = 3), statistically significant differences are marked with asterisks (*** p<0.001), n.s. = not significant.</p