High Affinity Binding of Indium and Ruthenium Ions by Gastrins

<div><p>The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga³⁺ were fitted by a 2 site model with dissociation constants (K_d) of 3.3 x 10⁻⁷ and 1.1 x 10⁻⁶ M. Although the absorption of gastrin did not change upon the addition of In³⁺ ions, the changes in absorbance on Fe³⁺ ion binding in the presence of indium ions were fitted by a 2 site model with K_d values for In³⁺ of 6.5 x 10⁻¹⁵ and 1.7 x 10⁻⁷ M. Similar results were obtained with Ru³⁺ ions, although the K_d values for Ru³⁺ of 2.6 x 10⁻¹³ and 1.2 x 10⁻⁵ M were slightly larger than observed for In³⁺. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In³⁺ or Ru³⁺ ions, and that the affinity of the first site for In³⁺ or Ru³⁺ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.</p></div

Proposed structural models of FeIII2Ggly and RuIII2Ggly.

<p>The model for Fe^III₂Ggly (A) is based on the EXAFS data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g005" target="_blank">Fig 5B</a>, and is consistent with previous NMR and visible spectroscopic studies of Ggly and mutant peptides.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.ref008" target="_blank">8</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.ref009" target="_blank">9</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.ref012" target="_blank">12</a>] The two Fe^III ions are coordinated by the carboxylate side chains likely from glutamates 6, 7, 8, 9 and 10, with glutamate 7 acting as a ligand to both Fe^III ions. One or more oxygens also act as bridging ligands between the two Fe^III ions. The peptide backbone and non-coordinating side chains have been omitted for simplicity. The model for Ru^III₂Ggly (B) is based on the EXAFS data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g005" target="_blank">Fig 5H</a>, and differs from the model for Fe^III₂Ggly in the presence of a Ru≡Ru bond and a chloride ion ligand.</p

Ruthenium ions compete with ferric ions for the gastrin binding sites.

<p>Addition of aliquots of ruthenium chloride (black ▼) to 10 μM Gamide or Gly in the buffer described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g002" target="_blank">Fig 2</a> legend resulted in an increase in absorbance at 280 nm which was significantly less than the changes seen on addition of aliquots of ferric chloride (red π). However in the presence of 5.30 (green ⬛) or 26.48 μM (blue •) ruthenium chloride the changes in absorbance seen on addition of aliquots of ferric chloride were considerably different from the changes seen in the absence of ruthenium chloride. The points are means from three separate experiments; bars represent the SEM. The lines were constructed with the dissociation constants and maximum absorbance values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.t001" target="_blank">Table 1</a>) obtained by fitting the data to the 2 site competitive model shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g001" target="_blank">Fig 1</a> with the program BioEqs.</p

Indium ions compete with ferric ions for the gastrin binding sites.

<p>Addition of aliquots of indium nitrate (black ▼) to 10 μM Gamide or Ggly in the buffer described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g002" target="_blank">Fig 2</a> legend resulted in little change in absorbance at 280 nm when compared to the changes seen on addition of aliquots of ferric chloride (red π). However in the presence of 3.99 (green ⬛) or 39.85 μM (blue •) indium nitrate the changes in absorbance seen on addition of aliquots of ferric chloride were considerably different from the changes seen in the absence of indium nitrate. The points are means from three separate experiments; bars represent the SEM. The lines were constructed with the dissociation constants and maximum absorbance values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.t001" target="_blank">Table 1</a>) obtained by fitting the data to the 2 site competitive model shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g001" target="_blank">Fig 1</a> with the program BioEqs.</p

Binding of metal ions by Gamide and Ggly.

<p>The affinity of, and the percentage absorbance change at 280 nm on, ferric or gallium ion binding to Gamide or Ggly were determined by fitting the mean data obtained in the absorbance experiments, described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g002" target="_blank">Fig 2</a> legend, to the models shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g001" target="_blank">Fig 1</a> with the program BioEqs. In the case of indium or ruthenium ions the corresponding values were obtained from fitting ferric ion titrations in the presence of various concentrations of indium or ruthenium ions, as described in the legends to Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g004" target="_blank">4</a>, respectively.</p

EXAFS spectra.

<p>EXAFS spectra.</p

Models of metal ion binding.

<p>In the 2 site model gastrin binds two metal ions with dissociation constants K_d1M and K_d2M. In the 2 site competitive model gastrin binds two ferric ions with dissociation constants K_d1Fe and K_d2Fe, and two metal ions (M) to the same two sites with dissociation constants K_d1M and K_d2M. The dissociation constant K_d3M describes the formation of the mixed FeGastrinM complex.</p

Glaucarubinone Combined with Gemcitabine Improves Pancreatic Cancer Survival in an Immunocompetent Orthotopic Murine Model

<p><i>Background</i>: Pancreatic cancer continues to have a poor survival rate with an urgent need for improved treatments. Glaucarubinone, a natural product first isolated from the seeds of the tree <i>Simarouba glauca</i>, has recently been recognized as having anti-cancer properties that may be particularly applicable to pancreatic cancer. <i>Methods</i>: The effect of glaucarubinone on the growth and migration of murine pancreatic cancer cells was assessed by ³H-thymidine incorporation assay. The survival impact of glaucarubinone alone and in combination with gemcitabine chemotherapy was assessed using an immunocompetent orthotopic murine model of pancreatic cancer. <i>Results</i>: Glaucarubinone inhibited the growth of the murine pancreatic cancer cell lines LM-P and PAN02. Treatment with either glaucarubinone or gemcitabine reduced proliferation <i>in vitro</i> and the combination was synergistic. The combination treatment improved survival two-fold compared to gemcitabine treatment alone (<i>p</i> = 0.046) in PAN02 cells. <i>Conclusions</i>: The synergistic inhibition by glaucarubinone and gemcitabine observed <i>in vitro</i> and the improved survival <i>in vivo</i> suggest that glaucarubinone may be a useful adjunct to current chemotherapy regimens.</p

Basal HIF1α protein expression, proliferation rates and migration/invasion rates in human PC cell lines. (

<p>A) Basal HIF1α protein concentrations in the human PC cell lines LNCaP, DU145 and PC3 under normoxic conditions were analyzed by Western blot. (B) Proliferation was assayed by cell counting after 24 and 48 hours. (C) Migration/invasion rates were measured by Transwell assays at 24 hours. Values in (A) and (C) are expressed as the fold increase compared to LNCaP cells, while the values in (B) are expressed as a percentage of the time 0 value. All values are the mean ± SEM of at least three separate treatments. (D) Survival rates of PC cells exposed to cytotoxic conditions. The survival of PC3 cells (which have higher basal HIF1α protein) when exposed to oxidative stress with hydrogen peroxide (H₂O₂) or chemotoxicity with 5-fluorouracil (5-FU) was compared to the survival of LNCaP cells (which have lower HIF1α expression). Survival was assessed by counting cell numbers at 24 hours. Values are expressed as a percentage of the untreated control and are the mean ± SEM of at least three separate treatments. #, P<0.05 versus treated LNCaP cells.</p

The translation efficiency of the HIF1α 5′UTR-luciferase reporter in prostate cancer cells.

<p>(A) <i>Firefly</i> and <i>Renilla</i> luciferase activities in prostate cancer cells following transfection of a HIF1α 5′UTR-luciferase construct and the pTK-Renilla control reporter vector were determined using a dual luciferase assay. (B) Real-time PCR (RT-PCR) analysis of luciferase mRNA in PC cells transfected with the HIF1α 5′UTR-luciferase construct. Following transfection, RNA was isolated, and luciferase mRNA expression detected by real time RT-PCR and normalized by 18S mRNA expression. (C) Translational efficiency represents the ratio of <i>Firefly</i>/<i>Renilla</i> luciferase activity, divided by the relative luciferase mRNA concentration in PC cells. The translational efficiency of luciferase mRNA driven by the 5′UTR region of HIF1α in PC3 cells is higher than in LNCaP cells. Values are the mean ± SEM of at least three separate experiments. *, P<0.05 versus treated LNCaP cells.</p