SN09-2 induces apoptosis of PC3 cells.

<p>(A) PC3 cells treated with 10 µM SN09-2 on cover slips were incubated with Cy3-conjugated annexin V, and briefly stained with Hoechst33342. Fluorescence images were taken under a fluorescence microscope. BF: cell morphology under light microscope (B, C) Annexin V-positive cells were increased in a dose- and time-dependent manner (B) PC3 cells treated with SN09-2 were harvested at different time points, incubated with FITC-conjugated annexin V, and applied to FACS analysis (C) PC3 cells were treated with different concentrations of SN09-2 for 12 h before annexin V staining. Data are means ± S.E. *, <i>p<</i>0.05; **, <i>p<</i>0.01, versus control.</p

SN09-2 stimulates cytochrome c release from mitochondria and caspase-3 activation.

<p>(A–D) Western blotting of cytochrome c in the cytosolic and mitochondrial fractions (A, C) PC3 cells were treated with different concentrations of SN09-2 for 24 h and fractionated into cytosol and mitochondria. 20 µg lysates from each fraction were used for SDS-PAGE and western blotting with anti-cytochrome c antibodies. (B, D) Cells treated with 10 µM SN09-2 were harvested at different time points, and then fractionated. (C, D) The signal intensity of each blot was measured and shown as graphs (E) PC3 cells treated with 10 µM SN09-2 were harvested at different time points, and used for western blotting with anti-caspase-3 or a cleaved form of caspase-3 antibodies. The amount of loaded proteins was normalized with anti-β-actin antibodies. (F) SN09-2-treated PC3 cells on cover slips were labeled with antibodies for active cleaved caspase-3 and anti-β-actin, and then briefly stained with Hoechst33342. Fluorescence signals were observed under a confocal microscope.</p

Dose-response of inhibition by GnRH-II antagonists.

<p>Either bfGnRHR-II or gmGnRHR-II was transfected with SRE-luc reporter into CV-1 cells. Cells were treated with the antagonists of different concentration in the presence of GnRH-II (1 nM for bfGnRHR-II, 10 nM gmGnRHR-II).</p

SN09-2 decreases mitochondrial membrane potential and induces ROS generation.

<p>(A) After treatment with 10 µM SN09-2 for 3 days, PC3 cells on cover slips were incubated with JC-1, fixed with 4% paraformaldehyde, briefly stained with Hoechst33342, and observed under the confocal microscope. (B) SN09-2-treated PC3 cells were detached from the dish and applied to FACS analysis to examine the fluorescence color change. (C) PC3 cells treated with SN09-2 for 3 h were incubated with DCF-DA, and applied to FACS analysis (D) Positive control of intracellular ROS. PC3 cells were treated with H₂O₂ for 5 min, labeled with DCF-DA, and then applied to FACS analysis. (E) Geometric means of fluorescence signals in PC3 cells treated with different concentrations of SN09-2 are shown as graphs. Data are means ± S.E. **, <i>p<</i>0.01, versus control.</p

SN09-2 inhibits the growth of PC3 xenografts in nude mice.

<p>(A, B) Effect of SN09-2 on PC3 cell growth in nude mice (n = 6 for each group). PC3 cells were injected into the flanks of the mice. After 10 days, the mice were subcutaneously injected with different doses of SN09-2 for 25 consecutive days. Data represent fold increase of tumor volumes over those for the first day injection. (B) Tumor volumes of each group are presented on day 25 after SN09-2 injection. Data are means ± S.E. **, <i>p<</i>0.01, versus control. C: control.</p

Subcellular localization of SN09-2 in PC3 cells.

<p>Cells were incubated with FITC-conjugated SN09-2 and MitoTracker. After washing, the cells were briefly labeled with Hoechst33342 (for nucleus staining). The images were taken under a confocal microscope. Red: MitoTracker, Green: FITC-SN09-2, Blue: Hoechst33342</p

SN09-2 inhibits prostate cancer cell growth.

<p>(A) Molecular sequences of GnRH-II, Trp-1, and SN09-2 (B) PC3 cells were incubated in RPMI media containing various concentrations of FBS and exposed to 10 µM SN09-2 for 4 days. The number of viable cells was counted under a light microscope. (C) PC3, Du145, LNCaP, HeLa, and DLD1 cells were incubated with 5% FBS media containing 10 mM SN09-2 or DMSO for 3 days. (D) PC3 cells were treated with different concentrations of Trp-1 or SN09-2 for 3 days, and then viable cells were counted under the light microscope. The cell number of each group was compared with the DMSO-treated group. CTL: DMSO-treated. (E) PC3 cells were treated with various concentrations of SN09-2 for 3 days. Using culture supernatants from each group, LDH activity was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099723#s2" target="_blank">Materials and Methods</a>. Data are means ± S.E. *, <i>p<</i>0.05; **, <i>p<</i>0.01, versus control.</p

Activities of GCG family peptides on GCRP receptor.

<p>HEK293T cells were co-transfected with CRE-luc and plasmids containing chGCRPR (A), xeGCRPR (B), or mdGCRPR (C) in 48-well plates. Forty-eight hours after transfection, cells were treated with the indicated concentrations of peptides (▪ for GCRP; • for chGIP; ◊ for chGLP2; ○ for chGCG; △ for huGLP1; ▾ for Exe-4). Species-specific responses of chGCRPR (D), xeGCRPR (E), and mdGCRPR (F) were determined by treating cells with increasing concentrations of GCRP (▾ for chGCRP; ? for xeGCRP; • for mdGCRP) for 6 h, and luciferase activity was examined.</p

Amino acid sequence alignment of GCRP and neighbor-joining phylogenetic tree for related peptides of vertebrates.

<p><b><i>A,</i></b> The mature peptide sequences of GCRP were predicted and aligned along with the GIP, GCG, GLP1, GLP2, and Gila monster exendins. Conserved residues for GCRPs and related peptides are indicated by different colors as proposed by the ClustalX-2.1 program. The GCRP-specific motif at positions 16–18 is highlighted by red dots above the sequences. <b><i>B,</i></b> Neighbor-joining phylogenetic tree for GCRP-related peptides of human (hu), mouse (mo), chicken (ch), anole lizard (an), <i>Xenopus (xe)</i>, zebrafish (zf), medaka (md), fugu (fu), stickleback (sb), and tetraodon (to) along with Gila monster exendins and human SCT, GHRH, VIP, and PACAP. The mature peptide sequences were aligned on ClustalX-2.1, and a tree was constructed with MEGA 5.05. Bootstrap numbers represent 1,000 replicates.</p

Ligand potency (EC₅₀ value) and efficacy (E_max value) for GCRPR.

<p>Results are presented as mean ± S.E. of at least three independent experiments. NR, no response; ND, not determined.</p