Plasma Gh and Igf-1 levels in GH3 and GH3-FTY mice.

<p>BALB/c-nu mice were inoculated with GH3 or GH3-FTY cells. Control mice were inoculated by the medium only. Plasma levels of Gh (<b>A</b>) and Igf-1 (<b>B</b>) at 8 weeks after inoculation. Data were compared using the one-way ANOVA with Tukey’s post-hoc test. *<i>P</i><0.05, ***<i>P</i><0.001. ns, not significant.</p

Body weight, weight change, body length, and liver weight in GH3 and GH3-FTY mice.

<p>(<b>A</b>) Actual appearance of control, GH3, and GH3-FTY mice. Arrowheads indicate xenograft tumor. (<b>B</b>) Time course of body weight changes. A significant difference between GH3-FTY and control mice was observed from 7 weeks after inoculation. No significant difference was observed between GH3 and control mice during the observation period. Statistical comparison of changes in body weight (<b>C</b>), body length (<b>D</b>), and liver weight (<b>E</b>) in control, GH3, and GH3-FTY mice. Data were compared using the one-way ANOVA with Tukey’s post-hoc test. *<i>P</i><0.05, **<i>P</i><0.01 vs control. †<i>P</i><0.05, ††<i>P</i><0.01 vs GH3. ns, not significant.</p

Screening of <i>Aip</i>-disrupted GH3 clones generated by CRISPR/Cas9.

<p>The detailed mutations of clones 1 and 2 are described in the Results section and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164131#pone.0164131.s001" target="_blank">S1 Fig</a></b>. Clone 1 corresponds to GH3-FTY cells intensively investigated in the present study. (<b>A</b>) Western blot analysis of Aip protein in GH3 and mutant GH3 clones (clones 1 and 2). Fifteen μg of protein from cell lysates was subjected to SDS-PAGE and immunoblotted with antibodies against Aip and beta-actin. Each target protein was visualized using VersaDoc 5000 (BIO-RAD, Tokyo, Japan). (<b>B, C</b>) The relative expression of <i>Gh</i> mRNA and <i>Prl</i> mRNA to <i>Actb</i> mRNA is shown, respectively. Data were compared using the unpaired two-tailed <i>t</i>-test. **<i>P</i><0.01 (clone 1 or 2) vs GH3. (<b>D)</b> The relative expression of <i>Aip</i> mRNA to <i>Actb mRNA</i> in cultured clone 1 (GH3-FTY cells) and clone 2 is shown. Data were compared using the unpaired two-tailed <i>t</i>-test. * <i>P</i><0.05 (clone 1 or 2) vs GH3.</p

Augmented Growth Hormone Secretion and Stat3 Phosphorylation in an Aryl Hydrocarbon Receptor Interacting Protein (AIP)-Disrupted Somatotroph Cell Line

<div><p>Aryl hydrocarbon receptor interacting protein (<i>AIP</i>) is thought to be a tumor suppressor gene, as indicated by a mutational analysis of pituitary somatotroph adenomas. However, the physiological significance of <i>AIP</i> inactivation in somatotroph cells remains unclear. Using CRISPR/Cas9, we identified a GH3 cell clone (termed GH3-FTY) in which <i>Aip</i> was genetically disrupted, and subsequently investigated its character with respect to growth hormone (Gh) synthesis and proliferation. Compared with GH3, GH3-FTY cells showed remarkably increased Gh production and a slight increase in cell proliferation. Gh-induced Stat3 phosphorylation is known to be a mechanism of Gh oversecretion in GH3. Interestingly, phosphorylated-Stat3 expression in GH3-FTY cells was increased more compared with GH3 cells, suggesting a stronger drive for this mechanism in GH3-FTY. The phenotypes of GH3-FTY concerning Gh overproduction, cell proliferation, and increased Stat3 phosphorylation were significantly reversed by the exogenous expression of <i>Aip</i>. GH3-FTY cells were less sensitive to somatostatin than GH3 cells in the suppression of cell proliferation, which might be associated with the reduced expression of somatostatin receptor type 2. GH3-FTY xenografts in BALB/c nude mice (GH3-FTY mice) formed more mitotic somatotroph tumors than GH3 xenografts (GH3 mice), as also evidenced by increased Ki67 scores. GH3-FTY mice were also much larger and had significantly higher plasma Gh levels than GH3 mice. Furthermore, GH3-FTY mice showed relative insulin resistance compared with GH3 mice. In conclusion, we established a somatotroph cell line, GH3-FTY, which possessed prominent Gh secretion and mitotic features associated with the disruption of <i>Aip</i>.</p></div

Western blot analysis of p-Stat3 (Tyr705) and Stat3 in cultured GH3 and GH3-FTY cells.

<p>(<b>A</b>) Western blot analysis of p-Stat3 (Tyr705) and Stat3. Twenty μg protein from cell lysates was separated by SDS-PAGE and immunoblotted with antibodies against p-Stat3, Stat3, and beta-actin. (<b>B</b>) Statistical evaluation of Stat3/ beta-actin expression between cultured GH3 and GH3-FTY cells. Data were compared using the unpaired two-tailed <i>t</i>-test. ns, not significant. The intensity of each detected band was analyzed using the image analysis software Quantity One (BIO-RAD), and the Stat3/ beta-actin ratio was calculated. (<b>C</b>) Statistical evaluation of p-Stat3/ beta-actin expression between cultured GH3 and GH3-FTY cells. Data were compared using the unpaired two-tailed <i>t</i>-test. **<i>P</i><0.01. (<b>D)</b> Western blot analysis of p-Stat3 and Stat3. The effect of lentivirus (LV)-mediated forced expression of exogenous <i>Aip</i> (LV-AIP) or <i>gfp</i> (LV-GFP) as a control on p-Stat3 expression was examined. Twenty μg protein from cell lysates was subjected to SDS-PAGE and immunoblotted with antibodies against p-Stat3 and beta-actin. (<b>E)</b> Statistical evaluation of p-Stat3. GFP and Aip indicate LV-GFP and LV-Aip, respectively. Data were compared using the unpaired two-tailed <i>t</i>-test. **<i>P</i><0.01. ns, not significant. (<b>F</b>) <i>Il6r</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR. (<b>G</b>) <i>Il6r</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR before and after lentivirus (LV)-mediated exogenous expression of <i>Aip</i> (LV-Aip) or <i>gfp</i> (LV-GFP) as a control. GFP and Aip indicate LV-GFP and LV-Aip, respectively. Data were compared using the unpaired two-tailed <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.01. ns, not significant.</p

Gh secretion in the cultured medium, <i>Gh</i> mRNA levels before and after exogenous <i>Aip</i> expression, and intracellular cAMP content in cultured GH3 and GH3-FTY cells.

<p>(<b>A</b>) Gh secretion in the media from cultured GH3 and GH3-FTY cells was measured and expressed as a fold increase. (<b>B</b>) <i>Gh</i> mRNA levels in cultured GH3 and GH3-GTY cells were calculated relative to <i>Actb</i> (internal control) using the ∆Ct method. (<b>C</b>) The effect of lentivirus (LV)-mediated forced expression of exogenous <i>gfp</i> as a control or <i>Aip</i> on <i>Gh</i> mRNA levels. GFP and Aip indicate LV-GFP and LV-Aip, respectively. (<b>D</b>) The intracellular cAMP content of both cultured cells was measured as described in the Methods. Data were compared using the unpaired two-tailed <i>t</i>-test. **<i>P</i><0.01. ns, not significant.</p

Analysis of tumors in GH3 or GH3-FTY-xenografted mice.

<p>(<b>A</b>) Actual appearance of tumors removed from control mice, GH3 mice, and GH3-FTY mice. (<b>B</b>) Tumor volume and (<b>C</b>) tumor weight 8 weeks after inoculation. Data were compared using the two-tailed unpaired <i>t</i>-test. *<i>P</i><0.05. (<b>D</b>) Typical tissue histology of somatotroph cell tumors in GH3 or GH3-FTY mice by HE staining (upper two panels) and immunohistochemical analysis (lower two panels) at 4 weeks. Bar, 50 μm. Staining of Gh (green) and nucleus (DAPI, blue) was observed using confocal microscopy. A larger N/C ratio and anisonucleosis in GH3-FTY cells relative to GH3 cells were observed. More prominent Gh vesicles were observed in GH3-FTY compared with GH3 mice. Bar, 20 μm. (<b>E</b>) Typical tissue histology of tumors in GH3 and GH3-FTY mice by HE staining at 8 weeks. Arrowheads indicate abnormal mitotic cells. (<b>F</b>) Ki67 scores of both tumors were statistically compared. Data were compared using the two-tailed unpaired <i>t</i>-test. *<i>P</i><0.05.</p

Comparison of <i>Sstr2</i>, and <i>Zac1</i> mRNA expression and Sstr2 expression levels by western blot in cultured GH3 and GH3-FTY cells.

<p>(<b>A</b>) <i>Sstr2</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR. (<b>B</b>) Western blot analysis of Sstr2 and beta-actin in cultured GH3 and GH3-FTY cells. Twenty μg protein from cell lysates was separated by SDS-PAGE and immunoblotted with antibodies against Sstr2 and beta-actin (upper panel). Statistical evaluation of Sstr2/ beta-actin expression between cultured GH3 cells and GH3-FTY cells (lower panel). (<b>C</b>) <i>Zac1</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR. (<b>D</b>) <i>Sstr2</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR after lentivirus (LV)-mediated exogenous expression of <i>Aip</i> (LV-Aip) or <i>gfp</i> (LV-GFP) as a control. GFP and Aip indicate LV-GFP and LV-Aip, respectively. (<b>E</b>) <i>Zac1</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR after lentivirus (LV)-mediated exogenous expression of <i>Aip</i> (LV-Aip) or <i>gfp</i> (LV-GFP) as a control. GFP and Aip indicate LV-GFP and LV-Aip, respectively. Data were compared using the unpaired two-tailed <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.01. ns, not significant.</p

Exendin–4 and metformin decrease the number of Ki67 and P504S-positive cells, and increase GLP-1R expression.

<p>Paraffin-embedded tumor sections (5 μm) were subjected to immunohistochemistry for (A) Ki67, (C) P504S and GLP-1R, and (F) AR, and counterstained with DAPI. Magnification, ×400. (B) Ki67, (D) P504S, (E) GLP-1R, and (G) AR-positive cells were quantified by analyzing the fraction of stained cells in the tumor relative to the total number of nuclei. Values are expressed as a percentage of positive cells. Unpaired <i>t</i>-tests were performed to calculate statistical significance (*<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. control).</p

Exendin–4 and metformin inhibit prostate cancer cell proliferation additively, but not migration.

<p>(A) LNCaP cells, (B) PC 3 cells and (C) DU145 cells were maintained in media supplemented with 10% FBS with or without metformin (0.1–10mM). After 0, 24, 48, 72 and 96 h, the cells were harvested, and cell proliferation was analyzed by cell counting using a hemocytometer. Unpaired <i>t</i>-tests were performed to calculate statistical significance (*<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. control). (D) LNCaP cells, (E) PC3 cells and (F) DU145 cells were maintained as described in A-C, but with the indicated treatments. Unpaired <i>t</i>-tests were performed to calculate statistical significance (*<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. control). (G) LNCaP cells, (H) PC3 cells and (I) DU145 cells were plated at a density of 5000 cells/well in 96-well plates in media supplemented with 10% FBS and incubated with saline (control), Ex–4 (10nM), metformin (0.1mM), or both Ex–4 (10nM) and metformin (0.1mM) for 24 h. BrdU solution was added during the last 2 h, and cells were harvested for measurement of DNA synthesis using a microplate reader at 450–620 nm. Mean data are expressed as a ratio of the control cell proliferation. Unpaired <i>t</i>-tests were performed to calculate statistical significance (*<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. control, ^#<i>P</i> < 0.05 vs. Ex–4, ^††<i>P</i> < 0.01 vs. metformin). (J) LNCaP cells and (K) PC3 cells were seeded as 1.5×10⁵ in each chambers for the migration assay. After chemo attraction with 10% FBS with or with saline (control), Ex–4 (10 nM), metformin (0.1 mM) or both Ex–4 (10 nM) and metformin (0.1 mM), cells were stained and examined at OD 570nm. Unpaired <i>t</i>-tests were performed to calculate statistical significance.</p