Role of PFK15 in cell cycle progression.

<p>(a) MKN45 and AGS cells were fixed and stained by PI following dedicated concentrations of PFK15 treatment after 24 h. Then the cell cycle distribution was analyzed using flow cytometer. PFK15 treatment remarkably prevented MKN45 cells from proceeding to S phase, thus resulting in cell cycle arrest at G0/G1 phase. (b) PFK15 treatment significantly increased the percentage of cells in G1 phase, indicating a time dependent effect on tested cells up to 48 h post-treatment. Data were analyzed by means ± SD and were representative of three independent experiments. *P<0.05, compared with controls.</p

Mechanism of PFK15 induced apoptosis.

<p>(a) After treated with various concentrations of PFK15 for 24 h, MKN45 and AGS cells were collected and the protein changes were analyzed by western blot assay. (b) The optical density of Bcl-2 and Bax protein levels were quantified to controls by ImageJ software. PFK15 decreased the ratio of Bcl-2/Bax in a concentration-dependent manner. Columns, mean; bars, SD. *P<0.05, compared with controls.</p

NSK-01105, a Novel Sorafenib Derivative, Inhibits Human Prostate Tumor Growth via Suppression of VEGFR2/EGFR-Mediated Angiogenesis

<div><p>The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i> models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited <i>ex vivo</i> angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities.</p></div

The effect of NSK-01105 and sorafenib in prostate cancer xenograft models.

<p>*, <i>p</i><0.05, compared with control. Significant difference was calculated by one-way analysis of variance.</p><p>The effect of NSK-01105 and sorafenib in prostate cancer xenograft models.</p

Pyruvate activated Bcl-xL-cytochrome c-caspase9/3 pathway.

<p>Cells were pretreated with vehicle or pyruvate (from 1 to 20 mM) for 30 minutes at 37°C. After preincubation, cells were challenged with vehicle or 30 mM glutamate for 3 hours. Bcl-xL, Bcl-2, Bax, cytochrome c, caspase-9 and caspase-3 levels were determined by western blot analysis. β-actin was used as an internal control. B, C, D, E, F, G and H represented Bcl-xL, Bcl-2, Bax, Bcl-xL/Bax ratio, cytochrome c, cleaved caspase-9 and cleaved caspase-3 level, respectively. The protein/Actin ratio in control cells was set as 100%. All experiments were repeated at least 3 times. *<i>P</i><0.05 vs control; ^#<i>P</i><0.05 vs glutamate.</p

PFK15 inhibited the invasion of MKN45 and AGS cells.

<p>(a, b) Cells were treated with the indicated concentrations of PFK15 for 12 h in the upper chambers and the lower chambers were filled with 10% FBS medium. Then, the invaded cells were stained with crystal violet and observed under a microscope with 200× magnification. Data were obtained from six randomly chosen fields and were normalized to the control group. (c) Proteins related with cell migration and invasion were detected. Phospho-FAK levels were downregulated in 7 and 9 μM while phospho-Cadherin E levels upregulated in both cell lines. Columns, mean; bars, SD. *P<0.05, compared with controls.</p

Role of PFK15 in cell apoptosis.

<p><b>(a)</b> MKN45 and AGS cells were treated with various concentrations of PFK15 and collected after 24 h. Then cells were analyzed by flow cytometry after stained with Annexin V and PI. The percentages of early apoptotic (Annexin V⁺/ PI^-) and late apoptotic (Annexin V⁺/ PI⁺) cells were quantified. (b) PFK15 treatment significantly increased the percentage of apoptotic cells, indicating a time dependent effect on tested cells up to 48 h post-treatment. Data were analyzed by means ± SD and were representative of three independent experiments. *P<0.05, compared with controls.</p

PFK15 suppressed tumor growth in gastric tumor xenograft models.

<p>MKN45 tumor-bearing mice were treated with PFK15 intraperitoneally at 25 mg/kg or vehicle every three days for 15 days. PFK15 had satisfactory inhibition effects against MKN45 tumor growth with an IR of 56.10% compared with vehicle group (a). Tumor volumes and body weight were measured twice a week (b, c). Columns, mean; bars, SD. *p<0.05, compared with vehicle controls.</p

NSK-01105 inhibited the activation of VEGFR2-mediated signaling pathways in HUVECs.

<p>VEGF stimulated VEGFR-2 autophosphorylation in HUVECs. (a) NSK-01105 and sorafenib suppressed the activation of VEGFR2 and its downstream key kinases FAK and eNOS at 5 µM in HUVECs. (b) The ratios of the optical density between target molecules and β-actin. The optical density was quantified by IPP software. Columns, mean; bars, SD (n = 3). *, compared with vehicle controls, <i>P</i><0.05; #, compared with VEGF controls, <i>P</i><0.05.</p

NSK-01105 inhibited VEGF-induced cell viability in HUVECs.

<p>(a) Chemical structures of the NSK-01105 and Sorafenib. (b) NSK-01105 inhibited the VEGF-induced viability of endothelial cells. Stimulated with VEGF (10 ng/mL), HUVECs showed a high rate of viability. VEGF-induced viability of HUVECs was significantly inhibited by NSK-01105 and sorafenib at concentrations of 5, 10 and 20 µM for 24 h. Columns, mean; bars, SD (n = 6). *, compared with vehicle controls, <i>P</i><0.05; #, compared with VEGF controls,<i>P</i><0.05.</p