Fig 1.
Effect of PFK15 on cell viability, F2,6P2 production and glucose uptake.
(a) Cells grown in exponential phase were exposed to the indicated concentrations of PFK15 and, after 24 h, the effects on viability were determined by trypan blue exclusion. *p<0.05, compared with the GES-1 groups. (b) Time-dependent effects of PFK15 on MKN45 and AGS cell viability under the concentration of 10 μM. *p<0.05, compared with the control group of MKN45 cell line; #p<0.05, compared with the control group of AGS cell line. (c,d) To identify acute effects on known metabolic effects of PFKFB3 inhibition, MKN45 and AGS cells were exposed to PFK15 for 12 h and the F2,6P2 and 2-Deoxyglucose uptake (Glucose Assay Kit) were measured. (e) The rescue effect of F2,6P2 on PFK15 induced cell proliferation suppression. The marked reduction of cell proliferation caused by 10 μmol/L of PFK15 in MKN45 or AGS cells could be partially rescued by F2,6P2 addition. Data are represented as the means ± SD from three independent experiments. *p<0.05, compared with controls.
Fig 2.
Role of PFK15 in cell cycle progression.
(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.
Fig 3.
Mechanism of PFK15-induced cell cycle arrest in G0/G1 phase.
Cells were treated with various concentration of PFK15 for 24 h. The cell cycle associated proteins were checked by western bolt assay. PFK15 treatment significantly decreased expressions of cyclin D1, cyclin E1, phosphorylated Rb and the nuclear transfactor E2F-1 levels while increased non-phosphorylated Rb protein levels in both MKN45 and AGS cell lines.
Fig 4.
Role of PFK15 in cell apoptosis.
(a) 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.
Fig 5.
Mechanism of PFK15 induced apoptosis.
(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.
Fig 6.
PFK15 inhibited the invasion of MKN45 and AGS cells.
(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.
Fig 7.
PFK15 suppressed tumor growth in gastric tumor xenograft models.
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.