Fig 1.
Structures of Aripiprazole and OPC-14857.
Fig 2.
Effects of Aripiprazole (ARP) and OPC-14857 (OPC) on glioblastoma cells.
Inhibitory effects of cell proliferation on U251 (A), T98G (B), U87 (C) and HEK293 (D) cells. IC₅₀ values of ARP and OPC in each cell line (E). The cells were seeded in a 96-well plate and incubated at 37°C for 24 h. Subsequently, the cells were treated with the test compound and incubated at 37°C for 24 h. Cell viability was determined using Cell Counting Kit-8, according to the instructions provided by the manufacturer. The symbol * (p < 0.05) indicates significant differences between ARP and OPC by one-way analysis of variance and student t-test. Data are shown as the mean ± SEM (n = 3).
Fig 3.
Effect of ARP (30 µM) or OPC (30 µM) on cell migration in U251 cells.
U251 cells were seeded at a density of 80,000 cells in a 35 mm dish and incubated at 37°C for 48 h to 90%–100% confluence. The cells were treated with ARP (30 µM) or OPC (30 µM). The cells were scratched on a petri dish using a tip and observed under a microscope (0 h and 24 h).
Fig 4.
Recovery activity of cell death inhibitors against ARP (A, 30 μM) or OPC (B, 30 μM) and other compounds in U251 cells.
U251 cells were treated with ARP (A) or OPC (B) and several cell death inhibitors (Z-VAD, Nec-1, Fer-1, and IM-54) for 24 h. Cell viability was determined using a water-soluble tetrazolium assay. Each error bar represents the mean ± SEM (n = 4). Statistical analysis was performed using one-way analysis of variance and subsequent Dunnett’s test in each compound with or without ARP or OPC. The symbol * (p < 0.05) indicates significant differences between non-inhibitor and each cell death inhibitor treatment by one-way analysis of variance and subsequent Dunnett’s test. The symbol ns indicates not significant compared with the non-inhibitor. The 100% viable cells correspond to untreated cells (0.1% DMSO).
Fig 5.
Effects of ARP or OPC on the formation and distribution of actin filaments.
A) Red shows F-actin stained by phalloidin-rhodamine conjugated and green shows G-actin stained by Alexa Fluor™ 488 DNase I. The right column shows the merged staining of F-actin and G-actin. Cells with membrane-localized F-actin are indicated by arrows. B) The fluorescence intensity ratio of ARP and OPC in F-actin and G-actin staining. The F-actin to G-actin (F/G) ratio was calculated using approximately 5 images. Statistical analyses were performed using Dunnett’s test. The Symbol *** (p < 0.001, n = 5) indicates significant differences between DMSO and ARP or OPC (30 µM).
Fig 6.
Effects of ARP and OPC on the cell cycle in U251 cells.
After treatment with DMSO (A), ARP (B), or OPC (C), the number of cells in each cell cycle phase was measured using flow cytometry with propidium iodide staining. A-C) Cell cycle distribution was analyzed using flow cytometry. D) Histograms represent the percentage of cells distributed in each cell cycle when the total cell count was 100%. Error bars represent mean ± SEM (n = 3). The symbols *** (p < 0.001), ** (p < 0.01), and * (p < 0.05) indicate significant differences between each drug and DMSO treatment by one-way analysis of variance followed by Dunnett’s test.
Fig 7.
Combined experiment of DOX (75 nM) and ARP or OPC (5 μM) in U251 cells.
U251 cells were treated with DOX in the presence or absence of ARP or OPC for 48 h. Cell viability was determined using a water-soluble tetrazolium assay. Error bars represent mean ± SEM (n = 4). Statistical analyses were performed using Dunnett’s test. ** (p < 0.01) indicates significant differences between DOX alone and DOX plus ARP or OPC.
Fig 8.
Comparison of the expression levels of signaling proteins related to cell survival and apoptosis following treatment with ARP or OPC.
U251 cells were exposed to ARP (20 µM) or OPC (20 µM) for 24 h, after which the cells were detached, disrupted by ultrasonication, and the proteins were extracted for Western blot analysis. The results of western blotting were shown in A) survivin and p53, D) Akt, p-Akt and caspase-3, H) Src. Each protein expression was expressed relative to the DMSO control (set to 1) and normalized to GAPDH as a loading control, B) survivin, C) p53, E) p-Akt, F) Akt, G) Caspase-3, I) Src. In this experiment, proteins were transferred from the gel to the membrane, after which the membrane was sectioned and individually probed with antibodies. The images shown in this figure are unprocessed. Statistical analyses were performed using Dunnett’s test. Error bars represent mean ± SEM (n = 3). The symbols * (p < 0.05) and ** (p < 0.01) indicate significant differences between DMSO and ARP or OPC. The symbol ns indicates not significant compared with the non-inhibitor.