G. and models, despite the fact that the absorption rate was different.10 Having a high content of oligomeric PA, our former study showed that BLPs exhibited strong cellular antioxidant and antiproliferative activities the HepG2 Necrostatin 2 racemate cell model.14 However, their anti-cancer function remains unknown and is worth further investigation. CSCs produce a tumor mass continuous self-renewal, which can be regulated by several signaling pathways, such as Wnt/-catenin, Hedgehog, Notch, < 0.05, < 0.01 and < 0.001. 3. Results 3.1 Spheroid (SP) cells Necrostatin 2 racemate isolated from OVCAR-3 ovarian cancer cells exhibited stem cell-like properties With self-renewal ability, CSCs grow as nonadherent spheres under stem-cell-selective conditions, which is different from monolayer cancer cells. OVCAR-3 cells were seeded in ultra-low-attachment culture plates in Mammocult complete medium at a density of 2 104 cells per well for 7 days. Cell aggregates were formed and some cells died due to the low serum conditions (Fig. 1A). After the first 7-day incubation period, the first generation of OVCAR-3 SP cells was collected and the rest of the cells were isolated by centrifugation at 800 rpm for 5 min and were re-seeded on ultra-low-attachment culture plates for the second and third generation cultures. To determine whether the SP cells generated from the OVCAR-3 cells exhibited CSC-like properties, the cells were investigated by ALDH assay and western blot assay. ALDH has been reported to be a CSC marker and its activity was determined by flow cytometry with the Aldefluor assay kit in the present study. As shown in Fig. 1C Necrostatin 2 racemate & D, the percentage of ALDH+ cells in adherent (AD) cells and the first, second and third generations of SP cells was 0.87 0.03%, 6.31 0.77%, 9.76 0.17% and 23.50 1.93%, respectively. The third generation of SP cells exhibited the highest population of ALDH+ and thus were collected for further investigation. Ovarian CSCs were reported to express stem cell-related genes, such as SOX, OCT-4, < 0.01, (***) < 0.001, compared with the control. (E) Relative expression of Oct-4 and Sox-2 in OVCAR-3 SP cells. Oct-4, Sox-2 and GAPDH protein expressions were detected by western blot analysis and quantified by ImageJ software. Results are representative of three independent experiments and are expressed as mean SD. (**) < 0.01, two-tailed Students < 0.01). The cell viability reduced from 81.4 2.0% to 44.4 0.7% after treating with BLPs from 2 HDM2 to 20 g mL?1 for 24 h. The IC50 value was about 16.4 g mL?1. This indicated that BLPs have a great potential to inhibit the growth and proliferation of OVCAR-3 SP cells. Open in a separate window Fig. 2 BLPs inhibited the viability of OVCAR-3 SP cells in a dose dependent manner. Results Necrostatin 2 racemate are representative of three independent experiments and are expressed as mean SD. (**) < 0.01, compared with the control. 3.3 BLPs regulated CSC-like characteristics in OVCAR-3 SP cells Sphere formation and colony formation, which are the characteristics of ovarian CSCs, were investigated in the present study to explore how BLP treatment affected OVCAR-3 SP cells. After treating with BLPs, the sphere formation ability of OVCAR-3 SP cells decreased significantly (Fig. 3A) by showing a much smaller size and diameter of the spheres of BLP treatment groups compared with those of the control group. Furthermore, BLPs also dose-dependently reduced the number of colonies formed by Necrostatin 2 racemate OVCAR-3 SP cells (Fig. 3B & C). BLPs at 10 g mL?1 reduced approximately 79.6% of colonies compared with the control group, which suggested that BLPs induced cytotoxicity and proliferation inhibition in cells. These results were consistent with Fig. 2 and revealed that BLPs attenuated the stem cell characteristics in OVCAR-3 SP cells. Open in a separate window Fig. 3 BLPs regulated CSC-like characteristics in OVCAR-3 SP cells. (A) BLPs inhibited the sphere formation ability of OVCAR-3 SP cells. Single cells were seeded in 6-well ultra-low attachment plates in Mammocult complete medium at a density of 1000 cells per well. Spheres were photographed after 7 days of incubation. All assays were.