E numbers in the substratum of the membrane per view under high magnification (200? in

E numbers in the substratum of the membrane per view under high magnification (200? in the control group compared with the 25 nM CEP-37440MedChemExpress CEP-37440 oleandrin group of U2OS cells were 41.1 ?5.7 vs. 25.8 ?6.1 (P = 0.033), and the corresponding numbers of SaOS-2 cells were 65.8 ?12.3 vs. 39.4 ?10.0 (P = 0.045) (Fig. 4f).Oleandrin suppressed the activity of Wnt/-catenin signaling pathwayPrevious studies reported that the abnormal activation of the Wnt signaling pathway plays an important role in OS pathogenesis. In this study, we explored whetherMa et al. Journal of Experimental Clinical Cancer Research (2015) 34:Page 7 ofFig. 3 The apoptosis analysis of U2OS and SaOS-2 cells by flow cytometry (FCM). a/b The total apoptotic rates of U2OS (a) and SaOS-2 (b) cells after treatment with 50 nM oleandrin for 0, 24, and 48 h. c/d The quantitative results of the apoptosis analysis of U2OS (c) and SaOS-2 (d) cells. n = 3. Mean ?SD. **P < 0.01 vs. 0 h. #P < 0.05, ##P < 0.01 vs. 24 holeandrin had an effect on the Wnt/-catenin signaling pathway, and a dual-luciferase assay was used to evaluate this effect in U2OS cells. Fig. 5a shows that without LiCl, an inhibitor of GSK-3, oleandrin was able to suppress the activities of Wnt/-catenin signaling by downregulating the TOP/FOP flash ratio in a concentration-dependent manner (25 nM or 50 nM vs. control: P = 0.017 or P = 0.001, 25 nM vs. 50 nM: P = 0.043). In addition, after pretreatment with LiCl, the TOP/FOP flash ratio first increased but then declined in a concentration-dependent manner after oleandrin treatment (25 nM or 50 nM vs. control: P = 0.073 or P = 0.005, 25 nM vs. 50 nM: P = 0.070). Similarly, Fig. 5b also shows that oleandrin could downregulate the TOP/FOP flash ratio in a time-dependent manner with LiCl (24 or 48 h vs. 0 h: P = 0.004 or P PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28192408 = 0.000, 24 vs. 48 h: P = 0.005) or without LiCl (24 or 48 vs. 0 h: P = 0.017 or P = 0.002, 24 vs. 48 h: P = 0.120).Oleandrin downregulated the target gene expression of the Wnt/-catenin pathway at both the mRNA and protein levelsprotein expression changes of the downstream target genes, which included c-myc, survivin, cyclin D1, MMP2 and MMP-9, using semi-quantitative RT-PCR and western blot assays. As we expected, the results of the RT-PCR showed that oleandrin significantly downregulated the mRNA levels of these genes to different degrees dependent on treatment time (Fig. 5c, d). In accordance with the RT-PCR results, after treatment with oleandrin for 24 and 48 h, the protein expression of the target genes was reduced, which indicated that oleandrin had a remarkable inhibiting effect on the downstream molecules of the Wnt/ -catenin signaling pathway (Fig. 6a, b).Oleandrin inhibited the protein expression of -catenin and reduced its nuclear localizationTo study the effect of oleandrin on the Wnt/-catenin signaling pathway, we detected the mRNA and totalAs a key transcriptional factor, the expression and nuclear accumulation of -catenin directly influenced the activity of the Wnt signaling pathway and regulated the transcription and expression of the target genes. Therefore, we explored the regulating effect of oleandrin on -catenin by western blot analysis of the total cytoplasmic and nuclear protein extracts. As shown in Fig. 6c, d, oleandrin treatment led to significantly decreased totalMa et al. Journal of Experimental Clinical Cancer Research (2015) 34:Page 8 ofFig. 4 The changes of OS cell migration and invasion abilities after treatment with oleandrin. a/b The m.