All chemicals and materials used in this study were listed in Table S1. 4.2. the ERK1/2, Rb/E2F1, cell cycle pathways, and the expressions of FGFR1-4 proteins, suggesting that cordycepin can be used as a novel anticancer drug for testicular cancers. = 4. values were calculated using two-way ANOVA with Tukeys Batimastat (BB-94) multiple comparisons post-tests. * < 0.05, *** < 0.001 compared to the control group (0 ng/mL FGF9) at each dose of cordycepin; < 0.001 compared to the group with 0 M cordycepin and 0 ng/mL FGF9 treatments; ### < 0.001 compared to the group with 0 M cordycepin and 50 ng/mL FGF9 treatments. 2.2. Cordycepin Inhibited FGF9-Induced ERK1/2 and pRb/E2F Pathway in MA-10 Cells We next investigated whether cordycepin could suppress the signaling pathway induced by FGF9 in MA-10 cells. The results showed that FGF9-induced phosphor-ERK1/2 (p-ERK1/2) expression was significantly inhibited by cordycepin at 0.25 and 12 h after treatment (Figure 3A). At 24 h after Batimastat (BB-94) FGF9 treatment, the phosphorylation of ERK1/2 was not elevated. However, the basal protein levels of p-ERK1/2 were significantly reduced by cordycepin (Figure 3A). The effects of cordycepin on the p-Rb/E2F pathway and the downstream signaling of ERK1/2 were also examined. Cordycepin (25, 50 and 100 M) significantly inhibited FGF9-induced phosphorylation of Rb at 0.25 and 12 h, but not at 24 h after treatments (Figure Batimastat (BB-94) 3B), and also inhibited FGF9-induced E2F1 expression 12 h after treatments (Figure 3C). These data indicated that cordycepin could inhibit FGF9-induced Rb phosphorylation and E2F1 overexpression, and subsequently suppress cell proliferation in MA-10 cells. Open in a separate window Figure 3 Cordycepin suppressed FGF9-induced expression of p-ERK1/2, p-Rb and E2F1 in MA-10 cells. Western blot analysis for the expression of (A) total ERK1/2, p-ERK1/2 (Thr202/Tyr204), (B) p-Rb and (C) E2F1 in MA-10 cells treated without or with FGF9 (50 ng/mL) and different concentrations of cordycepin (0, 25, 50 and 100 M) for 0.25, 12 and 24 h, respectively. Quantitative analysis of Western blotting using ImageJ software. Values are shown as the mean SEM, = 4. values were calculated using two-way ANOVA with Tukeys multiple comparisons post-tests. * < 0.05 compared to the control group (0 ng/mL FGF9) at each dose of cordycepin; < 0.001 compared to the group with 0 M cordycepin and 0 ng/mL FGF9 treatments; # < 0.05, ## < 0.01, ### < 0.001 compared to the group with 0 M cordycepin and 50 ng/mL FGF9 treatments. 2.3. Cordycepin Reduced the Expression of Cyclins and CDKs in FGF9-Treated MA-10 Cells According to our previous study, which showed that FGF9 did increase the expressions of cyclins and CDKs to promote cell cycle progression for MA-10 cell proliferation [31], the effects of ATF3 cordycepin on cell cycle progression in FGF9-treated MA-10 cells were investigated. Consistent with previous data [31], FGF9 could induce cyclin D1, cyclin E1 and cyclin A1 at 12 h after treatment (Figure 4ACD), and up-regulate cyclin B1 at 24 h after treatment (Figure 4A,E). In the 12 h FGF9-treated group, the FGF9-induced overexpression of cyclin D1, cyclin E1 and cyclin A1 could be reversed by cordycepin in a dose-dependent manner (Figure 4ACD), whereas the expression of cyclin B1, had not yet been induced by FGF9 and was also down-regulated by cordycepin (Figure 4A,E). In the 12 h control group, the expression of cyclin A1 and cyclin B1 were also significantly reduced by cordycepin (Figure 4A,D,E). At 24 h after Batimastat (BB-94) treatment, FGF9-induced cyclin B1 could be significantly suppressed by 100 M cordycepin (Figure 4A,E). In addition, cordycepin did reduce protein basal levels of cyclin B1 and E1 proteins whether treated with FGF9 or not at 12 h after treatment (Figure 4A,D,E). These data illustrated that cordycepin could affect cell cycle progression by downregulating cyclin D1, cyclin E1, cyclin A1 and cyclin B1 proteins in FGF9-treated MA-10 cells. Open in a separate window Open in a separate window Figure Batimastat (BB-94) 4 Cordycepin suppressed FGF9-induced expression of cyclin D1, cyclin E1, cyclin A1 and cyclin B1 in MA-10 cells. (A) Western blot analysis of cyclin D1, cyclin E1, cyclin A1 and cyclin B1 expression in MA-10 cells treated without or with FGF9 (50 ng/mL) and different concentrations of cordycepin (0, 25, 50 and 100 M) for 0.25, 12 and.
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