When they were subjected to serum withdrawal for 48 hours, the apoptotic activity of the cells increased 6.7 ± 1.7-fold and 4.3 ± 1.1-fold, respectively [determined by fluorescence-activated cell sorting (FACS) analyses; n = 3]. As a result, 96 hours of serum withdrawal reduced the numbers of viable HCC-1.2 cells (0.3 ± 0.1-fold) and Hep3B cells (0.6 ± 0.1-fold) with respect to unstarved
controls (determined by the EZ4U assay; n = 3). This indicates that the amounts of secreted FGF18 and presumably other FGF8 subfamily members in the medium were not selleck products sufficient for the cells to cope completely with the proapoptotic stimulus of serum withdrawal. The addition of 10 ng of recombinant FGF8, FGF17, or FGF18 per mL of the medium increased the viability of the starved cells significantly, suppressed their apoptotic activity, and enhanced the fraction of HCC-1.2 cells in the S-phase or G2/M-phase of the cell cycle (Fig. 3). In Hep3B cells, however, the rescue effect of the FGFs may predominantly
be due to the inhibition of apoptosis because significant effects on the cell cycle were not evident. In conclusion, the increased production of FGF8 subfamily members with a lack of serum and and/or oxygen may enhance the survival of malignant hepatocytes. In this study, serum deprivation clearly reduced the levels of phosphorylated extracellular signal-regulated kinase (pERK) and phosphorylated S6 (pS6) and elevated the level of phosphorylated glycogen synthase kinase 3β (pGSK3β) in both HCC-1.2 and Hep3B this website cells (representative data are shown in Fig. 4). This may reflect a lack of extracellular signal-regulated kinase 1 (ERK1) stimulation by the growth factor–depleted serum-free medium, reduced MAP kinase signaling and translational activity in the cells, and concomitant inactivation of glycogen synthase kinase 3β (GSK3β). Treatment of the starved cells with FGF8, FGF17, or FGF18 partly reversed the effect of serum withdrawal and elevated the level of pERK within minutes. FGF8 instead reduced pGSK3β and elevated pS6, whereas
FGF17 and FGF18 left the phosphorylated form Interleukin-2 receptor of S6 and pGSK3β more or less unchanged. To assess the role of FGF18 in the survival and malignant behavior of HCC-1.2, HepG2, and Hep3B cells, the expression of this growth factor was knocked down by siRNA. As demonstrated in Fig. 5, small interfering RNA targeting fibroblast growth factor 18 (siFGF18) somewhat elevated apoptotic activity, significantly reduced viability, and impaired the cells’ potential to form clones at a low density (clonogenicity) and in soft agar. siSCR exerted no significant effect on FGF18 expression, viability, or clone formation (not shown). This is strong evidence that FGF18 is of essential importance for the survival and tumorigenic phenotype of the cells. We asked whether FGF8, FGF17, and FGF18 also affect cells of the tumor stroma.