Western blot analysis validated the selleck catalog presence of FGFR2 and FRS2�� in all cell lines tested (Figure 2D). The expression of p-FRS2�� (Y196), a docking site for Grb2-Sos complexes, was decreased with dovitinib treatment in both sensitive and resistant cell lines, indicating inhibition of FGFRs by dovitinib. Decreased p-FRS2�� expression by dovitinib treatment was associated with marked decrease in the phosphorylation (activation) of AKT, GSK-3�� and Erk in both sensitive and resistant cell lines, suggesting that Akt and Erk signalling inhibition were pharmacodynamics downstream effects by dovitinib but did not predict anti-cancer effect. The expression of Bcl-2 family members were analysed and no major changes in expression of Bcl-2 and Bcl-xL proteins were observed following treatment.
Interestingly, Mcl-1 was downregulated with dovitinib treatment in sensitive cell lines but no significant changes in Mcl-1 level was observed in resistant cell lines. In the sensitive but not resistant cell lines, dovitinib treatment decreased Bid expression, a key BH3 domain-only protein, with associated increase in cleaved Bid (tBid). No changes were observed in other BH3 domain-only proteins (Bim, PUMA and Bad, data not shown). This suggests that dovitinib treatment induced Bid cleavage by caspase 8 to tBid, which translocated to mitochondria and induced apoptosis via cytochrome c release. Cyclin D1, a cell proliferation marker, was decreased more significantly following dovitinib treatment in sensitive cells and not the resistant cells.
To investigate whether the activity of FGFR, VEGFR2 and PDGFR�� signalling affect dovitinib’s pro-apoptotic effect, we contrasted FGFR1�C4 expression (Figure 2B), and phosphorylation ratio of FRS2�� (Figure 2D), VEGFR2 and PDGFR�� (Figure 2E) between untreated sensitive and resistant cells. There was significantly elevated FGFR signalling activity in untreated dovitinib-sensitive cells, as measured by higher FRS2 phosphorylation ratio, than resistant cells (P=0.0079) but not that of VEGFR2 and PDGFR�� (Figure 2F); and, there was no correlation between dovitinib sensitivity and FGFR1�C4 expression (Supplementary Figure S1). AKT/Mcl-1 axis mediates dovitinib’s pro-apoptotic effect in sensitive but not resistant cells The PI3K/Akt and MAPK pathways are key mediators of FGF signalling with the former being a primary transmitter of anti-apoptotic signals in cancer cells (Beenken and Mohammadi, 2009; Wesche et al, 2011).
To investigate whether Akt signalling had a functional role in mediating dovitinib-induced apoptosis, sensitive cell lines were stably transfected with a constitutively active AKT1 (CA-AKT1) and two single clones for each were selected for analysis Brefeldin_A (Figure 3A). Overexpression of CA-AKT1 dramatically increased the expression of Mcl-1 and phosphorylated GSK-3��, indicating the AKT-dependent regulation of Mcl-1.
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