Biochemical and cellular effects of c-Src kinase-selective pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors

Activation of Stat3 by receptor tyrosine kinases and cytokines regulates survival in human non-small cell carcinoma cells

“…A previous report also demonstrated the ability of PD173955, a selective pharmacological inhibitor of Src-kinase, to reduce in vitro activity of Yes and Src, induce mitotic arrest in breast carcinoma cells, and induce apoptosis (Moasser et al, 1999). One question that remains is which members of the family of Src are important in nonsmall cell lung carcinoma cells, since these inhibitors can inhibit the activity of multiple family members (Blake et al, 2000;Kraker et al, 2000). This has been more extensively studied in small cell lung cancers, where Lck and Yes have been implicated in the ability of stem cell factor/Kit mediation proliferation (Krystal et al, 1998).…”

“…Previous work had identified this compound selective against c-Src and Lck, while having minimal affect on autophosphorylation of receptors for bFGF, PDGF, and EGF (Kraker et al, 2000). Cells were treated with increasing concentrations of PD180970 and cells assayed for phosphorylated and total Src protein after 24 h. PD180970 results in a dose-dependent reduction in phosphorylated Src with major reductions seen at 500 nM and above (Figure 5a).…”

“…Since our results above implicated both EGF and IL-6 in the activation of Stat3 and other reports suggested a role of Src in the pathogenesis of human nonsmall cell lung cancers, we determined the tyrosine kinase requirements for ligand-induced activation of Stat3 by EGF, IL-6, and HGF. A549 cells were serum deprived for 24 h and pretreated for 1.5 h with DMSO (control), PD180970 (a specific inhibitor of Src-kinase), PD158780 (a specific inhibitor of EGF-R tyrosine kinase activity), PD0183805 (an irreversible pan-HER kinase inhibitor), and AG490 (a specific inhibitor of JAK) (Fry, 1999;Levitzki, 1999;Kraker et al, 2000). Cells were subsequently stimulated with either EGF or IL-6 as before and harvested 10 to 20 min after stimulation and nuclear extracts were prepared for EMSA.…”

“…To better define a role of Stat3 in growth and survival of lung cancer cells, as well as identify tyrosine kinases which result in constitutive Stat3 activation, we treated cells with pharmacological inhibitors of tyrosine kinases for up to 3 days and assessed their affect on cell viability. A549 and H358 human lung cancer cells growing in 5% FBS were plated at low density and treated with DMSO as control, PD180970 (Src-kinase inhibitor), PD158780 (EGF-R kinase inhibitor), PD0183805 (pan-HER kinase inhibitor), or AG490 (JAK kinase inhibitor) (Fry, 1999;Levitzki, 1999;Kraker et al, 2000;Garcia et al, 2001). Fresh media with fresh inhibitors were exchanged every 24 h and viable cells were counted every 24 h after drug treatment for 72 h. As demonstrated in Figure 4a, inhibition of both Src and JAK by the pharmacological inhibitors leads to reduced cell viability in both A549 and H358 lung carcinoma cells.…”

“…Dishes were incubated at 371C for times indicated prior to preparation of the nuclear extracts. For treatment with PD158780 (Fry, 1999), PD180970 (Kraker et al, 2000), and AG490 (Levitzki, 1999), stock solutions of the compounds in 100% DMSO were diluted directly into the media to indicated concentrations followed by incubation at 371C. For cell growth and viability assays, media plus inhibitors were changed every 24 h.…”