Consistently, our biochemical studies, which showed limited side effects on cell survival, also demonstrated that inhibi tion of Jak2/Stat3 pathway www.selleckchem.com/products/Nilotinib.html did not reduce the secretion of IL 6 in A549 cells, but inhibition of NF B and PI3 K/Akt pathways did. Our knock down studies of AS2, MCF 7/ADR, and KC CPT100 cells and our pharmacological inhibition experi ments with seven established cell lines and 20 clinical samples revealed that Stat3 did in fact affect expression of IL 6 in most of the cancer cells we tested. In Stat3 null mouse embryonic Inhibitors,Modulators,Libraries fibroblasts, S3F up regulated IL 6 mRNA expression suggesting that unphosphorylated Stat3 plays a role in regulating IL 6 expression. In our study, however, treatment with A490 or over expression of S3F inhibited Stat3 phos phorylation and reduced IL 6 expression in the Stat3 active AS2 cells.
Similarly, AG490 treatment also decreased the IL 6 secretion in various drug resis tant cancer cells exhibiting constitutively active Stat3. We hypothesized that unphosphory lated Stat3 may have a basal activity in the regulation of IL 6 expression but tyrosine phosphorylated Stat3 has better activity in the induction of IL 6 expression. To date, no Stat3 binding site has yet been Inhibitors,Modulators,Libraries identified in IL 6 promoter. Using prediction Inhibitors,Modulators,Libraries software, we were also unable to find any specific Stat3 binding site 5 kb upstream from the transcriptional start site of IL 6 pro moter. However, in the promoter experiments, we showed that a transient transfection of S3C plasmide into AS2 cells increased IL 6 promoter luciferase activity.
On the contrary, the transient transfection Inhibitors,Modulators,Libraries of S3F plasmid or treatment with AG490 reduced IL 6 promoter luciferase activity in AS2 cells. These results suggest that Stat3 might regulate IL 6 transcription at the promoter level. Stat3 has been reported to induce Inhibitors,Modulators,Libraries the expression of AP 1 proteins and C/EBPa, b and. The AP 1 and C/EBP transcrip tional factors are major regulators of IL 6 expression. Therefore, Stat3 may increase the expression of IL 6 indirectly through the regulation of these transcriptional factors. However, it may do so directly by interacting with other transcription factors and co localizing to IL 6 promoter at non consensus sites. For example, Stat3 has been shown to interact directly with NF B forming a complex that synergistically promotes target genes expression.
Stat3 could also cooperate with C/ EBPs, CREB, or AP 1 to regulate target gene expression by binding together to either its consensus sites or the non consensus regions. Regardless of how Stat3 contributes to the regulation of IL 6 expression, Stat3 DNA binding activity is required. Our study demonstrates that over expression of S3D suppresses IL 6 expression in AS2 cells. That S3D is unable to bind to DNA suggests that Stat3 DNA bind ing activity plays an important role in the regulation of IL 6 expression.