| STAT3 and NF-κB are two essential members in the series of cancer associated signaling that have been well understood. These two signal pathways are involved in almost all the processes of tumor development, including cell proliferation, survival, angiogenesis and metastasis. STAT3 and NF-κB are usually constitutively activated in many malignant cancers, like colon cancer, breast cancer, glioma, lung cancer, prostate cancer and gastric cancer, etc. Numerous evidences have been provided that specific ablation/inhibition of STAT3 and/or NF-κB signaling usually results attenuated cell growth, proliferation and metastasis. Meanwhile, aberrant activated STAT3 and NF-κB signals in other types of cells in tumor microenvironment have also been proven to promote tumor development. Specific inhibition of STAT3 and NF-κB activities in these cells will suppress tumor development and improve prognosis significantly sometimes. Therefore, identification of selective inhibitors of STAT3 and NF-κB signaling by high-throughput screening, will provide candidate compounds for the development of anti-cancer drugs.We then developed and utilized a useful system for STAT3 and NF-κB dual signaling-targeting anticancer drugs screening. Comparing to normal-tissue-derived cell line, we analyzed and picked out several cancer cell lines which showed both constitutively activated STAT3 and NF-κB signals by EMSA and Western-Blot analyses. A ratio of 16:1 between STAT3 and NF-κB response elements was chosen, which made the luciferase reporter vector finally match one of these cell lines, A549, for the construction of high-throughput drug screening system. Stable cell lines we got for drug screening could response to both STAT3 and NF-κB signaling changes significantly, and these cells lines could be further used to screen STAT3 inhibitors, NF-κB inhibitors, as well as inhibitors which could suppress both STAT3 and NF-κB signaling. We got a number of compounds which showed inhibitory activity (i.e. STAT3 and/or NF-κB inhibition) by screening extracts from medicinal herbs, as well as small molecule library from natural products. Screening methods were optimized after several rounds of drug screening.Preliminary validation was performed at the cellular level for the effects of small molecules from natural products after screening. The inhibition effects of these compounds on STAT3 and NF-κB signaling were mainly investigated by Western-Blot. One of these compounds from natural products, named Brevilin A, showed significant STAT3 inhibition while without immediate-direct toxicity. Further investigation revealed that, in STAT3 signaling dependent DU145 and MDA-MB-468 cells, Brevilin A could effectively inhibit STAT3 activation and down-regulate expressions of its target genes involved in cell survival and cell cycle, like c-Myc and CyclinD1, finally led to cell growth arrest and cell apoptosis. However, Brevilin A showed less effects on normal human cells.Then we found that Brevilin A specifically blocked the upstream JAKs activity. Brevilin A inhibited cytokine (e.g., IL-6 and IFNs) induced JAK-STAT activation; Following investigations revealed that Brevilin A blocked JAKs-JHl tyrosine kinase domain overexpression induced STATs activation (including STAT3 and STAT1), while Src overexpression induced substrate phosphorylation was not affected. In vitro kinase assay with Tyk2-JH1 and recombinant STAT3 protein further proved this inhibition between the JH1 kinase domain and substrate protein. Inhibition of Brevilin A on cell migration and Matrigel clone formation in vitro revealed its novel effects on some processes of tumorigenesis.The roles of Brevilin A targeting on JAKs activity indicated that Brevilin A may not only be used as a STAT3 inhibitor but also a compound blocking other JAK-STAT hyperactivation. Thus, these findings provided a strong impetus for the development of selective JAK-STAT inhibitors and therapeutic drugs in order to improve survival of patients with hyperactivated JAKs and STATs. |
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