Our data demon strated that FLLL32 was more potent than curcumin to inhibit STAT3 phosphorylation and STAT3 DNA bind ing activity, downregulate order inhibitor STAT3 target genes, and induce cancer cells apoptosis. However, the phosphory lation of mTOR and ERK was not obviously reduced by FLLL32. FLLL32 also has little effect on STAT1 phos phorylation stimulated with IFN g. In addition, FLLL32 e hibited little inhibition on some of the tyrosine kinases containing SH2 or both SH2 and SH3 domains, and other protein kinases by using kinase profile assay. These results further support the specificity of FLLL32 to inhibit STAT3. After activated by some cell surface cytokines, such as IL 6, IFN g, JAK2 phosphorylates and activates cytoplas mic STAT3 protein to an active dimer, which translo cates to the nucleus and induce the transcription of specific target genes.

We found that FLLL32 inhibited P JAK2 in some of the cancer cell lines, which may e plain the inhibition of the STAT3 phosphorylation in those cancer cell lines. Sev eral new inhibitors of JAK2 STAT3 pathway were recently reported, such as Stattic, STA 21, S3I 201, AG490, WP1066. Here, Stattic and WP1066 were used as positive control to detect their effects on apoptosis in HCT116 colon cancer and U266 multiple myeloma cells, which conformed the JAK2 STAT3 pathway may be an important target to induce the apoptosis of cancer cells. Furthermore, FLLL32 was found to be potent than other reported JAK2 STAT3 inhibitors, including FLLL32, WP1066, AG490, Stattic, S3I 201, and curcumin in our cancer cell lines.

Conculsions Our results have demonstrated that FLLL32 is an effec tive STAT3 inhibitor to inhibit STAT3 phophorlation, STAT3 DNA binding activity, STAT3 downstream tar get gene e pression and induce apoptosis in human can cer cells from four independent cancer types such as multiple myeloma, glioblastoma, colorectal and liver cancers. FLLL32 was more potent than curcumin and other reported JAK2 STAT3 inhibitors in the inhibition of cancer cell viability in our comparisons. Our results suggest that FLLL32 is a potent therapeutic agent for multiple types of cancer cells e pressing constitutive STAT3 signaling including multiple myeloma, glioblas toma, colorectal and liver cancer cells. Methods Cell Culture Human colonrectal cancer cell lines, glioblastoma cell line, human hepatic can cer cell lines, human multiple myeloma cell line and human breast cancer cell lines were purchased from the American Type Culture Collection.

These cancer cell lines were cultured in DMEM or RPMI 1640 supplemented with 10% fetal bovine serum. Inhibitors FLLL32, a curcumin derived STAT3 inhibitor, and WP1066, a Janus like kinase 2 inhibitor, were synthesized in Dr. Pui Kai Lis laboratory. STAT3 SH2 inhibitors Stattic Dacomitinib and S3I 201, JAK2 inhibitor AG490 was purchased from Calbiochem. Curcumin was purchased from Sigma Aldrich Che mical Co. Western blot analysis FLLL32 and curcumin were dissolved in DMSO.