A small molecule, LLL12 inhibits constitutive STAT3 and IL-6-induced STAT3 signaling and exhibits potent growth suppressive activity in human multiple myeloma cells.

We characterized the effects of a newly developed signal transducers and activators of transcription 3 (STAT3) inhibitor, LLL12 in multiple myeloma (MM) cells. LLL12 specifically inhibited STAT3 phosphorylation, nuclear localization, DNA binding activity, down-regulated STAT3 downstream genes, and induced apoptosis in MM cells. Importantly, LLL12 significantly inhibited STAT3 phosphorylation, induced apoptosis in primary MM cells which came from patients that were clinically resistant to lenalidomide and bortezomib. LLL12 is a potent inhibitor of cell proliferation with IC50 values ranging between 0.26 and 1.96 µM in MM and primary MM cells. LLL12 also inhibited STAT3 phosphorylation induced by interleukin-6 (IL-6) and interferon-α but not STAT1, STAT2, STAT4 and STAT6 phosphorylation induced by interferon-α, interferon-γ and IL-4 indicating the selectivity of LLL12 for STAT3. The selectively of LLL12 on STAT3 was further demonstrated on 21 protein kinases, which LLL12 had IC50 values ≥ 73.92 µM. In addition, the pretreatment of LLL12 blocked the promotion of the cell proliferation and resistance to lenalidomide by IL-6. Furthermore, LLL12 significantly blocked tumor growth of MM cells in mouse model. Our results indicate that LLL12 blocks constitutive STAT3 and IL-6 induced STAT3 signaling and may be a potential therapeutic agent for MM.

A novel small molecule inhibits STAT3 phosphorylation and DNA binding activity and exhibits potent growth suppressive activity in human cancer cells.

BACKGROUND: Targeting Signal Transducer and Activator of Transcription 3 (STAT3) signaling is an attractive therapeutic approach for most types of human cancers with constitutively activated STAT3. A novel small molecular STAT3 inhibitor, FLLL32 was specifically designed from dietary agent, curcumin to inhibit constitutive STAT3 signaling in multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells. RESULTS: FLLL32 was found to be a potent inhibitor of STAT3 phosphorylation, STAT3 DNA binding activity, and the expression of STAT3 downstream target genes in vitro, leading to the inhibition of cell proliferation as well as the induction of Caspase-3 and PARP cleavages in human multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cell lines. However, FLLL32 exhibited little inhibition on some tyrosine kinases containing SH2 or both SH2 and SH3 domains, and other protein and lipid kinases using a kinase profile assay. FLLL32 was also more potent than four previously reported JAK2 and STAT3 inhibitors as well as curcumin to inhibit cell viability in these cancer cells. Furthermore, FLLL32 selectively inhibited the induction of STAT3 phosphorylation by Interleukin-6 but not STAT1 phosphorylation by IFN-γ. CONCLUSION: Our findings indicate that FLLL32 exhibits potent inhibitory activity to STAT3 and has potential for targeting multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells expressing constitutive STAT3 signaling.

Novel STAT3 phosphorylation inhibitors exhibit potent growth-suppressive activity in pancreatic and breast cancer cells.

The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently detected in most types of human cancer where it plays important roles in survival, drug resistance, angiogenesis, and other functions. Targeting constitutive STAT3 signaling is thus an attractive therapeutic approach for these cancers. We have recently developed novel small-molecule STAT3 inhibitors, known as FLLL31 and FLLL32, which are derived from curcumin (the primary bioactive compound of turmeric). These compounds are designed to bind selectively to Janus kinase 2 and the STAT3 Src homology-2 domain, which serve crucial roles in STAT3 dimerization and signal transduction. Here we show that FLLL31 and FLLL32 are effective inhibitors of STAT3 phosphorylation, DNA-binding activity, and transactivation in vitro, leading to the impediment of multiple oncogenic processes and the induction of apoptosis in pancreatic and breast cancer cell lines. FLLL31 and FLLL32 also inhibit colony formation in soft agar and cell invasion and exhibit synergy with the anticancer drug doxorubicin against breast cancer cells. In addition, we show that FLLL32 can inhibit the induction of STAT3 phosphorylation by IFNalpha and interleukin-6 in breast cancer cells. We also show that administration of FLLL32 can inhibit tumor growth and vascularity in chicken embryo xenografts as well as substantially reduce tumor volumes in mouse xenografts. Our findings highlight the potential of these new compounds and their efficacy in targeting pancreatic and breast cancers that exhibit constitutive STAT3 signaling.

A novel small molecule, LLL12, inhibits STAT3 phosphorylation and activities and exhibits potent growth-suppressive activity in human cancer cells.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 microM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.

Dietary agent, benzyl isothiocyanate inhibits signal transducer and activator of transcription 3 phosphorylation and collaborates with sulforaphane in the growth suppression of PANC-1 cancer cells.

The Signal Transducer and Activator of Transcription (STAT) proteins comprise a family of latent transcription factors with diverse functions. STAT3 has well established roles in cell proliferation, growth and survival, and its persistent activation has been detected with high frequency in many human cancers. As constitutive activation of STAT3 appears to be vital for the continued survival of these cancerous cells, it has emerged as an attractive target for chemotherapeutics. We examined whether the inhibitory activities of bioactive compounds from cruciferous vegetables, such as Benzyl isothiocyanate (BITC) and sulforaphane, extended to STAT3 activation in PANC-1 human pancreatic cancer cells. BITC and sulforaphane were both capable of inhibiting cell viability and inducing apoptosis in PANC-1. Sulforaphane had minimal effect on the direct inhibition of STAT3 tyrosine phosphorylation, however, suggesting its inhibitory activities are most likely STAT3-independent. Conversely, BITC was shown to inhibit the tyrosine phosphorylation of STAT3, but not the phosphorylation of ERK1/2, MAPK and p70S6 kinase. These results suggest that STAT3 may be one of the targets of BITC-mediated inhibition of cell viability in PANC-1 cancer cells. In addition, we show that BITC can prevent the induction of STAT3 activation by Interleukin-6 in MDA-MB-453 breast cancer cells. Furthermore, combinations of BITC and sulforaphane inhibited cell viability and STAT3 phosphorylation more dramatically than either agent alone. These findings suggest that the combination of the dietary agents BITC and sulforaphane has potent inhibitory activity in pancreatic cancer cells and that they may have translational potential as chemopreventative or therapeutic agents.

New curcumin analogues exhibit enhanced growth-suppressive activity and inhibit AKT and signal transducer and activator of transcription 3 phosphorylation in breast and prostate cancer cells.

Curcumin, the active component of turmeric, has been shown to protect against carcinogenesis and prevent tumor development in cancer. To enhance its potency, we tested the efficacy of synthetic curcumin analogues, known as FLLL11 and FLLL12, in cancer cells. We examined the impact of FLLL11 and FLLL12 on cell viability in eight different breast and prostate cancer cell lines. FLLL11 and FLLL12 (IC(50) values 0.3-5.7 and 0.3-3.8 micromol/L, respectively) were substantially more potent than curcumin (IC(50) values between 14.4-50 micromol/L). FLLL11 and FLLL12 were also found to inhibit AKT phosphorylation and downregulate the expression of HER2/neu. In addition, we demonstrate for the first time that FLLL11 and FLLL12 inhibit phosphorylation of signal transducer and activator of transcription (STAT) 3, an oncogene frequently found to be persistently active in many cancer types. The inhibition of STAT3 signaling was confirmed by the inhibition of STAT3 DNA binding and STAT3 transcriptional activity. Furthermore, FLLL11 and FLLL12 were more effective than curcumin in inhibiting cell migration and colony formation in soft agar as well as inducing apoptosis in cancer cells. These results indicate that FLLL11 and FLLL12 exhibit more potent activities than curcumin on the inhibition of STAT3, AKT, and HER-2/neu, as well as inhibit cancer cell growth and migration, and may thus have translational potential as chemopreventive or therapeutic agents for breast and prostate cancers.

New structural analogues of curcumin exhibit potent growth suppressive activity in human colorectal carcinoma cells.

BACKGROUND: Colorectal carcinoma is one of the major causes of morbidity and mortality in the Western World. Novel therapeutic approaches are needed for colorectal carcinoma. Curcumin, the active component and yellow pigment of turmeric, has been reported to have several anti-cancer activities including anti-proliferation, anti-invasion, and anti-angiogenesis. Clinical trials have suggested that curcumin may serve as a potential preventive or therapeutic agent for colorectal cancer. METHODS: We compared the inhibitory effects of curcumin and novel structural analogues, GO-Y030, FLLL-11, and FLLL-12, in three independent human colorectal cancer cell lines, SW480, HT-29, and HCT116. MTT cell viability assay was used to examine the cell viability/proliferation and western blots were used to determine the level of PARP cleavages. Half-Maximal inhibitory concentrations (IC50) were calculated using Sigma Plot 9.0 software. RESULTS: Curcumin inhibited cell viability in all three of the human colorectal cancer cell lines studied with IC50 values ranging between 10.26 microM and 13.31 microM. GO-Y030, FLLL-11, and FLLL-12 were more potent than curcumin in the inhibition of cell viability in these three human colorectal cancer cell lines with IC50 values ranging between 0.51 microM and 4.48 microM. In addition, FLLL-11 and FLLL-12 exhibit low toxicity to WI-38 normal human lung fibroblasts with an IC-50 value greater than 1,000 microM. GO-Y030, FLLL-11, and FLLL-12 are also more potent than curcumin in the induction of apoptosis, as evidenced by cleaved PARP and cleaved caspase-3 in all three human colorectal cancer cell lines studied. CONCLUSION: The results indicate that the three curcumin analogues studied exhibit more potent inhibitory activity than curcumin in human colorectal cancer cells. Thus, they may have translational potential as chemopreventive or therapeutic agents for colorectal carcinoma.