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The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity.

Bill MA, Fuchs JR, Li C, Yui J, Bakan C, Benson DM, Schwartz EB, Abdelhamid D, Lin J, Hoyt DG, Fossey SL, Young GS, Carson WE, Li PK, Lesinski GB - Mol. Cancer (2010)

Bottom Line: In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors.In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-gamma, IL 2).Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-gamma production when cultured with K562 targets as compared to vehicle (DMSO).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Arthur G, James Cancer Hospital and Richard J, Solove Research Institute, The Ohio State University, 410 W, 10th Ave, Columbus, OH 43210, USA.

ABSTRACT

Background: We characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells.

Results: FLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-gamma-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-gamma, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-gamma production when cultured with K562 targets as compared to vehicle (DMSO).

Conclusions: These data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy.

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FLLL32 reduced STAT3 DNA binding and gene expression. (A) STAT3 DNA binding was measured in A375 cells following a 16 hour treatment with FLLL32 (2 μM or 4 μM) as described in the Methods section. Unlabeled target DNA were included to compete for binding as indicated, and a STAT3 specific Ab was used to were included confirm specificity (last lane). Cell lysates were evaluated concurrently by immunoblot to control for total level of STAT3 protein at the 16 hour time point. (B) FLLL32 reduced STAT3-regulated gene expression. Expression of STAT3-regulated genes cyclin D1 and survivin were evaluated following a 24 hour treatment with FLLL32 in melanoma cell lines. Membranes were probed with β actin as a loading control and all blots represent data from at least two independent experiments.
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Figure 2: FLLL32 reduced STAT3 DNA binding and gene expression. (A) STAT3 DNA binding was measured in A375 cells following a 16 hour treatment with FLLL32 (2 μM or 4 μM) as described in the Methods section. Unlabeled target DNA were included to compete for binding as indicated, and a STAT3 specific Ab was used to were included confirm specificity (last lane). Cell lysates were evaluated concurrently by immunoblot to control for total level of STAT3 protein at the 16 hour time point. (B) FLLL32 reduced STAT3-regulated gene expression. Expression of STAT3-regulated genes cyclin D1 and survivin were evaluated following a 24 hour treatment with FLLL32 in melanoma cell lines. Membranes were probed with β actin as a loading control and all blots represent data from at least two independent experiments.

Mentions: FLLL32 inhibited STAT3 phosphorylation at Tyr705 but not at Tyr727 in multiple human melanoma cell lines after a 24 hour treatment (Figure 1D). Prior studies indicated FLLL32 could inhibit Jak2 kinase activity in an in vitro cell-free assay [16]. However, we did not observe an appreciable alteration in Jak2 phosphorylation even at a concentration of 8 μM, suggesting that this compound likely acted directly against the STAT3 protein (Figure 1D). Time course studies also revealed that fulminant cell death occurred after 24 hours of continuous culture, yet exposure to FLLL32 at 2 - 4 μM for only 4 hours was sufficient to reduce pSTAT3 and induce cell death (Additional File 1: Figure S1A-B). FLLL32 did not appear to inhibit the phosphorylation of other key signaling pathways that are constitutively active in malignant cells (e.g. Src, Akt) at doses capable of inhibiting STAT3 phosphorylation after 24 hours. Consistent with reciprocal activation of the p38 MAPK and STAT3 pathways [29], FLLL32 treatment led to increased levels of total p38 MAPK protein once pSTAT3 decreased (Figure 1D). Importantly, FLLL32 was capable of reducing pSTAT3 levels, cyclin D1 expression and inducing apoptosis in primary human melanoma cell cultures derived from recurrent cutaneous melanoma tumors [17] (Figure 1E). Finally, treatment of basal pSTAT3-positive human melanoma cell lines with FLLL32 for 24 hours led to reduced STAT3 DNA binding as determined by gel shift assays and expression of the STAT3-regulated genes, cyclin D1 and survivin as measured by immunoblot (Figure 2A-B).


The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity.

Bill MA, Fuchs JR, Li C, Yui J, Bakan C, Benson DM, Schwartz EB, Abdelhamid D, Lin J, Hoyt DG, Fossey SL, Young GS, Carson WE, Li PK, Lesinski GB - Mol. Cancer (2010)

FLLL32 reduced STAT3 DNA binding and gene expression. (A) STAT3 DNA binding was measured in A375 cells following a 16 hour treatment with FLLL32 (2 μM or 4 μM) as described in the Methods section. Unlabeled target DNA were included to compete for binding as indicated, and a STAT3 specific Ab was used to were included confirm specificity (last lane). Cell lysates were evaluated concurrently by immunoblot to control for total level of STAT3 protein at the 16 hour time point. (B) FLLL32 reduced STAT3-regulated gene expression. Expression of STAT3-regulated genes cyclin D1 and survivin were evaluated following a 24 hour treatment with FLLL32 in melanoma cell lines. Membranes were probed with β actin as a loading control and all blots represent data from at least two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902420&req=5

Figure 2: FLLL32 reduced STAT3 DNA binding and gene expression. (A) STAT3 DNA binding was measured in A375 cells following a 16 hour treatment with FLLL32 (2 μM or 4 μM) as described in the Methods section. Unlabeled target DNA were included to compete for binding as indicated, and a STAT3 specific Ab was used to were included confirm specificity (last lane). Cell lysates were evaluated concurrently by immunoblot to control for total level of STAT3 protein at the 16 hour time point. (B) FLLL32 reduced STAT3-regulated gene expression. Expression of STAT3-regulated genes cyclin D1 and survivin were evaluated following a 24 hour treatment with FLLL32 in melanoma cell lines. Membranes were probed with β actin as a loading control and all blots represent data from at least two independent experiments.
Mentions: FLLL32 inhibited STAT3 phosphorylation at Tyr705 but not at Tyr727 in multiple human melanoma cell lines after a 24 hour treatment (Figure 1D). Prior studies indicated FLLL32 could inhibit Jak2 kinase activity in an in vitro cell-free assay [16]. However, we did not observe an appreciable alteration in Jak2 phosphorylation even at a concentration of 8 μM, suggesting that this compound likely acted directly against the STAT3 protein (Figure 1D). Time course studies also revealed that fulminant cell death occurred after 24 hours of continuous culture, yet exposure to FLLL32 at 2 - 4 μM for only 4 hours was sufficient to reduce pSTAT3 and induce cell death (Additional File 1: Figure S1A-B). FLLL32 did not appear to inhibit the phosphorylation of other key signaling pathways that are constitutively active in malignant cells (e.g. Src, Akt) at doses capable of inhibiting STAT3 phosphorylation after 24 hours. Consistent with reciprocal activation of the p38 MAPK and STAT3 pathways [29], FLLL32 treatment led to increased levels of total p38 MAPK protein once pSTAT3 decreased (Figure 1D). Importantly, FLLL32 was capable of reducing pSTAT3 levels, cyclin D1 expression and inducing apoptosis in primary human melanoma cell cultures derived from recurrent cutaneous melanoma tumors [17] (Figure 1E). Finally, treatment of basal pSTAT3-positive human melanoma cell lines with FLLL32 for 24 hours led to reduced STAT3 DNA binding as determined by gel shift assays and expression of the STAT3-regulated genes, cyclin D1 and survivin as measured by immunoblot (Figure 2A-B).

Bottom Line: In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors.In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-gamma, IL 2).Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-gamma production when cultured with K562 targets as compared to vehicle (DMSO).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Arthur G, James Cancer Hospital and Richard J, Solove Research Institute, The Ohio State University, 410 W, 10th Ave, Columbus, OH 43210, USA.

ABSTRACT

Background: We characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells.

Results: FLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-gamma-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-gamma, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-gamma production when cultured with K562 targets as compared to vehicle (DMSO).

Conclusions: These data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy.

Show MeSH
Related in: MedlinePlus