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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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The FLLL32 curcumin analog induced apoptosis in human melanoma cells. (A) The molecular structure of curcumin indicates that the molecule exists in two distinct tautomeric forms: 1) a diketone form and 2) a keto-enol form. FLLL32 was designed as a novel structural analog of curcumin that approximates a modified version of the molecule when locked into the keto-form. (B) Annexin V/PI staining of human metastatic melanoma cells following a 48 hour treatment with FLLL32. Error bars show 95% prediction limits based on the model fit at the estimated IC50 from two or more independent experiments. The non-responsive 1106 MEL and 1259 MEL cell lines were pSTAT3-negative. (C) Annexin V/PI staining of representative pSTAT3+ melanoma cells treated with either 20 μM curcumin or 2 μM FLLL32. Data are presented as the mean percentage of apoptotic cells. Error bars represent the standard deviation from at least two individual experiments. (D) Immunoblot analysis (left panel) or immunoprecipitation for total Jak2 protein (right panel; blot with Jak2 or pJak2 antibodies) of pSTAT3-positive A375 and Hs294T cells following 24 hour treatment. (E) FLLL32 treatment reduced pSTAT3, the STAT3-regulated gene, cyclin D1 and induced apoptosis in primary human cells derived from recurrent cutaneous melanoma tumors. These primary melanoma cell cultures have been previously described by our group [17]. Cells were treated for 48 hours with the indicated concentrations of FLLL32 or curcumin (20 mM) as a biologic control and analyzed by immunoblot. 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 1: The FLLL32 curcumin analog induced apoptosis in human melanoma cells. (A) The molecular structure of curcumin indicates that the molecule exists in two distinct tautomeric forms: 1) a diketone form and 2) a keto-enol form. FLLL32 was designed as a novel structural analog of curcumin that approximates a modified version of the molecule when locked into the keto-form. (B) Annexin V/PI staining of human metastatic melanoma cells following a 48 hour treatment with FLLL32. Error bars show 95% prediction limits based on the model fit at the estimated IC50 from two or more independent experiments. The non-responsive 1106 MEL and 1259 MEL cell lines were pSTAT3-negative. (C) Annexin V/PI staining of representative pSTAT3+ melanoma cells treated with either 20 μM curcumin or 2 μM FLLL32. Data are presented as the mean percentage of apoptotic cells. Error bars represent the standard deviation from at least two individual experiments. (D) Immunoblot analysis (left panel) or immunoprecipitation for total Jak2 protein (right panel; blot with Jak2 or pJak2 antibodies) of pSTAT3-positive A375 and Hs294T cells following 24 hour treatment. (E) FLLL32 treatment reduced pSTAT3, the STAT3-regulated gene, cyclin D1 and induced apoptosis in primary human cells derived from recurrent cutaneous melanoma tumors. These primary melanoma cell cultures have been previously described by our group [17]. Cells were treated for 48 hours with the indicated concentrations of FLLL32 or curcumin (20 mM) as a biologic control and analyzed by immunoblot. Membranes were probed with β actin as a loading control and all blots represent data from at least two independent experiments.

Mentions: The molecular structure of curcumin indicates that the molecule exists in two distinct tautomeric forms: 1) a diketone form and 2) a keto-enol form, which each have unique properties relevant for drug design (Figure 1A). We developed a series of analogs based on curcumin in its diketone form which were predicted by computational modeling to interact with the SH2 domain of STAT3 [16] and inhibit STAT3 homodimerization (unpublished observations, Dr. Pui-Kai Li, The Ohio State University). One analog, termed FLLL32, was selected as a candidate for inhibition of the Jak2-STAT3 pathway (Figure 1A). This analog has previously been shown to inhibit the Jak2-STAT3 pathway and elicit anti-tumor activity against pancreatic and breast cancer cells [16].


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)

The FLLL32 curcumin analog induced apoptosis in human melanoma cells. (A) The molecular structure of curcumin indicates that the molecule exists in two distinct tautomeric forms: 1) a diketone form and 2) a keto-enol form. FLLL32 was designed as a novel structural analog of curcumin that approximates a modified version of the molecule when locked into the keto-form. (B) Annexin V/PI staining of human metastatic melanoma cells following a 48 hour treatment with FLLL32. Error bars show 95% prediction limits based on the model fit at the estimated IC50 from two or more independent experiments. The non-responsive 1106 MEL and 1259 MEL cell lines were pSTAT3-negative. (C) Annexin V/PI staining of representative pSTAT3+ melanoma cells treated with either 20 μM curcumin or 2 μM FLLL32. Data are presented as the mean percentage of apoptotic cells. Error bars represent the standard deviation from at least two individual experiments. (D) Immunoblot analysis (left panel) or immunoprecipitation for total Jak2 protein (right panel; blot with Jak2 or pJak2 antibodies) of pSTAT3-positive A375 and Hs294T cells following 24 hour treatment. (E) FLLL32 treatment reduced pSTAT3, the STAT3-regulated gene, cyclin D1 and induced apoptosis in primary human cells derived from recurrent cutaneous melanoma tumors. These primary melanoma cell cultures have been previously described by our group [17]. Cells were treated for 48 hours with the indicated concentrations of FLLL32 or curcumin (20 mM) as a biologic control and analyzed by immunoblot. 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 1: The FLLL32 curcumin analog induced apoptosis in human melanoma cells. (A) The molecular structure of curcumin indicates that the molecule exists in two distinct tautomeric forms: 1) a diketone form and 2) a keto-enol form. FLLL32 was designed as a novel structural analog of curcumin that approximates a modified version of the molecule when locked into the keto-form. (B) Annexin V/PI staining of human metastatic melanoma cells following a 48 hour treatment with FLLL32. Error bars show 95% prediction limits based on the model fit at the estimated IC50 from two or more independent experiments. The non-responsive 1106 MEL and 1259 MEL cell lines were pSTAT3-negative. (C) Annexin V/PI staining of representative pSTAT3+ melanoma cells treated with either 20 μM curcumin or 2 μM FLLL32. Data are presented as the mean percentage of apoptotic cells. Error bars represent the standard deviation from at least two individual experiments. (D) Immunoblot analysis (left panel) or immunoprecipitation for total Jak2 protein (right panel; blot with Jak2 or pJak2 antibodies) of pSTAT3-positive A375 and Hs294T cells following 24 hour treatment. (E) FLLL32 treatment reduced pSTAT3, the STAT3-regulated gene, cyclin D1 and induced apoptosis in primary human cells derived from recurrent cutaneous melanoma tumors. These primary melanoma cell cultures have been previously described by our group [17]. Cells were treated for 48 hours with the indicated concentrations of FLLL32 or curcumin (20 mM) as a biologic control and analyzed by immunoblot. Membranes were probed with β actin as a loading control and all blots represent data from at least two independent experiments.
Mentions: The molecular structure of curcumin indicates that the molecule exists in two distinct tautomeric forms: 1) a diketone form and 2) a keto-enol form, which each have unique properties relevant for drug design (Figure 1A). We developed a series of analogs based on curcumin in its diketone form which were predicted by computational modeling to interact with the SH2 domain of STAT3 [16] and inhibit STAT3 homodimerization (unpublished observations, Dr. Pui-Kai Li, The Ohio State University). One analog, termed FLLL32, was selected as a candidate for inhibition of the Jak2-STAT3 pathway (Figure 1A). This analog has previously been shown to inhibit the Jak2-STAT3 pathway and elicit anti-tumor activity against pancreatic and breast cancer cells [16].

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