Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties

Abstract

Nuclear factor kappa B (NF-κB) is a key signaling molecule in the elaboration of the inflammatory response. Data indicate that curcumin, a natural ingredient of the curry spice turmeric, acts as a NF-κB inhibitor and exhibits both anti-inflammatory and anti-cancer properties. Curcumin analogs with enhanced activity on NF-κB and other inflammatory signaling pathways have been developed including the synthetic monoketone compound 3,5-Bis(2-fluorobenzylidene)-4-piperidone (EF24). 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31) is a structurally-related curcumin analog whose potency for NF-κB inhibition has yet to be determined. To examine the activity of EF31 compared to EF24 and curcumin, mouse RAW264.7 macrophages were treated with EF31, EF24, curcumin (1-100 μM) or vehicle (DMSO 1%) for 1h. NF-κB pathway activity was assessed following treatment with lipopolysaccharide (LPS) (1 μg/mL). EF31 (IC(50)~5 μM) exhibited significantly more potent inhibition of LPS-induced NF-κB DNA binding compared to both EF24 (IC(50)~35 μM) and curcumin (IC(50) >50 μM). In addition, EF31 exhibited greater inhibition of NF-κB nuclear translocation as well as the induction of downstream inflammatory mediators including pro-inflammatory cytokine mRNA and protein (tumor necrosis factor-α, interleukin-1β, and interleukin-6). Regarding the mechanism of these effects on NF-κB, EF31 (IC(50)~1.92 μM) exhibited significantly greater inhibition of IκB kinase β compared to EF24 (IC(50)~131 μM). Finally, EF31 demonstrated potent toxicity in NF-κB-dependent cancer cell lines while having minimal and reversible toxicity in RAW264.7 macrophages. These data indicate that EF31 is a more potent inhibitor of NF-κB activity than either EF24 or curcumin while exhibiting both anti-inflammatory and anticancer activities. Thus, EF31 represents a promising curcumin analog for further therapeutic development.

Figure 1. Chemical structures of curcumin and the monoketone analogs

Figure 2. EF31 is a potent inhibitor of LPS-induced NF-κB DNA binding activity

A) Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and treated with LPS (1 or 10 µg/mL) or saline. Nuclear proteins were collected at different time-points (5 minutes– 60 minutes) as described in section 2.3. NF-κB DNA-binding activity was measured using a DNA-binding ELISA. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM). * p < 0.05 vs. 5 minutes- LPS (1 µg/mL) treated group. B) Mouse RAW264.7 macrophages were pre-treated with curcumin, EF24, EF31 (1, 5, 10, 30, 50, or 100 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) for 15 minutes. Nuclear proteins were collected and NF-κB DNA-binding was measured using a DNA-binding ELISA. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. * p < 0.05, *** p < 0.001 vs. curcumin at same concentration; †† p < 0.01, ††† p < 0.001 vs. EF24 at same concentration. C) Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and pre-treated with EF31 (10 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) or saline for 15 or 30 minutes. Whole cell extracts and western blot analyses were conducted for unphosphorylated and phosphorylated IkB and NFkB p65 as described in section 2.9. Results shown are representative of three separate experiments.

Figure 3. EF31 impairs LPS-induced NF- κB nuclear translocation

A) Mouse RAW264.7 macrophages were grown in 8-well chamber slides and pre-treated with curcumin, EF24, EF31 (5, 10, or 50 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) for 15 minutes. Cells were then fixed and processed as described in section 2.4. Images were obtained using a LSM510 confocal microscope. Scale bar = 5µm. B) The induction of NF-κB nuclear translocation by LPS was quantified by measuring nuclear p65 fluorescence intensity as described in section 2.4. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. *** p < 0.001 vs. curcumin at same concentration; †† p < 0.01 vs. EF24 at same concentration; a p < 0.001 vs. vehicle.

Figure 4. EF31 inhibits LPS-induced pro-inflammatory cytokine mRNA expression

Mouse RAW264.7 macrophages were grown in 6-well plates and pre-treated with curcumin, EF24, EF31 (5, 10, or 50 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) for 3 hours. Whole cell mRNA was then collected as described in section 2.5 to assess TNF-alpha (A), IL-1 beta (B), and IL-6 (C) gene expression using RT-PCR. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. *** p < 0.001 vs. curcumin at same concentration; † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. EF24 at same concentration.

Figure 5. EF31 inhibits LPS-induced production of pro-inflammatory cytokine proteins

Mouse RAW264.7 macrophages were grown in 6-well plates and pre-treated with curcumin, EF24, EF31 (5, 10, or 50 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) for 16 hours. Medium was collected and assessed for concentrations of TNF-alpha (A), IL-1 beta (B), and IL-6 (C) protein using ELISAs. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. curcumin at same concentration; †† p < 0.01, ††† p < 0.001 vs. EF24 at same concentration. (Of note, LPS + DMSO treated samples yielded protein levels of approximately 1,400 pg/mL for TNF-α, 15 pg/mL for IL-1β, and 11,000 pg/mL for IL-6)

Figure 6. Mechanism for EF31-dependent inhibition of the NF-κB pathway

IκKβ recombinant protein was pre-incubated with EF24, EF31 (0.0977, 0.391, 1.56, 6.25, 25, 100 µM), or vehicle (DMSO 1%) for 30 minutes. Inhibition of IκKβ activity was measured using a Z’-Lyte kinase assay kit as described in section 2.7. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. ** p < 0.01, *** p < 0.001 vs. EF24 at same concentration.

Figure 7. Inhibition of NF-κB DNA-binding by EF31 is reversible

Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and pre-treated with curcumin (50 µM), EF24(50 µM), EF31 (10 µM), or vehicle (DMSO 1%) for 1 hour. Cells were then washed and medium was replaced and treated with LPS (1 µg/mL) or saline. Nuclear proteins were collected at different time-points (15 minutes– 6 hours) as described in section 2.3. NF-κB DNA-binding activity was measured using a DNA-binding ELISA. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM). * p < 0.05 vs. vehicle + LPS at 15 minutes.

Figure 8. EF31 shows no reduction in cell viability at concentrations that inhibit NF-κB

Mouse RAW264.7 macrophages were grown in 96-well plates and pre-treated with curcumin, EF24, EF31 (1, 5, 10, or 50 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) or saline for 15 minutes. Cell viability/ proliferation was then measured as described in section 2.8. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent of vehicle. ** p < 0.01, *** p < 0.001 vs. curcumin at same concentration in the saline treated groups; a p < 0.05 vs. EF24 at same concentration in the saline treated groups; †† p < 0.01 vs. curcumin at same concentration in the LPS treated groups; bb p < 0.01 vs. EF24 at same concentration in the LPS treated groups.

Figure 9. EF31 shows potent toxicity on NF-κB dependent cancer cell lines

Cancer cell lines were grown in 96-well plates and treated with curcumin, EF31 (1, 5, 10, or 50 µM), or vehicle (DMSO 1%) for 48 hours. Cell viability/ proliferation was measured as described in section 2.8. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent of vehicle. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. curcumin at same concentration.

Figure 10. EF31 inhibits MAPK transcription factor DNA-binding activity

Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and pretreated with curcumin (50 µM), EF31 (10 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) or saline for 15 minutes. Nuclear proteins were collected as described in section 2.3 and transcription factor DNA-binding was measured using a DNA-binding ELISA for ATF-2 (top) and c-Jun (bottom). Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM). *** p < 0.001 vs. curcumin + LPS; ††† p < 0.001 vs. vehicle + LPS; aa p < 0.01, aaa p < 0.001 vs. vehicle + saline.

Figure 11. EF31 inhibits lipopolysaccharide-induced phosphorylation of MAPKs p38, JNK, and ERK

Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and pre-treated with EF31 (10 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) or saline for 15 or 30 minutes. Whole cell extracts and western blot analyses were conducted for unphosphorylated and phosphorylated (p) p38 (A), JNK (B), and ERK (C) and as described in section 2.9. Results shown are representative of three separate experiments.