Synthesis and identification of new 4-arylidene curcumin analogues as potential anticancer agents targeting nuclear factor-κB signaling pathway

Abstract

A series of curcumin analogues including new 4-arylidene curcumin analogues (4-arylidene-1,7-bisarylhepta-1,6-diene-3,5-diones) were synthesized. Cell growth inhibition assays revealed that most 4-arylidene curcumin analogues can effectively decrease the growth of a panel of lung cancer cells at submicromolar and low micromolar concentrations. High content analysis technology coupled with biochemical studies showed that this new class of 4-arylidene curcumin analogues exhibits significantly improved NF-κB inhibition activity over the parent compound curcumin, at least in part by inhibiting IκB phosphorylation and degradation via IKK blockage; selected 4-arylidene curcumin analogues also reduced the tumorigenic potential of cancer cells in a clonogenic assay.

Figure 1. The structures of 1,3-diketones curcumin analogs (1-6), monoketone curcumin analogs (7-10), 4-arylidene crucumin analogs (13-37) and 4-hydroxymethylene curcumin analogs (38-40)

Figure 2

Inhibition of lung cancer cell viability by curcumin and its analogs. Cells were grown in 384 well plates and were treated with curcumin, 17, 21 or 35 as indicated for 72 hr. Cell viability was assessed by the Alamar Blue method and expressed as % control (DMSO). Results with a panel of lung cancer cells are shown (lung adenocarcinoma: A549 H1944, H1792; squamous cell carcinoma: H157; and squamous cell carcinoma: H157). Results from one representative experiment are shown.

Figure 3

Inhibition of the TNFα induced NF-κB activation by new curcuminoids, 17, 18, 20, and 21. Results from one representative experiment are shown. (A) Example images of NF-κB subcellular localization. A549 cells were treated with compounds, or vehicle (DMSO) for 30 min, followed by stimulation with TNFα (10 ng/ml) for 30 min. In the vehicle (DMSO) treatment, NF-κB is located at cytoplasm. Upon TNFα treatment, NF-κB is activated and translocated to nucleus. Pre-incubation of the cells with increasing concentrations of compounds, with compound 21 as an example, dose-dependently inhibited the TNFα-induced NF-κB translocation to the nucleus. (B) Dose-response curves of the inhibitory effect of test compounds on TNFα-induced NF-κB activation. The inhibitory effect of the compound on TNFα induced NF-κB translocation (activation) was expressed as: % of Control = ΔFI _(compound)/ ΔFI _{(TNFα only control)}. ΔFI is the measured NF-κB fluorescence (green) intensity difference between nucleus and cytoplasm. Data showed are average values from triplicate samples with SD.

Figure 4

Effect of curcumin analogs on in vivo activity of IKK. (A) In vivo IKK activity as revealed by phosphorylation of IκB. A549 cells were pretreated with test compounds for 30 min prior to adding TNFα (10 ng/ml). Whole cell lysates were prepared after 7 min of TNFα treatment and analyzed for the phosphorylation state of IκB with anti-pS³² antibody via Western blotting. Then, antibodies on the membrane were stripped and the membrane re-probed for total IκB with antiserum against IκB as indicated. (B) IκB stability assay. A549 cells were pretreated with test compounds at indicated dosage prior to the addition of TNFα. Cells were cultured in the presence of TNFα (10 ng/ml) for an additional 20 minutes, lysed and analyzed for total IκB levels by Western blot.

Figure 5

Effect of curcumin analogs on the catalytic activity of recombinant IKK in vitro. . Recombinant IKKβ was incubated with increasing concentrations of test compounds as indicated. Addition of Mg/[γ-³²P] cocktail with purified GST-I-κB started the reactions, which were continued for 30 min at 30°C. Proteins were separated by SDS-PAGE and processed for radiolabeled GST-I-κB (³²P- I-κB) and stained total GST-I-κB (GST- I-κB). Controls include reactions without IKKβ (lane 1 from left) or without compound (lane 2).

Figure 6

Compound 17, 21, and 35 inhibit the colony formation of A549 cells. A549 cells were seeded in 12-well plate at density of 200 cells/well and incubated overnight. The cells were then treated with compounds, or vehicle (DMSO) for 3 days. The medium and compound were replaced for every 3 days. After a total of 9 day incubation, cells were fixed and stained with SRB. The image of the plate was scanned and the colonies were counted. (B) The colonies were counted and normalized to DMSO control. The data shown are average of triplicate samples with SD.

Figure 7

Binding of compound 17 and ATP with the IKKβ catalytic domain homology model. Left, overlay of compound 17 (colored by atom type) and ATP (purple) in the ATP binding pocket, the molecules were represented in stick; Right, Surface representation of the IKKβ catalytic domain homology model with compound 17 (colored by atom type) and ATP (purple) docking into the ATP binding pocket, the kinase surface was mapped by lipophilic potential (from orange to blue, lipophilic potential decrease), and Z-Clipping to remove the N terminal region for visualization facility. Compound 17 adopts a propeller-shaped conformation in the pocket. Typical hydrogen bonds of compound 17 (green dotted line) and ATP (brown dotted line) with the homology model were shown.

Scheme 1. Synthesis of compounds 1-6 and 13-37

Scheme 2. Synthesis of compounds 7-10

Scheme 3. Synthesis of compound 38-40