Characterization of a novel curcumin analog P1 as potent inhibitor of the NF-κB signaling pathway with distinct mechanisms

Abstract

Aim: Curcumin has shown promising anticancer activity, which relies on its inhibition on NF-κB pathway. In this study, we characterized the pharmacological profile of a novel curcumin analog P1 and elucidate the related mechanisms. Methods: HEK293/NF-κB cells, stably transfected with an NF-κB-responsive luciferase reporter plasmid, were generated for highthroughput screen (HTS). Eight cancer cell lines, including PC3, COLO 205, HeLa cells etc. were tested. Cell viability was assessed using the sulforhodamine B (SRB) assays. Cell apoptosis was evaluated using FACS, immunocytochemistry, and Western blotting. H 2 -DCFDA and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS). The mitochondrial function was evaluated using mitochondrial oxygen consumption assay. Results: P1, a tropinone curcumin, was found in HTS targeting the NF-κB pathway. Its IC 50 value in inhibition of TNF-α-induced NF-κB activation was 0.8 μmol/L, whereas its IC 50 values in inhibiting the growth of A549 and HeLa cells were 1.24 and 0.69 μmol/L, respectively, which was 20-to 30-fold more potent than curcumin. The inhibition of P1 on the NF-κB pathway was further addressed in HeLa cells. The compound up to 10 µmol/L did not affect the binding of NF-κB to DNA, but markedly inhibited NF-κB nuclear translocation, IκB degradation and IκB kinase phosphorylation. The compound (1 and 3 µmol/L) concentration-dependently induced ROS generation, whereas curcumin up to 20 µmol/L had no effect. P1-induced ROS generation was mainly localized in mitochondria, and reversed by NAC. Moreover, the compound significantly enhanced TNF-α-induced apoptosis. Conclusion: P1 is a novel curcumin analog with potent anticancer activities, which exerts a distinct inhibition on the NF-κB pathway.

Cephalotanins A–D, Four Norditerpenoids Represent Three Highly Rigid Carbon Skeletons from Cephalotaxus sinensis

“…Stable HE K293/NF‐ κ B cells were seeded into 96‐well cell culture plates (Corning, NY, USA) and allowed to grow for 24 h. The cells were then treated with compound in 6 doses at a dilution ratio of 1:3, followed by stimulation with 10 ng mL −1 TNF‐ α . After 6 h, the luciferase substrate (Promega, Madison, WI, USA) was added to each well, and the released luciferin signal was detected using an EnVision microplate reader (Perkin–Elmer, Waltham, MA, USA).…”

“…Stable HE K293/NF-kBc ells [18] were seeded into 96-well cell culture plates (Corning, NY,U SA) and allowed to grow for 24 h. The cells were then treated with compound in 6d oses at ad ilution ratio of 1:3, followed by stimulation with 10 ng mL À1 TNF-a.A fter 6h,t he luciferase substrate (Promega, Madison, WI, USA) was added to each well, and the released luciferin signal was detected using an EnVision microplate reader (Perkin-Elmer,W altham, MA, USA). IC 50 values were derived from an onlinear regression model (curvefit) based on as igmoidal dose response curve (variable slope) and computed using Graphpad Prism version 5.02 (Graphpad Software).…”

Cancer Stem Cells in Prostate Cancer: Implications for Targeted Therapy

“…It has been shown that the activation of NF-κB signaling in PCa cells correlates with PCa progression, chemoresistance, recurrence, and metastasis [82,83] . Thus, inhibiting NF-κB can suppress chemoresistance, and mediate antitumor responses while enhancing the sensitivity of tumor cells to other anticancer drugs [82] . Several studies have focused on targeting the NF-κB pathway on its own or in combination with conventional agents [84] .…”

Alkaloid Constituents of Ficus hispida and Their Antiinflammatory Activity

“…HEK293/NF-κB cells were prepared as reported [16]. The luciferase assay procedure was performed according to the reported methods [17].…”