Characterization of a novel curcumin analog P1 as potent inhibitor of the NF-κB signaling pathway with distinct mechanisms
Abstract
Yan-min PENG1, Jian-bin ZHENG2, Yu-bo ZHOU1, *, Jia LI1, *
1National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 2School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
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 high-throughput 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. H2-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 IC50 value in inhibition of TNF-α-induced NF-κB activation was 0.8 μmol/L, whereas its IC50 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.
Keywords: curcumin; P1; anticancer agent; high-throughput screen; NF-κB; HeLa cell; mitochondria; ROS
This work was supported by grants from the National Natural Science Foundation of China (No 91029716, 81021062, and 81072667).
* To whom correspondence should be addressed.
E-mail jli@mail.shcnc.ac.cn (Jia LI); ybzhou@mail.shcnc.ac.cn (Yu-bo ZHOU)
Received 2012-10-22 Accepted 2013-01-10
Keywords:
1National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 2School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
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 high-throughput 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. H2-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 IC50 value in inhibition of TNF-α-induced NF-κB activation was 0.8 μmol/L, whereas its IC50 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.
Keywords: curcumin; P1; anticancer agent; high-throughput screen; NF-κB; HeLa cell; mitochondria; ROS
This work was supported by grants from the National Natural Science Foundation of China (No 91029716, 81021062, and 81072667).
* To whom correspondence should be addressed.
E-mail jli@mail.shcnc.ac.cn (Jia LI); ybzhou@mail.shcnc.ac.cn (Yu-bo ZHOU)
Received 2012-10-22 Accepted 2013-01-10