The novel acanthoic acid analog, NPI-1387 inhibits tumor cell proliferation, reduces NF-κB DNA binding activity and functions upstream in the NF-κB signaling pathway

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Acanthoic acid is a pimaradiene diterpene isolated from the Korean medicinal plant, Acanthopanax koreanum Nakai (Araliaceae). The root and stem bark of Acanthopanax koreanum have been used in traditional medicine as a tonic and sedative as well as in the treatment of rheumatism and diabetes in Korea. In vitro studies have demonstrated that acanthoic acid inhibited the production of proinflammatory cytokines such as TNF-α and IL-1. Experiments performed in mice also demonstrated that it was active in several inflammation models. To study structure-activity relationships, two cell-based assays were used to screen a library of semi-synthetic acanthoic acid analogs. Among these analogs, NPI-1387 inhibited LPS-induced TNF-α synthesis in the murine macrophage-like RAW264.7 cell line most potently. In addition, NPI-1387 also reduced TNF-α induced nuclear factor-κB (NF-κB) activation in a HEK293 NF-κB/Luciferase reporter cell line, suggesting that NPI-1387 is an inhibitor in the NF-κB signaling pathway. NF-κB is a key transcription factor that regulates survival in many cells and elevated levels of activated NF-κB have been shown to protect cancer cells (i.e. multiple myeloma and prostate) from apoptosis. We therefore tested the effects of NPI-1387 on TNF-α or LPS-induced NF-κB DNA binding activity in the multiple myeloma cell line RPMI 8226 or the prostate carcinoma cell line PC-3 by using electrophoretic mobility shift assays (EMSA). Our results show that NPI-1387 inhibited TNF-α or LPS-induced NF-κB DNA binding activity in both cell lines. In addition, NPI-1387 was most potent at inhibiting the proliferation of the multiple myeloma RPMI 8226 cell line (IC₅₀= 5.1 ± 1μM), while in PC-3 clonogenicity assays, exposure of 6hr to 20μM of NPI-1387 was sufficient to completely abolish PC-3 colony formation. To elucidate the molecular target(s) of NPI-1387 in the NF-κB signaling pathway, RAW264.7 cells were treated with NPI-1387 before LPS stimulation and western blot and fluorescence microscopy analyses were performed. Results demonstrated that NPI-1387 not only inhibited the phosphorylation of endogenous IRAK1 and its downstream target IκBα in a dose-dependent manner, but also inhibited the nuclear translocation of activated IRAK1 and NF-κB suggesting that NPI-1387 may function as an upstream inhibitor in the NF-κB pathway. This inhibitory effect of NPI-1387 on the NF-κB activation pathway provides the basis for potential future therapeutic applications in cancer and inflammatory diseases.