Nuphar lutea thioalkaloids inhibit the nuclear factor κappaB pathway, potentiate apoptosis and are synergistic with cisplatin and etoposide

We screened thirty-four methanolic plant extracts for inhibition of the constitutive nuclear factor κB (NF-κB) activity by a NF-κB-luciferase reporter gene assay. Strong inhibition of NF-κB activity was found in extracts of leaf and rhizome from Nuphar lutea L. SM. (Nuphar). The inhibitory action was narrowed down to a mixture of thionupharidines and/or thionuphlutidines that were identified in chromatography fractions by one- and two-dimensional NMR analysis. Dimeric sesquiterpene thioalkaloids were identified as the major components of the mixture. The Nuphar alkaloids mixture (NUP) showed a dose dependent inhibition of NF-κB activity in a luciferase reporter gene assay as well as reduction of nuclear NF-κB subunits expression as tested by western blots and immunohistochemistry. Decreased DNA binding was demonstrated in electro mobility shift assays. NUP inhibited both inducible and constitutive NF-κB activation and affected the canonical and alternative pathways. Suppression of NF-κB was not cell type specific. Induction of apoptosis by the alkaloid mixture was demonstrated by time-dependent and dose-dependent cleavage of procaspase-9 and PARP. Synergistic cytotoxicity of the active mixture with cisplatin and etoposide was demonstrated. Overall, our results show that NUP inhibits the NF-κB pathway and acts as a sensitizer to conventional chemotherapy, enabling the search for its specific target and application against cancer and inflammation.