Antitumor activity and macrophage nitric oxide producing action of medicinal herb, Crassocephalum crepidioides

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Abstract
Background: Crassocephalum crepidioides, a plant distributed in Okinawa Islands, is known in folk medicine; however, its anticancer activity has not been investigated. The aim of this study was to determine thein vitroandin vivoantitumor activities ofC. crepidioideson murine Sarcoma 180 (S-180) and related molecular mechanisms.Methods: The antitumor effect ofC. crepidioideswas evaluated in S-180-cell-bearing mice. Cell growth was assessed using a colorimetric assay. Nitrite and nitrate levels were measured by colorimetry. The expression levels of inducible NO synthase (iNOS) in murine RAW264.7 macrophages was assessed by reverse transcriptase-polymerase chain reaction. Activation of iNOS promoter was detected by reporter gene. Activation of nuclear factor-κB (NF-κB) was evaluated by electrophoretic mobility shift assay. The role of NF-κB signaling was analyzed using inhibitors of NF-κB and dominant-negative mutants, and Western blot analysis.Results: C. crepidioidesextract delayed tumor growth in S-180-bearing mice. However, it did not inhibit S-180 cell growthin vitro. Supernatant of culturedC. crepidioides-stimulated RAW264.7 macrophages was cytotoxic to S-180 cells. This cytotoxicity was associated with nitric oxide (NO) production. NF-κB signaling pathway was crucial for the transcriptional activation of iNOS gene. Isochlorogenic acid, a component ofC. crepidioides, induced NF-κB activation and iNOS expression.Conclusions: The results highlight the oncolytic and immunopotentiation properties ofC. crepidioidesmediated through NF-κB-induced release of NO from macrophages.

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