Andrographolide decreased VEGFD expression in hepatoma cancer cells by inducing ubiquitin/proteasome-mediated cFos protein degradation

Background: Andrographolide (Andro) is reported to inhibit hepatoma tumor growth in our previous studies. This study aims to further search the critical signals involved in such Andro-induced inhibition. Methods: The anti-tumor effect of Andro was evaluated in vivo. Cancer PathwayFinder RT2 Profiler (TM) PCR array was used to find the altered genes. Real-time PCR was used to detect the mRNA expression. Protein expression was detected by Western-blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. Activator protein-1 (AP-1) transcriptional activity was detected by luciferase reporter assay. Results: Andro (10 mg/kg) inhibited hepatoma tumor growth in vivo. The expression of four genes decreased in Andro-treated tumor tissues. Among which, vascular endothelial growth factor (VEGFD) was the highest decreased gene. The decreased VEGFD expression was further confirmed by real-time PCR and immunohistochemical staining assay. Andro decreased VEGFD mRNA and protein expression in hepatoma Hep3B and HepG2 cells. Andro also decreased VEGFD amount in Hep3B cell supernatant Andro decreased cFos protein expression and its translocation into nucleus, and also reduced AP-1 luciferase activity. Further results showed that Andro induced polyubiquitination of cFos. Proteasome inhibitor MG132 reversed the decreased expression of cFos protein, and the decreased mRNA and protein expression of VEGFD. SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), reversed the decreased expression of cFos and VEGFD induced by Andro. Conclusions: Andro decreased VEGFD expression in hepatoma cancer cells via inducing c-fos protein degradation, which will contribute to its anti-cancer activity, and JNK plays some roles in regulating this process. (C) 2015 Elsevier B.V. All rights reserved.