Hinokiflavone induces apoptosis via activating mitochondrial ROS/JNK/caspase pathway and inhibiting NF-κB activity in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common malignancy with limited treatment options. Hinokiflavone (HF), a natural biflavonoid, has shown to inhibit the proliferation of melanoma, whereas its antitumour effect against HCC and the underlying mechanisms remain elusive. Here, we aimed at evaluating its antitumour effect against HCC in both in vitro and in vivo. Cell counting kit 8, colony formation assay, PI/RNase staining and Western blotting revealed that HF inhibited the proliferation of HCC cells via G0/G1 cell cycle arrest with p21/p53 up-regulation. DAPI staining, Annexin V-FITC/PI staining and Western blotting confirmed that HF triggered caspase-dependent apoptosis. Moreover, HF increased the levels of mitochondrial reactive oxygen species (mtROS) and activated c-Jun N-terminal kinase (JNK) pathway, as measured by MitoSOX Red staining and Western blotting. After respectively inhibiting mtROS (Mito-TEMPO) and JNK (SP600125), HF-induced apoptosis was reversed. Additionally, Western blotting documented that HF suppressed nuclear factor kappa B (NF-κB) activity and the anti-apoptotic genes downstream, contributing to cell apoptosis. Finally, in vivo studies demonstrated that HF significantly impaired tumour growth in HCC xenograft. Collectively, these findings suggested that HF induced apoptosis through activating mtROS/JNK/caspase pathway and inhibiting NF-κB signalling, which may represent a novel therapeutic agent for treating HCC.