Although shikonin, a naphthoquinone derivative, has showed anti-cancer activity, its precise molecular anti-tumor mechanism remains to be elucidated. In this study, we investigated the effects of shikonin on human hepatocellular carcinoma (HCC) in vitro and in vivo. Our results showed that shikonin induced apoptosis of Huh7 and BEL7402 but not nontumorigenic cells. ROS generation was detected, and ROS scavengers completely inhibited shikonin-induced apoptosis, indicating that ROS play an essential role. Although the JNK activity was significantly elevated after shikonin treatment, JNK was not linked to apoptosis. However, downregulation of Akt and RIP1/NF-κB activity was found to be involved in shikonin-induced apoptosis. Ectopic expression of Akt or RIP1 partly abrogated the effects of shikonin, and Akt inhibitor and RIP1 inhibitor synergistically induced apoptosis in conjunction with shikonin treatment. ROS scavengers blocked shikonin-induced inactivation of Akt and RIP1/NF-κB, but Akt or RIP1/NF-κB did not regulate ROS generation, suggesting that Akt and RIP1/NF-κB signals are downstream of ROS generation. In addition, the results of xenograft experiments in mice were consistent with in vitro studies. Taken together, our data show that shikonin, which may be a promising agent in the treatment of liver cancer, induced apoptosis in HCC cells through the ROS/Akt and RIP1/NF-κB pathways.
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