| Cancer is one of the biggest health issues around the world. Most cases of hepatocellular carcinoma (HCC) occur in China, with high mortality rate and bad prognosis. The treatments of hepatocellular carcinoma contain majorly surgical resection, radiotherapy, chemotherapy, and so on. As there are limited types of drugs on clinical treatment for HCC now, it is very important to develop novel anti-HCC drugs. The traditional Chinese medicine (TCM) is a great source for anti-tumor agents. Shikonin a naphthoquinone derived from the Chinese medicinal plant Lithospermum erythrorhizon, shows anti-cancer capability. However, the detailed mechanism is still unclear. In the presented research, we investigated the inhibition effect of shikonin on HCC in vivo and in vitro.Programmed cell death (PCD) is an essential mechanism for chemotherapeutics killing tumor cells effectively, which mainly contains apoptosis and autophagy. Our results indicated that a low dose of shikonin (2-2.5μM) treatment for a short time (12hours) induced HCC and normal liver cells autophagy, which was determined by LC3conversion assay, GFP-LC3puncta formation, and AV staining.3-MA could block early autophagosome formation induced by shikonin. Autophagic flux was confirmed by LC3turnover assay and degradation of p62. On the other hand, results from Western blot analysis of apoptosis-related proteins and Annexin-V FITC/PI staining plus FACS analysis referred that shikonin treatment at a high dose (6-8μM) and for a long time (24hours) might induce HCC cells but not normal cells apoptotic cell death. Shikonin-induced apoptosis could be blocked by z-VAD, a CASPASE pan-inhibitor, thus it was CASPASE-dependent. Our results showed that shikonin could trigger HCC cell lines apoptosis and autophagy in vitro.As ROS was reported to be involved in mechanisms of some anti-cancer drugs inducing apoptosis, we next investigated the mechanism and found ROS accumulation after shikonin treatment. The ROS scavengers NAC and Tiron completely blocked apoptosis and autophagy. Therefore, the ROS accumulation played an essential role in shikonin-induced apoptosis and autophagy.Akt and RIP/NF-κB are important in cell fate decision. In our studies on the mechanism of shikonin-induced apoptosis, downregulation of Akt and RIP/NF-κB activity was found to be involved in shikonin-induced apoptosis. Because ectopic expression of Akt or RIP partly abrogated the effects of shikonin, and downregulation by Akt inhibitor and RIP inhibitor synergistically induced apoptosis in conjunction with shikonin treatment. Moreover, ROS scavengers blocked shikonin-induced inactivation of Akt and RIP/NF-κB, but Akt or RIP/NF-κB did not regulate ROS generation, suggesting that Akt and RIP/NF-κB signals are downstream of ROS generation.On the other hand, we found that activated the ERK and ATG7are connected to shikonin-induced autophagy, since downregulation of these two pathways led to block of autophagy, and ERK inhibitor enhanced shikonin-induced autophagy. Meanwhile, RIP pathway was involved in autophagy as well, as RIP inhibitor plus shikonin cotreatment triggered enhanced autophagy, and RIP overexpression inhibited autophagy. ROS accumulation was found to be an upper stream event of RIP and ERK pathways as well.Moreover, experiments in vivo revealed similar results: shikonin treatment in Huh7tumor xenograft model of nude mice caused suppression of tumor growth with a low toxicity to animals. Shikonin induced tumor cells autophagy at a low dose (2.5-5mg/kg body weight) and apoptosis at a high dose (5-10mg/kg body weight in vivo, via the similar pathways as in vitro.Taken together, our findings suggest that shikonin is an inducer of autophagy and apoptosis, and may be a promising agent in the treatment of liver cancer. The molecular mechanisms of shikonin are induction of apoptosis through ROS/Akt and ROS/RIP/NF-κB pathways, and triggering autophagy via. ROS/ERK、ROS/RIP、 ATG7pathways. |
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