Icaritin promotes apoptosis and inhibits proliferation by down-regulating AFP gene expression in hepatocellular carcinoma.
BACKGROUND
Icaritin, an active ingredient of the Chinese herb Epimedium, plays an anti-tumor role in liver cancer by inhibiting the proliferation of hepatocellular cells and promoting their apoptosis. In China, phase II and a large phase III clinical trial of icaritin reagent for the treatment of hepatocellular cancer is under-going, but the specific mechanism of icaritin action was unclear. Alpha-fetoprotein (AFP), an oncofetal protein, produced in the healthy fetal liver and yolk sac. Intracellular AFP promoted cellular proliferation and inhibited cellular apoptosis in hepatocellular carcinoma (HCC). The study was aimed to investigate the effect of icaritin on HCC through p53/AFP pathway.


METHODS
Real-time RT PCR and western blot were used to detect p53 and AFP expression levels in HCC cells treated with icaritin. The mechanism of icaritin affecting p53 expression was verified by ubiquitination experiment, and the binding activity of icaritin on p53 in AFP promoter region was verified by luciferase experiment. EdU, MTT and flow cytometry were used to determine whether icaritin affected HCC cellular proliferation and apoptosis through p53/ AFP pathway. Expression levels of p53 and AFP in xenograft mouse model were determined by western blotting.


RESULTS
Our results showed icaritin inhibited AFP expression at mRNA and protein level. AFP was also identified as the target gene of the p53 transcription factor. Icaritin abrogated murine double minute (Mdm) 2-mediated p53 ubiquitination degradation to improve the stability of p53. Up-regulated p53 protein levels then transcriptionally inhibited the AFP promoter. Icaritin-mediated decrease of AFP through Mdm2/p53 pathways inhibited HCC cellular proliferation and promoted HCC cellular apoptosis.


CONCLUSION
Our findings revealed the mechanism of icaritin in promoting apoptosis and inhibiting proliferation in liver cancer cells. The regulatory mechanism of icaritin in AFP protein down-regulation provides a theoretical and experimental basis for further research into new drugs for the treatment of liver cancer.
['Li|Hui|H|http://orcid.org/0000-0002-5740-2655', 'Liu|Yujuan|Y|', 'Jiang|Wei|W|', 'Xue|Junhui|J|', 'Cheng|Yuning|Y|', 'Wang|Jiyin|J|', 'Yang|Ruixiang|R|', 'Zhang|Xiaowei|X|']
D000818:Animals D017209:Apoptosis D006528:Carcinoma, Hepatocellular D049109:Cell Proliferation D015536:Down-Regulation D005419:Flavonoids D015972:Gene Expression Regulation, Neoplastic D056945:Hep G2 Cells D006801:Humans D008113:Liver Neoplasms D008297:Male D051379:Mice D015398:Signal Transduction D016159:Tumor Suppressor Protein p53 D023041:Xenograft Model Antitumor Assays D000509:alpha-Fetoproteins
Icaritin AFP Hepatocellular carcinoma p53 ubiquitination Hepatocellular proliferation