Abstract
BackgroundIcaritin, 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.MethodsReal-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.ResultsOur 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.ConclusionOur 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.
Highlights
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
AFP protein expression gradually decreased with increasing doses of icaritin in HepG2 cells and SMMC7721 cells (Fig. 1a), becoming most apparent after icaritin treatment for 20 h at a dose of 20 μM. qRTPCR showed that AFP mRNA was down-regulated after icaritin treatment for 20 h at a dose of 20 μM in HepG2 and SMMC7721 cells (Fig. 1b)
These results indicated that icaritin inhibited AFP expression at the transcriptional level
Summary
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. Intracellular AFP promoted cellular proliferation and inhibited cellular apoptosis in hepatocellular carcinoma (HCC). We confirmed that cytoplasmic AFP blocked retinoic acid/retinoic acid receptor-mediated expression of GADD153, GADD45A, and Fn14 and that downregulation of these genes led to the abnormal growth of HCC cells [5,6,7,8]. These results showed the importance of serum or cytoplasmic AFP in promoting cellular proliferation and inhibiting cellular apoptosis in HCC. In recent years, AFP has been used as an immunotherapy target for HCC [9]
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