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.

Effect of Diphtheria Toxin-Based Gene Therapy for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a major global malignancy, responsible for >90% of primary liver cancers. Currently available therapeutic options have poor performances due to the highly heterogeneous nature of the tumor cells; recurrence is highly probable, and some patients develop resistances to the therapies. Accordingly, the development of a novel therapy is essential. We assessed gene therapy for HCC using a diphtheria toxin fragment A (DTA) gene-expressing plasmid, utilizing a non-viral hydrodynamics-based procedure. The antitumor effect of DTA expression in HCC cell lines (and alpha-fetoprotein (AFP) promoter selectivity) is assessed in vitro by examining HCC cell growth. Moreover, the effect and safety of the AFP promoter-selective DTA expression was examined in vivo using an HCC mice model established by the hydrodynamic gene delivery of the yes-associated protein (YAP)-expressing plasmid. The protein synthesis in DTA transfected cells is inhibited by the disappearance of tdTomato and GFP expression co-transfected upon the delivery of the DTA plasmid; the HCC cell growth is inhibited by the expression of DTA in HCC cells in an AFP promoter-selective manner. A significant inhibition of HCC occurrence and the suppression of the tumor marker of AFP and des-gamma-carboxy prothrombin can be seen in mice groups treated with hydrodynamic gene delivery of DTA, both 0 and 2 months after the YAP gene delivery. These results suggest that DTA gene therapy is effective for HCC.

Regulation of α-Fetoprotein Expression by Nkx2.8

ABSTRACT The α-fetoprotein (AFP) gene is an important model of developmental gene silencing and neoplastic gene reactivation. Nkx2.8 is a divergent homeodomain factor originally cloned through its binding to the promoter-coupling element (PCE), a regulatory region upstream of the AFP promoter that mediates stimulation by distant enhancers. Nkx2.8 is the only developmentally regulated factor that has been associated with AFP gene expression. Fetoprotein transcription factor, an orphan nuclear receptor, has also been shown to bind the PCE but is not developmentally regulated. The binding specificities of both families of transcription factor were determined, and overlapping sites for each were defined in the PCE. After modification of nuclear extract and gel shift analysis procedures, Nkx2.8 was identified in six AFP-positive cell lines. Transient-transfection analysis did not show transcriptional stimulation by Nkx2.8 or other active NK2 factors, which only interfered with gene expression. However, two sets of analysis demonstrated the relationship of Nkx2.8 to AFP expression: chromatin immunoprecipitation demonstrated that Nkx2.8 bound to the active AFP promoter, and antisense inhibition of Nkx2.8 mRNA translation selectively reduced expression of both the endogenous human AFP gene and transfected reporters containing the rat AFP promoter.