Icaritin promotes apoptosis and inhibits proliferation by down-regulating AFP gene expression in hepatocellular carcinoma

Icaritin with a purity of up to 99% was a gift from Dr. Kun Meng (Shenogen Biomedical Co., Ltd.) A stock solution was dissolved in dimethyl sulfoxide (DMSO) at various concentrations (2.5, 5, 10, 20, and 40 mM) and stored at − 20 °C.

The human HepG2 cell line (AFP-positive and p53-wild-type HCC cell line) was obtained from China Infrastructures of Cell Line Resource and SMMC7721 cell line (AFP-positive and p53-wild-type HCC cell line) was provided by Prof. Fengmin Lu [22] (Peking University Health Science Centre, China). HepG2 and SMMC7721 cells separately maintained in high glucose Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and RPMI 1640 medium (Invitrogen) supplemented with 10% FBS, respectively. PLC cells (AFP-positive and p53-mutant-type HCC cell line) and L02 cells (a normal human liver cell line that produces no detectable AFP) were provided by Prof. Fengmin Lu [8] (Peking University Health Science Centre, China). and maintained in high glucose Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Gibco). Cells were grown at 37 °C in 5% CO₂. Medium was changed to phenol red-free medium with 10% FBS before icaritin was added. All cells were confirmed to be negative for mycoplasma by Mycoplasma Detection Kit (Solarbio, Beijing, China).

Pluc-1 contains 1.8 kb of DNA from the human AFP gene upstream of the translational start site. Luc1–700mut/luc1-900mut contained a mutated p53 site and luc1–700/900mut contained two mutated p53 sites. Mutated primer DNA sequences are listed as follows:

Cells were transiently transfected with plasmids using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol. At 28 h after transfection, cells were treated with icaritin for 20 h, then harvested and lysed for the detection of AFP promoter activity. The lentivirus vector pLL3.7-shp53 expresses short hairpin (sh) RNA targeting p53 mRNA (5′-CCACTTGAUGGAGAGTATT-3′) as previously described [23]. The vector was used to create p53 knockdown cells.

Western blot was performed for the analysis of AFP and p53 expression in HCC cell lines. Briefly, cells were lysed in lysis buffer, and 30 μg of protein was utilized for each western blot. Primary antibodies were against AFP (145501–1-AP, Proteintech), p53 (sc-126, Santa Cruz Biotechnology), Mdm2 (sc-965, Santa Cruz Biotechnology), Flag (F1804, Sigma-Aldrich), p-p53(sc-377,561, Santa Cruz Biotechnology), Arf^p14(sc-53,639, Santa Cruz Biotechnology), pro-caspase-3 and cleaved-caspase-3 (14,220, CST), PTEN (9188, CST) and GAPDH (KM9002, Sungene Biotech). The secondary antibodies IRDye 800-conjugated anti-mouse IgG antibody (610–132-121) and DyLight 800-conjugated affinity-purified anti-rabbit IgG (611–145-002) were purchased from Rockland. Immunocomplexes were visualized by Odyssey infrared imaging system (LI-COR Bioscience, Lincoln, NE).

Total RNA was isolated using the RNAsimple Total RNA kit (Tiangen). The cDNA was synthesized using ReverAid First Strand cDNA Synthesis kit (Thermo Scientific) and then analyzed by real-time PCR analysis with Maxima SYBR Green qPCR Master Mix (Thermo Scientific). The relative content of AFP and p53 mRNA was presented as a fold-change compared with the control. Primer DNA sequences are listed as follows:

Cells for the immunoprecipitation assay were lysed in IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) containing Protease Inhibitor Cocktail (Sigma). Cell extracts were incubated with specified antibodies or control IgG overnight at 4 °C with constant rotation. Protein A Sepharose (GE) was then added to the complexes for 2 h, which were washed three times with IP lysis buffer, and subsequently resolved by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) followed by western blotting analysis.

A total of 2 μg of normal IgG or antibodies against p53 was used to perform the ChIP assay using Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore) following ChIP assay instructions. PCR was used to detect binding between p53 and the AFP promoter. Quantitative (q)PCR was performed to determine the influence of icaritin on the capacity of p53 binding to DNA. Data processing was achieved according to our previously published articles [23]. Primer DNA sequences are listed as follows:

To detect p53 ubiquitination levels, HepG2 and SMMC7721 cells were cotransfected with various plasmids or treated with 20 μM icaritin. A total of 14 h after transfection and icaritin treatment, cells were treated with 10 μM MG132 (Merck Millipore) for 6 h, then whole cell lysates prepared by Flag-lysis buffer (50 mM Tris-HCl pH 7.8, 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100, 0.2% Sarkosyl, 1 mM DTT, 10% glycerol, and fresh protease inhibitors) were immunoprecipated with an anti-p53 antibody and resolved by SDS–PAGE followed by western blot analysis.

The protein half-life assay was used to detect the influence of icaritin on p53 post-transcriptional regulation. HepG2 and SMMC7721 cells were treated with icaritin as indicated in individual experiments. A total of 20 h after treatment, 100 μg/ml of cycloheximide was added to the dishes, and this treatment was terminated after 0, 15, 30, 45, 60, or 120 min as indicated. Whole cell lysates were collected and 20 μg or 40 μg of total protein from each sample was analyzed by western blot with an anti-p53 antibody. P53 protein quantification was determined using IMAGE-J software, normalized to GAPDH.

Flow cytometry was performed to determine the effect of icaritin and AFP on apoptosis. After treatment of HepG2 cells (+/− p53) and SMMC7721 cells (+/− p53) for 24 h with icaritin, apoptosis induced by icaritin was analyzed by flow cytometry. Cells were collected and re-suspended in 70% ethanol after washing. They were thenstained using an Annexin V/propidium iodide (PI) Apoptosis Detection Kit (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer’s instructions. Relative fluorescent intensities of PI staining were measured using a FACScan-420 flow cytometer (Becton Dickinson). The extent of cellular apoptosis was determined according to DNA analysis. This experiment was repeated at least three times.

HepG2 and SMMC7721 cells were stably transfected with either control plasmid or vector pLL3.7-shp53 following selection with puromycin (0.4 μg/ml) and/or G418 (400 μg/ml), cells were seeded into 96-well plates at a density of 2000 cells/well. After culturing for 2, 4, 6, 8, 12, 20, or 24 h, 15 μl of MTT solution (5 mg/ml) was added to each well, followed by further incubation at 37 °C for 4 h. Medium was then removed and 200 μl of DMSO was added to each well to dissolve the formazan crystals. Absorbance at 490 nm was determined using a microplate reader.

DNA synthesis in HepG2 and SMMC7721 cells was performed by the EdU incorporation assay (RIBOBIO) following the manufacturer’s instructions. Cells were incubated with an EdU-labeling solution for 2 h at 37 °C and fixed with 4% paraformaldehyde for 30 min. After permeabilization, cells were reacted with the reaction solution for 30 min. Subsequently, cell nuclei were stained with 1 × Hoechst 33342 for 30 min, then photographed under a fluorescent microscope (Olympus). Finally, the proliferation rate was calculated.

The results of multiple observations are presented as the mean ± standard deviation of at least three separate experiments. Statistical significance was determined using the Student’s t test (SPSS 17.0 software). P values p < 0.01 or 0.05 were considered statistically significant.

Cytoplasmic AFP has been shown to promote tumor cell proliferation, inhibit cell apoptosis, and to play an important role in HCC occurrence and development. To determine the effect of icaritin on AFP expression, western blotting and qRT-PCR were used in icaritin-treated and -untreated hepatoma cells (HepG2 cells and SMMC7721 cells). As shown in Fig. 1, icaritin inhibited AFP expression at both the mRNA and protein level. 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. qRT-PCR 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.

To analyze the activity of elements in the 5′ regulatory region of AFP and the impact of icaritin on the AFP promoter, we constructed three AFP promoter–luciferase reporters with the native AFP segment (1871 bp, 448 bp, or 215 bp from the translational start site), designated pLuc1, pLuc2, and pLuc3, respectively. The three plasmids were transfected into HepG2 cells following 20 μM icaritin treatment. Analysis with the luciferase activity assay showed that icaritin repressed the pLuc1 promoter compared with the other two constructs (Fig. 2a), possibly by acting as a silencer.

P53-mediated repression of AFP expression has been reported to involve direct interaction with a p53 DNA binding element or chromatin structure alteration at the core promoter [24]. Therefore, pLuc-1 was analyzed and predicted to contain two new p53 affinity sites at − 730 to − 760 bp and − 910 to − 940 bp in the 5′ regulatory region of AFP (Fig. 2b). The ChIP assay was used to validate the p53 affinity sites. The result of ChIP assay showed icaritin enhanced the ability of p53 binding to the promoter of AFP in vivo (Fig. 2b). Three p53-binding defective mutants (luc1–700mut, luc1-900mut, and luc1–700/900mut) containing one or two point mutations in the p53 binding element that abolish p53 binding were used to investigate sequence-specific repression of the AFP promoter. Icaritin treatment decreased native AFP promoter activity, but had no effect on that of p53-binding defective mutants (Fig. 2c). Western blot demonstrated that p53 protein levels and phosphorylated p53 were up-regulated and AFP was down-regulated after 20 μM icaritin treatment for 20 h in HepG2 and SMMC7721 cells (Fig. 2d). While icaritin had no effect on the expression of p53 positive regulator ARF^p14. This indicated Arf^p14 was not involved in the up-regulation of p53 induced by icaritin. When p53 protein was knocked-down, the expression of AFP was increased. Moreover, icaritin up-regulated p53 expression and suppressed AFP expression compared with p53 knockdown group (Fig. 2d). These results further demonstrated that p53 negatively regulated AFP expression through direct binding to the AFP promoter, and that icaritin down-regulated AFP expression through up-regulating p53 protein expression.

The above results indicated that p53 protein expression was increased after icaritin treatment. We used qRT-PCR to determine whether icaritin affected p53 expression at the transcriptional level. As shown in Fig. 3a, icaritin treatment had no effect on p53 mRNA in HepG2 and SMMC7721 cells, suggesting that p53 protein may be regulated by icaritin at the post-transcriptional level. Therefore, we investigated p53 protein stability using a half-life assay. As shown in Fig. 3b, the half-life of p53 was prolonged after icaritin treatment. We next sought to elucidate the potential mechanism by which icaritin enhances p53 protein stability using a p53 ubiquitination assay. HepG2 cells were harvested 14 h after the addition of icaritin following treatment with 10 μM proteasome inhibitor MG132 for 6 h, and ubiquitinated p53 was detected by western blotting. As shown in Fig. 3c, icaritin inhibited both endogenous p53 ubiquitination degradation (left panel) and the ubiquitination degradation of overexpressed exogenous p53 (right panel). Together, these results suggest that icaritin enhanced p53 stability by inhibiting p53 ubiquitination and degradation.

The E3 ubiquitin ligase Mdm2 is a major negative regulator of p53 expression, so we next tested whether icaritin up-regulated p53 protein expression by repressing Mdm2 expression. As shown in Fig. 4a, icaritin treatment down-regulated Mdm2 protein expression, while CoIP revealed that icaritin reduced the interaction between Mdm2 and p53 (Fig. 4b). Then we examined the effect of Mdm2 protein on inhibtion of p53 ubiquitination caused by icaritin. We introduced exogenous Mdm2 and Ub into HepG2 and SMMC7721 cells, and added icaritin after 28 h. Cells were harvested 14 h after the addition of icaritin following treatment with 10 μM proteasome inhibitor MG132 for 6 h, and ubiquitinated p53 was detected by western blot. As shown in Fig. 4c, icaritin inhibited p53 ubiquitination, while Mdm2 protein overexpression rescued the inhibition in HepG2 and SMMC7721 cells. These results indicate that icaritin stabilized p53 by inhibiting Mdm2-mediated p53 ubiquitination.

To further determine the role of the p53/AFP pathway in the effect of icaritin on hepatoma cells, western blot, MTT assays, EdU assays, and flow cytometric analysis were performed. In Fig. 5a, activated caspase 3 and PTEN were up-regulated after icaritin treatment. MTT and EdU assays showed that icaritin decreased the proliferation of HepG2 and SMMC7721 cells compared with control cells treated with DMSO (Fig. 5b and c). However, icaritin had little effect on the proliferation of L02 human liver cells (Fig. 5b and c). These results implied that icaritin has a selective anticancer effect. To investigate the role of p53 protein in icaritin-induced cellular proliferation inhibition, we constructed p53 knockdown HepG2/SMMC7721 cells. As shown in Fig. 5b and c, when p53 was knocked down by shRNA the viability and proliferation of HCC cells was increased; this effect was rescued by icaritin. We also verified that p53 knockdown inhibited icaritin-induced apoptosis (Fig. 5d). Taken together, these data showed that icaritin decreased HCC cell proliferation and promoted HCC cell apoptosis through up-regulating p53 protein expression.

To determine the role of AFP in icaritin-induced cellular proliferation, we constructed AFP-overexpressing HepG2/SMMC7721 cells treated with icaritin. Figure 5b and c showed that icaritin inhibited HCC cell proliferation and AFP overexpression rescued this inhibition. We also used flow cytometry to reveal that icaritin increased the percentage of apoptotic HCC cells compared with the DMSO group and AFP overexpression inhibited the apoptosis induced by icaritin. However, icaritin had little effect on the apoptosis of L02 human liver cells (Fig. 5d). These results suggest that icaritin promoted cell apoptosis by increasing p53 protein expression and down-regulating AFP protein expression. In order to assess the effect of icaritin on the tumorigenic behavior of liver cancer cells in vivo, we tested xenograft growth with HepG2 cells in a nude mouse model. As shown in Fig. 5e, Icaritin inhibited tumorigenesis of HepG2 cells. We extracted tumor tissue protein and performed western blot. The data showed p53 was upregulated and AFP was reduced in icaritin-treated HCC xenografts. According mentioned above, icaritin inhibited cell proliferation and promoted cell apoptosis by regulating the p53–AFP signaling pathway. Compared to icaritin, the effect of standard chemotherapy drug gemcitabine (GEM) on p53-AFP axis was tested. The data showed GEM had no any effect on p53-AFP axis (Fig.S1).