Activated p53 with Histone Deacetylase Inhibitor Enhances L-Fucose-Mediated Drug Delivery through Induction of Fucosyltransferase 8 Expression in Hepatocellular Carcinoma Cells

<div><p>Background</p><p>The prognosis of advanced hepatocellular carcinoma (HCC) is dismal, underscoring the need for novel effective treatments. The α1,6-fucosyltransferase (fucosyltransferase 8, FUT8) has been reported to accelerate malignant potential in HCC. Our study aimed to investigate the regulation of FUT8 expression by p53 and develop a novel therapeutic strategy for targeting HCC cells using L-fucose-mediated drug delivery.</p><p>Methods</p><p>Binding sites for p53 were searched for within the <i>FUT8</i> promoter region. FUT8 expression was assessed by immunoblotting. Chromatin immunoprecipitation (ChIP) assays were performed to analyze p53 binding to the <i>FUT8</i> promoter. The delivery of Cy5.5-encapsulated L-fucose-liposomes (Fuc-Lip-Cy5.5) to a <i>Lens Culinaris</i> agglutinin-reactive fraction of α-fetoprotein (AFP-L3)-expressing HCC cells was analyzed by flow cytometry. The induction of FUT8 by histone deacetylase inhibitor (HDACi) -inducing acetylated -p53 was evaluated by immunoblotting. Flow cytometric analysis was performed to assess whether the activation of p53 by HDACi affected the uptake of Fuc-Lip-Cy5.5 by HCC cells. The cytotoxicity of an L-fucose-bound liposome carrying sorafenib (Fuc-Lip-sorafenib) with HDACi was assessed <i>in vivo</i> and <i>in vitro</i>.</p><p>Results</p><p>The knock down of p53 with siRNA led to decreased FUT8 expression. ChIP assays revealed p53 binds to the <i>FUT8</i> promoter region. Flow cytometric analyses demonstrated the specific uptake of Fuc-Lip-Cy5.5 into AFP-L3-expressing HCC cells in a p53- and FUT8-dependent manner. HDACi upregulated the uptake of Fuc-Lip-Cy5.5 by HCC cells by increasing FUT8 via acetylated -p53. The addition of a HDACi increased apoptosis induced by Fuc-Lip-sorafenib in HCC cells.</p><p>Conclusions</p><p>Our findings reveal that <i>FUT8</i> is a p53 target gene and suggest that p53 activated by HDACi induces Fuc-Lip-sorafenib uptake by HCC cells, highlighting this pathway as a promising therapeutic intervention for HCC.</p></div

Combination therapy with HDACi enhances the cytotoxicity of Fuc-Lip-sorafenib.

<p>(A) AFP-L3 expressing HepG2, and JHH7 cells, and AFP-L3 non-expressing JHH6 cells were treated for 2 h with F0-Lip-sorafenib or F50-Lip-sorafenib. The cells were then, washed, incubated for 48 h, and cell viability was measured by BrdU assay. The percentage of viable cells is shown compared with untreated cells. (B) HCC cells were treated with F0-Lip-sorafenib or F50-Lip-sorafenib (5 μM) for 2 h with or without SAHA (1 μM) and, then washed and incubated for 48 h. Cell viability was measured by BrdU assay. Experiments were carried out in triplicate and repeated three times. NT: no treatment. * <i>P</i> < 0.05.</p

p53 and FUT8 regulate the introduction of Fuc-Lip-Cy5.5 into HCC cells.

<p>(A) Expression levels of p53 and FUT8 in HCC cells were examined by western blot. (B) Quantification of AFP-L3 values. (C) Determination of the introduction of Fuc-Lip-Cy5.5 into HCC cells. After siRNA transfection, HepG2 and JHH7 cells were exposed to 1 μM SAHA for 6 h, then treated with F50-Lip-Cy5.5 for 30 min and analyzed by flow cytometry. One siRNA was used for p53 (si-p53) and two were used for FUT8 (si-FUT8-1 and si-FUT8-2). Experiments were carried out in triplicate and repeated at least three times.</p

Identification of the p53-DNA binding site within the <i>FUT8</i> genomic promoter.

<p>(A) Mapping of p53-binding DNA sequences on the <i>FUT8</i> genomic locus. Based on DNA sequence results, each candidate shows high homology to the p53-DNA binding consensus sequence (as indicated by%). Red points indicate mismatched residues compared with the consensus sequence, whereas candidate sites are written in blue. (B) Protein expression of p53 and FUT8 in HepG2 cells was analyzed by western blot after 24- and 48-h of infection with Ad-p53. (C) FUT8 protein expression after p53 knock-down in HepG2 cells was determined by western blot. (D) ChIP assay of HepG2 cells infected with Ad-p53 and Ad-LacZ. The PCR product for the p21 binding site of p53 was used as a positive control. I: 10% input, L: Ad-LacZ infection, P: Ad-p53 infection. NT: no treatment. Experiments were carried out in triplicate and repeated at least three times.</p

Combination of HDACi and Fuc-Lip-sorafenib augments tumor suppression <i>in vivo</i>.

<p>(A) Treatment schedule for the HepG2 xenograft mouse model. F0-Lip-sorafenib (1 mg/kg), or F50-Lip-sorafenib solution (1 mg/kg) was administered via tail vein injection twice a week for seven weeks. SAHA (1 mg/kg) was administered daily intraperitoneally. (B) Tumor volumes for each treatment group. At every treatment schedule, tumor volumes were measured according to the formula = length x width² / 2. (C) Tumor tissue was prepared on day 45 after the start of treatment. HE and TUNEL staining in HepG2 tumors are shown. Veh: Vehicle. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Acetylated-p53 by HDACi up-regulates FUT8 expression and incorporation of Fuc-Lip-Cy5.5 into cells.

<p>(A) HepG2,JHH7 and JHH6 cells were exposed to SAHA for 6 h and then the expression levels of p53, Acetylated-p53 (Ac-p53), and FUT8 proteins were analyzed by western blot. (B) Efficacies of incorporation of Fuc-Lip-Cy5.5 into cells. Cells were treated with SAHA (0, 1, 5 μM) for 6 h, then incubated with Fuc-Lip-Cy5.5 for 30 min, and subsequently incorporation of Cy5.5 was analyzed by flow cytometry. The percentages of Cy5.5-positive cells are shown. F0, F0-Lip-Cy5.5: F25, F25-Lip-Cy5.5: F50, F50-Lip-Cy5.5. Experiments were carried out in triplicate and repeated twice.</p