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

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 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

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

Targeting Anticancer Drug Delivery to Pancreatic Cancer Cells Using a Fucose-Bound Nanoparticle Approach

<div><p>Owing to its aggressiveness and the lack of effective therapies, pancreatic ductal adenocarcinoma has a dismal prognosis. New strategies to improve treatment and survival are therefore urgently required. Numerous fucosylated antigens in sera serve as tumor markers for cancer detection and evaluation of treatment efficacy. Increased expression of fucosyltransferases has also been reported for pancreatic cancer. These enzymes accelerate malignant transformation through fucosylation of sialylated precursors, suggesting a crucial requirement for fucose by pancreatic cancer cells. With this in mind, we developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specifically to cancer cells. L-fucose-bound liposomes containing Cy5.5 or Cisplatin were effectively delivered into CA19-9 expressing pancreatic cancer cells. Excess L-fucose decreased the efficiency of Cy5.5 introduction by L-fucose-bound liposomes, suggesting L-fucose-receptor-mediated delivery. Intravenously injected L-fucose-bound liposomes carrying Cisplatin were successfully delivered to pancreatic cancer cells, mediating efficient tumor growth inhibition as well as prolonging survival in mouse xenograft models. This modality represents a new strategy for pancreatic cancer cell-targeting therapy.</p> </div

Production and physicochemical properties of L-fucose-bound liposomes.

<p>(<b>A)</b> Liposome preparation scheme showing sugar chains. HSA, BS³, Tris, and DTSSP denote the following, respectively: human serum albumin; bis(sulfosuccinimidyl) suberate; Tris(hydroxymethyl) aminomethane; 3,3-dithiobis (sulfosuccinimidylpropionate). (<b>B)</b> Electron microscopic image of L-fucose-bound liposome. Scale bar shows 50 nm. (<b>C, D)</b> Physicochemical characterization of Fuc-Liposome-Cy5.5. Average particle size <b>(C)</b> and zeta-potential <b>(D)</b> of liposomes that were prepared in water was determined by dynamic light scattering spectrophotometry.</p

Pretreatment with D-mannose does not affect accumulation of F50-Liposome-Cy5.5.

<p>(<b>A</b>) Fuc-Liposome- or Liposome-Cy5.5 was administered via the tail vein (50 µl/mouse). The tumor regions of MIA PaCa-2, BxPC-3 and AsPC-1 cells (the back side of a bilateral flank lesion) in the mouse was observed and Cy5.5 accumulation was quantified using the IVIS imaging system at 96 hours after injection. D-mannose (1000-fold of the L-fucose) was injected simultaneously with liposome injection. (<b>B</b>) Total flux of the tumor and liver was calculated by using Living Image software according to the manufacturer’s instructions.</p

Receptor-mediated uptake of Fuc-Liposomes by pancreatic cancer cells.

<p>(<b>A</b>) Incorporation of ¹⁴C-labeled-L-fucose in AsPC-1 cells. Cells were incubated in the presence or absence of excess L-fucose (excess cold) in the ¹⁴C-labeled-L-fucose-containing medium for the indicated time, then ¹⁴C-labeled-L-fucose incorporation was measured. (<b>B</b>) BxPC-3 cells were incubated with or without chroloquine for 24 hours, treated with F50-Liposome-Cy5.5 for 2 hours at 37°C, and then analyzed by flow cytometry. (<b>C, D</b>) ¹⁴C-labeled-L-fucose binding assay using AsPC-1 cells. Scatchard plot analysis revealed 3.25×10⁶ receptors/cell, a K_d of 28.74 nM, and a Bmax of 5.49 pmol/10⁶ cells. Methods are described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039545#s4" target="_blank">Materials and Methods</a></i>.</p