3β-Hydroxysterol Δ24-Reductase on the Surface of Hepatitis C Virus-Related Hepatocellular Carcinoma Cells Can Be a Target for Molecular Targeting Therapy

<div><p>In our previous study, we demonstrated that 3β-hydroxysterol Δ24-reductase (DHCR24) was overexpressed in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), and that its expression was induced by HCV. Using a monoclonal antibody against DHCR24 (2-152a MAb), we found that DHCR24 was specifically expressed on the surface of HCC cell lines. Based on these findings, we aimed to establish a novel targeting strategy using 2-152a MAb to treat HCV-related HCC. In the present study, we examined the antitumor activity of 2-152a MAb. In the presence of complement, HCC-derived HuH-7 cells were killed by treatment with 2-152a MAb, which was mediated by complement-dependent cytotoxicity (CDC). In addition, the antigen recognition domain of 2-152a MAb was responsible for the unique anti-HCV activity. These findings demonstrate the feasibility of using 2-152a MAb for antibody therapy against HCV-related HCC. In addition, surface DHCR24 on HCC cells exhibited a functional property, agonist-induced internalization. We showed that 2-152a MAb-mediated binding of a cytotoxic agent (a saponin-conjugated secondary antibody) to surface DHCR24 led to significant cytotoxicity. This suggests that surface DHCR24 on HCC cells can function as a carrier for internalization. Therefore, surface DHCR24 could be a valuable target for HCV-related HCC therapy, and 2-152a MAb appears to be useful for this targeted therapy.</p></div

Specific uptake mediated by cell-surface DHCR24.

<p>(<i>a</i>) HCC cell lines (HuH-7, Hep3B, and PLC/PRF/5) and HeLa cells were incubated with 2-152a MAb at 4°C (a temperature that inhibits endocytosis) or 37°C (physiological temperature) for 2 h, and then incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. (<i>b</i>) HuH-7 cells were seeded at a density of 5 × 10³ cells/well in 96-well tissue culture plates. After incubation for 24 h, serial dilutions of 2-152a MAb or mouse IgG were added in the presence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was then assessed using the BrdU ELISA assay kit. Average viability was calculated relative to the viability of untreated cells, which was set at 100%. (<i>c</i>) HeLa, Hep3B, and PLC/PRF/5 cells were treated with 2-152a MAb or mouse IgG (10 μg/mL) in the presence or absence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was determined using a BrdU ELISA assay kit. Percent viability was calculated relative to the viability of untreated cells, which was set at 100%. *, <i>p</i> < 0.05.</p

Complement-dependent cytotoxicity (CDC) induced by 2-152a MAb.

<p>HeLa, HepG2, and HuH-7 cells (1 × 10⁶) were incubated with 2-152a MAb or mouse IgG (final concentration, 1 or 10 μg/mL) in the presence of guinea pig complement (the complement:cell ratio was 1:8 for HeLa and HepG2 and 1:20 for HuH-7) for 30 minutes at 30°C. Cell viability was measured using the WST-8 cell counting kit, and cell killing was calculated by comparing death in the experimentally treated cells with that in the untreated cells. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01.</p

Overexpression of DHCR24 on the cell surface of HCC cell lines was decreased by treatment with U18666A and cyclosporin A.

<p>(<i>a</i>) Cell lysates of each cell line (containing 50 μg of protein) were separated by 10% SDS-PAGE and analyzed by western blotting with 2-152a MAb and an anti-actin MAb. Normal hepatic cell lines: NKNT, and TTNT. HB-derived cell line: HepG2. HCC-derived cell lines: HuH-7, Hep3B, and PLC/PRF/5. (<i>b</i>) HuH-7 cells were treated with U18666A (final concentration, 1 μM) for 48 h, and then the surface expression of DHCR24 was analyzed by flow cytometry. (<i>c</i>) HuH-7 cells were treated with cyclosporin A (final concentration, 5 or 10 μM) or solvent (cremophor) for 48 h, and then the surface expression of DHCR24 was analyzed by flow cytometry.</p

152a ChAb can bind to surface DHCR24 and shows anti-HCV activity.

<p>(<i>a</i>) HuH-7 (1 × 10⁶) cells were incubated with the light or heavy chain of the chimeric Ig (152a Ch-L, 152a Ch-H) or 152a ChAb (1 μg/mL, respectively) at 4°C for 2 h, and then with an Alexa Fluor 488-conjugated goat anti-human IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. (<i>b</i>) HuH-7 (1 × 10⁶) cells were incubated with 152a scFv-hIgG1-Fc at 4°C for 2 h, and then incubated with Alexa Fluor 488-conjugated goat anti-human IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. (<i>c</i>) Subgenomic HCV replicon FLR3-1 cells were plated in a 96-well plate at a density of 5 × 10³ cells/well and allowed to adhere overnight. Then, the supernatant was removed, and the cells were treated with the light or heavy chain of the chimeric Ig (152a Ch-L, 152a Ch-H) or 152a ChAb (1 μg/mL, respectively) for 72 h. HCV replication was evaluated by measuring luciferase activity using the Bright-Glo Luciferase Assay System. *, <i>p</i> < 0.05. (<i>d</i>) Simultaneously, the viability of FLR3-1 cells was evaluated by measuring the absorbance (OD at 450 nm) using the WST-8 Cell Counting Kit. Experiments were performed 3 times with triplicate wells.</p

Specific uptake mediated by cell-surface DHCR24.

<p>(<i>a</i>) HCC cell lines (HuH-7, Hep3B, and PLC/PRF/5) and HeLa cells were incubated with 2-152a MAb at 4°C (a temperature that inhibits endocytosis) or 37°C (physiological temperature) for 2 h, and then incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. (<i>b</i>) HuH-7 cells were seeded at a density of 5 × 10³ cells/well in 96-well tissue culture plates. After incubation for 24 h, serial dilutions of 2-152a MAb or mouse IgG were added in the presence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was then assessed using the BrdU ELISA assay kit. Average viability was calculated relative to the viability of untreated cells, which was set at 100%. (<i>c</i>) HeLa, Hep3B, and PLC/PRF/5 cells were treated with 2-152a MAb or mouse IgG (10 μg/mL) in the presence or absence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was determined using a BrdU ELISA assay kit. Percent viability was calculated relative to the viability of untreated cells, which was set at 100%. *, <i>p</i> < 0.05.</p

Construction of a chimeric antibody and scFv consisting of the 2-152a MAb antigen-binding domain and the human IgG constant domain.

<p>(<i>a</i>) The 2-152a VL and VH cDNAs were isolated from hybridoma #2-152a and cloned into pFUSE2ss-CLIg-hk and pFUSEss-CHIg-hG1, respectively. HEK293 cells were co-transfected with the expression vector harboring the chimeric Ig (152a Ch-L; pFUSE2ss-152aVL-CLIg-hk, 152a Ch-H; pFUSEss-152aVH-CHIg-hG1). The 152a Chimera Ab or chimeric Ig (152a Ch-L, 152a Ch-H) was secreted into the culture medium and then purified from the culture medium by using protein A/G/L sepharose. (<i>b</i>) The 152a scFvfragments (152a VL-VH, 152a VH-VL) were constructed by SOE-PCR and cloned into pFUSE-hIgG1e4-Fc2. HEK293 cells were transfected with the 152a scFv-hIgG1-Fc expression vectors (pFUSE-scFv152a(VLVH)-hIgG1-Fc and pFUSE-scFv152a(VHVL)-hIgG1-Fc). 152a scFv-hIgG1-Fc fusion protein was secreted into the culture medium, and then the protein was purified from the culture medium by using protein A/G/L sepharose. (<i>c</i>) Schematic diagram of scFv152a-hIgG1-Fc. The scFv fragments (152a VL-VH or 152a VH-VL) derived from 2-152a MAb were fused to the Fc portion of human IgG1.</p

Colocalization of NS5A and Core protein with exosomal proteins at the plasma membrane.

<p>(A) The HCV-infected cells (at day 10 p.i.) were co-stained with anti-Core (red) (left panel) or -NS5A (red) (middle panel) or -NS4B (red) (right panel) and anti-CD81 (green). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) and DAPI (gray), respectively. Enlarged views of parts of every image (insets) are shown. PM, plasma membrane; Bars, 10 µm. (B, C) Immuno-EM of plasma membrane co-labeled with antibodies against Core (6 nm) (B) or NS5A (6 nm) (C) and CD81 (18 nm) are shown. Arrowheads, gold-labeled CD81. Arrows, gold-labeled Core and NS5A. Bars, 200 nm and 50 nm (B and C, left and right panels, respectively).</p

Nonstructural Protein 5A Is Incorporated into Hepatitis C Virus Low-Density Particle through Interaction with Core Protein and Microtubules during Intracellular Transport

<div><p>Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD) through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.</p></div

Double immunofluorescence localisation of B220 (Green) and NF-κB p65 (Red) in HCV-Tg mice and the fractionation analysis of mouse tissues.

<p><b>A</b>: Co-localisation of NF-κB p65 immunoreactivity with B220 is indicated by arrows. (a–b) Cells double-positive for B220 and NF-κB in the control mouse (CD19cre). (c–d) Cells double-positive for B220 and NF-κB in the asymptomatic HCV-Tg mouse (RzCD19cre). (e–f) Cells double-positive for B220 and NF-κB in the lymphomatous HCV-Tg mouse (RzCD19cre). <b>B</b>: Quantitative analysis of the ratio of double-positive cells among B220-positive cells in each HCV-Tg mouse. Bar graph indicates the percentage of cells with NF-κB p65 nuclear translocation in B220-positive cells. <b>C</b>: Bar graph shows the ratio of double-positive cells within the B220-positive cells in normal, asymptomatic and lymphomatous HCV-Tg mice. Ho: Hoechst33342 Data are presented as means ± S.E., * P<0.05, ** P<0.01, *** P<0.001. <b>D</b>: Western blot analysis: tissues from the spleen of controls (224–2, 3) or HCV-Tg mice without BCL (217–3, 224–4, 232–3) or with BCL (56–5, 69–5) were fractionated into nuclear and cytoplasmic fractions. NF-κB p50 and p65 were detected by antibodies. Relative ratios of quantitation by imager are indicated. GAPDH was detected as a loading control of the cytoplasmic fraction.</p