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

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

* months from treatment initiation.

RPR ratio compared with RPR at treatment initiation is shown. (XLSX)</p

Association between the slope of the ratio of the post-treatment RPR titer compared to the baseline titer and other characteristics using linear regression analysis.

Association between the slope of the ratio of the post-treatment RPR titer compared to the baseline titer and other characteristics using linear regression analysis.</p

Flow chart of the study procedures.

IntroductionAlthough the rapid plasma reagin (RPR) test is used to determine treatment efficacy for syphilis, animal studies show that it decreases gradually after an initial increase even without treatment. Pre-treatment changes in RPR titer in humans and its relationship with post-treatment changes in RPR titer are not well known.MethodsWe retrospectively analyzed the clinical records of syphilitic patients who underwent automated RPR (Mediace) testing twice before treatment (i.e., at diagnosis and treatment initiation) within 1–3 months at an HIV/AIDS referral hospital in Japan between 2006 and 2018. The RPR values were expressed as the ratio to the value at treatment initiation. The mean monthly relative change in the RPR after treatment was calculated on the log2 scale for each patient and analyzed by multivariable linear regression.ResultsSixty-eight patients were identified. The median age was 45 (interquartile range [IQR], 38–50), 98.5% (67/68) were men, and 97.1% (66/68) had HIV. The median RPR titer ratio at treatment initiation/diagnosis was 0.87 (IQR, 0.48–1.30). The RPR titer decreased more than twofold in 26.5% (18/68) and more than fourfold in 10.3% (7/68) before treatment. In the multivariable analysis, higher age (predicted monthly RPR relative change on the log2 scale 0.23/10 years [95% confidence interval [CI], 0.090–0.37]), history of syphilis (0.36 [95% CI, 0.07–0.65]), and a lower ratio of RPR at treatment initiation/diagnosis (−0.52/every 10-fold increase [95% CI, −0.81 to −0.22]) were associated with a slower RPR decrease after treatment.ConclusionsIn a mostly HIV patient population, RPR titer can show more than four-fold spontaneous increase or decrease within 1–3 months. Pre-treatment spontaneous decrease of RPR titer was associated with a slower decrease in post-treatment RPR titer.</div

Time series of CH₄ and CO concentrations for 2010 and 2011.

<p>(a) Variability in XCH₄ concentrations (ppb) in the southern Amazon region (dark green circles), central South America region (light green circles), and background region (ocean area between 0°–20°S and 20°–50°W; red line). Daily data of the background region were constructed from discrete XCH₄ data using a Humming window with a window-width of 80 days. The standard errors (one sigma) of XCH₄ data are given by the gray boundary. The discontinuity at the background region for May 22–June 20 in 2011 reflects a lack of XCH₄ data. (b) Monthly variability in XCH₄ values (ppb) with the background subtracted (ΔXCH₄) in the southern Amazon region (dark green box-and-whiskers) and the central South America region (light green box-and-whiskers). Upper and lower values of the boxes are monthly quartiles of ΔXCH₄, bars through the boxes are medians, and whiskers represent the range of the 10^th and 90^th percentiles (data are not shown for months in which the number of data points was <25). (c) Same as (b), but showing surface CO concentrations (ppb) from the MOPITT L3 product.</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

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

Total annual CH₄ flux (upper) and relative contributions of anthropogenic (magenta), natural (blue), and biomass-burning (red) fluxes (lower) in the southern Amazon region (left) and central South America region (right) for 2010 and 2011 (in Tg CH₄ region⁻¹ yr⁻¹).

<p>Error bars represent flux uncertainties.</p