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Inhibitory effect of antibodies against the cell adhesion molecule gicerin on gastric cancer progression

  • Authors:
    • Saaya Ueno
    • Nao Uemura
    • Kazuhide Adachi
    • Yasuhiro Tsukamoto
  • View Affiliations / Copyright

    Affiliations: Laboratory of Animal Hygiene, Department of Agricultural and Life Science, Faculty of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606‑8522, Japan
    Copyright: © Ueno et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 321
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    Published online on: September 17, 2025
       https://doi.org/10.3892/mmr.2025.13687
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Abstract

Gicerin is a member of the immunoglobulin superfamily, which functions as a cell adhesion molecule that is expressed in developing and regenerating tissues, as well as in various types of tumors. In malignant neoplasms, gicerin has been implicated in promoting cellular invasion and metastasis. The present study aimed to investigate whether or not anti‑gicerin antibodies could inhibit the progression of the human gastric cancer cell line NUGC‑4 in a murine model. First, immunofluorescence staining was employed to confirm gicerin expression in the NUGC‑4 cells. The cells were subsequently pretreated with either anti‑gicerin antibodies or pre‑immune IgG and transplanted subcutaneously into nude mice, where the extent of tumor growth and local invasion were both assessed. In a separate hematogenous metastasis model, mice received a tail‑vein injection of NUGC‑4 cells pretreated in an identical manner and pulmonary metastases were evaluated. Anti‑gicerin antibody pretreatment led to a significant suppression of subcutaneous tumor formation, histological invasiveness and lung nodule formation compared with the controls. Taken together, these findings suggested that the antibody‑mediated inhibition of gicerin reduced both local tumor progression and hematogenous spread in gastric cancer, highlighting gicerin as a promising therapeutic target that potentially may be effective when used in combination with conventional chemotherapy.
View Figures

Figure 1

Generation of mouse anti-gicerin
antibody and validation of its binding activity. (A) The procedure
for generating and purifying the murine anti-gicerin antibody is
summarized. (a) BALB/c mice were immunized with recombinant gicerin
protein, followed by IgG purification from serum. The IgG fraction
was subsequently affinity-purified using a Sepharose column
conjugated with gicerin protein. (b) As shown by SDS-PAGE, the
gicerin recombinant protein immunized as the antigen for antibody
production (lane 1) appeared as a single band at ~120 kDa, while
the final purified mouse IgG (lane 2) exhibited a single band at
~150 kDa. Binding activity of mouse IgG to gicerin proteins by
ELISA. (c) Recombinant proteins of gicerin were bound to plate at 2
µg/ml overnight at 4°C. Samples were blocked with 1% blocking
buffer (Block Ace) in distilled water for 2 h. Serial dilutions of
pre-immune or anti-gicerin IgG were incubated at room temperature
for 2 h. After washing with PBS, HRP-conjugated polyclonal antibody
against mouse IgGs were reacted for 1 h at room temperature. Then,
3,3′,5,5′-tetramethylbenzidine was added to each well and plates
were read at 450 nm utilizing a Multiscan JX (Thermo Fisher
Scientific, Inc.). ELISA demonstrated that the purified antibody
exhibited strong binding activity to gicerin protein, with an ELISA
titer of 12,800 when compared to pre-immune IgG. (B) Neutralizing
activity of anti-gicerin IgG in cell adhesion. (a) In the cell
aggregation assay, L-929 cells expressing gicerin formed
multicellular aggregates upon the addition of pre-immune IgG,
indicating cell-cell adhesion. (b) When the anti-gicerin IgG was
added to the suspension cultures, a marked inhibition of cell-cell
aggregation was observed. Scale bars, 100 µm; magnification, ×200.
(c) Dose-response curves were generated by plotting the aggregation
index against the logarithmic antibody concentrations. Nonlinear
regression analysis determined that the IC50 of
anti-gicerin IgG was 49.5 ng/ml, indicating its potent neutralizing
effect on cell adhesion. IC50, half-maximal inhibitory
concentration.

Figure 2

Gicerin expression in NUGC-4 cells.
(A) Indirect immunofluorescence staining of NUGC-4 cells was
performed and observed under a fluorescence microscope (as shown by
the B-excitation filter). No fluorescence was detected when
pre-immune IgG was applied (a, phase contrast; b,
immunofluorescence). By contrast, strong green fluorescence was
observed on the cell surface following staining with anti-gicerin
IgG (c, phase contrast; d, immunofluorescence), indicating
membrane-associated expression of gicerin. scale bars, 50 µm;
magnification, ×400. (B) Western blot analysis of membrane
fractions from B16 (lane 2), HUVEC (lane 3) and NUGC-4 cells (lane
4) following SDS-PAGE. The recombinant gicerin protein used as the
immunogen was also subjected to electrophoresis as a positive
control and to verify the specificity and reactivity of the
antibody (lane 1). Notably, a doublet band of ~120 kDa was observed
in lane 4, consistent with the expression of both the short and
long isoforms of gicerin in NUGC-4 cells.

Figure 3

Tumor growth and histopathological
features following subcutaneous injection into nude mice. (A)
NUGC-4 cells treated with either pre-immune IgG or anti-gicerin IgG
were subcutaneously injected into the dorsal neck region of nude
mice (n=5 per group). Tumor volume was measured over time using the
formula: AxB2/2, where A and B represent the maximum and
minimum tumor diameters, respectively. Data are mean ± SD (n=5 mice
per group). Two-tailed unpaired Student's t-tests were performed
between pre-immune IgG and anti-gicerin IgG at each time point.
Significance indicators: ns (P≥0.05), *P<0.05, **P<0.01. (B)
On day 15 post-injection, tumor tissues were examined both
histologically and using immunofluorescence analysis. (a) Gicerin
expression was detected in the tumor cell membranes, especially in
epithelial-like structures, as well as in neovascular structures
(denoted by the arrows) scale bars, 100 µm; magnification, ×200.
Compared with mice injected with pre-immune IgG (b and c), those
injected with anti-gicerin IgG displayed smaller tumor masses and
reduced tumor areas histologically with hematoxylin and eosin
staining (arrows) (d and e). (C) In the pre-immune IgG group,
histological evidence of tumor invasion into the dermis (a),
adipose tissue (b), subcutaneous muscle (c) and undifferentiated
adenocarcinoma (d) regions was noted (arrows indicate invasion into
the muscle). (e-g) By contrast, mice treated with anti-gicerin IgG
showed markedly reduced tumor invasiveness. (h) Furthermore, signs
of cellular degeneration, structural disintegration, pyknosis and
karyorrhexis were frequently observed within tumors treated with
anti-gicerin antibody. Scale bars, 100 µm; magnification: a and e:
×100; b, f and g: ×50; and c, d and h, ×200.

Figure 4

Pulmonary metastasis following tail
vein injection in nude mice. (A) NUGC-4 cells pretreated with
either pre-immune IgG or anti-gicerin IgG were injected into the
tail veins of nude mice (n=5 per group). On day 15, lung tissues
were harvested and examined histologically. (a) In the pre-immune
IgG group, multiple metastatic foci were observed, characterized by
epithelial-like tumor cell proliferation and frequent hemorrhaging.
(b) By contrast, few metastatic lesions were detected in the
anti-gicerin IgG group. Scale bar, 100 µm; magnification, ×100. (B)
The number of metastatic foci per unit area in lung tissue was
quantified histologically. Data are shown as mean ± SD (n=5 mice
per group). Between-group comparison was performed with a
two-tailed unpaired Student's t-test; the difference was
statistically significant. Significance indicators: ns (P≥0.05),
*P<0.05, **P<0.01.

Figure 5

(A) Inhibition of NUGC-4 cell
adhesion to gicerin protein by anti-gicerin antibody. Only the
right half of each culture dish was coated with recombinant gicerin
protein. NUGC-4 cell adhesion was assessed under a phase-contrast
microscope. (a) The cells predominantly adhered to the
gicerin-coated region, indicating that NUGC-4 cells exhibit
homophilic binding through gicerin molecules expressed on their
surface. Adhesion was not inhibited by pre-immune IgG. (b) By
contrast, treatment with anti-gicerin IgG markedly suppressed the
attachment of NUGC-4 cells to the gicerin-coated area. These
findings demonstrate that anti-gicerin IgG effectively blocks the
homophilic adhesion ability of NUGC-4 cells via gicerin. Scale
bars, 100 µm; magnification ×200. (B) Adhesion of NUGC-4 cells to
HUVEC monolayers. (a) Immunofluorescence staining revealed gicerin
expression on both adherent NUGC-4 cells and HUVEC feeder cells.
NUGC-4 cells treated with either (b) pre-immune IgG or (c)
anti-gicerin IgG were seeded on to confluent HUVEC monolayers.
Compared with the pre-immune IgG group, the number of NUGC-4 cells
attached to HUVEC layers was noticeably reduced in the anti-gicerin
IgG group. Scale bar, 100 µm. Magnification: a, ×200; and b and c,
×50. (C) The number of NUGC-4 cells adhering to HUVECs was markedly
lower in the anti-gicerin IgG group compared with the pre-immune
control. Adherent cells were counted in randomly selected 0.3
mm2 areas under light microscopy. Data are mean ± SD
(n=5 independent replicates per group). Group differences were
analyzed by one-way ANOVA followed by Tukey's HSD test across PBS,
pre-immune IgG and anti-gicerin IgG conditions. Anti-gicerin IgG
differed markedly from the other groups (***P<0.001), whereas
other pairwise comparisons were not significant unless indicated.
(D) Inhibition of NUGC-4 cell migration by anti-gicerin antibody.
NUGC-4 cells were seeded on culture dishes pre-coated with gicerin
protein and incubated until they reached confluence. (a) A linear
scratch was made across the cell monolayer using the tip of a
yellow pipette to create a cell-free area. (b) Culture media
containing either pre-immune IgG or anti-gicerin IgG was then added
and cell migration into the scratched area was monitored over time
using phase-contrast microscopy. (b) In the presence of pre-immune
IgG, numerous cells migrated into the scratch area by 48 h and (c)
the wound was completely closed by 96 h. (d) By contrast, dishes
treated with anti-gicerin IgG showed (e) markedly reduced cell
migration at 48 h and (f) a large cell-free area remained even
after 96 h. These findings indicate that anti-gicerin IgG inhibits
gicerin-mediated cell motility. The dashed straight line indicates
the edge of the wound at each time point after the initial scratch
procedure. Scale bar, 100 µm; magnification: ×100.

Figure 6

Complement-dependent cytotoxicity
assay. (A) NUGC-4 cells were incubated with either pre-immune IgG
or anti-gicerin IgG, followed by treatment with either fresh mouse
serum (complement-active) or heat-inactivated serum (heated at 56°C
for 30 min). After a 1 h incubation at 37°C, non-adherent cells
were removed and adherent cells were detached by treating the cells
with trypsin-EDTA. Cell viability was assessed using the trypan
blue exclusion method under light microscopy (a, with PBS; b, with
pre-immune IgG, c, with anti-gicerin IgG; d, with pre-immune IgG
and inactivated serum; e, with anti-gicerin IgG and inactivated
serum; f, pre-immune IgG and fresh serum; g, with anti-gicerin IgG
and fresh serum. (B) Viable (unstained) and nonviable
(blue-stained) cells were counted and the percentages of dead cells
were calculated. Data are mean ± SD (n=5 independent replicates per
group). Seven groups were compared by one-way ANOVA with Tukey's
HSD; only the cell + anti-gicerin IgG + fresh mouse serum group
differed markedly from all other groups (***P<0.001). (C)
Doubling times of NUGC-4 cells with anti-gicerin antibody. The
cells were cultured with PBS, pre-immune IgG and anti-gicerin IgG
and doubling times in each condition were calculated. As shown in
the graph, anti-gicerin antibody did not affect the proliferation
of NUGC-4 cells under in vitro conditions, similar to
pre-immune IgG. Data are presented as mean ± SD (n=4 independent
replicates per group). Differences among the three groups (PBS,
pre-immune IgG and anti-gicerin IgG) were evaluated by one-way
ANOVA followed by Tukey's HSD post hoc test; no statistically
significant differences were detected.
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Copy and paste a formatted citation
Spandidos Publications style
Ueno S, Uemura N, Adachi K and Tsukamoto Y: Inhibitory effect of antibodies against the cell adhesion molecule gicerin on gastric cancer progression. Mol Med Rep 32: 321, 2025.
APA
Ueno, S., Uemura, N., Adachi, K., & Tsukamoto, Y. (2025). Inhibitory effect of antibodies against the cell adhesion molecule gicerin on gastric cancer progression. Molecular Medicine Reports, 32, 321. https://doi.org/10.3892/mmr.2025.13687
MLA
Ueno, S., Uemura, N., Adachi, K., Tsukamoto, Y."Inhibitory effect of antibodies against the cell adhesion molecule gicerin on gastric cancer progression". Molecular Medicine Reports 32.6 (2025): 321.
Chicago
Ueno, S., Uemura, N., Adachi, K., Tsukamoto, Y."Inhibitory effect of antibodies against the cell adhesion molecule gicerin on gastric cancer progression". Molecular Medicine Reports 32, no. 6 (2025): 321. https://doi.org/10.3892/mmr.2025.13687
Copy and paste a formatted citation
x
Spandidos Publications style
Ueno S, Uemura N, Adachi K and Tsukamoto Y: Inhibitory effect of antibodies against the cell adhesion molecule gicerin on gastric cancer progression. Mol Med Rep 32: 321, 2025.
APA
Ueno, S., Uemura, N., Adachi, K., & Tsukamoto, Y. (2025). Inhibitory effect of antibodies against the cell adhesion molecule gicerin on gastric cancer progression. Molecular Medicine Reports, 32, 321. https://doi.org/10.3892/mmr.2025.13687
MLA
Ueno, S., Uemura, N., Adachi, K., Tsukamoto, Y."Inhibitory effect of antibodies against the cell adhesion molecule gicerin on gastric cancer progression". Molecular Medicine Reports 32.6 (2025): 321.
Chicago
Ueno, S., Uemura, N., Adachi, K., Tsukamoto, Y."Inhibitory effect of antibodies against the cell adhesion molecule gicerin on gastric cancer progression". Molecular Medicine Reports 32, no. 6 (2025): 321. https://doi.org/10.3892/mmr.2025.13687
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