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In vitro assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma

  • Authors:
    • Dong-Guk Park
    • Hyun-Ji Kim
    • Sak Lee
    • Hye-Mi Jiang
    • Seong-Doo Hong
    • Su-Jung Choi
    • Sung-Dae Cho
  • View Affiliations / Copyright

    Affiliations: Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 03080, Republic of Korea
    Copyright: © Park et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 97
    |
    Published online on: June 17, 2025
       https://doi.org/10.3892/or.2025.8930
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Abstract

STAT3 is abnormally activated in several types of cancer, and elevated nuclear levels of STAT3 are strongly associated with poor prognosis in oral squamous cell carcinoma (OSCC). Despite ongoing progress in developing targeted therapies, there is no Food and Drug Administration‑approved drug currently targeting STAT3 in OSCC. To evaluate the anticancer effects of BBI608, a potent STAT3 inhibitor, in two human OSCC cell lines (HSC‑3 and HSC‑4), various two‑dimensional (2D) or 3D in vitro analyses were performed, including western blot analysis, colony formation assay, DAPI staining, sub‑G1 population analysis and Annexin V/PI staining. The molecular mechanisms of BBI608 were also determined using cross‑linking assay, nuclear and cytoplasmic fractionation assay, reverse transcription‑quantitative PCR and chromatin immunoprecipitation assay. In the present study, it was observed that human HSC‑3 and HSC‑4 OSCC cells exhibited higher levels of phosphorylated (p)‑STAT3 compared with those in immortalized oral keratinocytes (iHOK cells). BBI608 inhibited cell proliferation in a concentration‑dependent manner and triggered caspase 3‑dependent apoptosis in HSC‑3 and HSC‑4 cells. Additionally, BBI608 reduced the nuclear translocation of p‑STAT3 in HSC‑3 and HSC‑4 cells compared with that in DMSO‑treated cells. Mechanistically, BBI608 modulated anti‑apoptotic STAT3 downstream genes: Survivin expression was regulated at the transcriptional level, while myeloid cell leukemia‑1 expression was modulated post‑translation via proteasomal degradation. Consistent with the results from 2D culture, BBI608 showed effective anticancer effects against OSCC spheroids in 3D culture. These results suggest that BBI608 effectively inhibits STAT3 activation in both 2D and 3D models, offering a promising therapeutic strategy and supporting its potential for repurposing in patients with OSCC who exhibit elevated STAT3 activity.
View Figures

Figure 1

Growth inhibitory activity and
apoptotic effects of BBI608 in OSCC cell lines. (A) Protein levels
of p-STAT3 in OSCC cell lines were analyzed by western blotting.
The expression levels of total STAT3 were normalized to those of
β-actin, while p-STAT3(Y705) levels were normalized to those of
total STAT3. (B) HSC-3 and HSC-4 cells were treated with BBI608 at
0.4 and 3.2 µM, respectively, for ~2 weeks. Colony formation assays
were performed to assess non-adherent, anchorage-independent
growth. (C) HSC-3 and HSC-4 cells were treated with BBI608 at 0.4
and 3.2 µM for 24 h, respectively. Representative images of DAPI
staining are shown, with condensed nuclei indicated by white
arrows. (D) Cell cycle evaluation of OSCC cell lines was performed
using flow cytometry analysis with PI staining. The horizontal axis
represents PI staining intensity, and the vertical axis represents
cell counts. (E) Apoptotic cell death was evaluated by Annexin V/PI
staining. The horizontal axis shows Annexin V intensity, and the
vertical axis shows PI intensity. (F) HSC-3 and HSC-4 cells were
treated with BBI608 at 0.6 and 3.2 µM for 24 h, respectively. The
expression levels of c-PARP and c-caspase 3, total PARP, total
caspase 3 and β-actin (loading control) were analyzed. The
expression levels of c-PARP and c-caspase 3 levels were normalized
to those of total PARP and caspase 3, respectively. (G) HSC-3 and
HSC-4 cells were pre-treated with Z-VAD-FMK at 10 and 5 µM for 1 h,
followed by treatment with BBI608 at 0.6 and 3.2 µM for 24 h,
respectively. All experiments were conducted in triplicate and the
results are presented as the mean ± SD. *P<0.05 vs. iHOK cells
(panel A) or as indicated (panels B-G); #P<0.05.
OSCC, oral squamous cell carcinoma; p, phosphorylated; c, cleaved;
PARP, poly(ADP-ribose) polymerase; cas, caspase.

Figure 2

Inhibitory effect of BBI608 on STAT3
signaling in oral squamous cell carcinoma cell lines. (A) Western
blot analysis showing the expression levels of p-STAT3, STAT3 and
β-actin. The expression levels of total STAT3 were normalized to
those of β-actin, while p-STAT3 levels were normalized to those of
total STAT3. (B) STAT3 dimerization was assessed by western blot
analysis following treatment with bismaleimidohexane (a
cross-linker) and normalized to β-actin. (C) Cytoplasmic and
nuclear fractionation was performed to examine the levels p-STAT3
in the nucleus after BBI608 treatment. α-tubulin and histone H3
were used as loading controls for the cytoplasmic and nuclear
fractions, respectively. The expression levels of total STAT3 were
normalized to those of α-tubulin and histone H3, while p-STAT3
levels were normalized to those of total STAT3. (D) Western blot
analysis showing the expression of STAT3-regulated anti-apoptotic
proteins and β-actin (loading control). All experiments were
performed in triplicate, and the results are presented as the mean
± SD. *P<0.05 vs. the (−) group or as indicated. Mcl-1,
myeloid cell leukemia-1; p, phosphorylated.

Figure 3

Effect of BBI608 on survivin
expression in oral squamous cell carcinoma cell lines. To
investigate changes in mRNA levels of survivin and Mcl-1 in
response to BBI608, HSC-3, and HSC-4 cells were treated with BBI608
at concentrations of 0.6 and 3.2 µM for 6 to 24 h. (A) RT-qPCR
analysis showed a decrease in survivin mRNA levels and an increase
in Mcl-1 mRNA levels following BBI608 treatment. The data were
normalized to the expression of the housekeeping gene GAPDH. (B)
Schematic diagram of STAT3-binding sites and ChIP primer locations
on the survivin promoter. HSC-3 and HSC-4 cells were treated with
BBI608 at 0.6 and 3.2 µM, respectively, for 24 h. To confirm the
presence of STAT3 at the survivin promoter region, a ChIP assay
coupled with RT-qPCR on the survivin promoter region was performed.
Data are presented as % of input. All experiments were performed in
triplicate, and the results are presented as the mean ± SD.
*P<0.05. TSS, transcription start site; RT-qPCR, reverse
transcription-quantitative PCR; Mcl-1, myeloid cell leukemia-1;
ChIP, chromatin immunoprecipitation; P1, survivin ChIP sense; P2,
survivin ChIP antisense.

Figure 4

Effect of BBI608 on Mcl-1 expression
in oral squamous cell carcinoma cell lines. (A) HSC-3 and HSC-4
cells were pretreated with CHX at concentrations of 50 and 100
ng/ml for 1 h, respectively. Following this, the cells were treated
with BBI608 at concentrations of 0.6 and 3.2 µM for the designated
periods of time, respectively. (B) HSC-3 and HSC-4 cells were
pretreated with MG-132 at concentrations of 300 and 500 nM for 1 h,
respectively, and then treated with BBI608 at 0.6 and 3.2 µM for 24
h. The data were normalized to the expression of β-actin. All
experiments were performed in triplicate, and the results are
presented as mean ± SD. *P<0.05 vs. DMSO or as indicated;
#P<0.05. CHX, cycloheximide; Mcl-1, myeloid cell
leukemia-1.

Figure 5

Inhibitory effects of BBI608 on the
growth of OSCC spheroids and STAT3 signaling. HSC-3 and HSC-4 cells
were treated with BBI608 at concentrations of 2.5 and 20 µM for 24
h, respectively. Cell viability of OSCC spheroids was assessed to
determine the cytotoxic effects of BBI608 using (A) Cell Counting
Kit-8 and (B) live/dead assays (magnification, ×100; scale bar, 100
µm). Apoptotic cell death in OSCC spheroids was evaluated by (C)
the expression of c-PARP and c-caspase 3 and (D) Annexin V/PI
staining. The horizontal axis represents Annexin V staining, while
the vertical axis represents PI staining. (E) Western blot analysis
indicated the expression levels of STAT3-regulated proteins along
with β-actin. Protein expression levels were normalized to β-actin,
except for p-STAT3 levels, which were normalized to those of total
STAT3. (F) The mRNA levels of survivin and Mcl-1 in OSCC spheroids
were analyzed using reverse-transcription-quantitative PCR. The
data were normalized to the expression of the housekeeping gene
GAPDH. All experiments were performed in triplicate, and the
results are presented as the mean ± SD. *P<0.05. OSCC, oral
squamous cell carcinoma; Mcl-1, myeloid cell leukemia-1; p,
phosphorylated; c, cleaved; PARP, poly(ADP-ribose) polymerase; cas,
caspase; BF, bright field.
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Spandidos Publications style
Park D, Kim H, Lee S, Jiang H, Hong S, Choi S and Cho S: <em>In vitro</em> assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma. Oncol Rep 54: 97, 2025.
APA
Park, D., Kim, H., Lee, S., Jiang, H., Hong, S., Choi, S., & Cho, S. (2025). <em>In vitro</em> assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma. Oncology Reports, 54, 97. https://doi.org/10.3892/or.2025.8930
MLA
Park, D., Kim, H., Lee, S., Jiang, H., Hong, S., Choi, S., Cho, S."<em>In vitro</em> assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma". Oncology Reports 54.2 (2025): 97.
Chicago
Park, D., Kim, H., Lee, S., Jiang, H., Hong, S., Choi, S., Cho, S."<em>In vitro</em> assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma". Oncology Reports 54, no. 2 (2025): 97. https://doi.org/10.3892/or.2025.8930
Copy and paste a formatted citation
x
Spandidos Publications style
Park D, Kim H, Lee S, Jiang H, Hong S, Choi S and Cho S: <em>In vitro</em> assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma. Oncol Rep 54: 97, 2025.
APA
Park, D., Kim, H., Lee, S., Jiang, H., Hong, S., Choi, S., & Cho, S. (2025). <em>In vitro</em> assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma. Oncology Reports, 54, 97. https://doi.org/10.3892/or.2025.8930
MLA
Park, D., Kim, H., Lee, S., Jiang, H., Hong, S., Choi, S., Cho, S."<em>In vitro</em> assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma". Oncology Reports 54.2 (2025): 97.
Chicago
Park, D., Kim, H., Lee, S., Jiang, H., Hong, S., Choi, S., Cho, S."<em>In vitro</em> assessment of BBI608 in 2D and 3D culture models for drug repositioning in oral squamous cell carcinoma". Oncology Reports 54, no. 2 (2025): 97. https://doi.org/10.3892/or.2025.8930
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