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Article Open Access

KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells

  • Authors:
    • Natalia Podhorska
    • Jan Hrabeta
    • Marie Belhajova
    • Tomas Eckschlager
  • View Affiliations / Copyright

    Affiliations: Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, 150 06 Prague 5, Czech Republic
    Copyright: © Podhorska et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 79
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    Published online on: February 25, 2026
       https://doi.org/10.3892/or.2026.9084
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Abstract

Chemoresistance is a major cause of cancer therapy failure. Increasing evidence points to the importance of histone lysine demethylase function, whose dysregulation has been described in several types of cancer. KDM5, a family of histone lysine demethylases, may carry out a key role in the downregulation of tumor‑suppressors or upregulation of oncogenes and in the development of drug tolerance. The present study examined the expression of KDM5D in cell lines derived from high‑risk neuroblastoma. The present study found that KDM5D expression was lost in all cisplatin‑chemoresistant neuroblastoma cell lines compared with sensitive parental cells. In addition, the cisplatin‑chemoresistant neuroblastoma cell line had increased expression of the ubiquitin ligase cullinaA 4A (CUL4A) compared with the sensitive parental cells. CUL4A carries out a role in cellular processes and its aberrant regulation has been observed in a number of types of cancer. The present study shows that silencing of KDM5D causes a more aggressive phenotype of neuroblastoma by promoting cell proliferation and migration, evading cell death, promoting S phase of the cell cycle and desensitizing sensitive cells to cisplatin via the gene CUL4A. In addition, ectopic expression of KMD5D in a cisplatin‑resistant cell line reversed these phenomena. The results suggest that KDM5D and/or CUL4A may be a biomarkers of chemoresistance to cisplatin and a potential therapeutic target in neuroblastoma.

View Figures

Figure 1

Loss of KDM5D expression in
CDDP-resistant neuroblastoma cell line UKF-NB-3CCDDP.
PrestoBlue assay results showing cytotoxicity of CDDP after 72 h.
(A) UKF-NB-3 has an IC50 of 13.33±0.97 µM and
CDDP-resistant UKF-NB-3CDDP has an IC50 of
32.39±0.96 µM. (B) KDM5D mRNA expression was lost in CDDP-resistant
UKF-NB-3CDDP. Treatment with 20 µM CDDP for 72 h
significantly decreased KDM5D levels in UKF-NB-3. The mRNA
expression was detected by reverse transcription-quantitative PCR.
(C) KDM5D protein expression was detected by flow cytometry in
UKF-NB-3 and CDDP-resistant UKF-NB-3CDDP cell lines and
showed the same pattern of expression as mRNA levels. Data are
shown as mean ± standard deviation from three independent
experiments. Statistical significance was determined using one-way
ANOVA with Tukey's post hoc test. **P<0.01; ***P<0.001 (ANOVA
with Tukey's post hoc). NB3, UKF-NB-3; NB3 CDDP, CDDP-resistant
cell line UKF-NB-3CDDP.

Figure 2

KDM5D knockdown affects histone H3K4
trimethylation in CDDP-sensitive neuroblastoma cell line UKF-NB-3.
(A) Reverse transcription-quantitative PCR showed decreased
expression of KDM5D mRNA in UKF-NB-3 cell line transfected with
siKDM5D #1 (12.5 nM) or siKDM5D #2 (25 nM) compared to control NC
#1 (12.5 nM) or NC #2 (25 nM) (P<0.001). (B) Reduced expression
of KDM5D protein after siKDM5D transfection (48 h) was detected by
flow cytometry (P<0.001). (C) Flow cytometry histogram showed
decreased expression of KDM5D protein after siKDM5D transfection
(48 h) and treatment with CDDP (48 h). (D) Flow cytometry showed
decreased expression of KDM5D protein in control after treatment
with CDDP in UKF-NB-3 (P<0.001) but did not further decrease the
expression of KDM5D in transfected cells. (E) Flow cytometry
histogram showed increased expression levels of histone H3K4
trimethylation after siKDM5D transfection (48 h). (F) Silencing of
KDM5D increased the level of histone H3K4me3 trimethylation
(P<0.01) and treatment with CDDP in UKF-NB-3 did not increase
the level of H3K4me3 in control but further increased the level of
histone H3K4me3 trimethylation in transfected cells (P<0.001).
Data are shown as mean ± standard deviation from three independent
experiments. Statistical significance was determined using one-way
ANOVA with Tukey's post hoc test. *P<0.05; **P<0.01;
***P<0.001. CTR, control; si, short interfering RNA; SEC,
control with only the secondary antibody; NC, control cells with
non-coding RNA; siKDM5D, control cells transfected with siKDM5D;
FC, flow cytometry.

Figure 3

Aberrant expression of KDM5D affects
histone H3K4 trimethylation in the CDDP-resistant neuroblastoma
cell line UKF-NB-3CDDP. (A) Flow cytometry showed
increased levels of KDM5D protein in the UKF-NB-3CDDP
cell line transfected with the KDM5D ORF-clone plasmid pCMV-3Tag1,
expressing the KDM5D gene (pl-KDM5D #, 8 ng/µl; or pl-KDM5D #2, 16
ng/µl) compared with control cells transfected with the control
plasmid pCMV6-AC-GFP (mock #1, 8 ng/µl; or mock #2, 16 ng/µl),
while control cells did not express KDM5D (P<0.001). (B) Flow
cytometry histogram showed increased expression of KDM5D protein
after KDM5D overexpression (48 h). (C) Flow cytometry proved
increased protein level of KDM5D in KDM5D transfected
UKF-NB-3CDDP cells and showed that CDDP treatment in
transfected cells decreased protein expression of KDM5D
(P<0.001). (D) Flow cytometry histogram showed that histone H3K4
trimethylation decreased after KDM5D overexpression (48 h). (E)
KDM5D overexpression decreased histone H3K4 trimethylation in
UKF-NB-3CDDP (P<0.001) but treatment with CDDP
increased H3K4me3 trimethylation in transfected cells (P<0.05).
Data are shown as mean ± standard deviation from three independent
experiments. Statistical significance was determined using one-way
(A) and two-way ANOVA (C, E) with Tukey's post hoc test
**P<0.01; ***P<0.001. CTR, control; pl-ORF-clone plasmid;
SEC, control with only the secondary antibody; Mock/NC, control
cells with non-coding RNA; pl-KDM5D, control cells transfected with
pl-KDM5D; FC, flow cytometry.

Figure 4

KDM5D expression affects cell
sensitivity to CDDP in UKF-NB-3 and UKF-NB-3CDDP.
PrestoBlue assay results showed that incubation with CDDP (48 h)
after (A) KDM5D silencing (48 h) in UKF-NB-3 caused reduced
inhibition of cell viability induced by CDDP in silenced cells
compared with NC after treatment with increasing concentrations of
CDDP (P<0.001). (B) Overexpression of KDM5D in
UKF-NB-3CDDP made the cells more sensitive to CDDP
compared with mock (P<0.5; P<0.001). Data are shown are mean
± standard deviation from three independent experiments.
Statistical significance was determined using two-way ANOVA with
Tukey's post hoc test. *P<0.05; **P<0.01; ***P<0.001
(ANOVA with Tukey's post hoc test). CTR, control; si, short
interfering RNA; pl, ORF-clone plasmid.

Figure 5

Expression of KDM5D increases CDDP
induced apoptosis in neuroblastoma cells. (A) Cleaved caspase-3 in
CDDP sensitive cell line UKF-NB-3 and (B) CDDP resistant cell line
UKF-NB-3CDDP. Sensitive cell line UKF-NB-3 had increased
levels of activated caspase-3 after CDDP treatment (P<0.001),
where silencing of KDM5D led to decreased levels compared with NC
(P<0.001). In CDDP-resistant cell line UKF-NB-3CDDP,
CDDP treatment after transfection with plasmid with KDM5D gene,
increased activated caspase-3 level (P<0.01) compared with mock.
Data are shown as mean ± standard deviation from three independent
experiments. Statistical significance was determined using two-way
ANOVA with Tukey's post hoc test **P<0.01; ***P<0.001. si,
short interfering RNA; pl, ORF-clone plasmid; Mock/NC, control
cells with non-coding RNA; siKDM5D/pl-KDM5D, control cells
transfected with siKDM5D/pl-KDM5D; FC, flow cytometry.

Figure 6

Flow cytometry analysis of cell
cycle, proliferation and cell migration by xCELLigence in
CDDP-sensitive cell line UKF-NB-3 and CDDP-resistant cell line
UKF-NB-3CDDP. (A) Silencing of KDM5D in sensitive cell
line UKF-NB-3 led to increased S phase (P<0.01) and decreased
G2/M phase (P<0.05) compared with control or cells with NC. (B)
Transfection of plasmid with KDM5D in CDDP-resistant cell line
UKF-NB-3CDDP led to decreased S phase (P<0.001) and
increased G0/G1 phase (P<0.01) compared with control or mock
(P<0.001). (C) Monitoring of cell migration by xCELLigence after
transfection of siKDM5D in UKF-NB-3 or ORF cDNA plasmid with KDM5D
gene in UKF-NB-3CDDP. (D) Monitoring of cell
proliferation by xCELLigence after transfection of siKDM5D in
UKF-NB-3 or ORF cDNA plasmid with KDM5D gene in
UKF-NB-3CDDP. Data are shown as mean ± standard
deviation from three independent experiments. Statistical
significance was determined using one-way ANOVA (A) and two-way
ANOVA (B, C and D) with Tukey's post hoc test. *P<0.05;
**P<0.01; ***P<0.001 (ANOVA with Tukey's post hoc test). CTR,
control; NC, non-coding RNA; si, short interfering RNA; pl,
ORF-clone plasmid; NB3, UKF-NB-3; FC, flow cytometry.

Figure 7

KDM5D and CDDP treatment affect
expression of CUL4A in UKF-NB-3 and UKF-NB-3CDDP. Flow
cytometry analysis in (A) UKF-NB-3 and UKF-NB-3CDDP,
where sensitive cell line UKF-NB-3 showed lower expression of CUL4A
gene compared with the resistant cell line (P<0.05) and this
expression increased in sensitive cells after CDDP treatment
(P<0.001) compared with control. (B) Silencing of KDM5D
increased of CUL4A level (P<0.001). (C) In the CDDP-resistant
cell line UKF-NB-3CDDP, KDM5D overexpression decreased
CUL4A expression compared with mock and control plasmid
(P<0.001). CDDP treatment of overexpressed cells with KDM5D
increased the level of CUL4A (P<0.001), while in control cells
CDDP treatment did not change the level of CUL4A. Data are shown as
mean ± standard deviation from three independent experiments.
Statistical significance was determined using two-way ANOVA with
Tukey's post hoc test. *P<0.05; **P<0.01; ***P<0.001
(ANOVA with Tukey's post hoc test). CTR, control; NC, non-coding
RNA; si, short interfering RNA; pl, ORF-clone plasmid; NB3,
UKF-NB-3; Mock/NC, control cells; siKDM5D/pl-KDM5D, control cells
transfected with siKDM5D/pl-KDM5D; FC, flow cytometry.

Figure 8

Kaplan-Meier curves analyzing KDM5D
(A, B, C) and CUL4A (D, E, F) expression data and overall survival
of patients in the SEQC neuroblastoma dataset (498; RPM; seqcnb1):
(A) Male patients with stages 1, 2, 3 and 4S, KDM5AD expression.
(B) Male patients with stage 4, KDM5D expression. (C) All male
patients according to the International Neuroblastoma Staging
System, KDM5D expression. (D) Male patients with stages 1, 2, 3 and
4S, CUL4A expression. (E) Male patients with stage 4, CUL4A
expression. (F) All male patients according to the International
Neuroblastoma Staging System, CUL4A expression. Statistical
significance of survival differences was determined using the
log-rank (Mantel-Cox) test. R2: Genomics Analysis and Visualization
Platform revealed that low relative expression of KDM5D and high
expression of CUL4A is associated with worse survival.
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Copy and paste a formatted citation
Spandidos Publications style
Podhorska N, Hrabeta J, Belhajova M and Eckschlager T: KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells. Oncol Rep 55: 79, 2026.
APA
Podhorska, N., Hrabeta, J., Belhajova, M., & Eckschlager, T. (2026). KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells. Oncology Reports, 55, 79. https://doi.org/10.3892/or.2026.9084
MLA
Podhorska, N., Hrabeta, J., Belhajova, M., Eckschlager, T."KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells". Oncology Reports 55.4 (2026): 79.
Chicago
Podhorska, N., Hrabeta, J., Belhajova, M., Eckschlager, T."KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells". Oncology Reports 55, no. 4 (2026): 79. https://doi.org/10.3892/or.2026.9084
Copy and paste a formatted citation
x
Spandidos Publications style
Podhorska N, Hrabeta J, Belhajova M and Eckschlager T: KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells. Oncol Rep 55: 79, 2026.
APA
Podhorska, N., Hrabeta, J., Belhajova, M., & Eckschlager, T. (2026). KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells. Oncology Reports, 55, 79. https://doi.org/10.3892/or.2026.9084
MLA
Podhorska, N., Hrabeta, J., Belhajova, M., Eckschlager, T."KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells". Oncology Reports 55.4 (2026): 79.
Chicago
Podhorska, N., Hrabeta, J., Belhajova, M., Eckschlager, T."KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells". Oncology Reports 55, no. 4 (2026): 79. https://doi.org/10.3892/or.2026.9084
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