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D‑salicin induces oxidative stress‑mediated ERK1/2 suppression and apoptosis in endometrial cancer cells

  • Authors:
    • Neziha Senem Ari
    • Ayşe Çakir Gündoğdu
  • View Affiliations / Copyright

    Affiliations: Department of Histology and Embryology, Faculty of Medicine, Kütahya Health Sciences University, Kütahya 43100, Türkiye
    Copyright: © Ari et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 311
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    Published online on: May 22, 2026
       https://doi.org/10.3892/ol.2026.15666
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Abstract

Endometrial cancer is a common gynecologic malignancy for which therapeutic options remain limited in advanced or treatment‑resistant disease. Natural compounds that selectively perturb cancer‑associated redox signaling and survival pathways may offer novel anticancer strategies. The present study investigated the anticancer effects of D‑salicin in the endometrial adenocarcinoma‑derived Ishikawa cell line, with a focus on oxidative stress and extracellular signal‑regulated kinase (ERK)1/2 signaling, and assessed cancer selectivity using normal human dermal fibroblasts (HDF). D‑salicin selectively reduced Ishikawa cell viability in a concentration‑dependent manner while largely sparing HDF. This cytotoxic effect was accompanied by marked intracellular reactive oxygen species (ROS) accumulation, increased lipid peroxidation (MDA) and depletion of total glutathione (GSH), indicating induction of oxidative stress. Elevated oxidative burden was associated with caspase‑3 activation and concentration‑dependent suppression of ERK1/2 phosphorylation, both of which were attenuated by N‑acetylcysteine (NAC) co‑treatment. To determine redox dependence, NAC was incorporated as an antioxidant co‑treatment. D‑salicin induced a significant, concentration‑dependent reduction in Ishikawa cell viability (P<0.0001), whereas HDF viability remained largely preserved across the tested range (P>0.05). In Ishikawa cells, D‑salicin markedly increased intracellular ROS in a dose‑dependent manner (P≤0.01) and this effect was markedly attenuated by NAC co‑treatment (P≤0.01), indicating an NAC‑sensitive oxidative component. Consistent with redox disruption, D‑salicin increased MDA levels and depleted total GSH in Ishikawa cells (P≤0.01). Elevated oxidative stress was accompanied by increased caspase‑3 levels (P≤0.01), supporting engagement of apoptotic signaling. Moreover, D‑salicin markedly suppressed ERK1/2 phosphorylation in Ishikawa cells (P<0.0001), while ERK signaling was not markedly altered in HDF cells (P>0.05). Notably, NAC co‑treatment partly restored p‑ERK1/2 levels under the IC50 condition (P<0.01 vs. D‑salicin alone), supporting a redox‑linked contribution to ERK pathway modulation. Collectively, these findings indicated that D‑salicin exerts selective anticancer effects in endometrial cancer cells by inducing oxidative stress and disrupting ERK1/2‑mediated survival signaling, with NAC‑sensitive modulation consistent with involvement of a redox‑responsive ROS‑ERK axis.
View Figures

Figure 1

Effects of D-salicin and NAC on cell
viability in Ishikawa cells. (A) Viability of Ishikawa cells
following 72 h treatment with increasing concentrations of
D-salicin (0–80 µM), as determined by the MTT assay. D-salicin
induced a concentration-dependent decrease in cell viability. (B)
Cell viability of Ishikawa cells following 72 h exposure to NAC
(0.5–10 mM), as determined by the MTT assay. Data are expressed as
percentage of control and presented as mean ± SD. Statistical
analysis was performed using one-way ANOVA followed by Tukey's
multiple comparisons test. *P<0.05, **P<0.01,
***P<0.001, ****P<0.0001. NAC,
N-acetylcysteine.

Figure 2

Effects of D-salicin and NAC on cell
viability in HDF cells. (A) Cell viability of HDF cells following
72 h treatment with increasing concentrations of D-salicin (0–80
µM), as determined by the MTT assay. No significant reduction in
viability was observed across the tested concentration range. (B)
Cell viability of HDF cells following 72 h exposure to NAC (0.5–10
mM), as determined by the MTT assay. No significant reduction in
viability was observed across the tested concentration range. NAC,
N-acetylcysteine; HDF, human dermal fibroblasts.

Figure 3

Effects of D-salicin and NAC
co-treatment on intracellular ROS levels in Ishikawa and HDF cells.
(A) Ishikawa cells and (B) HDF cells were treated with D-salicin at
IC25 (26.7 µM), IC50 (48.8 µM), and
IC75 (69.9 µM) for 72 h in the absence (−NAC) or
presence (+NAC) of N-acetylcysteine (NAC, 0.5 mM). Control
represents untreated cells, and NAC represents cells treated with
NAC alone (0.5 mM). For D-salicin-treated groups, ‘-NAC’ and ‘+NAC’
indicate the absence or presence of NAC at each corresponding
D-salicin concentration. Intracellular ROS levels were measured
using the DCFDA assay and expressed as RFU, normalized to the
untreated control. In Ishikawa cells, D-salicin induced a
dose-dependent increase in ROS levels, which was markedly
attenuated by NAC co-treatment. In HDF cells, D-salicin induced
only modest changes in ROS levels, and NAC co-treatment did not
markedly alter this response. Data are presented as mean ± SD (n≥3
independent experiments). Statistical analysis was performed using
one-way ANOVA followed by Tukey's multiple comparisons test. Hash
symbols (#) indicate comparisons between matched
D-salicin groups in the absence and presence of NAC at the
corresponding concentration [e.g., 26.7 (−NAC) vs. 26.7 (+NAC)].
*P<0.05, **P<0.01, ****P<0.0001 vs.
untreated control; #P<0.05,
####P<0.0001. NAC, N-acetylcysteine; ROS,
reactive oxygen species; HDF, human dermal fibroblasts; RFU,
relative fluorescence units.

Figure 4

Effects of D-salicin on lipid
peroxidation (MDA) and GSH levels in Ishikawa and HDF cells. (A)
MDA concentrations in Ishikawa cells following 72 h exposure to
D-salicin at IC25 (26.7 µM), IC50 (48.8 µM),
and IC75 (69.9 µM). (B) MDA concentrations (ng/ml) in
HDF cells under identical treatment conditions. (C) GSH
concentrations (µg/ml) in Ishikawa cells following 72 h D-salicin
exposure at IC25, IC50, and IC75.
(D) GSH concentrations (µg/ml) in HDF cells under identical
conditions. MDA and GSH were quantified in cell lysates by ELISA,
and concentrations were calculated from standard curves. Data are
presented as mean ± SD from three independent experiments (n=3).
Each condition was assayed in technical triplicate, and experiments
were independently repeated at least three times (n≥3 biological
replicates). Statistical analysis was performed using one-way ANOVA
followed by Tukey's multiple comparisons test. *P<0.05,
**P<0.01, ***P<0.001, ****P<0.0001.
MDA, malondialdehyde; GSH, glutathione; HDF, human dermal
fibroblasts.

Figure 5

Effect of D-salicin on caspase-3
protein levels in Ishikawa and HDF cells. (A) Caspase-3 protein
levels in Ishikawa cells following 72 h treatment with D-salicin at
IC25 (26.7 µM), IC50 (48.8 µM), and
IC75 (69.9 µM). D-salicin induced a
concentration-dependent increase in caspase-3 levels. (B) Caspase-3
protein levels in HDF cells treated with D-salicin under identical
conditions. No significant changes were observed across treatment
groups. Data are presented as mean ± SD. Statistical analysis was
performed using one-way ANOVA followed by Tukey's multiple
comparisons test. **P<0.01, ****P<0.0001. HDF,
human dermal fibroblast.

Figure 6

Effect of D-salicin on ERK1/2
phosphorylation in Ishikawa and HDF cells. (A) Representative
immunofluorescence images of p-ERK1/2 expression in Ishikawa cells
treated with D-salicin (26.7, 48.8, and 69.9 µM). p-ERK1/2
immunoreactivity (red) decreased in a concentration-dependent
manner compared with control cells. Nuclei were counterstained with
DAPI (blue). (B) Representative immunofluorescence images of HDF
cells following D-salicin treatment under identical conditions. No
apparent changes in p-ERK1/2 fluorescence intensity were observed
across treatment groups. These representative immunofluorescence
images are shown alongside the corresponding quantitative analysis
to illustrate the observed changes in p-ERK1/2 expression. Bar
graphs show quantitative analysis of p-ERK1/2 fluorescence
intensity. Data are presented as mean ± SD. Statistical
significance was determined by one-way ANOVA followed by Tukey's
multiple comparisons test (*P<0.05, ***P<0.001,
****P<0.0001). Scale bar, 100 µm. CTCF values were
calculated as absolute fluorescence intensity measurements, and
statistical comparisons were performed directly between groups. p-,
phosphorylated; CTCF, corrected total cell fluorescence.

Figure 7

Effect of NAC co-treatment on
D-salicin-induced suppression of ERK1/2 phosphorylation in Ishikawa
cells. (A) Representative immunofluorescence images showing
phospho-ERK1/2 (p-ERK1/2) expression in Ishikawa cells following 72
h treatment with control, NAC (0.5 mM), D-salicin
(IC50=48.8 µM), and D-salicin + NAC. p-ERK1/2
immunoreactivity is shown in red, and nuclei are counterstained
with DAPI (blue). D-salicin reduced p-ERK1/2 fluorescence intensity
compared with control, whereas NAC co-treatment partially restored
p-ERK1/2 signal. (B) Quantitative analysis of p-ERK1/2 fluorescence
intensity expressed as CTCF. Data are presented as mean ± SD from
three independent experiments. Statistical significance was
determined using one-way ANOVA followed by Tukey's multiple
comparisons test **P<0.01, ***P<0.001,
****P<0.0001). Scale bars, 100 µm. NAC, N-acetylcysteine;
p-, phosphorylated; CTCF, corrected total cell fluorescence; DSA,
D-salicin.
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Copy and paste a formatted citation
Spandidos Publications style
Ari N and Çakir Gündoğdu A: D‑salicin induces oxidative stress‑mediated ERK1/2 suppression and apoptosis in endometrial cancer cells. Oncol Lett 32: 311, 2026.
APA
Ari, N., & Çakir Gündoğdu, A. (2026). D‑salicin induces oxidative stress‑mediated ERK1/2 suppression and apoptosis in endometrial cancer cells. Oncology Letters, 32, 311. https://doi.org/10.3892/ol.2026.15666
MLA
Ari, N., Çakir Gündoğdu, A."D‑salicin induces oxidative stress‑mediated ERK1/2 suppression and apoptosis in endometrial cancer cells". Oncology Letters 32.1 (2026): 311.
Chicago
Ari, N., Çakir Gündoğdu, A."D‑salicin induces oxidative stress‑mediated ERK1/2 suppression and apoptosis in endometrial cancer cells". Oncology Letters 32, no. 1 (2026): 311. https://doi.org/10.3892/ol.2026.15666
Copy and paste a formatted citation
x
Spandidos Publications style
Ari N and Çakir Gündoğdu A: D‑salicin induces oxidative stress‑mediated ERK1/2 suppression and apoptosis in endometrial cancer cells. Oncol Lett 32: 311, 2026.
APA
Ari, N., & Çakir Gündoğdu, A. (2026). D‑salicin induces oxidative stress‑mediated ERK1/2 suppression and apoptosis in endometrial cancer cells. Oncology Letters, 32, 311. https://doi.org/10.3892/ol.2026.15666
MLA
Ari, N., Çakir Gündoğdu, A."D‑salicin induces oxidative stress‑mediated ERK1/2 suppression and apoptosis in endometrial cancer cells". Oncology Letters 32.1 (2026): 311.
Chicago
Ari, N., Çakir Gündoğdu, A."D‑salicin induces oxidative stress‑mediated ERK1/2 suppression and apoptosis in endometrial cancer cells". Oncology Letters 32, no. 1 (2026): 311. https://doi.org/10.3892/ol.2026.15666
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