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Saikosaponin D overcomes gemcitabine resistance in pancreatic cancer via AKT/mTOR pathway inhibition and synergistic induction of apoptosis and autophagy

  • Authors:
    • Rui Zheng
    • Yusi Liu
    • Sen Zhou
    • Guimei Liu
    • Xiaobin Liu
    • Yiyuan Yang
    • Yufu Zhang
  • View Affiliations / Copyright

    Affiliations: Department of Medical Immunology, Yan'an Medical College, Yan'an University, Yan'an, Shaanxi 716000, P.R. China, Department of Clinical Laboratory, The Affiliated Hospital of Yan'an University, Yan'an, Shaanxi 716000, P.R. China, Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Yan'an University, Yan'an, Shaanxi 716000, P.R. China
    Copyright: © Zheng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 28
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    Published online on: December 4, 2025
       https://doi.org/10.3892/or.2025.9033
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Abstract

Gemcitabine (GEM) is the first‑line chemotherapy drug for pancreatic cancer, but its efficacy is often limited by inherent drug resistance. Saikosaponin D (SSD), a bioactive triterpenoid saponin derived from the Bupleurum chinense root, exhibits anti‑inflammatory and antitumor properties; however, to the best of our knowledge, its role in pancreatic cancer and GEM sensitization remains unclear. The present study investigated the effects of SSD on the proliferation, apoptosis and autophagy of pancreatic cancer cells, and evaluated whether SSD can overcome GEM resistance to enhance its antitumor effects. Using MIA PaCa‑2 and AsPC‑1 cells, the sensitivity to SSD and GEM was assessed using Cell Counting Kit‑8 assays, H&E staining and colony formation assays. Optimal sub‑lethal concentrations of GEM (0.25 µmol/l), SSD (4 µmol/l) and their combination (0.25 µmol/l GEM + 4 µmol/l SSD) were identified. Apoptosis was evaluated through Hoechst 33258 staining and TUNEL assays, while autophagy was measured using the monodasylcadaverine method. Western blotting and immunocytochemical staining were used to analyze the expression levels of proteins related to apoptosis, AKT/mTOR signaling and autophagy. The results demonstrated that the SSD + GEM combination significantly inhibited pancreatic cancer cell proliferation in both MIA PaCa‑2 and AsPC‑1 cell lines, with proliferation being suppressed by nearly half. Similarly, the combination treatment induced apoptosis and enhanced autophagosome formation, suggesting potential synergistic effects when compared with GEM monotherapy. In conclusion, SSD synergistically enhanced the antitumor effects of GEM by inhibiting pancreatic cancer cell proliferation, and inducing apoptosis and autophagy. SSD may overcome GEM resistance by sensitizing cells through AKT/mTOR pathway inhibition.
View Figures

Figure 1

Dose-dependent effects of SSD on
pancreatic cancer cells. (A) Chemical structure of SSD. (B) MIA
PaCa-2 and (C) AsPC-1 cell viability after treatment with various
concentrations of SSD (0–12 µmol/l; 24–72 h; Cell Counting Kit-8
assay). The Ctrl group was treated with 0 µmol/l SSD. Morphological
changes in (D) MIA PaCa-2 and (E) AsPC-1 cells based on H&E
staining (magnification, ×40). Arrows point to SSD-induced
apoptotic alterations, including nuclear condensation, cytoplasmic
shrinkage and apoptotic bodies. Data are presented as the mean ± SD
(n=5). At the 48-h mark, S12 sharply reduced cell viability in MIA
PaCa-2 cells. The same effect was observed in AsPC-1 cells, where
S12 also significantly inhibited cell proliferation. The
statistical details for S12 are as follows: MIA PaCa-2 cells,
Padj<0.0001; η2=0.73; and AsPC-1 cells,
Padj<0.0001; η2=0.81. All P-values were
adjusted using the Benjamini–Hochberg method. Scale bar, 50 µm. The
significance indicators in (B and C) (*P<0.05, **P<0.01,
***P<0.001, ****P<0.0001) represent the comparison between
each SSD-treated group and the Ctrl group (0 µmol/l). Statistical
analysis was performed using one-way ANOVA followed by Tukey's post
hoc test. Ctrl, control; SSD, Saikosaponin D.

Figure 2

Dose- and time-dependent effects of
GEM on pancreatic cancer cells. (A) Chemical structure of GEM.
Viability of (B) MIA PaCa-2 and (C) AsPC-1 cells treated with
various concentrations of GEM (0–8 µmol/l; 24–72 h; Cell Counting
Kit-8 assay). The Ctrl group was treated with 0 µmol/l GEM.
Representative H&E staining images of (D) MIA PaCa-2 and (E)
AsPC-1 cells (magnification, ×40). Arrows indicate GEM-induced
morphological alterations characteristic of apoptosis. Data are
presented as the mean ± SD (n=5). All P-values were adjusted using
the Benjamini-Hochberg method. The significance indicators in (B
and C) (*P<0.05, **P<0.01, ***P<0.001) represent the
comparison between each GEM-treated group and the Ctrl group (0
µmol/l) at the same time point. Statistical analysis was performed
using one-way ANOVA followed by Tukey's post hoc test. Scale bar,
50 µm. Ctrl, control; GEM, gemcitabine.

Figure 3

Combinatorial effects of GEM and SSD
on pancreatic cancer cells. Viability of (A) MIA PaCa-2 and (B)
AsPC-1 cells treated with G0.25, S4, S6 or combinations (G0.25 +
S4/S6) for 24–72 h. (C) H&E staining showing morphological
changes (magnification, ×40). Scale bar, 50 µm. (D-F) Colony
formation assays. (D) Representative images, and quantification in
(E) MIA PaCa-2 and (F) AsPC-1 cells. (D-F) GEM, 0.25 µmol/l and
SSD, 4 µmol/l. The colony formation rate for each group is
expressed as a percentage relative to the untreated control (Ctrl)
group, which was set to 100%. Note that the absolute colony-forming
capacity of the Ctrl group under baseline culture conditions was
defined as the reference point. Data are presented as the mean ± SD
(n=5). The data demonstrate that the combination treatment (G0.25 +
S4) resulted in significantly enhanced antitumor effects compared
with GEM monotherapy (G0.25). With regard to cell viability, the
combination was significantly more effective
(Padj=0.003; effect size: η2=0.62; 95% CI,
0.41–0.79). A strong synergistic effect was also observed in the
colony formation assay, where the combination treatment led to a
marked reduction in clonogenic survival (Padj=0.001;
effect size, η2=0.68; 95% CI, 0.46–0.83). All P-values
are Benjamini-Hochberg-adjusted. ***P<0.001, ****P<0.0001 for
comparisons vs. Ctrl; #P<0.05,
##P<0.01, ###P<0.001,
####P<0.0001 for G0.25 + S4 vs. G0.25. Statistical
analysis was performed using two-way ANOVA with Tukey's post hoc
test (A and B) and one-way ANOVA with Tukey's post hoc test (E and
F). Ctrl, control; GEM, gemcitabine; SSD, Saikosaponin D; G0.25,
0.25 µmol/l GEM; SX, X µmol/l SSD.

Figure 4

Apoptosis induction by GEM and SSD
combination therapy. (A) Representative TUNEL/DAPI staining images
(green, apoptotic cells; blue, nuclei) in MIA PaCa-2 (top) and
AsPC-1 (bottom) cells treated with Ctrl, G0.25, S4 or the
combination (G0.25 + S4). Quantification of TUNEL-positive cells in
(B) MIA PaCa-2 and (C) AsPC-1 cells. (D) Hoechst 33258 staining
showing apoptotic nuclei with condensed chromatin. In the figure,
GEM represents 0.25 µmol/l GEM and SSD represents 4 µmol/l SSD.
Data are presented as the mean ± SD (n=5). The data show that the
combination treatment (G0.25 + S4) significantly enhanced the
induction of apoptosis compared with GEM monotherapy (G0.25). The
combination treatment significantly increased apoptosis, evidenced
by a substantial rise in TUNEL-positive cells
(Padj=0.002; η2=0.59; 95% CI, 0.36–0.77) and
a corresponding increase in condensed chromatin, a hallmark of
apoptosis, as shown by Hoechst 33258 staining
(Padj=0.004; η2=0.55; 95% CI, 0.31–0.74). All
P-values are Benjamini-Hochberg-adjusted. Scale bar, 50 µm.
**P<0.01, ***P<0.001, ****P<0.0001 for comparisons vs.
Ctrl; ###P<0.001 for G0.25 + S4 vs. G0.25.
Statistical analysis was performed using one-way ANOVA with Tukey's
post hoc test for multiple comparisons. Ctrl, control; GEM,
gemcitabine; SSD, Saikosaponin D.

Figure 5

Molecular mechanisms of apoptosis
induction by GEM and SSD combination treatment. (A)
Immunocytochemistry of MIA PaCa-2 and AsPC-1 cells treated with
Ctrl, G0.25, S4 or the combination (G0.25 + S4; magnification,
×40). Scale bar, 50 µm. (B) Western blot analysis of apoptosis
markers Bax (20 kDa), Bcl-2 (26 kDa), caspase-3 (32 kDa) and
cleaved caspase-3 (17 kDa), and β-actin (42 kDa).
Semi-quantification of immunoblots for (C) MIA PaCa-2 and (D)
AsPC-1 cells. In the figure, GEM represents 0.25 µmol/l GEM and SSD
represents 4 µmol/l SSD. Data are presented as the mean ± SD (n=5).
The combination treatment (G0.25 + S4) increased the expression of
pro-apoptotic Bax and cleaved caspase-3, and elevated the
c-Casp3/Casp3 ratio, while decreasing the expression of
anti-apoptotic Bcl-2 compared with GEM monotherapy (G0.25).
Specifically, Bax expression increased by 2.3-fold in MIA PaCa-2
cells and 3.1-fold in AsPC-1 cells. Conversely, Bcl-2 expression
was reduced to 45 and 38% of GEM monotherapy levels in MIA PaCa-2
and AsPC-1 cells, respectively. The c-Casp3/Casp3 ratio was
significantly enhanced by the combination treatment, indicating
robust activation of the apoptotic executioner pathway. All
P-values are Benjamini-Hochberg-adjusted. ***P<0.001,
****P<0.0001 for G0.25 + S4 vs. Ctrl; ##P<0.01,
###P<0.001, ####P<0.0001 for G0.25 + S4
vs. G0.25. Statistical analysis was performed using one-way ANOVA
with Tukey's post hoc test for multiple comparisons. Ctrl, control;
GEM, gemcitabine; SSD, Saikosaponin D; Casp3, caspase-3; c-Casp3,
cleaved caspase-3.

Figure 6

AKT/mTOR pathway inhibition by GEM
and SSD combination therapy. (A) Immunocytochemistry staining of
AKT/mTOR signaling proteins in MIA PaCa-2 and AsPC-1 cells treated
with Ctrl, G0.25, S4 or the combination (G0.25 + S4; magnification,
×40; insets show phosphoprotein details). Scale bar, 50 µm. (B)
Western blot analysis of p-AKT (60 kDa), AKT (56 kDa), p-mTOR (289
kDa), mTOR (289 kDa) and β-actin (42 kDa). Semi-quantification of
phosphorylation ratios in (C) MIA PaCa-2 and (D) AsPC-1 cells. In
the figure, GEM represents 0.25 µmol/l GEM and SSD represents 4
µmol/l SSD. Data are presented as the mean ± SD (n=5). Compared
with GEM monotherapy, the G0.25 + S4 combination led to a greater
reduction in both p-AKT/AKT and p-mTOR/mTOR ratios (both
Padj<0.0001), with large effect sizes
(η2=0.73 and 0.81, respectively). All P-values are
Benjamini-Hochberg-adjusted. ****P<0.0001 for G0.25 + S4 vs.
Ctrl; ##P<0.01, ###P<0.001 for G0.25 +
S4 vs. G0.25. Statistical analysis was performed using one-way
ANOVA with Tukey's post hoc test. Ctrl, control; GEM, gemcitabine;
SSD, Saikosaponin D; p-, phosphorylated.

Figure 7

Autophagy induction by GEM and SSD
combination treatment. (A) MDC staining (green) showing
autophagosome accumulation in MIA PaCa-2 and AsPC-1 cells treated
with Ctrl, G0.25, S4 or the combination (G0.25 + S4; magnification,
×40). (B) Immunocytochemistry of autophagy markers. (C) Western
blot analysis of Beclin 1 (60 kDa), LC3I (16 kDa), LC3II (14 kDa)
and β-actin (42 kDa). Semi-quantification of autophagy markers in
(D) MIA PaCa-2 and (E) AsPC-1 cells. In the figure, GEM represents
0.25 µmol/l GEM and SSD represents 4 µmol/l SSD. Data are presented
as the mean ± SD (n=5). Compared with GEM monotherapy, the G0.25 +
S4 combination significantly increased the LC3-II/I ratio
(Padj=0.001; η2=0.65; 95% CI, 0.43–0.81) and
Beclin 1 levels (Padj=0.002; η2=0.61; 95% CI,
0.38–0.78), indicating strong induction of autophagy. All P-values
are Benjamini-Hochberg-adjusted. Scale bar, 50 µm. **P<0.01,
***P<0.001 for G0.25 + S4 vs. Ctrl; #P<0.05,
##P<0.01 for G0.25 + S4 vs. G0.25. Statistical
analysis was performed using one-way ANOVA with Tukey's post hoc
test. Ctrl, control; GEM, gemcitabine; SSD, Saikosaponin D; MDC,
monodasylcadaverine.

Figure 8

Proposed mechanism of GEM and SSD
combination therapy. Co-treatment with GEM and SSD synergistically
inhibits the AKT/mTOR pathway, concurrently inducing apoptosis (via
Bax/Bcl-2 modulation and caspase-3 activation) and autophagy (via
Beclin 1 activation and LC3 conversion), thereby overcoming GEM
resistance in pancreatic cancer cells. GEM, gemcitabine; P,
phosphorylated; SSD, Saikosaponin D.
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Copy and paste a formatted citation
Spandidos Publications style
Zheng R, Liu Y, Zhou S, Liu G, Liu X, Yang Y and Zhang Y: Saikosaponin D overcomes gemcitabine resistance in pancreatic cancer via AKT/mTOR pathway inhibition and synergistic induction of apoptosis and autophagy. Oncol Rep 55: 28, 2026.
APA
Zheng, R., Liu, Y., Zhou, S., Liu, G., Liu, X., Yang, Y., & Zhang, Y. (2026). Saikosaponin D overcomes gemcitabine resistance in pancreatic cancer via AKT/mTOR pathway inhibition and synergistic induction of apoptosis and autophagy. Oncology Reports, 55, 28. https://doi.org/10.3892/or.2025.9033
MLA
Zheng, R., Liu, Y., Zhou, S., Liu, G., Liu, X., Yang, Y., Zhang, Y."Saikosaponin D overcomes gemcitabine resistance in pancreatic cancer via AKT/mTOR pathway inhibition and synergistic induction of apoptosis and autophagy". Oncology Reports 55.2 (2026): 28.
Chicago
Zheng, R., Liu, Y., Zhou, S., Liu, G., Liu, X., Yang, Y., Zhang, Y."Saikosaponin D overcomes gemcitabine resistance in pancreatic cancer via AKT/mTOR pathway inhibition and synergistic induction of apoptosis and autophagy". Oncology Reports 55, no. 2 (2026): 28. https://doi.org/10.3892/or.2025.9033
Copy and paste a formatted citation
x
Spandidos Publications style
Zheng R, Liu Y, Zhou S, Liu G, Liu X, Yang Y and Zhang Y: Saikosaponin D overcomes gemcitabine resistance in pancreatic cancer via AKT/mTOR pathway inhibition and synergistic induction of apoptosis and autophagy. Oncol Rep 55: 28, 2026.
APA
Zheng, R., Liu, Y., Zhou, S., Liu, G., Liu, X., Yang, Y., & Zhang, Y. (2026). Saikosaponin D overcomes gemcitabine resistance in pancreatic cancer via AKT/mTOR pathway inhibition and synergistic induction of apoptosis and autophagy. Oncology Reports, 55, 28. https://doi.org/10.3892/or.2025.9033
MLA
Zheng, R., Liu, Y., Zhou, S., Liu, G., Liu, X., Yang, Y., Zhang, Y."Saikosaponin D overcomes gemcitabine resistance in pancreatic cancer via AKT/mTOR pathway inhibition and synergistic induction of apoptosis and autophagy". Oncology Reports 55.2 (2026): 28.
Chicago
Zheng, R., Liu, Y., Zhou, S., Liu, G., Liu, X., Yang, Y., Zhang, Y."Saikosaponin D overcomes gemcitabine resistance in pancreatic cancer via AKT/mTOR pathway inhibition and synergistic induction of apoptosis and autophagy". Oncology Reports 55, no. 2 (2026): 28. https://doi.org/10.3892/or.2025.9033
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