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Article Open Access

Astragaloside IV targets TUBB4B to inhibit proliferation and promote apoptosis of pituitary tumor cells via the STMN1/ERK pathway

  • Authors:
    • Jiale Li
    • Yufei Qu
    • Wenge Zhang
    • Zexu Yang
    • Yangdong Zeng
    • Jisheng Xu
    • Kuncen Xie
    • Qi Liu
  • View Affiliations / Copyright

    Affiliations: Department of Neurosurgery, The First Affiliated Hospital of Shihezi University, Shihezi University, Shihezi, Xinjiang 832008, P.R. China, National Health Commission Key Laboratory of Prevention and Treatment of Central Asia High Incidence Diseases (Co‑construction), Shihezi University, Shihezi, Xinjiang 832008, P.R. China
    Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 151
    |
    Published online on: April 1, 2026
       https://doi.org/10.3892/ijmm.2026.5822
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Abstract

Pituitary tumors, as common intracranial neoplasms, present challenges in clinical management because of high postoperative recurrence rate, drug resistance and pronounced side effects. Astragaloside IV (AS‑IV), the primary active component of the traditional Chinese herb Astragalus membranaceus, has antitumor activity in numerous types of cancer. However, its effects and mechanisms in pituitary tumors remain unclear. The present study aimed to investigate the effects of AS‑IV on proliferation and apoptosis of pituitary tumor cells and to elucidate its molecular mechanisms. Cell Counting Kit‑8 (CCK‑8), TUNEL, EdU, immunohistochemistry (IHC), and Western blotting, among others, showed that AS‑IV significantly suppressed the viability of the rat pituitary tumor cell lines GH3 and MMQ in a concentration‑ and time‑dependent manner while inducing apoptosis. Tubulin β4B Class IVb (TUBB4B) was highly expressed in pituitary tumor tissue and its overexpression promoted cell proliferation while inhibiting apoptosis. AS‑IV bound TUBB4B with high affinity, forming a stable complex. TUBB4B regulation influenced pituitary tumor cell sensitivity to AS‑IV, with AS‑IV demonstrating enhanced antitumor efficacy in TUBB4B‑overexpressing tumors. TUBB4B activated the ERK/MAPK signaling pathway by upregulating Stathmin 1 (STMN1) expression, which promoted G1/S phase transition. The ERK‑specific inhibitor U0126 reversed this pro-proliferative effect. To the best of our knowledge, the present study is the first to reveal that AS‑IV inhibits pituitary tumor proliferation and promotes apoptosis by targeting TUBB4B to regulate the STMN1‑ERK signaling axis, providing a novel theoretical basis and potential strategies for traditional Chinese medicine treatment and molecular targeted research on pituitary tumors.
View Figures

Figure 1

AS-IV inhibits the proliferation and
promotes the apoptosis of GH3/MMQ cells in a concentration- and
time-dependent manner. (A) Chemical structure of AS-IV.
IC50 of AS-IV in (B) GH3 and (C) MMQ cells following 48
h treatment. Viability of the GH3 cells treated with 0-140
μM AS-IV at (D) 0, (E) 24, (F) 48 and (G) 72 h. Viability of
MMQ-treated cells treated with 0-350 μM AS-IV at (H) 0, (I)
24, (J) 48 and (K) 72 h. (L) TUNEL assay was used to assess the
apoptotic effect of AS-IV on GH3. (M) Number of apoptotic cells in
each group. ****P<0.0001, ***P<0.001,
**P<0.01 vs. control. ns, not significant; AS-IV,
astragaloside IV; IC50, half-maximal inhibitory
concentration; OD, optical density.

Figure 2

Transcriptomic sequencing reveals
that the TUBB4B gene is significantly downregulated in GH3 cells
treated with AS-IV and TUBB4B is highly expressed in pituitary
tumors. (A) Number of DEGs between the AS-IV and NC group. (B)
Volcano plot showing the distribution of DEGs. (C) Gene Ontology
enrichment analysis for the biological processes. (D) Heatmap of
the expression of significant DEGs. Gene set enrichment analysis
demonstrated that downregulated DEGs were enriched in (E) 'mitotic
cell cycle', (F) 'cyclin-dependent protein kinase holoenzyme
complex', (G) 'double-stranded RNA binding' and (H) 'structural
constituent of cytoskeleton' (ES<-0.5; P<0.05; FDR<0.25).
(I) Venn diagram of genes enriched in 'double-stranded RNA binding'
and 'structural constituent of cytoskeleton' showing that the
TUBB4B gene is at the intersection. (J) Expression of TUBB4B in
normal human pituitary glands and pituitary tumors; TUBB4B was
highly expressed in pituitary tumors (data from GSE136781,
P<0.0001). (K) Immunohistochemical staining of TUBB4B in tissue
samples from PTs with pituitary neuroendocrine tumors. TUBB4B,
tubulin β4B class IVb; AS-IV, Astragaloside IV; DEG, differentially
expressed genes; NC, negative control; ES, enrichment score; FDR,
false discovery rate; PT, pituitary tumor; Vim, vimentin.

Figure 3

TUBB4B promotes the proliferation and
inhibits the apoptosis of pituitary tumor cells. (A) Fluorescence
images of the GH3/MMQ cells transfected with lentivirus. Reverse
transcription-quantitative PCR was used to assess expression
efficiency of TUBB4B in lentivirus-transfected (B) GH3 and (C) MMQ
cell lines. (D) Cell Counting Kit-8 assay was used to assess the
viability of GH3 cells. (E) Western blotting was used to assess
expression efficiency of TUBB4B in lentivirus-transfected (F) GH3
and (G) MMQ cell lines. (H) Cell Counting Kit-8 assay was used to
assess the viability of MMQ cells. (I) EdU staining was used to
assess the effect of TUBB4B on the proliferation of GH3 cells. (J)
Colony formation of GH3 cells. (K) EdU-positive cells. (L) Number
of colonies. (M) Number of apoptotic GH3/MMQ cells. (N) Western
blot bands (O) Flow cytometry. Western blotting was used to assess
protein expression of (P) PCNA, (Q) Bcl-2, (R) Bax, (S)
pro-caspase-3 and (T) clv caspase-3 in GH3 and MMQ cell lines.
****P<0.0001, ***P<0.001,
**P<0.01, *P<0.05 vs. vector. TUBB4B,
tubulin β4B class IVb; OE, overexpression; shNC, short hairpin RNA
negative control; KD, knockdown; OD, optical density; clv, cleaved;
ns, not significant.

Figure 4

AS-IV exerts an inhibitory effect on
pituitary tumors by targeting and binding TUBB4B. (A) Surface
electrostatic potential of the TUBB4B protein. (B) Molecular
docking between AS-IV (green) and TUBB4B (blue). Yellow dashed
lines represent hydrogen bonds. (C) Molecular docking binding
energies of AS-IV and TUBB4B in different spatial conformations.
(D) RMSF, (E) RMSD, (F) Rg and (G) SASA between TUBB4B protein
residues and the AS-IV solution in the experimental system. (H)
Free energy landscape showing that the conformation of the
AS-IV-TUBB4B complex is dominant and more stable when the Rg was
2.07-2.09 nm and the RMSD was 0.03-0.17 nm. (I) Flow cytometry was
used to assess apoptosis of GH3 cells. (J) Immunofluorescence of
GH3 cells following AS-IV treatment. (K) EdU staining was used to
assess proliferation of GH3 cells. (L) Proportion of apoptotic GH3
cells. (M) Number of EdU-positive cells. (N) Western blotting was
used to assess the protein expression of (O) PCNA, (P) Bcl-2, (Q)
Bax, (R) pro-caspase-3 and (S) cleaved-caspase-3 in the GH3/MMQ
cell lines. ****P<0.0001, ***P<0.001,
**P<0.01, *P<0.05. AS-IV, Astragaloside
IV; TUBB4B, tubulin beta 4B class IVb; RMSF, root mean square
fluctuation; RMSD, Root Mean Square Deviation; Rg, radius of
Gyration; SASA, Solvent-Accessible Surface Area; OE,
overexpression; KD, knockdown; l. b., lower bound; u. b., upper
bound; clv, cleaved; ns, not significant.

Figure 5

OE-TUBB4B upregulates STMN1 and
activates the ERK pathway. (A) Effect of TUBB4B on GH3 cell cycle
progression. (B) Cell cycle distribution. (C) Reverse
transcription-quantitative PCR was used to assess the effects of
OE-TUBB4B. (D) Transcriptomic sequencing showing Kyoto Encyclopedia
of Genes and Genomes pathway enrichment of differentially expressed
genes. (E) Transcriptomic sequencing showing the protein-protein
interaction network of proteins that interact with TUBB4B (red box
indicates key pathway proteins that interact with TUBB4B). (F)
Interaction diagram between the TUBB4B and STMN1 proteins from
STRING. (G) Cell Counting Kit-8 assay was used to assess the
viability of GH3 cells treated with U0126. In GH3 cell lines
treated with U0126, the protein expression levels of (H) STMN1, (I)
p-STMN1 (I), and cPLA2 (J) were assessed by western blotting (K),
and levels of p-cPLA2 (L), ERK (M), p-ERK (N), CCND1 (O), JNK (P),
and p-JNK (Q) were also assessed. ****P<0.0001,
***P<0.001, **P<0.01,
*P<0.05 vs. vector. OE, overexpression; TUBB4B,
tubulin beta 4B class IVb; STMN1, stathmin 1; p-, phosphorylated;
cPLA2, cytosolic phospholipase A2; CCND1, cyclin D1; KD, knockdown;
OD, optical density; ns, not significant.

Figure 6

AS-IV inhibits proliferation and
promotes apoptosis of xenograft tumors in animals by targeting of
TUBB4B. (A) Xenograft tumors in nude mice. (B) Body weight and (C)
tumor volume growth curve of the nude mice. (D) Tumor weight. (E)
Representative hematoxylin and eosin staining images of tumors
(inset: 10X magnification). (F) Western blotting was used to assess
protein expression. (G) Representative IHC staining images of
TUBB4B, PCNA, Bcl-2, Bax and cleaved-caspase-3 proteins. Expression
levels of (H) PCNA, (I) Bcl-2 and (J) Bax. IHC staining scores for
(K) TUBB4B, (L) PCNA, (M) Bcl-2, (N) Bax and (O) clv-caspase-3
(Allred scoring system was used). ****P<0.0001,
***P<0.001, **P<0.01,
*P<0.05. AS-IV, astragaloside IV; TUBB4B, tubulin β4B
class IVb; IHC, immunohistochemistry; OE, overexpression; KD,
knockdown; ns, not significant; clv, cleaved.

Figure 7

Mechanism by which AS-IV regulates
proliferation and apoptosis in pituitary tumors by targeting
TUBB4B. AS-IV is chemically purified from the Chinese herbal
medicine Astragalus membranaceus. AS-IV interacts with
pituitary tumor cells by specifically binding TUBB4B, resulting in
the formation of a TUBB4B-AS-IV complex. This complex formation
decreases cytoskeletal stability while suppressing the expression
of the downstream pathway protein STMN1 and its phosphorylation.
Additionally, it inhibits the activation of the positive feedback
loop of the ERK pathway, suppressing pituitary tumor proliferation
and promoting apoptosis. AS-IV, astragaloside IV; TUBB4B, tubulin
beta 4B class IVb; cPLA, cytosolic phospholipase A; STMN1, stathmin
1; CCN, cyclin.
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Copy and paste a formatted citation
Spandidos Publications style
Li J, Qu Y, Zhang W, Yang Z, Zeng Y, Xu J, Xie K and Liu Q: Astragaloside IV targets TUBB4B to inhibit proliferation and promote apoptosis of pituitary tumor cells via the STMN1/ERK pathway. Int J Mol Med 57: 151, 2026.
APA
Li, J., Qu, Y., Zhang, W., Yang, Z., Zeng, Y., Xu, J. ... Liu, Q. (2026). Astragaloside IV targets TUBB4B to inhibit proliferation and promote apoptosis of pituitary tumor cells via the STMN1/ERK pathway. International Journal of Molecular Medicine, 57, 151. https://doi.org/10.3892/ijmm.2026.5822
MLA
Li, J., Qu, Y., Zhang, W., Yang, Z., Zeng, Y., Xu, J., Xie, K., Liu, Q."Astragaloside IV targets TUBB4B to inhibit proliferation and promote apoptosis of pituitary tumor cells via the STMN1/ERK pathway". International Journal of Molecular Medicine 57.6 (2026): 151.
Chicago
Li, J., Qu, Y., Zhang, W., Yang, Z., Zeng, Y., Xu, J., Xie, K., Liu, Q."Astragaloside IV targets TUBB4B to inhibit proliferation and promote apoptosis of pituitary tumor cells via the STMN1/ERK pathway". International Journal of Molecular Medicine 57, no. 6 (2026): 151. https://doi.org/10.3892/ijmm.2026.5822
Copy and paste a formatted citation
x
Spandidos Publications style
Li J, Qu Y, Zhang W, Yang Z, Zeng Y, Xu J, Xie K and Liu Q: Astragaloside IV targets TUBB4B to inhibit proliferation and promote apoptosis of pituitary tumor cells via the STMN1/ERK pathway. Int J Mol Med 57: 151, 2026.
APA
Li, J., Qu, Y., Zhang, W., Yang, Z., Zeng, Y., Xu, J. ... Liu, Q. (2026). Astragaloside IV targets TUBB4B to inhibit proliferation and promote apoptosis of pituitary tumor cells via the STMN1/ERK pathway. International Journal of Molecular Medicine, 57, 151. https://doi.org/10.3892/ijmm.2026.5822
MLA
Li, J., Qu, Y., Zhang, W., Yang, Z., Zeng, Y., Xu, J., Xie, K., Liu, Q."Astragaloside IV targets TUBB4B to inhibit proliferation and promote apoptosis of pituitary tumor cells via the STMN1/ERK pathway". International Journal of Molecular Medicine 57.6 (2026): 151.
Chicago
Li, J., Qu, Y., Zhang, W., Yang, Z., Zeng, Y., Xu, J., Xie, K., Liu, Q."Astragaloside IV targets TUBB4B to inhibit proliferation and promote apoptosis of pituitary tumor cells via the STMN1/ERK pathway". International Journal of Molecular Medicine 57, no. 6 (2026): 151. https://doi.org/10.3892/ijmm.2026.5822
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