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Article Open Access

Senescence‑associated IL‑33 secretion undermines sorafenib efficacy in hepatocellular carcinoma via immune evasion

  • Authors:
    • Yu-Xin Lin
    • Hsien Liu
    • Wei-Chiao Liao
    • Yi-Ching Wang
    • Bo-Cheng Zhang
    • Shu-Wen Wan
    • Chien-Chin Chen
    • Chih-Peng Chang
  • View Affiliations / Copyright

    Affiliations: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan, Division of General Surgery, Department of Surgery, Ditmanson Medical Foundation Chia‑Yi Christian Hospital, Chiayi 60002,Taiwan, Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan, Department of Pathology, Ditmanson Medical Foundation Chia‑Yi Christian Hospital, Chiayi 60002, Taiwan
    Copyright: © Lin et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 154
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    Published online on: September 15, 2025
       https://doi.org/10.3892/or.2025.8987
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Abstract

Hepatocellular carcinoma (HCC) is a highly lethal cancer with increasing incidence rates worldwide. The recommended treatments for advanced‑stage HCC are sorafenib and regorafenib; however, developing resistance to these medications significantly limits their effectiveness, and the underlying mechanisms are poorly understood. The present study demonstrated that interleukin‑33 (IL‑33) promotes sorafenib resistance via immune regulation. In vitro, western blotting and reverse transcription‑quantitative PCR showed that both sorafenib and regorafenib treatments led to an increase in the upregulation and secretion of IL‑33 through a positive feedback loop involving the IL‑33/transmembrane suppression of tumorigenicity 2 (ST2L) pathway. Senescence‑associated β‑galactosidase staining and western blotting revealed that sorafenib and regorafenib treatments induce cell senescence in HCC cells. Flow cytometric analysis indicated that he secreted IL‑33 enhanced programmed cell death ligand 1 (PD‑L1) expression in HCC cells by activating NF‑κB pathways in response to the treatments. In vivo, a HCC‑bearing subcutaneous mouse model revealed that blocking the IL‑33 signaling pathway with anti‑IL‑33 or anti‑ST2L neutralizing antibodies, combined with sorafenib, significantly reduced tumor size, growth rate, and weight. Additionally, there was a notable decrease in tumor PD‑L1 expression and an increase in intra‑tumor CD8+ T cells infiltration. Importantly, the enhanced therapeutic efficacy of the anti‑IL‑33 treatment in sorafenib‑treated HCC‑bearing mice was lost in immunocompromised mice. This indicates that the anti‑IL‑33 neutralizing antibody enhances the antitumor activity of sorafenib by modulating the immune response rather than directly affecting HCC cell proliferation. The findings of the present study suggested that IL‑33 plays a role in decreasing the therapeutic effectiveness of sorafenib and regorafenib in HCC cells. The present study highlights the potential of targeting the IL‑33/ST2L axis in combination with targeted therapies as a novel strategy to improve the limited efficacy of sorafenib and regorafenib.
View Figures

Figure 1

Sorafenib and regorafenib induce
IL-33 upregulation and secretion in hepatocellular carcinoma cells.
(A) Huh-7 cells were treated with sorafenib or regorafenib at the
indicated concentrations. Cell death was assessed using PI staining
through flow cytometry. Representative flow cytometry histograms of
PI staining in Huh7 cells under the indicated treatment conditions
are shown (left). Quantification of PI-positive cells (%) is
presented (right). (B and C) Huh-7 cells were treated with DMEM,
sorafenib (10 µM), or regorafenib (20 µM) for the indicated time.
(B) The protein expression of IL-33 was analyzed by western
blotting. (C) The mRNA level of IL33 was determined by
RT-qPCR. (D and E) Huh-7 cells were treated with rIL-33 at the
indicated concentrations for 48 h. (D, upper) The mRNA level of
IL33 was determined by RT-qPCR. (D, lower) The cell surface
expression of ST2L was measured by flow cytometry. (E) The protein
expression of IL-33 was analyzed by western blotting. (F) Huh-7
cells were treated with sorafenib (left) and regorafenib (right)
with or without α-IL-33 antibodies (10 µg/ml). The mRNA level of
IL33 was determined by RT-qPCR. *P<0.05, **P<0.01 and
***P<0.001. PI, propidium iodide; RT-qPCR, reverse
transcription-quantitative PCR; Sup, supernatant; WCL, whole cell
lysate.

Figure 2

Sorafenib and regorafenib trigger
cellular senescence in hepatocellular carcinoma cells. (A) Huh-7
cells were treated with sorafenib (1 and 10 µM, upper) and
regorafenib (10 and 20 µM, lower). Cell numbers were calculated
using a hemocytometer at the indicated time points. (B) Huh-7 cells
were treated with sorafenib (10 µM) and regorafenib (20 µM) for 96
h. The SA-β-gal activity was detected using a senescence detection
kit. (C) Huh-7 cells were treated with sorafenib (10 µM, left) and
regorafenib (20 µM, right) for the indicated time. The protein
expression of p16 and p21 was analyzed by western blotting.

Figure 3

Sorafenib and regorafenib enhance
PD-L1 expression via IL-33 signaling in hepatocellular carcinoma
cells. (A) Huh-7 cells were treated with sorafenib (10 µM) and
regorafenib (20 µM) for 96 h. The cell surface expression of PD-L1
was measured by flow cytometry. (B) Huh-7 cells were treated with
sorafenib (10 µM, upper) and regorafenib (20 µM, lower) with or
without treatment of α-IL-33 antibodies (10 µg/ml) for 96 h. The
cell surface expression of PD-L1 was measured by flow cytometry. (C
and D) Huh-7 cells were treated with rIL-33 at the indicated
concentrations for 96 h. (C) The mRNA level of CD274 was
determined by reverse transcription-quantitative PCR. (D) Huh-7
cells were treated with rIL-33 (100 ng/ml) with or without
treatment of JSH-23 (50 µM). The cell surface expression of PD-L1
was measured by flow cytometry. *P<0.05, **P<0.01 and
***P<0.001. PD-L1, programmed death ligand 1.

Figure 4

Blockage of IL-33/ST2L by
neutralizing antibodies enhances the antitumor efficacy of
sorafenib. (A) Schematic illustration of the treatment regimen in
the subcutaneous HCC-bearing mouse model. HCC-bearing mice were
treated with sorafenib (10 µg/g), α-IL-33 (10 µg/g), α-ST2L (10
µg/g), or a combination of sorafenib with α-IL-33 or α-ST2L. (B)
Tumor growth was measured starting from day 5 and once every 4
days. (C and D) Tumor size (C) and tumor weight (D) were measured
at the end of the experiment. (E and F) Paraffin-embedded HCC tumor
sections from each group were stained with (E) IL-33 and (F) PD-L1
and counterstained with hematoxylin. Magnified views of the inset
regions are shown on the right. (G) Intra-tumoral CD4+ T
cells (upper), CD8+ T cells (middle), and
CD8+CTLA4+ cells (lower) were measured by
flow cytometry. HCC, hepatocellular carcinoma. *P<0.05 and
**P<0.01.

Figure 5

The effectiveness of α-IL-33
neutralizing antibodies in enhancing the antitumor activity of
sorafenib is abolished in athymic mice. (A) Schematic illustration
of the treatment regimen in the subcutaneous hepatocellular
carcinoma-bearing nude mouse model. HCC-bearing nude mice were
treated with sorafenib (10 µg/g), α-IL-33 (10 µg/g), or a
combination of sorafenib with α-IL-33. (B) Tumor growth was
measured starting on day 4 and once every 4 days. (C and D) Tumor
size (C) and tumor weight (D) were measured at the end of the
experiment.
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Copy and paste a formatted citation
Spandidos Publications style
Lin Y, Liu H, Liao W, Wang Y, Zhang B, Wan S, Chen C and Chang C: Senescence‑associated IL‑33 secretion undermines sorafenib efficacy in hepatocellular carcinoma via immune evasion. Oncol Rep 54: 154, 2025.
APA
Lin, Y., Liu, H., Liao, W., Wang, Y., Zhang, B., Wan, S. ... Chang, C. (2025). Senescence‑associated IL‑33 secretion undermines sorafenib efficacy in hepatocellular carcinoma via immune evasion. Oncology Reports, 54, 154. https://doi.org/10.3892/or.2025.8987
MLA
Lin, Y., Liu, H., Liao, W., Wang, Y., Zhang, B., Wan, S., Chen, C., Chang, C."Senescence‑associated IL‑33 secretion undermines sorafenib efficacy in hepatocellular carcinoma via immune evasion". Oncology Reports 54.5 (2025): 154.
Chicago
Lin, Y., Liu, H., Liao, W., Wang, Y., Zhang, B., Wan, S., Chen, C., Chang, C."Senescence‑associated IL‑33 secretion undermines sorafenib efficacy in hepatocellular carcinoma via immune evasion". Oncology Reports 54, no. 5 (2025): 154. https://doi.org/10.3892/or.2025.8987
Copy and paste a formatted citation
x
Spandidos Publications style
Lin Y, Liu H, Liao W, Wang Y, Zhang B, Wan S, Chen C and Chang C: Senescence‑associated IL‑33 secretion undermines sorafenib efficacy in hepatocellular carcinoma via immune evasion. Oncol Rep 54: 154, 2025.
APA
Lin, Y., Liu, H., Liao, W., Wang, Y., Zhang, B., Wan, S. ... Chang, C. (2025). Senescence‑associated IL‑33 secretion undermines sorafenib efficacy in hepatocellular carcinoma via immune evasion. Oncology Reports, 54, 154. https://doi.org/10.3892/or.2025.8987
MLA
Lin, Y., Liu, H., Liao, W., Wang, Y., Zhang, B., Wan, S., Chen, C., Chang, C."Senescence‑associated IL‑33 secretion undermines sorafenib efficacy in hepatocellular carcinoma via immune evasion". Oncology Reports 54.5 (2025): 154.
Chicago
Lin, Y., Liu, H., Liao, W., Wang, Y., Zhang, B., Wan, S., Chen, C., Chang, C."Senescence‑associated IL‑33 secretion undermines sorafenib efficacy in hepatocellular carcinoma via immune evasion". Oncology Reports 54, no. 5 (2025): 154. https://doi.org/10.3892/or.2025.8987
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