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Article Open Access

Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling

  • Authors:
    • Kun Li
    • Jinwei Chen
    • Zhi Zheng
    • Yichun Gao
    • Jiayu Zhang
    • Wenya Ding
    • Tongguo Yang
    • Yuyang Gu
    • Xuhua Duan
    • Tiesuo Zhao
    • Huijie Jia
    • Pengfei Chen
    • Jianzhuang Ren
  • View Affiliations / Copyright

    Affiliations: Department of Interventional Radiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China, Department of Radiology, Shanxi Cancer Hospital, Shanxi Medical University, Taiyuan, Shanxi 030000, P.R. China, Xinxiang City Key Laboratory of Tumor Vaccine and Immunotherapy, Xinxiang Engineering Technology Research Center of Immune Checkpoint Drug for Liver‑Intestinal Tumors, Henan Medical University, Xinxiang, Henan 453000, P.R. China
    Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 69
    |
    Published online on: February 11, 2026
       https://doi.org/10.3892/or.2026.9074
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Abstract

Regorafenib, a multi‑kinase inhibitor, has limited efficacy in hepatocellular carcinoma (HCC) due to dose‑­dependent toxicity. The present study explored whether low‑dose Regorafenib combined with Nifuroxazide exerts enhanced anti‑tumor effects in HCC models. In vitro experiments with HepG2 cells showed the combination inhibited cell viability, proliferation and migration, induced apoptosis and reduced expression of key proteins, including phosphorylated signal transducer and activator of transcription 3 (STAT3). In vivo, H22 tumor‑bearing mice treated with the combination exhibited suppressed tumor growth without systemic toxicity, along with changes in apoptotic proteins, enhanced tumor‑infiltrating immune cells and improved systemic immune responses. These findings indicated that the combination exerts enhanced suppression of HCC by inhibiting STAT3 and remodeling anti‑tumor immunity, providing preclinical evidence for a safe and effective strategy.
View Figures

Figure 1

Transcriptome analysis reveals the
mechanisms underlying the enhanced efficacy of Regorafenib and
Nifuroxazide in HCC. (A) Gene expression heatmap of key genes
(including Vegfa, Bax and STAT3) in Regorafenib-treated compared
with control HCC cells. (B) GSEA-based enrichment of Hallmark
pathways in Regorafenib-treated HCC cells. (C) GSEA enrichment plot
for the Hallmark_IL6_Jak_STAT3_Signaling pathway. (D)
Representative results of the CCK-8 assay (for cell viability) of
HepG2 cells under different treatments. (E) Statistical analysis of
cell viability in Fig. 1D. (F)
Representative colony formation assay images of HepG2 cells under
different treatments. (G) Statistical analysis of the number of
colonies in Fig. 1F. (H)
Representative wound-healing assay images (0, 24 and 48 h) of HepG2
cells under different treatments. (I) Statistical analysis of wound
closure rate in Fig. 1H. Data are
presented as mean ± standard deviation (n=3). *P<0.05 vs. the
Control group; #P<0.05 vs. the Regorafenib group;
$P<0.05 vs. the Nifuroxazide group. HCC,
hepatocellular carcinoma; GSEA, Gene Set Enrichment Analysis.

Figure 2

Effects of different treatments on
apoptosis and the expression of related proteins. (A) Effects of
Nifuroxazide combined with Regorafenib on HepG2 cell apoptosis
detected by flow cytometry. (B) Statistical analysis of Fig. 2A. (C) Expression of related proteins
in cells detected by western blotting. (D) Statistical analysis of
Fig. 2C. Data are presented as mean
± standard deviation (n=3). *P<0.05 vs. the Control group;
#P<0.05 vs. the Regorafenib group;
$P<0.05 vs. the Nifuroxazide group. STAT3, signal
transducer and activator of transcription 3; PD-L1, programmed
death ligand 1; VEGF, vascular endothelial growth factor; p-,
phosphorylated.

Figure 3

Effects of different treatments on
tumor growth. (A) Treatment schedule: Mice were divided into four
groups; each mouse subcutaneously inoculated with 1×106
H22 cells prior to treatment. (B) Representative tumor images of
each group. (C) Statistical analysis of tumor weight from Fig. 3B. (D) Statistical analysis of
changes in tumor volume in mice. (E) Statistical analysis of
changes in mouse body weight. Data are presented as mean ± standard
deviation (n=6). *P<0.05 vs. the Control group;
#P<0.05 vs. the Regorafenib group;
$P<0.05 vs. the Nifuroxazide group.

Figure 4

Effects of different treatments on
cell apoptosis, proliferation, migration and the expression of
related proteins. (A) Expression of related proteins detected by
western blotting. (B) Statistical analysis of Fig. 4A. (C) Expression of Ki-67 in tumor
tissues detected by immunofluorescence. (D) Tumor cell apoptosis in
tumor tissues detected by TUNEL assay. (E) Statistical analysis of
Fig. 4C. (F) Statistical analysis
of Fig. 4D. Data are presented as
mean ± standard deviation (n=3). *P<0.05 vs. the Control group;
#P<0.05 vs. the Regorafenib group;
$P<0.05 vs. the Nifuroxazide group. TUNEL, terminal
deoxynucleotidyl transferase dUTP nick end labelling; STAT3, signal
transducer and activator of transcription 3; PD-L1, programmed
death ligand 1; VEGF, vascular endothelial growth factor; p-,
phosphorylated.

Figure 5

Effects of various treatments on
immune cells in tumor tissues. (A-D) The expression of
CD3+CD4+ T cells,
CD3+CD8+ T cells, CD86+
macrophages and CD206+ macrophages in mouse tumor
tissues. (E) Statistical analysis of Fig. 5A-D. Data are presented as mean ±
standard deviation (n=3). *P<0.05 vs. the Control group;
#P<0.05 vs. the Regorafenib group;
$P<0.05 vs. the Nifuroxazide group.

Figure 6

Effects of different treatments on
immune cells. (A-H) Distribution and statistical analysis of
CD3+CD4+ T cells,
CD3+CD8+ T cells, CD86+ cells and
CD206+ cells in mouse spleens. (I-L) Distribution and
statistical analysis of CD3+CD4+ T cells and
CD3+CD8+ T cells in peripheral blood. Data
are presented as mean ± standard deviation (n=3). *P<0.05 vs.
the Control group; #P<0.05 vs. the Regorafenib group;
$P<0.05 vs. the Nifuroxazide group.

Figure 7

Proposed mechanism by which
Regorafenib and Nifuroxazide exert enhanced suppression of
hepatocellular carcinoma by inhibiting STAT3 and immune remodeling.
Low-dose Regorafenib combined with Nifuroxazide exerts enhanced
anti-tumor effects through complementary mechanisms: Nifuroxazide
inhibits STAT3 phosphorylation, drives macrophage polarization
toward the pro-inflammatory M1 phenotype and promotes
CD8+ T cell activation, thereby reversing the
immunosuppressive tumor microenvironment. Concurrently, low-dose
Regorafenib exerts anti-angiogenic effects by suppressing VEGF
expression and downregulates PD-L1 expression on HCC cells,
directly impeding tumor cell proliferation and migration.
Collectively, this dual-action enhanced effect enhances immune
surveillance, disrupts tumor vascular supply and amplifies the
killing efficacy against HCC cells. STAT3, signal transducer and
activator of transcription 3; VEGF, vascular endothelial growth
factor; PD-L1, programmed death ligand 1; HCC, hepatocellular
carcinoma.
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Copy and paste a formatted citation
Spandidos Publications style
Li K, Chen J, Zheng Z, Gao Y, Zhang J, Ding W, Yang T, Gu Y, Duan X, Zhao T, Zhao T, et al: Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling. Oncol Rep 55: 69, 2026.
APA
Li, K., Chen, J., Zheng, Z., Gao, Y., Zhang, J., Ding, W. ... Ren, J. (2026). Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling. Oncology Reports, 55, 69. https://doi.org/10.3892/or.2026.9074
MLA
Li, K., Chen, J., Zheng, Z., Gao, Y., Zhang, J., Ding, W., Yang, T., Gu, Y., Duan, X., Zhao, T., Jia, H., Chen, P., Ren, J."Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling". Oncology Reports 55.4 (2026): 69.
Chicago
Li, K., Chen, J., Zheng, Z., Gao, Y., Zhang, J., Ding, W., Yang, T., Gu, Y., Duan, X., Zhao, T., Jia, H., Chen, P., Ren, J."Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling". Oncology Reports 55, no. 4 (2026): 69. https://doi.org/10.3892/or.2026.9074
Copy and paste a formatted citation
x
Spandidos Publications style
Li K, Chen J, Zheng Z, Gao Y, Zhang J, Ding W, Yang T, Gu Y, Duan X, Zhao T, Zhao T, et al: Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling. Oncol Rep 55: 69, 2026.
APA
Li, K., Chen, J., Zheng, Z., Gao, Y., Zhang, J., Ding, W. ... Ren, J. (2026). Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling. Oncology Reports, 55, 69. https://doi.org/10.3892/or.2026.9074
MLA
Li, K., Chen, J., Zheng, Z., Gao, Y., Zhang, J., Ding, W., Yang, T., Gu, Y., Duan, X., Zhao, T., Jia, H., Chen, P., Ren, J."Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling". Oncology Reports 55.4 (2026): 69.
Chicago
Li, K., Chen, J., Zheng, Z., Gao, Y., Zhang, J., Ding, W., Yang, T., Gu, Y., Duan, X., Zhao, T., Jia, H., Chen, P., Ren, J."Regorafenib and Nifuroxazide exert enhanced suppression of hepatocellular carcinoma by inhibiting STAT3 and immune remodeling". Oncology Reports 55, no. 4 (2026): 69. https://doi.org/10.3892/or.2026.9074
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