Nifuroxazide suppresses PD-L1 expression and enhances the efficacy of radiotherapy in hepatocellular carcinoma
- Department of Immunology, School of Basic Medical Sciences, Xinxiang Medical University, China
- Xinxiang Engineering Technology Research Center of immune checkpoint drug for Liver-Intestinal Tumors, Xinxiang Medical University, China
- Henan International Joint Laboratory of Immunity and Targeted Therapy for Liver-Intestinal Tumors, Xinxiang Medical University, China
- Zhengzhou Central Hospital Affiliated to Zhengzhou University, China
- The Biomedical Translational Research Institute, Faculty of Medical Science, Jinan University, China
- Department of Oncology, The Third Affiliated Hospital of Xinxiang Medical University, China
- Department of Gynecology, Xinxiang Central Hospital, China
- The Fourth Clinical College, Xinxiang Medical University, China
Figures
Figure 1 with 1 supplement
The effect of radiotherapy in combination with nifuroxazide on the proliferation, migration, and apoptosis of HepG2 cells.
(A) The effect of the radiotherapy in combination with nifuroxazide on the viability of HepG2 cells by CCK-8 assay. (B) The effect of the radiotherapy in combination with nifuroxazide on the proliferation of HepG2 cells by cell clone formation assay. (C) The effect of the radiotherapy in combination with nifuroxazide on the migration of HepG2 cells by Wound-Healing assay. (D) The effect of the radiotherapy in combination with nifuroxazide on the migration of HepG2 cells by transwell assay. (E) The effect of the radiotherapy in combination with nifuroxazide on the apoptosis of HepG2 cells by flow cytometry assay. One-way analysis of variance (ANOVA) was carried out and the data were presented as mean± SD (n=3). Compared with the control group, *p<0.05; compared with ‘4 Gy’ group, #p<0.05.
Figure 1—figure supplement 1
The effect of radiotherapy and nifuroxazide on the proliferation, migration, and apoptosis of HepG2 cells.
(A) The detection of radiotherapy combined with nifuroxazide on the viability of HepG2 cells using the CCK-8 assays. (B) The effect of radiotherapy and nifuroxazide on the migration of HepG2 through wound healing assays. (C) The influence of radiotherapy and nifuroxazide on the apoptosis of HepG2 cells using apoptosis assays.
Figure 2
The effect of radiotherapy in combination with nifuroxazide on the expressions of tumor-associated proteins in cells.
After radiotherapy, HepG2 cells were treated with nifuroxazide at the different dose. At 24 hr or 48 hr after being incubated, the expression of tumor-associated proteins was detected by Western blot. (A–E) The expression of Stat3, p-Stat3, PCNA, Ki67, and cyclin D1 related to cell proliferation was analyzed by Western blot. (F–N) The expression of cytochrome C, pro-caspase 3, c-caspase 3, pro-caspase 9, c-caspase 9, Bax, Bcl-2, PARP, and c-PARP related with cell apoptosis was analyzed by Western blot. One-way analysis of variance (ANOVA) was carried out and the data are presented as mean ± SD (n=3). Compared with the control group, *p<0.05; compared with ‘4 Gy’ group, #p<0.05.
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Figure 2—source data 1
Original file for the Western blot analysis in Figure 2A-F (anti-p-Stat3, anti-Stat3, anti-MMP2, anti-PCNA, anti-Ki67, anti-Cyclin D1, anti-Cytochrome C, and anti-tubulin).
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig2-data1-v1.zip
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Figure 2—source data 2
Original file for the Western blot analysis in Figure 2G-L (anti-pro-caspase3, anti-c-caspase3, anti-pro-caspase9, anti-c-caspase9, anti-Bax, anti-BCL-2, and anti-tubulin).
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig2-data2-v1.zip
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Figure 2—source data 3
Original file for the Western blot analysis in Figure 2M, N (anti-PARP, anti-C-PARP, and anti-tubulin).
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig2-data3-v1.zip
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Figure 2—source data 4
PDF containing Figure 2A-N and original scans of the relevant Western blot analysis (anti-p-Stat3, anti-Stat3, anti-MMP2, anti-PCNA, anti-Ki67, anti-Cyclin D1, anti-Cytochrome C, anti-pro-caspase3, anti-c-caspase3, anti-pro-caspase9, anti-c-caspase9, anti-Bax, anti-BCL-2, anti-PARP, anti-C-PARP, and anti-tubulin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig2-data4-v1.pdf
Figure 3
The effects of radiotherapy in combination with nifuroxazide on tumor growth and survival of tumor-bearing mice.
At 7 days after establishing the tumor model, the mice are received distinct treatments. (A) Treatment scheme of different methods. (B) Tumor pictures and tumor weight of tumor-bearing mice under different treatments are measured for statistical analysis (n=5). (C) The tumor volume changes of tumor-bearing mice under different treatments (n=10). (D) The survival of tumor-bearing mice under different treatments (n=10). (E) Pathological pictures of tumor-bearing mice under different treatments. The ruler in the top row of the images represents 200 nanometers; the ruler in the bottom row of the images represents 50 nanometers. (F) Statistical analysis of pathological score (n=3). One-way analysis of variance (ANOVA) was carried out and the data are presented as mean ± SD except in Figure 3D, which was analyzed by the Kaplan-Meier method. Compared with the PBS group, *p<0.05; compared with the radiotherapy group, $p<0.05; compared with the nifuroxazide group, #p<0.05.
Figure 4
The effects of radiotherapy in combination with nifuroxazide on the cell proliferation or apoptosis in tumor tissues.
(A–C) The expression of Ki6, PCNA, or c-caspase-3 in tumor tissues is detected by immunohistochemistry. The ruler in the top row of the images represents 200 nanometers; the ruler in the bottom row of the images represents 50 nanometers. (D) The cell apoptosis on tumor tissues is detected by TUNEL assay. (E–N) The protein expression of Stat3, p-Stat3, Ki67, PCNA, cyclin D1, cytochrome C, Bcl-2, Bax, pro-caspase 3, and c-caspase 3 in tumor tissues is detected by Western blot. One-way analysis of variance (ANOVA) was carried out and the data are presented as mean ± SD (n=3). Compared with the PBS group, *p<0.05; compared with the radiotherapy group, #p<0.05; compared with the nifuroxazide group, $p<0.05.
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Figure 4—source data 1
Original file for the Western blot analysis in Figure 4E–N (anti-p-Stat3, anti-Stat3, anti-MMP2, anti-Ki67, anti-PCNA, anti-Cyclin D1, anti-Cytochrome C, anti-BCL-2, anti-Bax, anti-c-caspase3, anti-pro-caspase3, and anti-tubulin).
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig4-data1-v1.zip
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Figure 4—source data 2
PDF containing Figure 4E–N and original scans of the relevant Western blot analysis (anti-p-Stat3, anti-Stat3, anti-MMP2, anti-Ki67, anti-PCNA, anti-Cyclin D1, anti-Cytochrome C, anti-BCL-2, anti-Bax, anti-c-caspase3, anti-pro-caspase3, and anti-tubulin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig4-data2-v1.pdf
Figure 5 with 1 supplement
The effects of radiotherapy in combination with nifuroxazide on the infiltration of immune cells in tumor tissues.
(A–C) Different isoforms of T lymphocytes infiltration in tumor tissues detected by immunofluorescence assay. (D) M1 macrophage infiltration in tumor tissues detected by immunofluorescence assay. (E) Statistical analysis about Semi-quantitative of Figure A-D. (F–I) The protein expression of CD4, CD8, CD86, and Granzyme in tumor tissues is detected by Western blot. One-way analysis of variance (ANOVA) was carried out and the data are presented as mean ± SD (n=3). Compared with the PBS group, *p<0.05; compared with the radiotherapy group, #p<0.05; compared with the nifuroxazide group, $p<0.05.
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Figure 5—source data 1
Original file for the Western blot analysis in Figure 5F–I (anti-p-CD4, anti-CD8, anti-CD86, anti-Granzyme B, and anti-tubulin).
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig5-data1-v1.zip
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Figure 5—source data 2
PDF containing Figure 5F–I and original scans of the relevant Western blot analysis (anti-p-CD4, anti-CD8, anti-CD86, anti-Granzyme B, and anti-tubulin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig5-data2-v1.pdf
Figure 5—figure supplement 1
The effect of combined nifuroxazide with radiotherapy on the immunocyte in tumor tissue of tumor-bearing mice.
(A) The proportion of CD4+ T lymphocytes in the tumor tissue of tumor-bearing mice after combination treatment. (B) The percentage of CD8+ T lymphocytes in the tumor tissue of tumor-bearing mice after combination treatment. (C) The proportion of Tregs+ T lymphocytes in the tumor tissue of tumor-bearing mice after combination treatment. (D) The percentage of Granzyme B+ lymphocytes in the tumor tissue of tumor-bearing mice after combination treatment. (E) The ratio of natural killer (NK) cells in the tumor tissue of tumor-bearing mice after combination treatment.
Figure 6
The effect of radiotherapy in combination with nifuroxazide on the ratios of immune cells in spleens.
The ratios of immune cells in the spleens of tumor-bearing mice were detected by flow cytometry. One-way analysis of variance (ANOVA) was carried out and the data are presented as mean ± SD (n=3). Compared with the PBS group, *p<0.05; compared with the radiotherapy group, #p<0.05; compared with the nifuroxazide group, $p<0.05.
Figure 7 with 1 supplement
Nifuroxazide in combination with the radiotherapy significantly increases the degradation of pd-l1 through the ubiquitination proteasome pathway.
(A) The expression of PD-L1 in cells combined treatment with radiotherapy and nifuroxazide is detected by Western blot. (B) The mRNA level of PD-L1 in cells is detected by PCR. (C–E) The effect of the radiotherapy combined with nifuroxazide on PD-L1 degradation through the ubiquitination-proteasome pathway. (F) The schematic diagram of the mechanism that nifuroxazide degraded PD-L1 through the ubiquitination-proteasome pathway. One-way analysis of variance (ANOVA) was carried out and the data are presented as mean ± SD (n=3). Compared with ‘0’ group, *p<0.05; compared with ‘4 Gy’ group, #p<0.05.
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Figure 7—source data 1
Original file for the Western blot analysis in Figure 7A and C–E (anti-PD-L1, anti-GSK3β, and anti-tubulin).
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig7-data1-v1.zip
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Figure 7—source data 2
PDF containing Figure 7A and C–E and original scans of the relevant Western blot analysis (anti-PD-L1, anti-GSK3β, and anti-tubulin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig7-data2-v1.pdf
Figure 7—figure supplement 1
The effect of nifuroxazide on PD-L1 expression in Huh7 cells.
(A) The effect of the combination therapy using nifuroxazide and radiotherapy on the expression of PD-L1 protein in Huh7 cells using Western blot analysis. (B) The effect of combined nifuroxazide with radiotherapy on the expression of PD-L1 gene in Huh7 cells via qPCR. (C) The effect of proteasome inhibitor MG132 on the expression of PD-L1.
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Figure 7—figure supplement 1—source data 1
Original file for the Western blot analysis in Figure 7—figure supplement 1A and C (anti-PD-L1, and anti-tubulin).
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig7-figsupp1-data1-v1.zip
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Figure 7—figure supplement 1—source data 2
PDF containing Figure 7—figure supplement 1A and C and original scans of the relevant Western blot analysis (anti-PD-L1, and anti-tubulin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig7-figsupp1-data2-v1.pdf
Figure 8
The effect of radiotherapy in combination with nifuroxazide on the expression of PD-L1 in tumor tissues.
(A) PD-L1 expression in tumor tissues of hepatocellular carcinoma (HCC) patients treated with radiotherapy are detected by immunofluorescence. (B) PD-L1 expression in tumor tissues of mice combined treatment with radiotherapy and nifuroxazide is detected by immunofluorescence. (C–D) The expression of PD-L1 or GSK3β in tumor tissues of mice combined treatment with radiotherapy and nifuroxazide is detected by Western blot. One-way analysis of variance (ANOVA) was carried out and the data were expressed as mean ± SD (n=3). Compared with the before-radiotherapy samples, ****p<0.0001; compared with the PBS group, *p<0.05; compared with the radiotherapy group, #p<0.05; compared with the nifuroxazide group, $p<0.05.
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Figure 8—source data 1
Original file for the Western blot analysis in Figure 8C–D (anti-PD-L1, anti-GSK3β, and anti-tubulin).
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig8-data1-v1.zip
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Figure 8—source data 2
PDF containing Figure 8C–D and original scans of the relevant Western blot analysis (anti-PD-L1, anti-GSK3β, and anti-tubulin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/90911/elife-90911-fig8-data2-v1.pdf
Figure 9
The synergistic antitumor mechanism of radiotherapy in combination with nifuroxazide in tumor-bearing mice.
NK cell: natural killer cell. Treg: regulatory T cells. TAM: tumor-associated macrophage. CD8+ CTL: CD8+ cytotoxic lymphocyte.
Additional files
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Supplementary file 1
The list of antibodies used for cytometry in this study.
- https://cdn.elifesciences.org/articles/90911/elife-90911-supp1-v1.xlsx
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Supplementary file 2
The list of antibodies used for western blotting in this study.
- https://cdn.elifesciences.org/articles/90911/elife-90911-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/90911/elife-90911-mdarchecklist1-v1.docx
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Nifuroxazide suppresses PD-L1 expression and enhances the efficacy of radiotherapy in hepatocellular carcinoma
eLife 12:RP90911.
https://doi.org/10.7554/eLife.90911.3
