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. 2020 Mar 26;17(7):939-945.
doi: 10.7150/ijms.42978. eCollection 2020.

The extracts of Astragalus membranaceus overcome tumor immune tolerance by inhibition of tumor programmed cell death protein ligand-1 expression

Hsu-Liang Chang  1 , Yi-Hsuan Kuo  2 , Li-Hsien Wu  2 , Chih-Min Chang  2   3 , Kai-Jen Cheng  2   4 , Yu-Chang Tyan  5 , Che-Hsin Lee  2   6   7   8   9
Affiliations

The extracts of Astragalus membranaceus overcome tumor immune tolerance by inhibition of tumor programmed cell death protein ligand-1 expression

Hsu-Liang Chang et al. Int J Med Sci. .

Abstract

A polysaccharide isolated from the radix of Astragalus membranaceus, called PG2, used in traditional Chinese medicine, with potential hematopoiesis inducing and immunomodulation activities. PG2 extracted from A. membranaceus has been demonstrated as a novel alternative medicine for cancer patients. Recently, we demonstrated that PG2 enhanced chemotherapy through bystander effect and reduced the expression of indoleamine 2, 3-dioxygenase 1 in tumor cells. Many tumors have been proven to have a high expression of programmed cell death protein ligand-1 (PD-L1), which binds with programmed cell death protein-1(PD-1) in immune cells, thus causing immune tolerance within the tumor microenvironment. With decreased expression of PD-L1, increased immune response can be observed, which might be helpful when developing tumor immunotherapy. The antitumor therapeutic effect mediated by PG2 may associate with an inflammatory immune response at the tumor site. However, the molecular mechanism that by which PG2 inhibits PD-L1 is still incompletely known. The expression of PD-L1 was decreased after tumor cells were treated with PG2. In addition, the cell signaling pathway in tumor cells was evaluated by Western blotting analysis after PG2 treatment. PG2 can downregulate the expression of PD-L1 on the cell surface via the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase beta-1 (p70S6K) pathway. In conclusion, our results indicate that PG2 inhibits PD-L1 expression and plays a crucial role in immunotherapy, which might be a promising strategy combined with other treatments.

Keywords: programmed cell death protein ligand-1; the extracts of Astragalus membranaceus (PG2); tumor immune tolerance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Effects of PG2 on cell viability in 4T1 and CT26 cells. (A) 4T1 and (B) CT26 cells were treated with indicated concentrations of PG2 for 24 h. Cell viability was measured by Cell Counting Kit-8 assay. (mean ± SD, n = 6)
Figure 2
Figure 2
PG2-mediated PD-L1 protein expression. PG2 reduced PD-L1 protein expression in (A) 4T1 and (B) CT26 cells in a dose-dependent manner. After treatment with PG2 (0-10 μg/ml) for 24 h, the expression of PD-L1 levels in 4T1 and CT26 cells were measured by Western blotting. PG2 reduced PD-L1 expression through AKT/mTOR/p70S6K signal pathways. PG2 reduced AKT/mTOR/p70S6K signaling pathways in (A) 4T1 and (B) CT26 cells in a dose-dependent manner. After treatment with PG2 (0-10 μg/ml) for 24 h, the expression of AKT/mTOR/p70S6K signaling pathways in 4T1 and CT26 cells were measured by Western blotting. Each experiment was repeated three times with similar results. PG2 reduced PD-L1 expression on tumor surface. PG2 reduced PD-L1 expression on the surface of (C) 4T1 and (D) CT26 cells. After treatment with PG2 (0-10 μg/ml) for 24 h, the expression of PD-L1 on the surface of (C) 4T1 and (D) CT26 cells were measured by flow cytometry. *, p<0.05; ***, p<0.001
Figure 3
Figure 3
PG2 reduces PD-L1 expression through AKT pathway. The (A) 4T1 and (B) CT26 (105) cells were transfected with constitutively active AKT plasmid (5μg) for 16 h prior to treated with PG2 (10µg/ml) for 24 h. The protein expression was measured by Western blotting. Each experiment was repeated three times with similar results.
Figure 4
Figure 4
PG2 affected AKT/mTOR signaling and apoptosis in immune cells. WEHI-3, EL4 cells and T lymphocytes were co-cultured with PG2 (10μg/ml)-treated (A) 4T1 and (B) CT26 cells prior to harvesting. The protein expression was analyzed by Western blotting. Each experiment was repeated three times with similar results.
Figure 5
Figure 5
The antitumor effects of PG2 in combination with cisplatin on tumors. Groups of 5-7 mice that inoculated subcutaneously with (A) 4T1 or (B) CT26 cells (106) at day 0 were intraperitoneal injected with PG2 (50 mg/kg) at day 7, day 9, day 14, day 16, day21, day 23 followed by cisplatin (2 mg/kg) at day 8, 15, and 22, or with either treatment alone. The control mice were treated with PBS. All of the mice were monitored for tumor weight at day 45. *, p<0.05; **, p<0.01; ***, p<0.001
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