Astragaloside IV-PESV Repressed T Cell Immunosuppression by Inhibiting PD-L1 Expression in Prostate Cancer through STAT3 Pathway

,


Introduction
Prostate cancer (PCa) has a high morbidity and mortality rate and is a major threat to men's health worldwide.Te morbidity and mortality of PCa continue to increase yearly due to its early symptoms that are not easily detected and limited screening conditions [1].From a clinical, morphological, and molecular perspective, PCa is a heterogeneous disease [2].Current treatment options include androgen receptor-(AR-) targeted drugs, chemotherapy, radionuclides, and the poly (ADP-ribose) inhibitor olaparib [3].Although androgen deprivation therapy is efective in advanced PCa, it still does not prevent the development of metastatic desmoplastic-resistant PCa [4].Te behaviors of cell proliferation, migration, invasion, and epithelialmesenchymal transition (EMT) are the main drivers of malignant growth, metastatic spread, and resistance to treatment in PCa [5,6].Furthermore, these behaviors of tumor cells can be linked to immune cells in the tumor microenvironment, further promoting tumor development by inhibiting immune cell function [7,8].Terefore, it is crucial to investigate novel immunotherapies to treat PCa.
Te interaction between the programmed cell death protein-1 (PD-1) and its ligand (PD-L1) is essential to suppress activated T lymphocytes [9].By regulating PD-L1, tumor cells promote tumor proliferation, invasion, angiogenesis, and EMT on the one hand and suppress CD8 + cytotoxic T cells mediating the immune escape process of tumor cells on the other hand [10][11][12][13].Terefore, targeting the PD-1/PD-L1 axis is a potential anticancer strategy whose clinical value has been demonstrated in various cancers, including non-small-cell lung cancer (NSCLC) [14], gastric cancer [15], and liver cancer [16].Studies have shown that STAT3 could act directly on PD-L1 promoter to induce PD-L1 expression in tumor cells and is involved in regulating PCa development [17,18].However, many patients, especially PCa, are inefective against PD-1/PD-L1 therapy, and therefore, there is an urgent need to seek a supportive strategy to improve conventional PD-1/PD-L1-targeted immunotherapy [19].
Our previous research found that astragaloside IV and polypeptide extract from scorpion venom (PESV) were the main active components of the astragalus-scorpion drug pair for treating PCa [20].He et al. reported that astragaloside IV enhanced PCa carboplatin sensitivity by inhibiting the AKT/ NF-κB signaling pathway [21].Zhang et al. revealed that PESV induced growth inhibition and apoptosis of PCa cells DU145 or could be used to treat PCa [22].Due to the wide range of pharmacological efects of traditional Chinese medicine (TCM), we continued to investigate the modulating efect of astragaloside IV-PESV on tumor immune microenvironment on this basis further to investigate the antitumor pharmacological mechanism of astragaloside IV-PESV and provide sufcient clinical antitumor application of astragaloside IV-PESV.

Materials and Methods
2.1.Animals.C57BL/6 male mice (six-week-old) were ordered from Hunan SJA Laboratory Animal Co. Ltd.After one week of acclimatization, RM-1 (a mouse PCa cell line derived from C57BL/6 mice) was inoculated subcutaneously (1 × 10 6 cells per mouse in an injection volume of 100 μL) into right axilla of mice to establish a homologous tumor mouse model [23].After tumor implantation, the tumors were measured and observed every three days.After the formation of tumors (6 d after implantation), mice were grouped and treated with astragaloside IV, PESV, or astragaloside IV-PESV mixture.Te groups were Control, astragaloside IV, PESV, and astragaloside IV-PESV, with 6 mice in each group.Astragaloside IV was administered at 40 mg/kg dose by gavage [24], and PESV was administered at a dose of 1.2 mg/ kg by intraperitoneal injection, both with saline as solvent.Te source and dosage of astragaloside IV and PESV are consistent with our previous study [20].Te control group was given the same volume of saline by gavage or intraperitoneal injection.In addition, ov-NC or ov-STAT3treated RM-1 was subcutaneously inoculated (1 × 10 6 cells per mouse) into the right axilla of mice.After the formation of tumors (6 d after implantation), mice were grouped and treated with astragaloside IV-PESV mixture.Te groups were ov-NC, ov-STAT3, astragaloside IV-PESV + ov-NC, and astragaloside IV-PESV + ov-STAT3, with 6 mice in each group.Te ov-NC and ov-STAT3 groups were given the same volume of saline by gavage or injection.Kinetics of tumor growth were analyzed every 3 days by caliper measurements.Te tumor volume was measured by using a vernier caliper to measure the longest axis (L) and shortest axis (W) in the tumor body (the position of the longest axis and shortest axis was determined visually) and then using a simplifed formula: tumor volume � (W × W × L)/2.According to the tumor growth, the experiment was ended after 21 d of tumor seeding, and the tumor tissues were removed and weighed.All animal experiments were approved by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine (No. 20210224026).

Flow
Cytometry.CD3+ CD8+ T cell levels in tumor tissues and peripheral blood were frst detected by fow cytometry.Te tumor was ground on a 70 μm flter to obtain a cell suspension.1500 rpm centrifugation was performed to remove the supernatant, and 5 mL erythrocyte lysis was added to lyse cells and then centrifuged.Te supernatant was removed, and the cells were re-suspended with a basal medium.For blood, 5 mL PBS was added to fresh blood and mixed with the liquid surface of 10 mL cell separation solution.Cells were centrifuged at 400 g (approximately 1500 rpm, 15 cm radius horizontal rotor) for 20 min.Ten, we collected the second layer of cells and added 6 mL of PBS bufer into the centrifuge tube, mixed thoroughly, and then centrifuged cells at 400 g (about 1500 rpm) for 20 min.5 mL of erythrocyte lysis solution was added, and then cells were lysed for 3 min and centrifuged.7 mL of PBS was added to resuspend cell precipitate and centrifuged.After the residue was washed twice, then lymphocytes were obtained.Subsequently, the abovetreated cells were centrifuged, the supernatant was discarded, the cell precipitate was resuspended with 100 μL of basal medium, and the corresponding CD3 (17-0031-82, eBioscience) and CD8 (17-0081-82, eBioscience) antibodies were added, mixed well, and incubated for 30 min and protected from light.At the same time, the non-stained and single-stained tubes were set up.Cells were washed with 1 mL of 0.5% BSA-PBS and centrifuged.Ten, the supernatant was discarded, and 150 μL of 0.5% BSA-PBS was used to resuspend cells for fow cytometry detection.
In addition, T cell apoptosis was detected by fow cytometry.Cells were collected by digestion with EDTA-free trypsin, and cells were centrifuged at 2000 rpm for 5 min each time to collect about 3.2 × 10 5 cells.500 μL binding bufer, 5 μL APC, and 5 μL propidium iodide were added and mixed well.Te reaction was performed away from light.Within 1 h, cells were observed and detected using fow cytometry.Assay.Cells were counted using a digestion method and then inoculated into 96-well plates at a density of 5 × 10 3 cells/well/100 μL.Subsequently, each well was supplemented with 10 μL of CCK-8 solution (NU679, Dojindo).After incubating the plates at 37 °C for 4 h, the absorbance values at 450 nm were examined and recorded.

Cell Counting
2.9.Transwell Assays.Te migration ability of cells was determined through transwell chambers (3428, Corning). 1 × 10 6 /mL of cells was resuspended in serum-free medium, and 100 μL of cell suspension was added to transwell chambers' upper chamber.Te lower chamber was incubated with a complete medium with 10% FBS for 48 h.Te culture medium in the chambers was discarded.Cells on the upper chamber were wiped of with a wet cotton swab, fxed with 4% paraformaldehyde, and stained with 0.5% crystal violet (AWC0333, Abiowell) for 5 min.Photos of cells on the upper chamber's outer surface were taken under an Olympus microscope (Japan).Cell invasion assays were performed with transwell chambers with matrix gel (#356231, Corning) [25].

Bioinformatics Prediction and Chromatin Immunoprecipitation (ChIP).
Te potential binding site of STAT3 in the promoter region of PD-L1 (ch9: 5449027-5449342) was frst predicted by the UCSC ChIP database (https://genome.ucsc.edu/index.html).Next, the potential binding of STAT3 to the PD-L1 promoter region was further verifed by ChIP experiments [26].Cells were cross-linked with 1.1% formaldehyde chromatin and sonicated for fragmentation.DNA fragment length was then examined, and DNA was immunoprecipitated with antibodies specifc to the target protein against STAT3.Immunoprecipitation products were collected after incubation with G protein agarose beads.Subsequently, uncrosslinking and DNA purifcation were performed.After DNA purifcation, PD-L1 promoter binding sites were assessed by qRT-PCR.Primer sequences are PD-L1-F: ATGCTCCTGCCAAATCAA, PD-L1-R: CGT TGTTTGCCTTTCCTT.
2.12.Docking Research.Docking studies of astragaloside IV and PESV with STAT3 and PD-L1 proteins were performed with AutoDock Vina 1.1.2software.PyMOL and LigPlot were then applied for visualization and analysis.

Statistical Analysis.
Measurement data were analyzed using GraphPad Prism8.0 software with mean ± standard deviation as the measure of statistical signifcance.Comparing groups was done using Student's t-test, and multigroup comparisons were done using one-way analysis of variance (ANOVA).A statistically signifcant diference was indicated by P < 0.05.

Overexpression of STAT3 Reversed Astragaloside IV-PESV Efects on Immune Escape In Vivo. Finally, we further validated the efects of ov-STAT3 on immune escape in vivo.
Compared with the ov-NC group, the tumor volume and weight were observably elevated in the ov-STAT3 group, and Ki-67 expression was increased.After administration of astragaloside IV-PESV, the tumor volume and weight were signifcantly reduced, and Ki-67 expression was decreased (Figures 6(a)-6(c)).Compared with the ov-NC group, overexpression of STAT3 downregulated CD8 levels in tumor tissues.After administration of astragaloside IV-PESV, CD8 levels increased (Figure 6(d)).After overexpression of STAT3, CD3+ CD8+ T cell levels in cancer tissues and peripheral blood were signifcantly reduced.After administration of astragaloside IV-PESV again, CD3+ CD8+ T cell levels increased (Figure 6(e)).In addition, IFNc, TNF-α, GZMB, and Perforin levels in peripheral blood were reduced after overexpression of STAT3 (Figure 6(f )).STAT3, p-STAT3, and PD-L1 levels were higher in the ov-STAT3 group than in the ov-NC group (Figure 6(g)).Te levels of these indicators were reversed after further administration of astragaloside IV-PESV.Collectively, overexpression of STAT3 reversed astragaloside IV-PESV efects on immune escape.

Discussion
PCa is traditionally considered an immune "cold" tumor with a low mutational burden, low PD-L1 expression, sparse T-cell infltration, and an immunosuppressive tumor microenvironment [27].In the research, we explored the role of astragaloside IV-PESV in regulating tumor immune microenvironment in PCa based on STAT3/PD-L1 signaling pathway, combined with in vitro and in vivo experiments, and elucidated its molecular mechanism.We showed that astragaloside IV-PESV repressed T cell immunosuppression by inhibiting PD-L1 expression in PCa via the STAT3 pathway.Our study provides a new strategy for immunotherapy in PCa.Astragaloside IV, a natural saponin isolated from Astragalus, is a cycloalkane-type triterpene glycoside compound with antioxidant, anti-infammatory, antiviral, antibacterial, and immunomodulatory efects [28].Additionally, astragaloside IV also exerts antitumor efects, including breast [29], liver [30], lung [31], and gastric [32] cancers.In PCa, astragaloside IV also plays an important role [20,21].Scorpion has an activating efect, and PESV could inhibit the proliferation of NSCLC cells [33], induce apoptosis in lung cancer cells [34], and inhibit the development of H22 liver cancer transplantation tumors in mice [35].In addition, PESV could also induce apoptosis in PCa cells DU145 [22].Our previous study found that combining astragaloside IV and PESV exerted the antitumor efects in PCa [20].On this basis, we then explored the modulatory efect of astragaloside IV-PESV on the cancer immune microenvironment.In the present study, we frst verifed the binding of astragaloside IV and PESV to STAT3 and PD-L1 by molecular docking.We then explored the efect of astragaloside IV-PESV on tumor immunity and the STAT3/ PD-L1 pathway.PD-L1 is closely related to the immune escape process of tumor cells [36].A blockade of PD-1/PD-L1 is usually considered to have a great deal of efect depending upon the expression of PD-L1, the availability of cytotoxic T lymphocytes, and the magnitude of tumor mutations [37].Cancer cells evade immune detection by inactivating T cells via PD-1/PD-L1 interactions [38].Anti-PD-1/PD-L1 therapy aims to restore the antitumor response of cytotoxic T cells by blocking the interaction between PD-L1 on cancer cells and PD-1 on cytotoxic T cells [39].Terefore, exploring the underlying mechanisms of PD-L1 regulation in cancer cells is one way to target the reduction of PD-L1 levels and thus make drug-resistant cancer cells respond to PD-1/ PD-L1 antibody therapy.Zhang et al. reported that Chinese medicine CFF-1 inhibited the PD-1/PD-L1 checkpoint signaling pathway via EGFR/JAK1/STAT3 pathway and played an antitumor immune role in inhibiting tumor growth and metastasis in PCa [40]. Lee et al. revealed that ginseng saponin metabolite compound K could induce apoptosis in PCa cells by inhibiting the STAT3/PD-L1 signaling pathway [41].Tis demonstrates the antitumor potential of TCM in PCa.Our study showed that in vivo, astragaloside IV-PESV repressed immunosuppression and STAT3/PD-L1 pathway.In vitro, astragaloside IV-PESV suppressed PD-L1 expression by inhibiting STAT3 signaling to modulate immunity.In vivo and in vitro experiments validated that the antitumor and immunomodulatory efects of astragaloside IV-PESV were associated with the STAT3/ PD-L1 pathway.Tus, next, we further explored the role of overexpression of STAT3 in the anti-PCa of astragaloside IV-PESV.
STAT3 mediates various gene expressions that play key roles in many cellular and biological processes, including cell proliferation, angiogenesis, and infammation [42].STAT3 signaling pathway regulates immune factor expression, recruits immunosuppressive cells, and creates the immunosuppressive environment [43].Xu et al. suggested that combined inhibition of JAK1, 2/PD-L1, and STAT3/PD-L1 pathway may heighten immune cytolytic activity of natural killer cells in response to hypoxia-induced denervationresistant PCa cells [44].Xu et al. revealed that inhibiting the IL-6-JAK/STAT3 pathway in denervation-resistant PCa cells enhanced natural killer cell-mediated cytotoxicity by altering PD-L1/NKG2D ligand levels [45].We demonstrated that overexpression of STAT3 reversed the inhibitory efects of astragaloside IV-PESV on PCa cell function and promoted T cell activation in vitro.In vivo experiments further illuminated that overexpression of STAT3 reversed astragaloside IV-PESV efects on immune escape.
However, this study has some limitations.In this study, to explore the regulatory efect of astragaloside IV-PESV on the tumor immune microenvironment, we only studied T cells.Te role of other immune cells such as macrophages, dendritic cells, natural killer (NK) cells, myeloid-derived suppressor cells (MDSC), and CD4 to CD8 ratio was not studied.In the future, to further investigate the regulatory efect of Astragaloside IV-PESV on the tumor immune microenvironment, we will explore the role of other immune cells such as macrophages, dendritic cells, NK cells, MDSC, and CD4 to CD8 ratio.

Conclusions
In this study, we initially assessed the relationship of astragaloside IV and PESV with STAT3 and PD-L1 using molecular docking.We explored the role of astragaloside IV-PESV in regulating the tumor immune microenvironment in PCa.Trough ex vivo experiments, we found that astragaloside IV-PESV repressed T cell immunosuppression by inhibiting PD-L1 expression in PCa through the STAT3 pathway.Our fndings suggest an antitumor mechanism of astragaloside IV-PESV in PCa and also provide novel targets and strategies for treating PCa.

Data Availability
Te data used to support the fndings of this study are included within the article and supplementary information fle.

Figure 1 :
Figure 1: Molecular docking validated astragaloside IV and PESV bound to STAT3 and PD-L1.(a) Molecular docking of the interaction of astragaloside IV with STAT3 and PD-L1.(b) Molecular docking of the interaction of PESV (LPDKVPIR peptide) with STAT3 and PD-L1.(c) Molecular docking of the interaction of PESV (VRDGYIADDK peptide) with STAT3 and PD-L1.

Table 1 :
Te results of docking research.