Ganodermanontriol regulates tumor-associated M2 macrophage polarization in gastric cancer

Cell grouping and intervention

Mouse mononuclear macrophage lines RAW264.7 (Procell Biotechnology, Wuhan, China) were chosen for study, which were cultured in a three-gas incubator using special media (Procell Biotechnology, Wuhan, China). After the cells grew to logarithmic phase, their viability was assayed by Trypan blue staining. The macrophages were divided into DMSO, IL-4, GAN-L and GAN-H (10, 20 μM) groups. The DMSO group served as the control, while in the IL-4 group, M2 polarization was induced by 10 ng/ml recombinant IL-4 protein (Abcam, USA) [13]. In the GAN groups, the macrophages were pretreated with GAN for 12 h, and then M2 polarization was induced by IL-4.

During further experiments, we co-cultured mouse gastric cancer MFC (Procell Biotechnology, Wuhan, China) with the RAW264.7 macrophages, in order to observe the M2 polarization effect induced by tumor cells. The cells were divided into DMSO, IL-4, MFC, GAN-L and GAN-H (10, 20 μM) groups. In the GAN groups, the cells were pretreated with GAN for 12 h, and then M2 polarization was induced by IL-4.

ELISA

In cellular experiments, the levels of M1 markers IFN-γ, TNF-α and IL-1β were detected, as well as the levels of M2 markers IL-10, Arg-1 and TGF-β1. After 48 d of cell induction, the media were isolated, centrifuged for 30 min, and then the supernatant was collected for subsequent experiments. The levels of IFN-γ, TNF-α, IL-1β, IL-10, Arg-1 and TGF-β1 in mouse tumor-bearing tissues were determined. The tissues were crushed with sterile scissors, ground into powder in liquid nitrogen, and lysed on ice for 30 min with 1 ml of NP-40 lysate (Beyotime Biotechnology, Shanghai, China). After adjusting the protein concentration, the supernatant was collected for detection. Cytokine levels were assayed as per the instructions of ELISA kit (Jiancheng Bioengineering Institute, Nanjing, China), and calculated by the standard curve method. The results were expressed as pg/ml.

Western-blotting

For the detection of p-STAT6 expressions in tissues and cells, the tumor tissues were ground in liquid nitrogen, while the cells were washed twice in PBS. The tissue homogenate and cells were lysed on ice for 30 min with 1 ml of NP-40 lysate to extract proteins, and then the protein concentrations were adjusted after collection of supernatants. Protein concentrations were detected by BCA assay, followed by the concentration adjustment and unification. After denaturation, the protein solutions were subjected to SDS-PAGE analysis and transferred onto membranes. The membranes were then blocked with 5% skim milk powder, incubated at 4°C overnight with TBST-diluted monoclonal antibody (Abcam, USA), and further with HRP-IgG (1:2,000 TBST dilution). Thereafter, chemiluminescent immunoassay was performed, and optical density (OD) was analyzed via Image Pro-Plus 6.0.

Tumor-bearing mouse model and intervention

Thirty 4-weeks-old female nude mice 20–25 g in body weight were chosen for study, which were divided into Control and GAN (10, 20 mg/kg) groups. After wiping the back of their right hind limbs with iodophor, 0.2 ml of cell suspension was injected to make the cell quantity 5 × 10⁶. One week later, nodular tissues appeared at the injection site of mice. The Control mice were given daily intragastric administration of normal saline, while those in the GAN groups were intragastrically administered with 10 and 20 mg/kg GAN, respectively, once daily for 30 d.

Immunohistochemical (IHC) staining

The expressions of CD206 in mouse tissues were detected. After preparing the tumor tissues into 4-μm frozen sections, they were treated with 0.01 mol/L citrate buffer, microwaved at 98°C for antigen retrieval, treated with 3% hydrogen peroxide, blocked in 2% BSA, and incubated at 4°C using TBST-diluted CD206 monoclonal antibodies (Abcam, USA). Thereafter, the sections were further incubated for 15 min with peroxidase-labeled streptomycin, and then washed thrice in phosphate buffer, visualized with DAB solution and microscopically observed for chromogenic reaction. Finally, the sections were restained with hematoxylin, sealed and observed under upright microscope.

Statistical methods

All measurement data were expressed as x̄ ± s, and processed using SPSS 17.0. After homogeneity of variance test, two groups of data were compared by two-independent samples t-test, while three or more groups of data were analyzed by one-way ANOVA. Subsequent pairwise comparisons between groups were all made by LSD method. All of the above test processes were two-sided, and differences were considered statistically significant when P < 0.05.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

GAN could inhibit IL-4-induced macrophage M2 polarization

We used IL-4 to induce the M2 activation in two kinds of macrophages. According to the results, IL-4 produced no effect on the expressions of M1 markers TNF-α, IL-1β or IFN-γ, whose levels were low, without showing differences from the Control group. Nonetheless, IL-4 could promote the expressions of M2 markers IL-10, TGF-β and Arg-1, as well as the expression of CD206 and the phosphorylation of STAT6. GAN could inhibit the IL-10, TGF-β, Arg-1 expressions and suppress the STAT6 phosphorylation. Detection of M1 markers revealed that the TNF-α, IL-1β and IFN-γ levels differed insignificantly between the RAW264.7 (Figure 1A–1C). GAN could inhibit the expressions of IL-10, TGF-β and Arg-1, showing significant differences from the IL-4 group (Figure 1D–1F). As suggested by assays of relative protein expressions, GAN inhibited the expression of p-STAT6 (Figure 1G, 1H).

Figure 1. GAN inhibited the IL-4-induced macrophage M2 polarization. (A–C) M1 macrophage markers (n = 3). TNF-α, IL-1β and IFN-γ levels in RAW264.7 differed insignificantly from those in J774A.1. (D–F) M2 macrophage markers (n = 3). GAN could inhibit the expressions of IL-10, TGF-β and Arg-1, showing significant differences from the IL-4 group. (G, H) Relative protein expressions (n = 3). Although GAN inhibited the p-STAT6 expression. ^*P < 0.05 vs. DMSO; ^#P < 0.05 vs. IL-4.

GAN could inhibit the MFC-induced macrophage M2 polarization

MFC co-culture with macrophages could promote the M2 polarization of macrophages, similar to the results induced by IL-4. The MFC co-culture had unobvious effect on M1 markers, nor did GAN produce any promoting effect on M1 markers, showing insignificant inter-group differences (Figure 2A–2C). The MFC-induced M2 polarization could promote the expressions of M2 markers, while GAN could inhibit such effect of MFC and lower the levels of M2 markers, showing significant differences from the MFC group (Figure 2D–2F). As suggested by assays of relative protein expressions, MFC could induce the STAT6 phosphorylation and upregulate the expression of p-STAT6, similar to IL-4. GAN could inhibit the STAT6 phosphorylation and lower the p-STAT6 level (Figure 2G, 2H).

Figure 2. GAN inhibited macrophage M2 polarization induced by MFC co-culture. (A–C) M1 macrophage markers (n = 3). MFC co-culture produced unobvious effects on M1 markers, nor did GAN produce any promoting effect on M1 markers, showing insignificant inter-group differences. (D–F) M2 macrophage marker (n = 3). GAN could inhibit the effects of MFC and lower the levels of M2 markers, showing significant differences from the MFC group. (G, H) Relative protein expressions (n = 3). GAN could inhibit the STAT6 phosphorylation and lower the p-STAT6 level. ^*P < 0.05 vs. DMSO; ^#P < 0.05 vs. IL-4.

GAN could inhibit the CD206 invasion in tumors

GAN could inhibit tumor growth and lower the levels of M2 markers in tumor tissues, although producing unobvious effects on M1 markers. It could also reduce the CD206 level. Assays of M1 markers revealed insignificant inter-group differences (Figure 3A–3C). GAN could inhibit the expressions of M2 markers, showing significant differences from Control (Figure 3D–3F). According to the IHC staining results, CD206 was strongly positively expressed in Control, while GAN could inhibit the expressions of CD206. (Figure 3G). Assays of relative protein expressions found that GAN could inhibit the phosphorylated expression of STAT6 (Figure 3H, 3I).

Figure 3. Effects of GAN on tumor-bearing mice. (A–C) M1 markers (n = 8). GAN produced unobvious effects on M1 markers, without showing inter-group differences. (D–F) M2 markers (n = 8). GAN could inhibit the expressions of M2 markers, showing significant differences from the Control. (G) IHC (n = 5). Expressions of CD206 were strongly positive in Control. (H, I) Relative protein expressions (n = 5). GAN could inhibit the phosphorylated expression of STAT6, leading to significantly lower p-STAT6 level than that in Control. ^*P < 0.05 vs. control.

Binding mode between GAN and STAT6

Through the small molecule-protein docking model, we found that GAN affected the phosphorylation process of STAT6 by binding to its ARG-324-329 (Figure 4A). Molecular dynamics simulations demonstrated that the free energy was reduced after GAN binding to STAT6, which were bound tightly (Figure 4B–4E).

Figure 4. Binding between GAN and STAT6. (A) Docking result between GAN and STAT6. (B–E) Molecular dynamics simulations between GAN and STAT6.