Organogermanium THGP Induces Differentiation into M1 Macrophages and Suppresses the Proliferation of Melanoma Cells via Phagocytosis

Abstract

M1 macrophages are an important cell type related to tumor immunology and are known to phagocytose cancer cells. In previous studies, the organogermanium compound poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) and its hydrolysate, 3-(trihydroxygermyl) propanoic acid (THGP), have been reported to exert antitumor effects by activating NK cells and macrophages through the induction of IFN-γ activity in vivo. However, the detailed molecular mechanism has not been clarified. In this study, we found that macrophages differentiate into the M1 phenotype via NF-κB activation under long-term culture in the presence of THGP in vitro and in vivo. Furthermore, long-term culture with THGP increases the ability of RAW 264.7 cells to suppress B16 4A5 melanoma cell proliferation. These mechanisms indicate that THGP promotes the M1 polarization of macrophages and suppresses the expression of signal-regulatory protein alpha (SIRP-α) in macrophages and CD47 in cancers. Based on these results, THGP may be considered a new regulatory reagent that suppresses tumor immunity.

Keywords: Ge-132; M1 macrophage; THGP; melanoma; organogermanium.

Figure 1

Morphological changes in macrophages after long-term culture with THGP. (a) Micrographs show RAW 264.7 cells cultured for 0, 1, 7, 10, 45 and 90 days in medium supplemented with or without 500 μM THGP through repeated passages. Scale bars: 20 μm. (b) Micrographs show RAW 264.7 cells cultured for 10 days in medium supplemented with or without 500 μM THGP. Arrows indicate spindle-like cells. Scale bars: 20 μm. (c) The percentage of spindle-like cells was calculated. (d) RAW 264.7 cells were maintained in medium containing 500 μM THGP for 0, 10, 20 or 40 days with repeated passages. Their cell proliferation rate was assessed using an MTS assay. (e) The graphs show side scatter (SSC-A) and forward scatter (FSC-A) of RAW264.7 cells analyzed using flow cytometry after treatment with or without THGP. (f) Their cell sizes were analyzed with a cell counter. (g) Photographs show the immunofluorescence staining for CD86 (green) or CD206 (red) and the cell nucleus stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) in RAW 264.7 cells cultured in the absence or presence of 500 μM THGP for 10 days or more. Scale bars: 20 μm. (h) The ratio of CD86-positive/CD206-positive cells is shown. The results are presented as the means ± S.D. (n = 6). ** p < 0.01 compared with the controls.

Figure 2

Effect of THGP on the M1/M2 polarization of RAW 264.7 cells during long-term culture. (a) The expression of M1 markers was analyzed using RT–PCR. RAW 264.7 cells were cultured for more than 10 days in the absence (Ctrl, white bar) or presence (THGP, black bar) of 500 μM THGP. (b) The expression of M2 markers was analyzed using RT–PCR. RPS18 was used as an internal control. (c–e) The expression levels of CD86 and CD206 were determined using Western blotting and quantified. β-actin was used as an internal control. (f) CD86 expression was observed under a fluorescence microscope after treatment with LPS to induce differentiation into M1 macrophages. Ctrl: untreated control cells, LPS: cells treated with 100 ng/mL LPS, LPS + THGP: cells treated with 100 ng/mL LPS and 500 μM THGP. The nucleus was stained with DAPI (blue), and CD86 is shown in red. Scale bars: 20 μm. (g) The fluorescence intensity of CD86 per cell was quantified in (f). (h) CD206 expression was observed under a fluorescence microscope after treatment with IL-4 to induce differentiation into M2 macrophages. Ctrl: untreated control cells, IL-4: cells treated with 10 ng/mL IL-4, IL-4 + THGP: cells treated with 500 μM IL-410 ng/mL and THGP. The nucleus was stained with DAPI (blue), and CD206 is shown in green. Scale bars: 20 μm. (i) The fluorescence intensity of CD206 per cell was quantified in (i). The results are presented as the means ± S.D. (n = 6). * p < 0.05 and ** p < 0.01 compared with the control.

Figure 3

Analysis of the mechanism of M1 macrophage differentiation induced by THGP. (a) The panels show RAW 264.7 cells treated with 500 μM THGP analyzed with an isotope microscope. Ge (elemental germanium) represents THGP, P (elemental phosphorus) represents the nucleus and CN (elemental carbon and nitrogen) represents the cytoplasm. Scale bars: 10 μm. (b) Volcano plot of the expression levels and p values in RAW 264.7 cells treated with THGP compared with untreated RAW 264.7 cells (Ctrl). Red plots represent the genes with a greater than 4-fold increase in expression in THGP-treated cells compared to the controls, and green plots represent the genes with a less than 4-fold decrease in expression in THGP-treated cells compared to the controls. (c) The enrichment analysis using Metascape showing the possible transcription factors that regulate the genes with differential expression in THGP-treated cells compared with Ctrl cells. (d) The expression heatmaps of the genes in the NF-κB pathway in cells cultured in the presence or absence of THGP. (e,f) The levels of p-38 and p-p38 were determined using Western blotting and quantified. β-actin was used as an internal control. (g–i) Levels of NF-κB in the cytosol and nucleus were quantified using Western blotting. β-actin was used as an internal control for the cytosol, and lamin B1 was used as an internal control for the nucleus. The results are presented as the means ± S.D. (n = 6). (j,k) The expression of CD86 on RAW 264.7 cells was determined using Western blotting after treatment with 500 μM THGP with or without 5 μM JSH-23, an NF-κB inhibitor, for 10 days. * p < 0.05 and ** p < 0.01 compared with the controls. N.S. = Not significant.

Figure 4

Evaluation of the functions of RAW 264.7 cells treated with THGP. (a) The graph shows the results of the pathway analysis of differentially expressed genes in THGP-treated cells compared with the control group using Metascape. (b) The graph shows the percentage of adherent cells among the total cells on the collagen-coated plate within 30 min after seeding. (c) The photographs show the migration of cells at 0 h and 24 h after scratching the cells. Scale bars: 200 μm. (d) The graph shows the rate of migration of cells in (c). (e) After RAW 264.7 cells were cultured for 10 days with 500 μM THGP, a phagocytosis assay was performed. In the photographs, green and blue signals represent phagocytosed FITC-beads and the cell nucleus (Hoechst 33452), respectively. Scale bars: 20 μm. (f) The fluorescence intensity was analyzed in the photographs shown in (e). The results are presented as the relative total intensity obtained from the total cells and are reported as the means ± S.D. (n = 6). ** p < 0.01 compared with the controls.

Figure 5

THGP increases the cytotoxicity of RAW 264.7 cells toward B16 4A5 melanoma cells. (a) The cytotoxicity of RAW 264.7 cells against B16 4A5 cells was evaluated via MTS assay. (b) The cytotoxicity of RAW 264.7 cells against B16-F10 LUC #2 cells was evaluated using a luciferase assay. (c) After staining with the dye CMFPTX, B16 4A5 cells (red) and RAW 264.7 cells (green) were cocultured and analyzed using flow cytometry. (d) Then, the percentages of B16 4A5 cells were determined. (e) The image shows apoptotic cells analyzed with PI and Hoechst staining. Total cells were stained blue with Hoechst 33258. RAW 264.7 cells were stained green with Cell Tracker Green. Apoptotic cells were stained red with propidium iodide. Apoptotic B16 4A5 cells are shown as blue⁺ green⁻ red⁺ cells (shown by white arrows). Live B16 4A5 cells are shown as blue⁺ green⁻ red⁻ cells. Apoptotic RAW 264.7 cells are shown as blue⁺ green⁺ red⁺ cells. Live RAW 264.7 cells are shown as blue⁺ green⁺ red⁻ cells. Scale bars: 20 μm. (f) The graph shows the percentage of apoptotic B16 4A5 cells cocultured with control RAW 264.7 cells or THGP-treated RAW 264.7 cells for 10 days or more. (g) The figure shows the growth rate of B16 4A5 cells cultured in conditioned medium supplemented with or without 500 μM THGP and in the mixture of the RAW 264.7 culture supernatant supplemented with or without 500 μM THGP and normal medium at a 1:1 ratio determined using the MTT assay. Ctrl: normal medium; THGP: normal medium containing 500 μM THGP; C-CM: untreated RAW 264.7 cell culture supernatant; T-TM: THGP-treated RAW 264.7 cell culture supernatant. n = 6. * p < 0.05 and ** p < 0.01 compared with the control. N.S.= Not significant.

Figure 6

Expression of genes related to phagocytosis in macrophages. (a) Bright-field images show the coculture of B16 4A5 cells and RAW 264.7 cells treated with or without THGP for 10 days or more. The images were taken at 20× magnification. (b) Fluorescence images obtained after the staining of RAW 264.7 cells (green) and B16 4A5 cells (blue). Ctrl represents B16 4A5 cells cocultured with THGP-untreated cells. THGP represents B16 4A5 cells cocultured with RAW 264.7 cells cultured with 500 μM THGP-containing medium for 10 days or more. Scale bars: 20 μm. (c) The percentage of B16 4A5 cells phagocytosed by macrophages was calculated. Phagocytosed cells were defined as those double positive for green and blue staining (indicated by the white arrows in Figure 6b). (d) SIRP-α expression in RAW 264.7 cells treated with or without THGP was analyzed using RT–PCR. (e,f) SIRP-α expression was determined using Western blotting and quantified in RAW 264.7 cells treated with or without THGP. β-actin was used as an internal control. (g–i) B16 4A5 cells were cultured in monoculture (Ctrl), C-CM or T-CM. Then, CD47 expression was analyzed using RT–PCR (g). CD47 expression was determined using Western blotting and quantified. β-actin was used as an internal control (h,i). * p < 0.05 and ** p < 0.01 compared with the controls.

Figure 7

Effect of feeding mice a Ge-132 diet on M1 macrophage polarization in murine intraperitoneal macrophages in vivo. (a) Fluorescence photographs reveal the morphology of primary intraperitoneal macrophages in mice fed a control or 0.05% Ge-132 diet for 30 days. Green represents the cytoskeleton stained with α-tubulin, and blue represents the nucleus stained with DAPI. RT–PCR was performed to determine the expression of the M1 markers CD86 (b) or CD80 (c) in primary intraperitoneal macrophages in mice fed a control diet or 0.05% Ge-132 diet for 30 days. (d) Photographs showing immunofluorescence staining for CD86 (green) and CD206 (red) along with DAPI (blue) in primary intraperitoneal macrophages from mice fed a control diet or 0.05% Ge-132 diet for 30 days. (e) M1 macrophage cells with high expression of CD86 and low expression of CD206 in (d). (f) M2 macrophage cells with low expression of CD86 and high expression of CD206 in (d). (g) A phagocytosis assay was performed. The green signal represents phagocytosed FITC-beads, and the blue signal represents the cell nucleus (Hoechst 33452). (h) Analysis of the fluorescence intensity in (g). The results show the relative total intensity obtained from the total cells. n = 6. ** p < 0.01 and * p < 0.05 compared with the controls. Scale bars: 20 μm.