Escherichia coli outer membrane vesicles inhibit rat glioma proliferation through cell endocytosis

doi:10.12122/j.issn.1673-4254.2026.08.02

Abstract

Abstract:

Objective To investigate the inhibitory effect of Escherichia coli outer membrane vesicles (E. coli-OMVs) on glioma proliferation and the underlying mechanism. Methods CCK-8 assay and EdU incorporation assay were used to determine the optimal concentrations of E. coli-OMVs and chlorpromazine (CPZ) for cell treatment. C6 glioma cells treated with 10 μg/mL E. coli-OMVs and 10 μmol/L CPZ were examined for PCNA and Ki67 expressions and cell cycle distribution using qRT-PCR, Western blotting, immunocytochemistry and flow cytometry. The expressions of β-catenin, cyclin D1, and c-Myc proteins in C6 cells were detected following treatment with OMVs in the presence or absence of CHIR99021. In a SD rat model bearing orthotopic glioma, the effects of intranasal PBS or OMVs (0.25, 0.5, 1.0 mg/kg) administration on tumor growth and PCNA and Ki67 expressions were observed using fluorescence imaging and immunohistochemistry. Results E. coli-OMVs within the concentrations of 2.5-10 μg/mL dose-dependently inhibited C6 cell viability, and such inhibitory effect was attenuated by 2.5, 5, and 10 μmol/L CPZ, which was the most effective at 10 μmol/L. The OMVs at 10 μg/mL significantly reduced EdU-positive cells, downregulated mRNA and protein expressions of PCNA and Ki67, and increased G0/G1-phase and decreased S-phase cell populations. These alterations were largely reversed by 10 μmol/L CPZ. Treatment with OMVs downregulated cellular expressions of β‑catenin, Cyclin D1, and c-Myc, which were partially restored by application of CHIR99021. In the tumor-bearing mice, intranasal OMVs administration dose-dependently decreased the relative fluorescence signals, slowed tumor growth, and reduced PCNA and Ki67 expressions. Conclusion E. coli-OMVs inhibit glioma cell proliferation and delay orthotopic tumor growth in rats likely via endocytic uptake of the OMVs to cause suppression of Wnt/β-catenin signaling and cell cycle arrest.

Key words: Escherichia coli, outer membrane vesicles, glioma cells, endocytosis, PCNA, Ki67

Chenxi HU, Xi GU, Pengcheng NIU, Yiting WANG, Liaoqiong FANG, Zhibiao WANG, Jin BAI. Escherichia coli outer membrane vesicles inhibit rat glioma proliferation through cell endocytosis[J]. Journal of Southern Medical University, 2026, 46(8): 1730-1742.

Figures/Tables 12

Fig.1 Isolation and characterization of Escherichia coli outer membrane vesicles (E. coli-OMVs). A: Transmission electron microscopy of purified OMVs (Scale bar=200 nm). B: Size distribution of OMVs measured by NTA. C: Western blotting of OMV markers OmpA and OmpC.

Fig.2 CCK-8 analysis of the effect of E. coli-OMVs on glioma cell viability. A: Glioma cell viability after 12-h co-culture with E. coli-OMVs. B: Glioma cell viability after 24-h co-culture with E. coli-OMVs. C: Glioma cell viability after 48-h co-culture with E. coli-OMVs (n=3). *P<0.05, **P<0.01, ***P<0.001 vs control group.

Fig.3 Effect of CPZ on uptake of E. coli-OMVs by glioma cells. A: CCK-8 analysis of C6 cell viability after treatment with different concentrations of CPZ (n=3). B: Representative fluorescence images of C6 cells incubated with fluorescently labeled E. coli-OMVs after pretreatment with different concentrations of CPZ (Scale bar=75 μm). C: Representative fluorescence images of C6 cells incubated with fluorescently labeled E. coli-OMVs for 0 (control), 2 and 4 h (Scale bar=10 μm). D: Representative fluorescence images of C6 cells pretreated with 10 μmol/L CPZ and then incubated with fluorescently labeled E. coli-OMVs for 0, 2 and 4 h (Scale bar=10 μm). ***P<0.001 vs PBS.

Fig.4 CPZ attenuates inhibitory effect of E. coli OMVs on glioma cell proliferation in vitro. A-C: Representative EdU staining images after 12, 24, and 48 h treatment (Scale bar=50 μm). D-F: Quantification of EdU-positive cells after 12, 24 and 48 h treatment (n=3). ***P<0.001 vs control. #P<0.05,##P<0.01, ###P<0.001 vs E. coli OMVs.

Fig.5 Effect of CPZ on PCNA and Ki67 transcriptional levels in E. coli-OMVs-treated glioma cells. A: qRT-PCR analysis of PCNA mRNA expression levels. B: qRT-PCR analysis of Ki67 mRNA expression levels (n=3). **P<0.01, ***P<0.001 vs PBS group; #P<0.05, ##P<0.01 vs E. coli-OMVs group.

Fig.6 Effect of CPZ on PCNA protein expression in E. coli-OMVs-treated glioma cells. A: Western blots showing PCNA protein levels. B: Quantitative analysis (grayscale values) of the protein bands (n=3). ***P<0.001 vs PBS group; ##P<0.01 vsE. coli-OMVs group.

Fig.7 Effect of CPZ on Ki67 protein expression in E. coli-OMVs-treated glioma cells. A: Immunofluorescence images of Ki67 expression (Scale bar=50 μm). B: Quantitative analysis of Ki67-positive cells (n=3). ***P<0.001 vs PBS group; ##P<0.01 vs E. coli-OMVs group.

Fig.8 Effect of CPZ on cell cycle distribution of E. coli-OMVs-treated glioma cells. A: Flow cytometry of C6 cell cycle distribution in PBS, E. coli-OMVs and CPZ+E. coli-OMVs groups. B: Quantitative analysis of the percentages of cells in G0/G1, S and G2/M phases (n=3). ***P<0.001 vs PBS group; ##P<0.01, ###P<0.001 vs E. coli-OMVs group.

Fig.9 E. coli-OMVs inhibit expressions of glioma cell proliferation-related proteins by regulating the Wnt/β-catenin signaling pathway. A: Western blots of β‑catenin, cyclin D1 and c-Myc proteins in PBS, OMVs, CHIR99021 and CHIR99021+OMVs groups. B: Quantitative analysis of β-catenin, cyclin D1 and c-Myc protein expression levels. (n=3). ***P<0.001 vs PBS group; ###P<0.01 vs E. coli- OMVs group.

Fig.10 Effect of intranasal administration of E. coli-OMVs on growth of orthotopic glioma in rats. A: In vivo bioluminescence images of orthotopic glioma-bearing rats on day 1, day 4, and day 7 after administration. B: Quantitative analysis of relative fluorescence intensity on day 4 (Day4/Day1). C: Quantitative analysis of relative fluorescence intensity on day 7 (Day7/Day1). D: Changes in body weight of rats in each group during treatment. OMVs: Outer membrane vesicles (n=3). **P<0.01, ***P<0.001 vs control group.

Fig.11 Effect of intranasal administration of E. coli-OMVs on histopathological changes in orthotopic glioma tissues of the rats (Scale bar=20 μm).

Fig.12 Effect of intranasal administration of E. coli-OMVs on expressions of proliferation-related proteins in orthotopic glioma tissues of the rats (Scale bar=20 μm). A: Immunohistochemical staining and quantitative analysis of PCNA expression in brain tumor tissues in each group. B: Immunohistochemical staining and quantitative analysis of Ki67 expression in brain tumor tissues in each group (n=3). ***P<0.001 vs PBS group.

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