RgpB contributes to chemoresistance in esophageal squamous cell carcinoma by preventing Cx43 degradation via inhibiting autophagosome-lysosome fusion

doi:10.12122/j.issn.1673-4254.2024.09.06

Abstract

Abstract:

Objective To investigate the mechanism through which RgpB, a virulence factor of Porphyromonas gingivalis (Pg), induces chemoresistance in esophageal squamous carcinoma. Methods The autophagy-regulating factors that interact with RgpB were screened by immunoprecipitation-mass spectrometry. The interaction between RgpB and the autophagy regulator TBC1D5 was investigated using co-immunoprecipitation. The impact of Pg infection on the expression of esophageal cancer cell membrane receptor molecule Cx43 was assessed using Western blotting. Immunofluorescence assay was used to analyze the relationship among Lamp1, Cx43 and TBC1D5. The effect of Pg infection on autophagosome-lysosome fusion was evaluated using autophagy double fluorescence technique. The effects of Pg infection and a Cx43 inhibitor on proliferation of esophageal cancer cells after chemotherapy were examined with plate cloning assay and CCK-8 method. Results Immunoprecipitation-mass spectrometry identified TBC1D5 as an autophagy regulator interacting with RgpB, and co-immunoprecipitation suggested that RgpB could directly bind to TBC1D5. In Pg-infected esophageal cancer cells, the expression of Cx43 on the cell membrane was significantly higher than that in non-infected cells. Immunofluorescence assay showed that the expression of Cx43 on the membrane of esophageal cancer cells increased significantly after Pg infection, which blocked autophagosome-lysosome fusion as shown by stubRFP-sensGFP-LC3 lentivirus study. Plate cloning assay and CCK-8 assay showed that the Cx43 inhibitor significantly attenuated the effect of Pg infection for promoting proliferation of esophageal cancer cells after chemotherapy. Conclusion Pg infection in esophageal cancer blocked autophagosome-lysosome fusion in the tumor cells, thereby preventing Cx43 from lysosomal degradation and leading to chemoresistance of esophageal cancer.

Key words: esophageal squamous cell carcinoma, Porphyromonas gingivalis, RgpB, Cx43, chemotherapy resistance

Yue DU, Xiusen ZHANG, Kexu ZHOU, Xing JIN, Xiang YUAN, Shegan GAO. RgpB contributes to chemoresistance in esophageal squamous cell carcinoma by preventing Cx43 degradation via inhibiting autophagosome-lysosome fusion[J]. Journal of Southern Medical University, 2024, 44(9): 1670-1676.

Figures/Tables 8

Fig.1 Silver staining results of RgpB immunoprecipitation.

Fig.2 Diagram of RgpB-related protein interaction network.

Fig.3 Interaction between RgpB and TBC1D5. A, B: Co-IP experiments demonstrating an interaction between both RgpB and TBC1D5.

Fig.4 Immunoprecipitation results of Cx43 detected by Western blotting. A: Effect of Pg infection and TBC1D5 knockdown on Cx43 protein expression in KYSE150 cells. B: Quantitative analysis of Western blotting results. ***P<0.001.

Fig.5 Changes in Lamp1, Cx43 and TBC1D5 expressions in Pg-infected KYSE150 cells detected by immunocytofluorescence staining (Scale bar=10 μm).

Fig.6 Effect of Pg infection on fusion of autophagosomes and lysosomes in KYSE150 cells. A: Transfection efficiency of stubRFP-sensGFP-LC3 in KYSE150 cells by fluorescence detection. B: Changes in autophagosome-lysosome fusion after Pg infection in stubRFP-sensGFP-LC3-KYSE150 cells detected by laser confocal microscopy (Scale bar=10 μm).

Fig.7 Effects of Pg infection and addition of Cx43 inhibitor on proliferation of KYSE150 cells examined by plate cloning assay.

Fig.8 Effect of Pg infection and Cx43 inhibitor on the proliferation of KYSE150 cells. ***P<0.001.

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