DU Y, Zhang X, Zhou K, Jin X, Yuan X, Gao S
Henan Provincial Key Laboratory of Microecology and Esophageal Cancer Prevention and Treatment, Henan Provincial Key Laboratory of Tumor Epigenetics, Luoyang 471003, China.
First Affiliated Hospital/School of Clinical Medicine, Henan University of Science and Technology, Luoyang 471003, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2024 Sep 20;44(9):1670-1676. doi: 10.12122/j.issn.1673-4254.2024.09.06.
To investigate the mechanism through which RgpB, a virulence factor of (Pg), induces chemoresistance in esophageal squamous carcinoma.
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.
Immunoprecipitation-mass spectrometry identified TBC1D5 as an autophagy regulator interacting with RgpB, and coimmunoprecipitation 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.
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.
探讨牙龈卟啉单胞菌（Pg）的毒力因子RgpB诱导食管鳞状细胞癌化疗耐药的机制。
通过免疫沉淀-质谱法筛选与RgpB相互作用的自噬调节因子。采用免疫共沉淀法研究RgpB与自噬调节因子TBC1D5之间的相互作用。利用蛋白质免疫印迹法评估Pg感染对食管癌细胞膜受体分子Cx43表达的影响。采用免疫荧光分析法分析溶酶体相关膜蛋白1（Lamp1）、Cx43和TBC1D5之间的关系。运用自噬双荧光技术评估Pg感染对自噬体-溶酶体融合的影响。采用平板克隆实验和CCK-8法检测Pg感染及Cx43抑制剂对化疗后食管癌细胞增殖的影响。
免疫沉淀-质谱法鉴定出TBC1D5是与RgpB相互作用的自噬调节因子，免疫共沉淀表明RgpB可直接与TBC1D5结合。在Pg感染的食管癌细胞中，细胞膜上Cx43的表达明显高于未感染细胞。免疫荧光分析显示，Pg感染后食管癌细胞膜上Cx43的表达显著增加，而stubRFP-sensGFP-LC3慢病毒研究表明这会阻断自噬体-溶酶体融合。平板克隆实验和CCK-8实验表明，Cx43抑制剂可显著减弱Pg感染对化疗后食管癌细胞增殖的促进作用。
食管癌中的Pg感染可阻断肿瘤细胞中的自噬体-溶酶体融合，从而阻止Cx43的溶酶体降解，导致食管癌化疗耐药。
