葡萄糖上调Fyn／Rho相关激酶信号通路促进膀胱肿瘤细胞问隧道纳米管形成

Glucose promotes the formation of tunneling nanotubes between bladder cancer cells by up - regu-lating Fyn/Rho -associated kinase signaling pathway

目的探讨葡萄糖诱导促进膀胱肿瘤细胞间隧道纳米管（TNTs）的形成及其机制。方法将高侵袭能力膀胱癌细胞124用Mito-Tracker标记后，与低侵袭能力膀胱癌细胞RT4混合培养24h，采用Actin-Tracker Green标记细胞骨架蛋白纤维型肌动蛋白（F-actin），观察两者之间是否形成隧道纳米管。RT4细胞和T24细胞分别培养12、24h后收集细胞培养液，采用液相色谱和质谱分析法进行代谢组学分析，筛选出两种细胞微环境中的差异因子。采用微环境中的差异因子诱导T24细胞，进一步验证差异因子是否能诱导增强124细胞形成TNTs的能力。Westernblot法检测细胞骨架调节蛋白Fyn、Rho相关激酶（ROCK）及磷酸化桩蛋白（p-paxillin）的表达水平差异。结果T24与RTg细胞之间混合培养可形成TNTs，其主要构成成分为F-肌动蛋白（F-actin）。代谢组学分析发现，葡萄糖为两细胞微环境的差异因子，能诱导促进T2d与RT4细胞之间形成TNTs（P=0．005）。T24与RT4细胞微环境之间葡萄糖浓度差异能上调细胞骨架相关蛋白Fyn、ROCK和p-paxillin的表达水平（P=0．001、0．004、0．010）。结论不同侵袭能力膀胱癌细胞微环境中葡萄糖浓度差异能促进两细胞之间TNTs的形成。提高膀胱癌细胞微环境中葡萄糖的水平，能上调细胞骨架相关蛋白Fyn／ROCK／p-paxillin信号通路的活性，是葡萄糖诱导促进膀胱肿瘤细胞之间TNTs形成潜在的机制。

Objective To investigate the effects of glucose in formation of tunneling nanotubes （TNTs） between bladder cancer cells and the potential mechanism. Methods The highly invasive blad- der cancer cells T24 were labeled with Mito - Tracker Deep Red, and then co - cultured with the less inva- sive bladder cancer cells RT4 for 24 hours. Actin - Tracker Green was used to label the cytoskeletal protein to detect the TNTs between T24 and RT4 cells. T24 and RT4 cells were cultured separately for 12 hours and 24 hours respectively, and the medium was collected and analyzed by liquid chromatography and mass spectrometry to screened out the difference factors between the micro - environment. T24 cells were cul- tured with the factors to confirm whether they could induce and enhance TNTs formation. The expression level of Fyn, Rho - associated kinase （ROCK） and p - paxillin proteins were detected and compared by Western blotting analysis. Results The formation of TNTs was found between T24 and RT4 cells. And the main component of TNTs was F- actin. Metabolomics analysis revealed that glucose was a differential fac- tor between the micro - environments of T24 and RT4 cells, which could induce the formation of TNTs be- tween T24 and RT4 cells （P = 0. 005 ）. Concentration gradient of glucose between T24 and RT4 cells can up- regulate the expression of Fyn/ROCK and p- paxillin proteins （P =0. 001, P =0. 004, P =0. 010）. Conclusion Concentration gradient of glucose in the micro - environment of bladder cancer cells can pro- mote the formation of TNTs between highly invasive and less invasive bladder cancer cells. The potential mechanism of the formation of TNTs induced by glucose might be explained by concentration gradient of glucose in the micro - environment of bladder cancer cells which can up - regulate the activity of Fyn/ ROCK signalinz pathway.