Glutamine Effecting On Bladder Cancer Proliferation Through Activating STAT3 Mediated By ROS And Glutaminolysis

Objectives To investigate the difference of metabolic dependence of glutamine(Gln)in non-muscle invasive bladder cancer(NMIBC)and muscle invasive bladder cancer(MIBC).Further,to explore how Gln regulates the proliferation and apoptosis of Gln-dependent bladder cancer cells.Methods The Oncomine database was used to search for difference of glutaminase(GLS)between NIMIBC and MIBC.The pathological results of bladder tumor resection after surgery in our hospital were used to indicate the clinical specimens of metastatic cell carcinoma.The expression of GLS in NMIBC and MIBC clinical pathological specimens wasdetected by immunohistochemistry.MTT,immunoblotting,apoptosis were used to compare the Gln-dependent differences of bladder cancer cell lines T24 and 5637,and analyse their cell proliferation,and apoptosis levels in the the Gln presence or not.For the Gln-dependent bladder cancer cell line T24,the cell cycle kit,MTT,microscopically Observation and other experimental methods were used to observe the growth state and proliferation level of T24 cells at different Gln concentrations.And then certify the growth state of cells cultured with glutamine(Gln+)and glutamine-free(Gln-)by glutamine analogue 6-diazo-5-oxo-L-norleucine(Don).In the glutamine-deficient condition with supplementing the intermediate products of glutamine tricarboxylic acid(TCA),dimethylglutamate(Glu)and dimethyl-2-oxoglutaric acid(?-?G)And the active oxygen scavenger acetylcysteine(NAC),ATP detection kit,reactive oxygen species(ROS)kit,immunofluorescence microscopy and MTT were used to compare the ATP,ROS content and proliferation of the different groups.The apoptosis and survival proportion of bladder cancer T24,in Gln(+),Gln(-),Gln(-)+?KG and Gln(-)+Glu,Gln+NAC treatment groups,were detected by flow cytometry.Western blotting was used to detect transcription factor signaling and activator of transcription 3(STAT3)and P-STAT3 protein and proliferation-associated proteins content in different Gln concentrations and Gln(+),Gln(-),Gln(-)+?KG,Gln(-)+Glu and Gln+NAC treatment groups.Results MIBC expressed a higher level of GLS than NMIBC.The GLS expressions of bladder tumor cells,T24 and 5637,with different degrees of Gln dependence were different.For Gln-dependent tumor cells T24,the higher the concentration of Gln,the higher the expression level of GLS,the faster the proliferation and the less apoptosis.Cytological experiments confirmed that T24 bladder cancer cell is significant dependent with glutamine which regulates the proliferation of the bladder cancer cell line T24 by acting as a TCA circulating metabolic intermediate and a ROS modulator.Compared with Gln(+),the proportion of S phase of T24 cells increased,intracellular ATP levels decreased,and ROS levels increased in Gln(-).By supplementing ?-?G and Glu,the ATP levels and proliferation survival levels of cells were significantly increased under Gln deficiency conditions.In addition,the expression of STAT3 protein in bladder cancer cell line T24 was decreased with the absence of Gln,and STAT3 expression was also affected by ATP and ROS content.Conclusion Bladder cancer is glutamine-dependent,and the higher the malignancy of bladder cancer,the greater the dependence of glutamine.Glutamine may promote the proliferation of glutamine-dependent bladder cancer by supplementing ATP production,neutralizing ROS,and activating the STAT3 pathway.