| Objectives: Laryngeal squamous cell carcinoma is one of the most common malignant tumors of the head and neck,because its onset is insidious and the early symptoms are not obvious,so most patients are in an advanced stage when they are discovered,and the cure rate is extremely low.Therefore,new treatments need to be explored to improve survival in advanced laryngeal squamous cell carcinoma.Fatty acid metabolism is one of the important biological processes in cells,CPT1 A is a key enzyme in fatty acid oxidation,and its specific inhibitor Etomoxir can significantly inhibit the progression of a variety of malignant tumors.In laryngeal squamous cell carcinoma,fatty acid metabolism can significantly affect its progression,and whether the protein expression of CPT1 A changes,and the role and mechanism of Etomoxir in laryngeal squamous cell carcinoma are not fully studied,so we detected the expression difference of CPT1 A in laryngeal squamous cell carcinoma tissues and adjacent normal tissues,and studied the inhibitory effect of Etomoxir on laryngeal squamous cell carcinoma and its mechanism in vitro experiments.In vivo nude mice,Etomoxir was verified to inhibit the growth of laryngeal squamous cell carcinoma,providing a new target for the treatment of laryngeal squamous cell carcinoma.Methods: In this study,10 samples of laryngeal squamous cell carcinoma and its adjacent normal tissues were collected,and the expression difference of CPT1 A in laryngeal squamous cell carcinoma and adjacent normal tissues was analyzed by immunohistochemistry.Western Blot experiments were used to screen out the two cell lines with the highest CPT1 A protein expression levels in the laryngeal squamous cell carcinoma cell lines TU138,TU212 and TU686.Different concentrations of CPT1 A specific inhibitor Etomoxir were added to two cell lines,and the effect of Etomoxir on the viability of laryngeal squamous cell carcinoma cells was detected by CCK8.Using plate clonal formation experiments,the effect of Etomoxir on cell proliferation ability over a long period of time was detected by observing the formation of cell cloning;The changes in cell migration and invasion ability after Etomoxir treatment were detected by wound healing experiment and Transwell invasion experiment.The effect of Etomoxir on cell cycle arrest and apoptosis was detected by flow cytometry.The Western Blot experiment was used to detect whether the amount of proteins such as p-AKT,AKT,pS6,S6,MMP2,Cyclin D1,caspase 3 and PARP changed with the change of Etomoxir solution concentration,and the mechanism of Etomoxir inhibition of the function of laryngeal squamous cell carcinoma cells was verified.In vivo experiments,the nude mouse xenograft model of laryngeal squamous cell carcinoma was established,and the experimental group was injected with Etomoxir solution intraperitoneally and the control group was injected with the same volume of PBS to analyze the effect of Etomoxir on the growth of laryngeal squamous cell carcinoma in vivo.Results:1.The immunohistochemical results of clinical samples showed that the expression of CPT1 A protein increased significantly in laryngeal squamous cell carcinoma tissues.2.Among the laryngeal squamous cell cancer cell lines TU138 cells,TU212 cells and TU686 cells,the highest expression of CPT1 A protein was TU212 cells,followed by TU138 cells,and the expression of CPT1 A protein in TU686 cells was the lowest.3.Through CCK8 experiment,it can be seen that with the increase of the concentration of Etomoxir solution,the activity of laryngeal squamous cell carcinoma cells gradually decreases within a certain range.Through plate cloning experiments,it can be seen that with the increase of the concentration of Etomoxir solution,the number of cell clones formed decreases,indicating that its proliferation potential is inhibited.Through scratch experiments,it can be seen that the scratch healing rate of Etomoxir dosing group is significantly reduced,indicating that the migration ability of cells in vitro is inhibited by Etomoxir.It can be seen from the Transwell invasion experiment that with the increase of the concentration of Etomoxir solution,the number of cells passing through the matrigel is getting smaller and smaller,indicating that its invasion is inhibited.Flow cytometry showed that with the increase of the concentration of Etomoxir solution,the cells located in the G0/G1 phase increased significantly,and the overall apoptosis rate of cells gradually increased,indicating that Etomoxir could promote apoptosis and exert the biological characteristics of cancer suppression.4.In laryngeal squamous cell cancer cells,with the increase of the concentration of Etomoxir solution,the protein expression of MMP2 and Cyclin D1 gradually decreases with the increase of the concentration of Etomoxir solution,and the protein expression of Caspase 3 and the ratio of PARP-CL/PARP-FL increase.5.In laryngeal squamous cell carcinoma,the amount of protein in total AKT and total S6 does not change with the concentration of Etomoxir solution,while the protein expression of p-AKT and p-S6 decreases.6.In the nude mouse xenograft model,compared with the control group,the tumor volume of the experimental group,namely the intraperitoneal injection of Etomoxir solution,was significantly reduced,which indicated that Etomoxir could significantly inhibit the growth of laryngeal squamous cell carcinoma.Conclusion:The immunohistochemical results of clinical samples showed that the expression of CPT1 A protein increased in laryngeal squamous cell carcinoma tissues,which indicated that CPT1 A was associated with poor clinical prognosis.In vitro experiments,Etomoxir can significantly reduce the viability of laryngeal squamous cell carcinoma cells through the AKT/mTOR signaling pathway,inhibit its proliferation potential,reduce its migration and invasion ability in vitro,and block the cell cycle in the G0/G1 phase and promote its apoptosis.In vivo experiments,compared with the control group,Etomoxir can significantly inhibit the growth and development of laryngeal squamous cell carcinoma in nude mouse xenograft models,providing a new strategy and target for the treatment of laryngeal squamous cell carcinoma. |
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