The Role And Mechanism Of SIRT1 Inhibitor EX527 On Invasion And Metastasis Of Drug-resistant Cancer Cells

BackgroundEsophageal cancer has become the eighth most common malignancy and the sixth leading cause of death in the world.Most patients with esophageal cancer are diagnosed late and have metastases.Tumor resistance is due to long-term or high-dose administration.Sirtuin1(SIRT1)belongs to the mammalian sirtuin family and plays an important role in the deacetylation of histones and non-histones.It has received extensive attention as a therapeutic target for human diseases,particularly in the treatment of cancer.In 2005,scientists at Elixir Pharmaceuticals discovered the first selective SIRT1 inhibitor,EX527,with an IC50 of 98 nM.So far,EX527 is still the most effective selective inhibitor of SIRT1.The combination of EX527 and sirtuins occurs after the formation of imidate intermediates and release of niacinamide.The main mechanism of EX527 inhibitors is to block the formation of ribose-sirtuin complexes.However,it is unclear whether EX527,as an inhibitor of SIRT1,has an effect on tumor-resistant metastasis and invasion.The study investigated the effects of EX527 on the migration and invasion of resistant esophageal cancer cells(EC109/PTX,TE-1/PTX).The experiment revealed that SIRT1 regulates the epithelial-mesenchymal transition(EMT)mechanism of drug-resistant esophageal cancer cells.It provides a theoretical basis for the clinical treatment of drug resistance and metastasis of esophageal cancer.Method(1)MTT assay validates the sensitivity of EC109/PTX and TE-1/PTX cells to paclitaxel.Scratches and Transwell assays were used to detect the migration of parental and drug resistant cells.Western blotting assay was used to detect the difference of EMT related protein expression between parental cells and drug resistant cells.Subcutaneous xenograft models of nude mice were established.The expression of EMT-related proteins in EC109,EC109/PTX,TE-1,and TE-1/PTX tumor tissues was detected by immunohistochemistry.Western blotting was used to detect the expression of SIRT1,H4K16 ac and P53 ac before and after drug resistance.(2)Inhibition of SIRT1 expression can inhibit cell EMT transformation.Inhibitor EX527 of SIRT1 can inhibit EC109/PTX and TE-1/PTX cell colony formation.Scratch and Transwell experiments showed that SiRNA or EX527 interfere SIRT1 can inhibit invasion and migration of drug-resistant cells.Western blotting,immunofluorescence and immunohistochemistry experiments showed that inhibition of SIRT1 can inhibit the expression of N-cadherin and snail protein and up-regulate the expression of E-cadherin.Subcutaneously inoculated with xenografts,the tumor volume of TE-1/PTX in nude mice can be inhibited by intraperitoneal injection of EX527(5 mg/kg).Result(1)Drug-resistant esophageal cancer cells EC109/PTX,TE-1/PTX have stronger migration and invasion ability.The MTT assay showed that the IC50 of EC109/PTX cells was 5.18 times greater than that of the parental cells.The IC50 of TE-1/PTX cells was 6.40 times greater than the parental cells.Scratches and Transwell experiments showed that the migration ability of cells was significantly stronger than their parental cells.Scratches and Transwell experiments showed that the migration ability of cells was significantly stronger than their parental cells.Western blotting experiments and immunofluorescence confirmed that the expression of EMT-related proteins N-cadherin,vimentin,and snail were significantly increased after cell resistance.A nude mouse model of subcutaneous transplantation tumor was established.The expression of EMT-related protein in tumor tissue was detected by immunohistochemistry.The expression of N-cadherin,vimentin,and snail were increased after drug resistance.There was no significant difference in the expression of SIRT1 in resistant esophageal cancer cells,but the expression of downstream protein SIRT1 H4K16 ac and P53 ac decreased after drug resistance,indicating that the activity of SIRT1 in deacetylation was enhanced.(2)Inhibition of SIRT1 expression can inhibit cell EMT transformation in resistant esophageal cancer cells.Inhibitor EX527 of SIRT1 can inhibit EC109/PTX and TE-1/PTX cell colony formation.SiRNA or EX527 interfered with SIRT1,Scratch and Transwell experiments revealed that invasion and migration ability was inhibited in resistant cells.After inhibition of SIRT1,Western blotting,immunofluorescence and immunohistochemistry experiments showed that the expression of N-cadherin and snail protein was inhibited while the expression of E-cadherin was up-regulated.The transplanted tumor was inoculated subcutaneously and intraperitoneal injection of EX527(5 mg/kg)inhibited the tumor volume of TE-1/PTX in nude mice.ConclusionIn summary,in vitro and in vivo experiments demonstrated that when the esophageal cancer cells(EC109,TE-1)are resistant,their invasion and migration ability increases as the process of Epithelial-mesenchymal transition.Inhibition of SIRT1 by inhibitor EX527 can significantly reduce the invasion and invasion of drug-resistant esophageal cancer cells.Further studies have revealed that interference with SIRT1 can regulate the process of epithelial-mesenchymal transition(EMT),change cell morphology,and regulate the invasion and metastasis of drug-resistant cells.