The Study Of The Effects Of SIRT1Inhibitor EX527on Cisplatin Sensitivity Of Human Lung Adenocarcinoma A549Cells In Vitro

Objective: The overall incidence of lung cancer has ranked first in China. Themalignant degree of lung cancer is high and the therapeutic effects are not very ideal, the5years survival rate remains low. Cisplatin, one of the commonly used chemotherapy drugs,is belonging to the cell cycle non-specific drugs. Cisplatin could induce DNA replicationobstacles, thereby inhibiting the tumor cell division. SIRT1, a class III histone deacetylase,might play an important role in the occurrence and development of lung cancer, but alsohave an effect on the chemotherapy sensitivity of lung cancer cells. EX527is a specificinhibitor of SIRT1. The study aims to study the effects of EX527on the sensitivity tocisplatin of human lung adenocarcinoma A549cells, and observe the changes of SIRT1and its target protein in this process.Method: The cell viability of A549cells treated with EX527and cisplatin wasassessed by MTT method, the cell proliferation of A549cells treated with EX527andcisplatin was examined by cell counting method, the ability of DNA synthesis of A549cells treated with EX527and cisplatin was examined by3H-TDR incorporation assay. ThemRNA and protein expression levels of SIRT1were evaluated by reverse transcriptionPCR and Western Blotting, respectively. The enzyme activity of SIRT1was measured bySIRT1activity assay kit. The cell cycle distribution and cell apoptosis induced by EX527and cisplatin were measued by flow cytometry using DAPI and Annexin V-FITC staining.The levels of reactive oxygen species was examined by DCFH-DA staining. Theacetylation levels and expression of SIRT1target protein were measured by WesternBlotting.Results:1. The cell viability of A549cells was decreased by cisplatin or EX527treatment alone in a concentration and time dependent manner. The cell viability of A549 cells treated with cisplatin and EX527was significantly lower than that of cisplatin-treatedgroup. EX527could enhance the proliferation and DNA synthesis inhibition function ofcisplatin on A549cells. EX527can enhance cisplatin induced G1phase arrest, inducemore Sub cells in G1phase, more apoptosis.2. SIRT1mRNA levels did not changesignificantly by cisplatin and/or EX527. The expression level of SIRT1protein wassignificantly elevated in A549cells treated with cisplatin, and EX527cannot inhibitcisplatin-induced the increasing expression of SIRT1protein. However, EX527can notonly reduce the activity of endogenous SIRT1in A549cells, but also significantlysuppressed SIRT1deacetylase activity increased by cisplatin.3. Cisplatin can increasereactive oxygen species in A549cells, and EX527can further enhance the level ofintracellular reactive oxygen species. EX527treatment increased the acetylation level ofp53, as well as the expression level of Foxo3a and p21.Conclusion: EX527can significantly enhance the cisplatin sensitivity of A549cells,demonstrated by decreased cell viability, decelerated cell proliferation and inhibited DNAsynthesis. EX527treatment could not change the SIRT1mRNA or protein expression, butdirectly inhibit the SIRT1deacetylase activity. Enhancement of cisplatin sensitivity ofA549cells induced by EX527might associate with G1cell cycle arrest, apoptosis and theincreased level of intracellular reactive oxygen species. EX527could increase theacetylation level of p53, thus enhance p53-mediated cell cycle arrest and apoptosis. EX527could increase the expression level of Foxo3a and p21, thus promote antitumor effects andsuppress the resistance to cisplatin.