Radiosensitivity Enhancement By Combined Treatment Of COX-2 Inhibitor And EGFR Inhibitor On A549 Human Lung Adenocarcinoma

Objective:To characterize the radiation-enhancing effects and underlying mechanisms of combined treatment with celecoxib,a cyclooxygenase-2 selective inhibitor,and erlotinib,an epidermal growth factor receptor(EGFR) tyrosine kinase inhibitor,in human lung adenocarcinoma A549 cell lines.Methods:Human lung adenocarcinoma A549 cell lines was cultured in vitro.The subjects were divided into 8 groups:control group(O group),celecoxib group(C group), erlotinib group(E group),celecoxib + erlotinib group(C + E group),radiation group (R group),radiation + celecoxib group(R + C group),radiation + erlotinib group(R + E group),radiation + celecoxib + erlotinib group(R + C + E group).The antiproliferative activity of celecoxib and erlotinib were determined by MTT analysis, and the IC₅₀ and IC₂₀ were caculated,respectively.The 20%inhibitory concentrations (IC₂₀) were needed for the next subjects.Clonogenic radiation survival assays after treatments with celecoxib and erlotinib with or without radiation were done on A549 cell lines.The combining effect was quantified by calculating the SF(Surviving Fraction).Alterations in apoptosis and cell cycle observed by Flow cytometry were measured and Western blot for Akt and phosphorylated Akt were conducted to identify the mechanisms underlying radiation-enhancing effects of celecoxib and erlotinib.Results:1.The effect of the dose-dependent growth inhibition was observed by the MTT analysis in human lung adenocarcinoma A549 cell lines.And the IC₅₀ and IC₂₀ were(50.13±2.41 )μmol/L and(40.32±1.26)μmol/L for celecoxib and (12.21±0.83)μmol/L and(5.15±0.14)μmol/L for erlotinib,respectively.2.Clonogenic radiation survival assays showed that the Dq,Do,SF₂ of(R+C+ E) group was lower than those in(R + C) group or(R + E) group.The SER of(R+ C+E) group was 2.217,the SER of(R+C) group was 1.299,the SER of(R+E) group was 1.503.The radiation-enhancing effects of drug combination were bigger than those of single drug.And the drug combination could enhance the radiosensitivity more significantly.3.The Flow cytometry showed that both celecoxib and erlotinib could induce G₁/G₀ phase arrest,and the reduction of S phase combined treatment with radiation, especially in(R+C+E) group.Both celecoxib and erlotinib could enhance radiation-induced apoptosis,and the apoptosis was highest in drug combination. So drug combined treatments could enhance radiation-induced apoptosis more significantly.4.Western blot showed that the expression of Akt protein had no difference in any group;both celecoxib and erlotinib could inhibit the expression of pAkt and radiation can enhance the expression of pAkt.In(R + C) group or(R + E) group, the expression of pAkt was lower than R group,and the expression of pAkt was lowest in(R+C+E) group.Conclusions:Both celecoxib and erlotinib could enhance the radiosensitivity,and the drug combination could enhance it more significantly.The mechanisms seemed to involve the of reduction of radiation-insensitive S phase and synergistic enhancement of radiation-induced apoptosis,possibly inhibiting PI3K/Akt signal transduction pathway was one of the mechanisms of radiosensitivity enhancement by combined treatment of celecoxib and erlotinib.