Enhancement Of Radiation Cytotoxicity By Blocking EGFR And COX-2 Pathways In A549 Human Lung Cancer Cell Line

Objective:The addition of molecular targeted agents to enhance the cytotoxicity of radiation isa promising strategy in cancer treatment. The combination of epidermal growth factorreceptor tyrosine kinase inhibitors or cyclooxygenase-2 inhibitors, such as erlotinib orcelecoxib with other anticancer agents, was reported to achieve synergistic cellgrowth inhibition in severalkinds of cancer cell lines. Therefore, we postulated thatthe addition of erlotinib and celecoxib to radiation, might further increase antitumoractivity.Methods:Assaying the antitumor effects of erlotinib and celecoxib, caculating the IC₅₀ andIC₂₀ in A549 human lung adenocarcinoma cell line respectively with MTTcolorimetry. Clonogenic cytotoxicity assays and clonogenic radiation survival assaysafter treatments with erlotinib and celecoxib with or without radiation were done onA549 human lung adenocarcinoma cell line. Synergisms after combined treatmentwith erlotinib, celecoxib, and radiation were investigated and analyzed according toan independent action model. Alterations in apoptosis and cell cycle were measuredto identify the mechanisms underlying the cell killing or radiation-enhancing effectsof erlotinib and celecoxib combined treatment with radiation using flow cytometry.Western blots for phosphorylated Akt, pAkt were conducted after treatment witherlotinib and/or celecoxib with or without radiation. A549 human lung cancerxenograft model was established on nude mice and was treatmented with erlotinibalone, celecoxib alone, radiation alone or the combination of these agents. Tumorprogression delay was assaied and the apoptic index and MVD were caculated.Results:1. Erlotinib and celecoxib showed cell proliferation inhibition effect to A549 cell linein vitro in a dose-dependent manner. The IC₅₀ and IC₂₀ of erlotinib and celecoxib was12.21±0.83μmol/L, 5.15±0.14μmol/L, 50.13±2.41μmol/L and 40.32±1.26μmol/L respectively.2. Combination treatment of erlotinib, celecoxib, and radiation was shown to causeclonogenic cell deaths in an additive manner in A549 cell line. 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 biggerthan those of single drug.3. The Flow cytometry showed that both celecoxib and erlotinib could induce G₁/G₀phase arrest, and the reduction of S phase combined treatment with radiation,especially in (R + C + E) group. Both celecoxib and erlotinib could enhanceradiation-induced apoptosis, and the apoptosis was highest in drug combination.4. Western blot showed that the expression of Akt protein had no difference in anygroup; both celecoxib and erlotinib could inhibit the expression of pAkt and radiationcan enhance the expression of pAkt. In (R + C) group or (R + E) group, theexpression of pAkt was lower than R group, and the expression of pAkt was lowest in(R + C + E) group.5. Tumor growth in the combined treatment group was significantly inhibitedcompared with the control, erlotinib or celecoxib group significantly. A significantdelay of tumor progression was observed in the combined treatment group comparedwith all other three groups.Conclusions:The current results strongly suggest that a cooperative effect of the combinedtreatment of on tumor progression is mediated through blocking both EGFR- andCOX-2-related pathways. The mechanisms underlying these additive effects seem toinvolve the synergistic enhancement of apoptosis and cooperative attenuation ofradiation-induced S arrest, possibly via inhibiting of PI3K/Akt signal transductionpathway, by the combined drug treatments.This combination regimen may provide apromising strategy for lung cancer therapy.