Experimental Study Of Radiosensitivity Mechanism Of Afatinib On Non-small Cell Lung Cancer A549 Cell Line

Background and PurposeLung cancer remains the most common cause of cancer death with the majority cases presented with advanced, unresectable or metastatic NSCLC. Radiotherapy has been reserved for about 64.3% patients with NSCLC. Radiotherapy for the treatment of cancer can cause a wide range of cellular effects, the most biologically potent of which is the double-strand break(DSBs) in DNA. There are two main pathways existed for DSB repair, non-homologous end joining (NHEJ) and homologous recombination (HR). The ability of cancer cells to effectively repair DSBs significantly influences the outcome of therapeutic regimens.In several cancers, including non-small cell lung cancer (NSCLC), the epidermal growth factor receptor (EGFR) is an important determinant of tumorigenesis and cancer progression. EGFR is an important target for cancer therapies. EGFR and HER2 play an important role in radioresistance, with EGFR positivity associated with a poor prognosis and unfavorable response to therapy. Afatinib(BIBW2992)is a novel representative of the new generation of tyrosine kinase inhibitors. This new compound is a potent and irreversible inhibitor of both the EGFR and HER2 kinases. This study was to determine whether afatinib can radiosensitize A549 cell line and explore its possible mechanism.MethodsAssaying the antitumor effects of afatinib, caculating the IC50 in A549 human lung adenocarcinoma cell line with MTT colorimetry. Clonogenic cytotoxicity assays and clonogenic radiation survival assays after treatment with afatinib with or without radiation were done on A549 human lung adenocarcinoma cell line. Synergisms after combined treatment with afatinib and radiation were investigated and analyzed according to an independent action model. Alteration in apoptosis and cell cycle were measured to indentify the mechanisms underlying the cell killing or radiation-enhancing effects of afatinib combined treatment with radiation using flow cytometry. Western blots for Bcl-2, Bax were conducted after treatment with afatinib with or without radiation.Results1. Afatinib showed cell proliferation inhibition effect to A549 cell line in vitro in a dose and time dependent manner. The IC50 of afatinib was (2.23±0.14) μmol/L.2. Combination treatment of afatinib and radiation was shown to cause clonogenic cell deaths in an additive manner in A549 cell line. The Dq、D0 and SF2 of combination group was lower than those in radiation group. The SER of combination group was 1.37.3. The Flow cytometry showed that afatinib could induce G0/G1 phase arrest, and the reduction of S phase combined treatment with radiation. Afatinib could enhance radiation-induced apoptosis. Western blot analysis results showed that irradiation can improve the expression of Bax protein and reduce the expression of Bcl-2 protein and this trend is even more obvious when afatinib was combined.ConclusionAn radiosensitizing effect on human lung adenocarcinoma A549 cells was obsverved in afatinib. The mechanism may be related to the cell cycle S phase, which was resistance to radiation, declined and increased radiation-induced apoptosis. We speculate that the mechanism of afatinib increased radiation-induced apoptosis involves Bax protein expression increased and Bcl-2 protein expression decreased.