Radiosensitization Enhanced By The Anti-EGFR Monoclonal Antibodies In Esophageal Cancer TE-13and TE-1cell Lines In Vitro

EGFR excessively expresses in many human solid tumor tissues. There is anestablished relationship between EGFR abnormal expression and tumor cellproliferation, differentiation, angiogenesis, metastases, irradiation resistance and poorprognosis. Nimotuzumab (h-R3) and cetuximab (C225) are monoclonal antibodies(mAbs) targeting the EGFR, and enhance the antitumor effect and sensitization ofradiation by blocking the activation of EGFR and EGFR-mediated downstreamsignaling pathways. However, little is known of whether the anti-EGFR mAbsnimotuzumab and cetuximab are different in modulation of radiosensitization and thelevels of EGFR expression in esophageal cancer cells affect the antitumor action ofcombined treatment with anti-EGFR antibodies and irradiation.Objective:We aim to compare the enhanced radiosensitization of nimotuzumab andcetuximab in esophageal cancer cell lines of differing levels of EGFR expression.Methods:1. The surface EGFR expression was analyzed by flow cytometry (FCM).2. The K-ras genetic state was analyzed by polymerase chain reaction (PCR) andDNA sequencing.3. The cell proliferation was evaluated by MTS assay and the percentage of cellproliferation was calculated.4. FCM analysis was applied to observe the cell apoptosis.5. The radiosensitivity enhanced by h-R3and C225was determined byclonogenic assay and survival curves were plotted with the multi-target, single-hitmodel. 6. The activation of EGFR signaling pathway proteins was measured using awestern blot analysis.Results:1. FCM and PCR-DNA sequencing demonstrated that TE-13and TE-1cellsexpressed high and low levels of EGFR, respectively, and harbored wild-type K-rasalleles.2. MTS and cell apoptosis assays showed that TE-1cell proliferative inhibitionand cell apoptosis were enhanced by irradiation alone and the combined treatment butnot by mAbs alone, and the effect of irradiation was not enhanced by the two mAbs. Incontrast, h-R3, C225, irradiation alone, and the combined treatment resulted in asignificant enhancement of TE-13cell proliferative inhibition and cell apoptosis, andthe effect of irradiation was further enhanced by the two mAbs. C225in combinationwith irradiation was more efficient than h-R3in combination with irradiation atinducing apoptosis in the TE-13cell line.3. Clonogenic assay revealed that, whereas both C225and h-R3had no effect onthe radiation sensitivity of TE-1cells, they increased the radiosensitivity of TE-13cells.C225was more efficient than h-R3at enhancing TE-13cell sensitivity to irradiation.4. Western blot analysis revealed that irradiation markedly induced the activationof the EGFR signaling pathway proteins in both TE-13and TE-1cells. The two mAbsdid not block the irradiation-induced activation of the EGFR signaling pathway proteinsin TE-1cells (which have a low level of EGFR expression), but they significantlyinhibited such activation in TE-13cells (which have a high level of EGFR expression),with C225more effective than h-R3.Conclusion:The two antibodies significantly enhanced the radiosensitization of the TE-13cells(which have a low level of EGFR expression), with C225more effective at enhancingcellular sensitivity to irradiation and radiation-induced apoptosis than h-R3. Thedisparity in modulation of radiosensitization by the two antibodies could be associatedwith their efficiencies at blocking the activation of EGFR signaling. The two antibodies did not affect the radioresponse of TE-1cells (which have a low level of EGFRexpression). This effect may be related to the level of EGFR expression on the cellsurface.