Clinically, RT is one of the most important therapeutic methods for ESCC, especially for those unresectable ones. Thus, new strategies that could enhance ESCC RT response and have no further therapeutic toxicities on normal tissues have been long-time needed. Previous studies reported that alteration in the expression and activity of growth factor receptors could not only directly perturb growth regulation, but also affect the sensitivity of cancer cells to various cytotoxic treatments, including RT [28, 29]. Several groups identified that EGFR inhibitors could improve RT response and local control of human tumors, providing a kind of additional agents in anti-cancer therapy [10, 11].
Nimotuzumab is a humanized IgG1 isotype monoclonal antibody of EGFR, which requires bivalent binding (i.e., binding with both antibody arms to two targets simultaneously) for stable attachment to cellular surface. Recently, it was reported that Nimotuzumab could substantially improve the radiosensitivity of brain tumor  and non-small cell lung cancer  cells with high or moderate levels of EGFR. In the present study, we determined to investigate the impact of Nimotuzumab on ESCC RT response and underling mechanisms. Our results demonstrated that although Nimotuzumab alone failed to inhibit ESCC cells growth, we did observe as well that Nimotuzumab dramatically enhanced radiation response of ESCC KYSE30 cell line (the cells with overexpression of EGFR) both in vitro and in vivo, as evidenced by increased radiation-inhibited cell growth and colony formation and radiation-mediated apoptosis. But similar results were not found in EGFR low-expressed ESCC cell line TE-1. These results revealed that Nimotuzumab only improved the radiosensitivity of ESCC cells with high expression of EGFR, suggesting a potential EGFR-dependent synergistic cytotoxicity of Nimotuzumab when combined with RT.
Previous reports documented that the anti-tumor effect of certain EGFR-specific mAbs is due to inhibition of ligand binding to EGFR and consequent inhibition of EGFR activation [32, 33]. Nimotuzumab was identified only selectively binds to tumor cells that express moderate to high levels of EGFR . To investigate the potential mechanisms of Nimotuzumab on promoting radiosensitivity of ESCC cells with high-level of EGFR, we first examined expression levels of EGFR and phosphorylated EGFR (i.e., p-EGFR, an active EGFR) before and after the administration of Nimotuzumab in ESCC cells. Our results showed that Nimotuzumab treatment alone did not result in any changes of the total EGFR levels in both EGFR overexpressed KYSE30 and EGFR low-expressed TE-1 ESCC cell lines. However, Nimotuzumab could down-regulate the levels of p-EGFR in KYSE30 cells. In TE-1 cells, in which the basic levels of EGFR were low and p-EGFR was not detected, the effect of p-EGFR downregulation cannot be observed in cells treated with Nimotuzumab. Furthermore, during the stimulation of EGF in both cells, we examined that the EGF-induced levels of p-EGFR in KYSE30 cells were dramatically inhibited by Nimotuzumab in a dose-dependent manner. But this down-regulation effect of p-EGFR by Nimotuzumab was not observed in TE-1 cell line. These observations clearly demonstrated that Nimotuzumab blocked the activation of EGFR in EGFR-overexpressed ESCC cells. Since previous studies documented that EGF-induced EGFR activation could decrease IGFBP-3 protein levels in ESCC TE-2 and TE-7 cell lines  and IGFBP-3 has been suggested as a key marker of radiosensitivity that enhances the susceptibility of ESCC to RT , in our study, we further examined the levels of IGFBP-3 in EGF-treated KYSE30 and TE-1 cell lines before and after Nimotuzumab administration. As anticipated, the treatment of Nimotuzumab could up-regulated the levels of IGFBP-3 in EGF-treated KYSE30 cells, concurrent with the decreased level of p-EGFR both in vitro and in vivo. Also, this phenomenon was not observed in EGF-treated TE-1 cell line. These data, collectively, suggested that Nimotuzumab might up-regulates IGFBP-3 expression in EGFR-overexpressed ESCC cells by suppressing the activation of EGFR.
IGFBP-3, a member of the IGFBP family, is the major carrier protein for insulin-like growth factors (IGF)-I or IGF-II in circulation [34, 35]. Depending on the experimental context, IGFBP-3 has been shown to possess growth-stimulatory, antiproliferative, or proapoptotic activities in vitro[36, 37]. In addition, IGFBP-3 has also been identified as a gene that is most highly up-regulated in EGFR-overexpressing esophageal cancer cell lines and primary esophageal tumors . In our previous work, we have demonstrated that IGFBP-3 upregulation after Nimotuzumab administration involved in ESCC cells chemosensitivity to DDP . To determine if IGFBP-3 participates in Nimotuzumab-enhanced radiosensitivity of ESCC cells with high expression of EGFR, we first knocked down the levels of IGFBP-3 in KYSE30 cells by specific shRNA against IGFBP-3 gene and found that silence of IGFBP-3 dramatically reduced ESCC cell radiosensitivity. Furthermore, even after the treatment of Nimotuzumab, the radiosensitivity of IGFBP-3-silenced KYSE30 cells was almost not enhanced. These results provided evidences that the level of IGFBP-3 in EGFR-overexpressing ESCC cells is responsible, at least in part, for the increased radiosensitivity by Nimotuzumab. Krause and colleagues previously suggested that IGFBP-3 is a novel EGFR downstream target molecule in primary and immortalized human esophageal epithelial cells, and moreover, EGF could suppress IGFBP-3 expression through activation of MAPK in an EGFR-tyrosine kinase-dependent manner . These data, taken together, prompt us to hypothesize that Nimotuzumab might up-regulate IGFBP-3 expression through EGFR-dependent pathway to enhance the radiosensitivity of ESCC cells. Clearly, further works are needed to clarify the regulation mechanisms of IGFBP-3 in ESCC and confirm our hypothesis in detail.
It is well known that, in the United States and Western countries, there has been a dramatic rise in the incidence of esophageal adenocarcinoma to equal or exceed the incidence of ESCC . Therefore, one limitation of the current study is no esophageal adenocarcinoma cell lines are investigated. Another limitation of this study is only 2 ESCC cell lines are studied. In order to minimize the bias on the study results, we have taken a series of effective measures such as a panel of EGFR high and EGFR low ESCC cell lines and adenocarcinoma cell lines used in further study.