The anti-epidermal growth factor receptor (EGFR) monoclonal antibody, C225, enhances radiation-induced apoptosis in primary glioma cell lines through mediation of MAPK/JNK/p38 signaling pathways

Abstract

Purpose: Increasing evidence suggests that signaling mediated by the epidermal growth factor receptor (EGFR) pathway contributes to radiation resistance. The anti-EGFR monoclonal antibody, C225, has been shown to enhance radiation response for several tumor types in preclinical models. Malignant gliomas are known to express, and quite frequently overexpress, EGFR. Our objectives in this study were to 1) Evaluate the efficacy of C225 as a radiation response modifier in EGFR-expressing glioma cell lines and to 2) Investigate the underlying molecular mechanisms mediating C225-induced enhancement of radiation response. Materials and Methods: Twelve EGFR-expressing glioma cells lines, established from patient tumors, were used for this study. Cells were incubated with C225, irradiated, and then evaluated for radiation response. Assays used to evaluate efficacy of C225-mediated radiosensitization included time-course apoptosis assays (Annexin V and TUNEL), viability assays (MTT), and clonogenic survival assays. The changes along MAPK (p44/p42)/JNK/p38-MAPK signal transduction pathways were then investigated using quantitative Western analysis with phospho-specific antibodies to determine the molecular mechanisms by which C225 mediates a given response. Results: C225 clearly enhanced radiation response for 7 of the 12 primary glioma cell lines studied. Enhancement of both immediate and delayed apoptotic responses was evident in these 7 responsive cell lines after C225 administration. The average apoptosis index at 6 hours post-RT+C225 for the 7 responsive lines was 9.5%, compared to 1.2% for the RT-only controls. A pattern of delayed apoptosis was evident in these 7 lines, with secondary apoptotic peaks (∼ 8.0%) occurring at 24 hours post-RT+C225. Time course viability measurements revealed a steady decrease in viable tumor cells in these responsive cell lines from 75% at 6 hours post-RT+C225 to 20% at 7 days. Clonogenic survival was also diminished in these 7 lines after C225 + RT compared to lines treated by RT alone (D0=2.0 vs. 2.8, respectively). The other 5 of 12 glioma cell lines demonstrated resistance to C225-mediated radiosensitization. Treatment of these 5 cell lines by combined RT + C225 failed to result in any enhancement of apoptosis or reduction in clonogenic survival compared to RT alone. C225 demonstrated distinct effects on the signaling pathways of responsive vs. resistant glioma cell lines. All 12 glioma cell lines demonstrated reduced levels of phospho-EGFR, demonstrating antagonism of EGFR by C225. JNK and p38 activation states, as measured by quantitative Western analysis using phospho-specific antibodies, were enhanced by 2.5-3.0 fold in the 7 responsive cell lines after RT + C225, compared to no measurable induction in the 5 resistant lines. The absence of JNK/p38 activation in the 5 resistant lines was reflected by a concomitant absence of phospho-MKK4 and MKK 3/6 induction after RT + C225, implying the presence of signaling deficits further upstream of JNK/p38 not directly regulated by EGFR activity. MAPK activity (p44/p42) was reduced 1.5-2.0 fold in the responsive lines compared to a more modest 0.5 fold in the resistant lines. Conclusion: C225 appears to enhance radiation response of a distinct subset of primary glioma cell lines through activation of JNK/p38 pathways and downregulation of signaling through MAPK. However, EGFR-expressing glioma cell lines resistant to C225-mediated enhancement of radiation-induced apoptosis demonstrated MAPK/JNK/p38 signaling that was not as directly impacted by inhibition of EGFR. It is critical to better understand the upstream signals mediating activation of these pathways to develop more effective strategies for enhancing radiation response.