Induction of different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells treated with various doses of Lu-Nimotuzumab.

INTRODUCTION

Radioimmunotherapy (RIT) is a major anti-cancer therapy in cancer management multimodalities. Lu-Nimotuzumab has been in the use for radioimmunotherapy of EGFR expressing tumors. This study focuses on understanding the differential cellular and molecular mechanisms of anti-tumor effects of Lu-Nimotuzumab on low EGFR expressing A549 and high EGFR expressing A431 tumor cells.

MATERIALS AND METHODS

Nimotuzumab labeled with Lu was characterized by SE-HPLC. Specificity of Lu-Nimotuzumab to EGFR expressed on A549 and A431 cells was confirmed by competitive assay using increasing amounts of unlabeled Nimotuzumab. Cellular responses of A549 (low EGFR) and A431 (high EGFR) in response to different doses of Lu-Nimotuzumab were determined by Viable count assay for cellular viability, cell-cycle analysis by DNA staining, apoptotic assay for cell death, and CFSE dilution assay for cellular proliferation capacity. The number of DNA DSBs formed was determined using γ-H2AX assay with PI staining. Transcription of genes involved in DNA damage response and repair (DRR) pathways was monitored by RT-qPCR.

RESULTS

Lu-Nimotuzumab characterized by SE-HPLC exhibited a radiochemical purity of 99.1 ± 0.6%. Cell binding competition studies with Lu-Nimotuzumab showed specific binding of 34.3 ± 1.7% with A431 cells and 18.4 ± 1.9% with A549 cells which decreased when co-incubated with unlabeled Nimotuzumab. Cytotoxicity and DNA damage (DNA DSBs) increased with an increase in the dose of Lu-Nimotuzumab. A549 displayed G2/M arrest while A431 showed G1 arrest. Apoptotic death was determined to be one of the modes of death of arrested A549 and A431 cells which increases with the increase in the dose of Lu-Nimotuzumab. Loss of proliferation capacity was higher in A431 showed by CFSE staining at different doses of Lu-Nimotuzumab. Transcription profile of most DRR genes in A431 and A549 showed a decrease in the transcription at 4 h followed by recovery at 16 h post-treatment. The degree of transcription of most DRR genes was similar, irrespective of Lu-Nimotuzumab dose.

CONCLUSION

Lu-Nimotuzumab induces different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells. This study would enable the development of integrative novel treatment strategies for radioimmunotherapy in low and high EGFR expressing tumors by Lu-Nimotuzumab with therapeutic gains.