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 ¹⁷⁷Lu-Nimotuzumab

Abstract

Introduction

Radioimmunotherapy (RIT) is a major anti-cancer therapy in cancer management multimodalities. ¹⁷⁷Lu-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 ¹⁷⁷Lu-Nimotuzumab on low EGFR expressing A549 and high EGFR expressing A431 tumor cells.

Materials and methods

Nimotuzumab labeled with ¹⁷⁷Lu was characterized by SE-HPLC. Specificity of ¹⁷⁷Lu-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 ¹⁷⁷Lu-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

¹⁷⁷Lu-Nimotuzumab characterized by SE-HPLC exhibited a radiochemical purity of 99.1 ± 0.6%. Cell binding competition studies with ¹⁷⁷Lu-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 ¹⁷⁷Lu-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 ¹⁷⁷Lu-Nimotuzumab. Loss of proliferation capacity was higher in A431 showed by CFSE staining at different doses of ¹⁷⁷Lu-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 ¹⁷⁷Lu-Nimotuzumab dose.

Conclusion

¹⁷⁷Lu-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 ¹⁷⁷Lu-Nimotuzumab with therapeutic gains.

Keywords:

• ¹⁷⁷Lu-Nimotuzumab
• cytotoxicity
• DNA damage
• apoptosis
• DRR genes

Acknowledgments

The authors are grateful to Dr A. Dash, Head, Radiopharmaceuticals Division and Dr P. K. Pujari, Associate Director, Radiochemistry & Isotope group, BARC for their support to this program. Acknowledgements are due to the staff of Radiochemicals Section, Radiopharmaceuticals Division for the regular supply of ¹⁷⁷Lu for the study.

Disclosure statement

All the authors confirm that no competing financial interests exist.

Additional information

Notes on contributors

ShishuKant Suman

Shishu Kant Suman, MSc in Biotechnology with a Post Graduate Diploma in Life Sciences with specialization in Nuclear Science, is a Scientific Officer in the Radiopharmaceutical Division of Bhabha Atomic Research Centre, Mumbai, India.

Rashmi Priya

Rashmi Priya, MSc in Life Sciences is a Scientific Officer associated with the Low Level Radiation Research Section of Radiation Biology & Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, India.

Mythili Kameswaran

Mythili Kameswaran, PhD in Applied Biology is the Senior Scientist from Radiopharmaceutical Division of Bhabha Atomic Research Centre, Mumbai, India and has been working extensively in the field of radioimmunotherapy and radioimmunodiagnosis of cancer.