Abstract 1858: EDNRB isoform 3 confers temozolomide resistance in human melanoma cells by modulating membrane potential, reactive oxygen species and mitochondrial Ca2+

Abstract

Abstract Background: The role of Endothelin receptor type B (EDNRB) isoform 3 involved in Temozolomide (TMZ) induced melanoma cell death has not yet been elucidated. Methods: The subcellular localization of EDNRB isoform 3 was determined by confocal and immunoblotting assays. Silencing EDNRB isoform 3 was performed by CRISPR/Cas9. Apoptosis was assessed by Annexin V/PI staining and caspases 3/7/9 activity. Mitochondrial membrane potential, reactive oxygen species and mitochondrial Ca2+ were measured by flow cytometry. Apoptosis protein array was applied. Results: Confocal and immunoblot analyses indicate mitochondrial localization of EDNRB isoform 3 and the first N-terminal (1-22) amino acids are sufficient for its mitochondrial targeting. EDNRB isoform 3 depleted A375 cells significantly inhibit apoptosis. EDNRB isoform 3 mediates chemoresistance appears to be acting via a mitochondrial apoptosis pathway as manifested by the prevention in mitochondrial depolarization, reduced reactive oxygen species, enhanced mitochondrial Ca2+ uptake and decreased caspase 9 activation. Additionally, apoptosis array shows that lack of EDNRB isoform 3 has relatively lower expression of phosphorylation of p53 at S392 and a slightly higher expression of Paraoxonase 2. Conclusion: Our findings raise the possibility of targeting EDNRB isoform 3 as new therapeutic strategy in combination with TMZ for melanoma treatment. Citation Format: Long Cui, Bo Liang, Xiaoting Chen, Chi Chiu Wang. EDNRB isoform 3 confers temozolomide resistance in human melanoma cells by modulating membrane potential, reactive oxygen species and mitochondrial Ca2+ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1858.