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EDNRB isoform 3 confers Temozolomide resistance in A375 melanoma cells by modulating membrane potential, reactive oxygen species and mitochondrial Ca2+

Authors Chen YS, Liu F, Luo YH, Fan Y, Xu FG, Li P, Zhou B, Pan XY, Wang CC, Cui L

Received 13 March 2019

Accepted for publication 10 July 2019

Published 5 August 2019 Volume 2019:11 Pages 7353—7367

DOI https://doi.org/10.2147/CMAR.S208604

Checked for plagiarism Yes

Review by Single-blind

Peer reviewers approved by Ms Justinn Cochran

Peer reviewer comments 2

Editor who approved publication: Dr Xueqiong Zhu


Yun Shan Chen,*,1 Fen Liu,*,1 Yi Hong Luo,1 Yue Fan,1 Fang Gui Xu,1 Pin Li,1 Bei Zhou,1 Xiu Yu Pan,1 Chi Chiu Wang,2–4 Long Cui1,4

1Department of Obstetrics and Gynaecology, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510623, Guangdong, People’s Republic of China; 2Reproduction and Development Laboratory, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong; 3School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong; 4Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong

*These authors contributed equally to this work

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/propium iodide 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 confers chemoresistance with 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 a new therapeutic strategy in combination with TMZ for melanoma treatment.

Keywords: melanoma, Temozolomide, mitochondrial targeting sequence, apoptosis, reactive oxygen species

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