Chen Yun Shan, Liu Fen, Luo Yi Hong, Fan Yue, Xu Fang Gui, Li Pin, Zhou Bei, Pan Xiu Yu, Wang Chi Chiu, Cui Long
Department of Obstetrics and Gynaecology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, Guangdong, People's Republic of China.
Reproduction and Development Laboratory, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong.
Cancer Manag Res. 2019 Aug 5;11:7353-7367. doi: 10.2147/CMAR.S208604. eCollection 2019.
The role of endothelin receptor type B (EDNRB) isoform 3 involved in Temozolomide (TMZ)-induced melanoma cell death has not yet been elucidated.
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 Ca were measured by flow cytometry. Apoptosis protein array was applied.
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 Ca 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.
Our findings raise the possibility of targeting EDNRB isoform 3 as a new therapeutic strategy in combination with TMZ for melanoma treatment.
内皮素B型受体（EDNRB）亚型3在替莫唑胺（TMZ）诱导的黑色素瘤细胞死亡中的作用尚未阐明。
通过共聚焦和免疫印迹分析确定EDNRB亚型3的亚细胞定位。采用CRISPR/Cas9技术沉默EDNRB亚型3。通过膜联蛋白V/碘化丙啶染色和半胱天冬酶3/7/9活性评估细胞凋亡。通过流式细胞术测量线粒体膜电位、活性氧和线粒体钙。应用凋亡蛋白阵列。
共聚焦和免疫印迹分析表明EDNRB亚型3定位于线粒体，其第一个N端（1-22）氨基酸足以使其靶向线粒体。EDNRB亚型3缺失的A375细胞显著赋予化学抗性，表现为线粒体去极化、活性氧减少、线粒体钙摄取增加和半胱天冬酶9激活减少。此外，凋亡阵列显示，缺乏EDNRB亚型3时，p53在S392处的磷酸化表达相对较低，对氧磷酶2的表达略高。
我们的研究结果提出了将靶向EDNRB亚型3作为一种新的治疗策略，与TMZ联合用于黑色素瘤治疗的可能性。
