Yu Ling, Gao Li Xia, Ma Xiao Qing, Hu Fang Xin, Li Chang Ming, Lu Zhisong
Institute for Clean Energy & Advanced Materials, Faculty of Materials and Energy, Southwest University, Chongqing 400715, China.
Integr Biol (Camb). 2014 Dec;6(12):1211-7. doi: 10.1039/c4ib00170b. Epub 2014 Nov 3.
The BRAF(V600E) inhibitor PLX4032 (Vemurafenib) is an FDA-approved new drug for the treatment of metastatic melanomas, which specifically inhibits the RAS/MEK/ERK signaling pathway to control cell proliferation and adhesion. However, no study has been carried out to investigate the role of intracellular oxidative balance in PLX4032-induced tumor growth inhibition. Herein, for the first time, superoxide (O2˙(-)) and nitric oxide (NO) generated from PLX4032-challenged melanoma cells were monitored using electrochemical sensors and conventional fluorescein staining techniques. Impacts of superoxide dismutase (SOD) and NG-monomethyl-L-arginine monoacetate (L-NMMA), a nitric oxide synthase inhibitor, were also examined to demonstrate the specificity of ROS/NO generation and its biological consequences. PLX4032 specifically triggers production of O2˙(-) and NO from BRAF(V600E) mutant A375 cells. SOD and L-NMMA could abolish the PLX4032-induced increase in intracellular O2˙(-) and NO production, thereby rescuing cell growth in BRAF mutant A375 cells (A375(BRAFV600E)). In addition, PLX4032 treatment could decrease the mitochondrial membrane potential in A375(BRAFV600E) cells. The results suggest that PLX4032 can selectively cause ROS production and depolarization of mitochondrial membranes, potentially initiating apoptosis and growth inhibition of PLX4032-sensitive cells. This work not only proposes a new mechanism for PLX4032-induced melanoma cell inhibition, but also highlights potential applications of electrochemical biosensors in cell biology and drug screening.
BRAF（V600E）抑制剂PLX4032（维罗非尼）是一种经美国食品药品监督管理局（FDA）批准用于治疗转移性黑色素瘤的新药，它能特异性抑制RAS/MEK/ERK信号通路，从而控制细胞增殖和黏附。然而，尚未有研究探讨细胞内氧化平衡在PLX4032诱导的肿瘤生长抑制中的作用。在此，首次使用电化学传感器和传统的荧光素染色技术监测了经PLX4032处理的黑色素瘤细胞产生的超氧阴离子（O2˙(-)）和一氧化氮（NO）。还研究了超氧化物歧化酶（SOD）和一氧化氮合酶抑制剂NG-单甲基-L-精氨酸单乙酸盐（L-NMMA）的影响，以证明ROS/NO产生的特异性及其生物学后果。PLX4032能特异性地触发BRAF（V600E）突变的A375细胞产生O2˙(-)和NO。SOD和L-NMMA可以消除PLX4032诱导的细胞内O2˙(-)和NO产生的增加，从而挽救BRAF突变的A375细胞（A375（BRAFV600E））的细胞生长。此外，PLX4032处理可降低A375（BRAFV600E）细胞的线粒体膜电位。结果表明，PLX4032可选择性地导致ROS产生和线粒体膜去极化，可能引发PLX4032敏感细胞的凋亡和生长抑制。这项工作不仅提出了PLX4032诱导黑色素瘤细胞抑制的新机制，还突出了电化学生物传感器在细胞生物学和药物筛选中的潜在应用。
