BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling

在MDA-MB-231人类乳腺癌细胞中，BCI-215抑制了细胞迁移能力，诱导了细胞凋亡而非初级坏死，并增强了细胞对淋巴因子激活的杀手细胞活性。从机制上看，BCI-215在无活性氧物种的情况下，诱导了细胞外信号调节激酶（ERK）、p38和c-Jun N末端激酶（JNK）的快速持续磷酸化，其毒性通过抑制p38得到部分解救，但抑制JNK或ERK无效。BCI-215还超激活了MKK4/SEK1，暗示了应激反应的激活[1]。

Peripheral blood mononuclear cells were obtained from healthy volunteers. Cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 1% glutamine, and 1% penicillin/streptomycin, and stimulated with 6,000 IU of Interleukin 2 for 24 hours. After incubation, cells were washed with PBS and counted. In parallel, MDA-MB-231 cells were pretreated in a 384-well plate with vehicle or BCI-215 (3 μM). After 24 hours in culture, medium was replaced and peripheral blood mononuclear cells (PBMCs) added in 2-fold serial dilutions starting with a 50-fold excess of PBMCs in triplicate. After 24 hours of coculture, cells were fixed with formaldehyde/Hoechst 33342, washed twice with PBS, and imaged on the ArrayScan II. Cancer cells were identified by their larger nuclei compared with PBMCs, setting a size gate in the Hoechst channel. In experiments with chemotherapeutics, cells carrying a biosensor consisting of a mitochondrial targeting sequence derived from cytochrome c oxidase VIII linked to GFP that is a surrogate for cytochrome c release from mitochondria were pretreated for 24 hours with cisplatin (2 μM) or doxorubicin (400 nM), exposed to LAK, and cancer cells were identified and quantified by green fluorescence. Cell densities were normalized to those in the absence of PBMCs. Mean cell densities from multiple independent experiments were averaged and plotted in GraphPad Prism version 7.00[1].