Laboratory Animal Center, Shanxi Province Cancer Hospital/Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, Shanxi, China.
Research Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi, China.
Clin Exp Pharmacol Physiol. 2023 Sep;50(9):738-748. doi: 10.1111/1440-1681.13800. Epub 2023 Jun 15.
Brazilin possesses anticancer effects, but the mechanisms are poorly understood. This study investigated the mechanisms of brazilin-induced cell death in the T24 human bladder cancer cell line. Low serum cell culture and the lactate dehydrogenase assay were used to confirm the antitumor effect of brazilin. Annexin V and propidium iodide double staining, transmission electron microscopy, fluo-3-AM assay for Ca mobilization and caspase activity assay were performed to identify the type of cell death after brazilin treatment. Mitochondria membrane potentials were measured using JC-1. Quantitative real-time polymerase chain reaction and western blot analyses were performed to verify the expression of the necroptosis-related genes and proteins receptor interacting protein 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL). The results showed that brazilin induced necrosis in T24 cells and upregulated the mRNA and protein levels of RIP1, RIP3 and MLKL and Ca influx. The necroptosis-mediated cell death was rescued by the necroptosis inhibitor necrostatin-1 (Nec-1), but not by the apoptosis inhibitor z-VAD-fmk. Brazilin repressed caspase 8 expression and decreased the mitochondrial membrane potentials; both effects were partially reversed by Nec-1. Brazilin induced physiological and morphological changes in T24 cells and RIP1/RIP3/MLKL-mediated necroptosis might be involved. In conclusion, the results confirm the involvement of necroptosis in brazilin-induced cell death and suggest that brazilin could be explored as an anticancer agent against bladder cancer.
巴西红厚壳素具有抗癌作用，但作用机制尚不清楚。本研究探讨了巴西红厚壳素诱导 T24 人膀胱癌细胞系细胞死亡的机制。采用低血清细胞培养和乳酸脱氢酶测定法证实巴西红厚壳素的抗肿瘤作用。采用 Annexin V 和碘化丙啶双重染色、透射电子显微镜、钙动员荧光-3-AM 测定法和半胱天冬酶活性测定法鉴定巴西红厚壳素处理后细胞死亡的类型。使用 JC-1 测定线粒体膜电位。采用定量实时聚合酶链反应和 Western blot 分析验证坏死相关基因和蛋白受体相互作用蛋白 1 (RIP1)、RIP3 和混合谱系激酶结构域样蛋白 (MLKL)的表达。结果表明，巴西红厚壳素诱导 T24 细胞坏死，并上调 RIP1、RIP3 和 MLKL 的 mRNA 和蛋白水平以及 Ca 内流。坏死抑制剂 necrostatin-1 (Nec-1) 挽救了由 necroptosis 介导的细胞死亡，但凋亡抑制剂 z-VAD-fmk 则没有。巴西红厚壳素抑制 caspase 8 的表达并降低线粒体膜电位；这两种作用均部分被 Nec-1 逆转。巴西红厚壳素诱导 T24 细胞发生生理和形态变化，RIP1/RIP3/MLKL 介导的 necroptosis 可能参与其中。总之，结果证实了 necroptosis 参与了巴西红厚壳素诱导的细胞死亡，并提示巴西红厚壳素可能作为一种抗癌剂用于膀胱癌的治疗。
