Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels
Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels
复制标题
甘草查耳酮 A 通过增加细胞内钙水平诱导 T24 膀胱癌细胞凋亡
DOI：
10.3892/mmr.2016.5334
复制
发表时间：
2016-07-01
影响因子：
3.4
通讯作者：
Zheng, Qiusheng
中科院分区：
文献类型：
--
作者：
Yang, Xinhui;Jiang, Jiangtao;Zheng, Qiusheng
据报道，甘草酮A(LCA)通过线粒体和内质网(ER)应激触发的信号通路，显著抑制T24人膀胱癌细胞的增殖，增加ROS水平，诱导T24人膀胱癌细胞凋亡。基于这些发现，本研究旨在探讨LCA诱导T24细胞凋亡的机制。体外培养的T24细胞经LCA处理后，用硫代罗丹明B比色法测定细胞存活率。用Annexin V/碘化丙啶染色的流式细胞仪和Hoechst 33258染色的荧光显微镜检测细胞凋亡。用Fluo-3AM染料标记法检测细胞内游离钙离子水平。用2‘，7’-二氯二氢荧光素二乙酸酯探针法测定细胞内ROS水平。用5，5‘，6，6’-四氯-1，1‘，3，3’-四乙基苯并咪唑碳菁碘测定线粒体膜电位。采用逆转录半定量和定量聚合酶链式反应技术，检测B细胞淋巴瘤(B细胞淋巴瘤)超大型相关X蛋白、细胞死亡相互作用介质、凋亡蛋白激活因子-1(APAF-1)、钙蛋白2、半胱氨酸天冬氨酸特异性蛋白酶(Caspase)-3、caspase-4和caspase-9的mRNA表达水平。LCA可抑制T24细胞的增殖并诱导其凋亡，增加细胞内钙离子水平和ROS的产生。此外，LCA还导致线粒体功能障碍，降低线粒体膜电位，上调APAF-1、caspase-9和caspase-3的mRNA表达水平。LCA对T24细胞的暴露也触发了calain 2和caspase-4的激活，导致了细胞凋亡。这些结果表明，LCA使细胞内钙离子水平升高，这可能与线粒体功能障碍有关。此外，内质网应激途径可能是LCA诱导T24膀胱癌细胞凋亡的重要机制。
Licochalcone A (LCA) has been reported to significantly inhibit cell proliferation, increase reactive oxygen species (ROS) levels, and induce apoptosis of T24 human bladder cancer cells via mitochondria and endoplasmic reticulum (ER) stress-triggered signaling pathways. Based on these findings, the present study aimed to investigate the mechanisms by which LCA induces apoptosis of T24 cells. Cultured T24 cells were treated with LCA, and cell viability was measured using the sulforhodamine B assay. Apoptosis was detected by flow cytometry with Annexin V/propidium iodide staining, and by fluorescent microscopy with Hoechst 33258 staining. The levels of intracellular free calcium ions were determined using Fluo-3 AM dye marker. Intracellular ROS levels were assessed using the 2',7'-dichlorodihydrofluorescein diacetate probe assay. The mitochondrial membrane potential was measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide. Furthermore, the mRNA expression levels of B-cell lymphoma (Bcl)-extra large, Bcl-2-associated X protein, Bcl-2-interacting mediator of cell death, apoptotic protease activating factor-1 (Apaf-1), calpain 2, cysteinyl aspartate specific proteinase (caspase)-3, caspase-4 and caspase-9 were determined using reverse transcription semiquantitative and quantitative polymerase chain reaction analyses. Treatment with LCA inhibited proliferation and induced apoptosis of T24 cells, and increased intracellular Ca2+ levels and ROS production. Furthermore, LCA induced mitochondrial dysfunction, decreased mitochondrial membrane potential, and increased the mRNA expression levels of Apaf-1, caspase-9 and caspase-3. Exposure of T24 cells to LCA also triggered calpain 2 and caspase-4 activation, resulting in apoptosis. These findings indicated that LCA increased intracellular Ca2+ levels, which may be associated with mitochondrial dysfunction. In addition, the ER stress pathway may be considered an important mechanism by which LCA induces apoptosis of T24 bladder cancer cells.