Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels

Yang,Xinhui; Jiang,Jiangtao; Yang,Xinyan; Han,Jichun; Zheng,Qiusheng

doi:10.3892/mmr.2016.5334

Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels

Authors:
- Xinhui Yang
- Jiangtao Jiang
- Xinyan Yang
- Jichun Han
- Qiusheng Zheng
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Affiliations: Department of Pharmacology, Key Laboratory of Xinjiang Endemic Phytomedicine Resources of Ministry of Education, School of Pharmacy, Shihezi University, Shihezi, Xinjiang 832002, P.R. China
Published online on: May 24, 2016 https://doi.org/10.3892/mmr.2016.5334
Pages: 911-919

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Abstract

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.

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