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

Abstract

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 Ca 2+ 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 Ca 2+ 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. IntroductionNatural herbal medicines have long been used to treat cancer. Biomedical research and cancer treatment clinical trials have provided evidence regarding the use of herbal medicines; therefore, they are increasingly being accepted as a complementary and alternative treatment (1). In addition, natural medicines have been reported to have an important role in human health, particularly certain well-studied plants, including Taxus chinensis (Taxus madia) (2), Radix Sophorae flavescentis (Sophora flavescens) (3), Alkanna tinctoria, Lithospermum erythrorhizon and licorice (Glycyrrhiza L) (4). Licorice is one of the most commonly prescribed herbs in Chinese Traditional Medicine, and has been used for >2,000 years. The effects of licorice have been reported on various diseases, ranging from microbial infection to cancer (5-7). However, since herbs usually contain numerous chemical compositions, the mechanism of action of these herbs is currently unclear. Recently, several chemical ing...

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