Esculetin, a natural coumarin compound, evokes Ca²⁺ movement and activation of Ca²⁺-associated mitochondrial apoptotic pathways that involved cell cycle arrest in ZR-75-1 human breast cancer cells

Esculetin (6,7-dihydroxycoumarin), a derivative of coumarin compound, is found in traditional medicinal herbs. It has been shown that esculetin triggers diverse cellular signal transduction pathways leading to regulation of physiology in different models. However, whether esculetin affects Ca²⁺ homeostasis in breast cancer cells has not been explored. This study examined the underlying mechanism of cytotoxicity induced by esculetin and established the relationship between Ca²⁺ signaling and cytotoxicity in human breast cancer cells. The results showed that esculetin induced concentration-dependent rises in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in ZR-75-1 (but not in MCF-7 and MDA-MB-231) human breast cancer cells. In ZR-75-1 cells, this Ca²⁺ signal response was reduced by removing extracellular Ca²⁺ and was inhibited by the store-operated Ca²⁺ channel blocker 2-aminoethoxydiphenyl borate (2-APB). In Ca²⁺-free medium, pre-treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) abolished esculetin-induced [Ca²⁺]_i rises. Conversely, incubation with esculetin abolished TG-induced [Ca²⁺]_i rises. Esculetin induced cytotoxicity that involved apoptosis, as supported by the reduction of mitochondrial membrane potential and the release of cytochrome c and the proteolytic activation of caspase-9/caspase-3, which were partially reversed by pre-chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Moreover, esculetin increased the percentage of cells in G2/M phase and regulated the expressions of p53, p21, CDK1, and cyclin B1. Together, in ZR-75-1 cells, esculetin induced [Ca²⁺]_i rises by releasing Ca²⁺ from the ER and causing Ca²⁺ influx through 2-APB-sensitive store-operated Ca²⁺ entry. Furthermore, esculetin activated Ca²⁺-associated mitochondrial apoptotic pathways that involved G2/M cell cycle arrest.