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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

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Tumor Biology

Abstract

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.

The summary of esculetin-evoked [Ca2+]i rises and -activated Ca2+-associated mitochondrial apoptotic pathways that involved cell cycle arrest. The natural coumarin derivative esculetin caused Ca2+ influx via 2-APB-sensitive store-operated Ca2+ entry and induced Ca2+ release from the endoplasmic reticulum. Moreover, esculetin activated the mitochondrial pathway of apoptosis in a Ca2+-associated manner that involved G2/M arrest.

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Acknowledgments

This work was supported by grants from Kaohsiung Veterans General Hospital (VGHKS104-116) to Chung-Ren Jan and Ministry of Science and Technology (MOST103-2314-B-075B-002) to Hong-Tai Chang.

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Authors and Affiliations

  1. Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, 813, Taiwan, China

    Hong-Tai Chang

  2. Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chia-Yi, 613, Taiwan

    Chiang-Ting Chou

  3. Chronic Diseases and Health Promotion Research Center, Chang Gung University of Science and Technology, Chia-Yi, 613, Taiwan

    Chiang-Ting Chou

  4. Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, 813, Taiwan

    You-Sheng Lin, Chung-Ren Jan & Wei-Zhe Liang

  5. Department of Pharmacy, Tajen University, Pingtung, 907, Taiwan

    Pochuen Shieh & Daih-Huang Kuo

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  1. Hong-Tai Chang
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Chang, HT., Chou, CT., Lin, YS. et al. 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. Tumor Biol. 37, 4665–4678 (2016). https://doi.org/10.1007/s13277-015-4286-1

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