Pharmacological modulation of Ca²⁺ signaling by calycosin promotes glioblastoma cell death.

Flavonoids are plant-derived polyphenols widely found in fruits, vegetables, and herbs, and have attracted growing interest for their neuroprotective and anti-cancer properties. Among them, calycosin (7-hydroxy isoflavone), a major bioactive component of Astragalus membranaceus, has demonstrated anti-inflammatory, antioxidant, and estrogenic activities. While its protective effects have been reported in cardiovascular and neurodegenerative models, its pharmacological role in glioblastoma and calcium ions (Ca²⁺) signaling remains largely unexplored. In this study, we investigated the effects of calycosin on intracellular Ca²⁺ levels ([Ca²⁺]i), cytotoxicity, and Ca²⁺-related signaling mechanisms in DBTRG-05MG human glioblastoma cells. Calycosin (25–75 µM) induced a concentration-dependent elevation in [Ca²⁺]i and significantly reduced cell viability. These cytotoxic effects were reversed by the intracellular Ca²⁺ chelator BAPTA-AM, indicating Ca²⁺-dependent toxicity. In Ca²⁺-free conditions, calycosin-induced Ca²⁺ entry was suppressed, and this was further attenuated by 2-aminoethoxydiphenyl borate (2-APB), a modulator of store-operated calcium entry (SOCE), and GF109203X, a protein kinase C (PKC) inhibitor. Thapsigargin experiments confirmed that calycosin mobilized Ca²⁺ from the endoplasmic reticulum (ER), while phospholipase C (PLC) inhibition by U73122 completely abolished the [Ca²⁺]i response. These results indicate that calycosin-induced cell death involves PLC-dependent ER Ca²⁺ release and SOCE-mediated Ca²⁺ influx, with PKC as a downstream effector. These findings highlight intracellular Ca²⁺ as a critical mediator of calycosin’s cytotoxic effects and suggest that calycosin, or related flavonoids, may serve as promising candidates for modulating Ca²⁺-dependent oncogenic signaling in glioblastoma therapy.