Dual Modulation of Intracellular Ca2+ Signaling by Hispidulin Through Endoplasmic Reticulum Mobilization and Extracellular Ca2+ Entry in Human Breast Cancer Cells

Hispidulin, a naturally occurring flavonoid found in various medicinal plants, exhibits anti-proliferative effects in multiple cancer models; however, its role in regulating intracellular calcium (Ca2+) signaling in human breast cancer cells remains unclear. Here, we investigated its effects on intracellular Ca2+ dynamics, cytotoxicity, and Ca2+-associated signaling in T-47D human breast cancer cells. Hispidulin (40–120 μM) induced a concentration-dependent increase in intracellular Ca2+ levels ([Ca2+]i) accompanied by reduced cell viability, and pretreatment with the intracellular Ca2+ chelator BAPTA-AM further enhanced cytotoxicity, indicating that disruption of Ca2+ homeostasis potentiates cell death. Removal of extracellular Ca2+ partially attenuated the response, with additional inhibition by nifedipine, a dihydropyridine-sensitive Ca2+ channel blocker, suggesting involvement of voltage-dependent Ca2+ influx. The Ca2+ response was also reduced by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of store-operated Ca2+ entry (SOCE), and by the protein kinase C (PKC) inhibitor GF109203X, implicating SOCE and PKC signaling. Thapsigargin-induced depletion of endoplasmic reticulum (ER) Ca2+ stores indicated that hispidulin mobilizes ER Ca2+, while phospholipase C (PLC) inhibition by U73122 completely abolished the [Ca2+]i increase. Collectively, these findings demonstrate that hispidulin regulates intracellular Ca2+ homeostasis via PLC-dependent ER Ca2+ release and extracellular Ca2+ influx through SOCE and nifedipine-sensitive Ca2+ entry components, leading to PKC activation. Enhanced cytotoxicity following Ca2+ chelation further underscores the importance of Ca2+ homeostasis in determining cellular responses to hispidulin.