The Mechanism of Ca²⁺ Movement in the Involvement of Baicalein-Induced Cytotoxicity in ZR-75-1 Human Breast Cancer Cells

Baicalein (5,6,7-trihydroxyflavone) (1) has been found to be active against a wide variety of cancer cells. However, the molecular mechanism underlying the effects of 1 on the induction of Ca²⁺ movement and cytotoxicity in human breast cancer cells is unknown. This study examined the relationship between 1-induced Ca²⁺ signaling and cytotoxicity in ZR-75-1 human breast cancer cells. The in vitro investigations reported herein produced the following results: (i) Compound 1 increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a concentration-dependent manner. The signal was decreased by approximately 50% by removal of extracellular Ca²⁺. (ii) Compound 1-triggered [Ca²⁺]_i increases were significantly suppressed by store-operated Ca²⁺ channel blockers 2-aminoethoxydiphenyl borate (2-APB) and the PKC inhibitor GF109203X. (iii) In Ca²⁺-free medium, compound 1-induced [Ca²⁺]_i increases were also inhibited by GF109203X. Furthermore, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) or 2,5-ditert-butylhydroquinone (BHQ) abolished 1-induced [Ca²⁺]_i increases. Inhibition of phospholipase C (PLC) with U73122 abolished 1-induced [Ca²⁺]_i increases. (iv) Compound 1 (20-40 μM) caused cytotoxicity, increased reactive oxygen species (ROS) production, and activated caspase-9/caspase-3. Furthermore, compound 1-induced apoptosis was significantly inhibited by prechelating cytosolic Ca²⁺ with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester) or by decreasing ROS with the antioxidant NAC (N-acetylcysteine). Together, baicalein (1) induced a [Ca²⁺]_i increase by inducing PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-dependent, 2-APB-sensitive store-operated Ca²⁺ channels. Moreover, baicalein (1) induced Ca²⁺-associated apoptosis involved ROS production in ZR-75-1 cells. (Figure Presented).