Clozapine, a neuroleptic agent, inhibits Akt by counteracting Ca²⁺/calmodulin in PTEN-negative U-87MG human glioblastoma cells

Clozapine (CZP), a dibenzodiazepine derivative with a piperazinyl side chain, is in clinical use as an antipsychotic drug. This study investigated the effect of CZP on the modulation of the PI3K/Akt/GSK-3β pathway in PTEN-negative U-87MG glioblastoma cells. Treatment with CZP rapidly inhibited the basal and EGF-induced phosphorylation of Akt. The inhibition of Akt resulted in the dephosphorylation of GSK-3β and increased GSK-3β kinase activity. A voltage-sensitive Ca²⁺ channel blocker and calmodulin (CaM) antagonists inhibited Akt phosphorylation, whereas elevation of the intracellular Ca²⁺ concentration prevented CZP-induced dephosphorylation of Akt and GSK-3β, suggesting that Ca²⁺/CaM participates in the inhibition of Akt by CZP in U-87MG cells. In addition, similar to LY294002, CZP arrested cell cycle progression at G0/G1 phase, which was accompanied by decreased expression of cyclin D1. The reduction in the cyclin D1 level induced by CZP was abrogated by the inhibition of GSK-3β, the inhibition of proteasome-dependent proteolysis, or an increase in the intracellular Ca²⁺ concentration. These results suggest that the antipsychotic drug CZP modulates the PI3K/Akt/GSK-3β pathway by counteracting Ca²⁺/CaM in PTEN-negative U-87MG glioblastoma cells.