Exploration of thioridazine-induced Ca²⁺signaling and non-Ca²⁺-triggered cell death in HepG2 human hepatocellular carcinoma cells

Thioridazine, belonging to first-generation antipsychotic drugs, is a prescription used to treat schizophrenia. However, the effect of thioridazine on intracellular Ca²⁺concentration ([Ca²⁺]_i) and viability in human liver cancer cells is unclear. This study examined whether thioridazine altered Ca²⁺signaling and viability in HepG2 human hepatocellular carcinoma cells. Ca²⁺concentrations in suspended cells were measured using the fluorescent Ca²⁺-sensitive dye fura-2. Cell viability was examined by WST-1 assay. Thioridazine at concentrations of 25-100 μM induced [Ca²⁺]_irises. Ca²⁺removal reduced the signal by 20%. Thioridazine (100 μM) induced Mn²⁺influx suggesting of Ca²⁺entry. Thioridazine-induced Ca²⁺entry was inhibited by 20% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate) and inhibitor (GF109203X) and by three inhibitors of store-operated Ca²⁺channels: nifedipine, econazole, and SKF96365. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺pump inhibitor thapsigargin (TG) abolished thioridazine-evoked [Ca²⁺]_irises. On the other hand, thioridazine preincubation completely inhibited the [Ca²⁺]_irises induced by TG. Furthermore, U73122 totally suppressed the [Ca²⁺]_irises induced by thioridazine via inhibition of phospholipase C (PLC). Regarding cytotoxicity, at 30-80 μM, thioridazine reduced cell viability in a concentration-dependent fashion. This cytotoxicity was not prevented by preincubation with 1,2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) (a Ca²⁺chelator). To conclude, thioridazine caused concentration-dependent [Ca²⁺]_irises in HepG2 human hepatoma cells by inducing Ca²⁺release from the endoplasmic reticulum via PLC-associated pathways and Ca²⁺influx from extracellular medium through PKC-sensitive store-operated Ca²⁺entry. In addition, thioridazine induced cytotoxicity in a Ca²⁺-independent manner.