Exploration of Thioridazine-Induced Ca^(2+) Signaling and Non-Ca^(2+)-Triggered Cell Death in HepG2 Human Hepatocellular Carcinoma Cells

10.4103/CJP.CJP_45_20

I-Shu Chen；Wei-Zhe Liang；Jue-Long Wang；Chun-Chi Kuo；Lyh-Jyh Hao；Chiang-Ting Chou；Chung-Ren Jan

Ca^(2+) ； fura-2 ； human hepatoma cells ； thioridazine

The Chinese Journal of Physiology

63卷4期（2020 / 08 / 31）

187 - 194

英文

Thioridazine, belonging to first-generation antipsychotic drugs, is a prescription used to treat schizophrenia. However, the effect of thioridazine on intracellular Ca^(2+) concentration ([Ca^(2+)]_i) and viability in human liver cancer cells is unclear. This study examined whether thioridazine altered Ca^(2+) signaling and viability in HepG2 human hepatocellular carcinoma cells. Ca^(2+) concentrations in suspended cells were measured using the fluorescent Ca^(2+)-sensitive dye fura-2. Cell viability was examined by WST-1 assay. Thioridazine at concentrations of 25-100 μM induced [Ca^(2+)]_i rises. Ca^(2+) removal reduced the signal by 20%. Thioridazine (100 μM) induced Mn^(2+) influx suggesting of Ca^(2+) entry. Thioridazine-induced Ca^(2+) 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 Ca^(2+) channels: nifedipine, econazole, and SKF96365. In Ca^(2+)-free medium, treatment with the endoplasmic reticulum Ca^(2+) pump inhibitor thapsigargin (TG) abolished thioridazine-evoked [Ca^(2+)]_i rises. On the other hand, thioridazine preincubation completely inhibited the [Ca^(2+)]_i rises induced by TG. Furthermore, U73122 totally suppressed the [Ca^(2+)]_i rises 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 Ca^(2+) chelator). To conclude, thioridazine caused concentration-dependent [Ca^(2+)]_i rises in HepG2 human hepatoma cells by inducing Ca^(2+) release from the endoplasmic reticulum via PLC-associated pathways and Ca^(2+) influx from extracellular medium through PKC-sensitive store-operated Ca^(2+) entry. In addition, thioridazine induced cytotoxicity in a Ca^(2+)-independent manner.

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