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

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

《The Chinese Journal of Physiology》 63卷4期 (2020/08) Pp. 187-194

https://doi.org/10.4103/CJP.CJP_45_20

摘要

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.

關鍵字

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

參考文獻

Varga B, Csonka Á, CsonkaA, Molnár J, Amaral L, Spengler G. Possible biological and clinical applications of phenothiazines. Anticancer Res 2017;37:5983-93.

Spengler G, Csonka Á, Molnár J, Amaral L. The anticancer activity of the old neuroleptic phenothiazine-type drug thioridazine. Anticancer Res 2016;36:5701-6.

Thanacoody RH. Thioridazine: The good and the bad. Recent Pat Antiinfect Drug Discov 2011;6:92-8.

Fenton M, Rathbone J, Reilly J, Sultana A. Thioridazine for schizophrenia. Cochrane Database Syst Rev 2007;3:CD001944.

Lawrie S, McIntosh A, Nadeem Z. Schizophrenia. Clin Evid 2005;14;1306-30.