|Year : 2020 | Volume
| Issue : 4 | Page : 187-194
Exploration of thioridazine-induced Ca2+ signaling and non-Ca2+-triggered cell death in HepG2 human hepatocellular carcinoma cells
I-Shu Chen1, Wei-Zhe Liang2, Jue-Long Wang3, Chun-Chi Kuo4, Lyh-Jyh Hao5, Chiang-Ting Chou6, Chung-Ren Jan7
1 Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
2 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung; Department of Pharmacy, Tajen University, Pingtung, Taiwan
3 Department of Rehabilitation, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
4 Department of Nursing, Tzu Hui Institute of Technology, Pingtung, Taiwan
5 Department of Endocrinology and Metabolism, Kaohsiung Veteran General Hospital Tainan Branch; Chung Hwa University of Medical and Technology, Tainan, Taiwan
6 Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chiayi Campus; Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital Chiayi Branch, Puzi City, Chiayi County, Taiwan
7 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
|Date of Submission||18-Jun-2020|
|Date of Acceptance||03-Aug-2020|
|Date of Web Publication||28-Aug-2020|
Dr. Chiang-Ting Chou
Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chiayi Campus, Puzi City, Chiayi County
Dr. Chung-Ren Jan
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung
Dr. Lyh-Jyh Hao
Department of Metabolism, Kaohsiung Veterans General Hospital Tainan Branch, Tainan
Source of Support: This work was supported by Kaohsiung Veterans General Hospital (VGHKS108-197) to I-Shu Chen., Conflict of Interest: None
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+]i rises. 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+]i rises. On the other hand, thioridazine preincubation completely inhibited the [Ca2+]i rises induced by TG. Furthermore, U73122 totally suppressed the [Ca2+]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 Ca2+ chelator). To conclude, thioridazine caused concentration-dependent [Ca2+]i rises 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.
Keywords: Ca2+, fura-2, human hepatoma cells, thioridazine
|How to cite this article:|
Chen IS, Liang WZ, Wang JL, Kuo CC, Hao LJ, Chou CT, Jan CR. Exploration of thioridazine-induced Ca2+ signaling and non-Ca2+-triggered cell death in HepG2 human hepatocellular carcinoma cells. Chin J Physiol 2020;63:187-94
|How to cite this URL:|
Chen IS, Liang WZ, Wang JL, Kuo CC, Hao LJ, Chou CT, Jan CR. Exploration of thioridazine-induced Ca2+ signaling and non-Ca2+-triggered cell death in HepG2 human hepatocellular carcinoma cells. Chin J Physiol [serial online] 2020 [cited 2020 Oct 25];63:187-94. Available from: https://www.cjphysiology.org/text.asp?2020/63/4/187/293587
I-Shu Chen & Wei-Zhe Liang contributed equally to this work.
| Introduction|| |
Thioridazine, belonging to typical first-generation antipsychotic drugs, is a prescription used to treat schizophrenia. It could also be used in psychotic disorders, depressive disorders, and geriatric psychoneurotic manifestations. However, cellular studies show that this drug exerts cytotoxic effects against various cell models., The mechanism is unclear, but it appears that thioridazine makes drug-resistant cancer cells more susceptible to cytotoxic compounds to which they were resistant originally. In cancer treatment, thioridazine was shown to act by having anti-proliferative activity via apoptosis-inducing pathways, and to block dopamine receptor 2 and thus could work as a potential drug for ovarian cancer therapy. Furthermore, it has been shown that thioridazine elicited potent anti-melanoma effects, in lung cancer stem-like cells and colorectal cancer stem cells.
In terms of the effect of thioridazine on cell viability in liver cancer cells, it has been shown that thioridazine caused a concentration-dependent cytotoxicity in three lines of hepatocellular carcinoma cells (HCCs; SNU449, LM3, and Huh7). However, its effect on Ca2+ signaling was unexplored. Ca2+ is a key second messenger in terms of triggering and modulating different cellular responses including plasticity, contraction, fertilization, apoptosis, protein functioning, and fluid secretion. When the cell is stimulated, intracellular Ca2+ level ([Ca2+]i) may increase via Ca2+ entry from external medium or Ca2+ release from intracellular stores such as the endoplasmic reticulum. One of the major pathways for Ca2+ entry is the store-operated Ca2+ entry (SOCE). The endoplasmic reticulum is the major internal Ca2+ store in most cell types. Ca2+ can be released from the endoplasmic reticulum through pathways such as phospholipase C (PLC) activation-mediated IP3 activation or inhibition of the Ca2+-ATP pumps on the endoplasmic reticulum membrane. The stimulation and regulation of a Ca2+ signal are complex involving many molecules.
Regarding the effect of thioridazine on Ca2+ signaling, it has been shown that thioridazine has Ca2+ channel blocking activity in the isolated rat. Furthermore, thioridazine was shown to have diltiazem-like effect on the dihydropyridine-binding site of the Ca2+ channel of rat myocardial membranes and induced Ca2+ influx in yeast and to induce concentration-dependent loss in viability of melanocytes and stimulate eryptosis via entry of extracellular Ca2+ and activation of p38 kinase. However, whether thioridazine induces Ca2+ release from organelles in HCCs is unexplored. HepG2 cell is a common model for studying HCCs. It has been shown that in this cell, [Ca2+]i rises and death can be induced by stimulation with reagents including tamoxifen and polyphyllin D. The effect of thioridazine on [Ca2+]i has not been explored previously in HepG2 cells. Since a Ca2+ signal can cause many cellular processes, it is worthy to investigate the pathways of thioridazine-induced [Ca2+]i rises. Moreover, the effect of thioridazine on viability was also explored. Thus, the goal of this study was to explore the mechanisms of this Ca2+ signal and the effect of thioridazine on viability and explore the relationship between these two effects.
| Materials and Methods|| |
All chemicals were purchased from Sigma-Aldrich® (St. Louis, MO, USA) unless otherwise stated. Fura-2/AM (aminopolycarboxylic acid/acetoxymethyl) and BAPTA/AM (1,2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/acetoxymethyl) were purchased from Molecular Probes® (Eugene, OR, USA). The chemicals for cell culture were purchased from Gibco® (Gaithersburg, MD, USA).
The human hepatoma HepG2 cells were purchased from the Bioresource Collection and Research Center (Taiwan). They were maintained in minimum essential medium (MEM) plus 100 mg/ml streptomycin, 100 U/ml penicillin, and 10% heat-inactivated fetal bovine serum (FBS). The cultured cells were maintained in an incubator at 37°C with a humidified atmosphere containing 5% CO2 and 95% air.
Experimental solutions for [Ca2+]i assays
Two solutions were used in [Ca2+]i measurements. Ca2+-containing medium contained 5 mM KCl, 140 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); pH 7.4. Ca2+-free medium contained comparable compounds, as Ca2+-containing medium apart from that CaCl2 was substituted with 2 mM MgCl2 and 0.3 mM ethylene glycol tetraacetic acid (EGTA). The main reagent thioridazine was dissolved in absolute ethanol as a 0.1 M stock solution. All the other reagents were dissolved in dimethyl sulfoxide (DMSO), ethanol, or water. Pioneering experiments showed that the concentration of organic solvents in the experimental solutions did not surpass 0.1% and did not influence viability or resting [Ca2+]i.
Cells that were grown to confluency on 6 cm dishes were used for [Ca2+]i experiments. Cells were trypsinized and made into a suspension in MEM at a concentration of 106 cell/ml. Then, trypan blue exclusion was applied to find out cell viability. The results suggest that the viability was routinely larger than 95% after the trypsin treatment. The Ca2+-selective fluorescent dye fura-2 was used to detect [Ca2+]i changes. Suspended cells were loaded with 2 μM fura-2/AM for 30 min at 25°C in MEM. Cells were subsequently rinsed with Ca2+-containing medium twice and were then made into a suspension in Ca2+-containing medium at a concentration of 107 cell/ml. [Ca2+]i measurements were conducted in a water-jacketed cuvette (25°C) with constant stirring. The cuvette had 1 ml of medium and 0.5 million cells. Fluorescence was detected with a Shimadzu RF-5301PC spectrofluorophotometer. The instrument was started instantly after 0.1 ml cell suspension was supplemented to 0.9 ml Ca2+-containing or Ca2+-free medium. The fluorescence was recorded by setting excitation signals at 340 nm and 380 nm and emission signal at 510 nm at 1-s intervals. At designated time point, individual reagent was administered to the cell-containing cuvette by breaking the recording for 2 s to open and close the cuvette-containing chamber. After completion of experiments, for the purpose of calibration of [Ca2+]i, the detergent Triton X-100 (0.1%) and CaCl2(5 mM) were added to the cuvette to acquire the maximal fura-2 fluorescence. Subsequently, the Ca2+ chelator EGTA (10 mM) was added to the cuvette to chelate Ca2+ to get the minimal fura-2 fluorescence. Control experiments suggested that after 20 min of fluorescence measurements, cells bathed in a cuvette still had a viability of 95%. [Ca2+]i was obtained according to the formula previously described. Mn2+ quenching of fura-2 fluorescence was performed in Ca2+-containing medium containing 50 μM MnCl2. For the experiments using Mn2+ quenching to detect Ca2+ influx, MnCl2 was administered to cell suspension in the cuvette 30 s before the fluorescence measurement began. The results were detected at the excitation signal at 360 nm (Ca2+ insensitive) and emission signal at 510 nm at 1-s intervals according to the literature published previously.
Cell viability analyses
The cell viability detecting tetrazolium compound 4-[3-[4-lodophenyl]-2-4 (4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1) was used to assay the cell viability in our experiments. The rational of measuring cell viability was based on the capability of live cells to cleave tetrazolium salts by dehydrogenases and cause color changes of WST-1. Increases in the amount of color were proportional to the number of live cells. Experiments were conducted according to the manufacturer's instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). For the viability experiments, cells seeded in 96-well plates at a density of 104 cell/well in culture medium for 24 h were exposed to 0-80 μM thioridazine. WST-1 (10 μl pure solution) was instantly administered to all the wells of the dish after treatment with thioridazine, and cells were incubated for 30 min in the culture incubator. For experiments applying the selective Ca2+ chelator BAPTA/AM to chelate cytosolic Ca2+ (i.e., to prevent rises in [Ca2+]i), cells were incubated with 5 μM BAPTA/AM for 1 h before treatment with thioridazine. The chemical-treated cells were rinsed once with Ca2+-containing medium and incubated with or without thioridazine further for 24 h. Then, changes in the intensity of absorbance were recorded. The absorbance of samples (A450) was determined using an enzyme-linked immunosorbent assay (ELISA) reader. Optical density was normalized to the absorbance of unstimulated cells in each plate and presented as a percentage of the control value.
The results were presented as mean ± standard error of the mean of three separate experiments. The data were assayed using one-way analysis of variances (ANOVA) via the Statistical Analysis System (SAS®, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were conducted by post hoc analysis applying the Tukey's honestly significantly difference (HSD) protocol. P value < 0.05 was thought statistically significant.
| Results|| |
Action of thioridazine on [Ca2+]i in HepG2 cells
The effect of thioridazine [Figure 1]a on resting [Ca2+]i was explored. [Figure 1]b indicates that the resting [Ca2+]i was 49 ± 2 nM. The data suggest that thioridazine evoked [Ca2+]i rises in a concentration-dependent fashion in Ca2+-containing medium at concentrations of 25–100 μM. At a concentration of 100 μM, thioridazine caused [Ca2+]i rises that reached a net increase of 40 ± 3 nM. Furthermore, [Figure 1]c depicts that in Ca2+-free medium, thioridazine induced concentration-dependent rises in [Ca2+]i also in a concentration-dependent manner. [Figure 2]d shows the concentration–response relationships of thioridazine-evoked [Ca2+]i rises. In sum, by fitting to a Hill equation, the EC50 value was found to be 30 ± 3 nM in Ca2+-containing or 35 ± 3 nM in Ca2+-free medium, respectively. [Figure 1]d implicates that removal of extracellular Ca2+ decreased the Ca2+ responses by around 25%.
|Figure 1: Effect of thioridazine on [Ca2+]i in fura-2-loaded HepG2 cells. (a) The chemical structure of thioridazine. (b) Thioridazine-induced [Ca2+]i rises in fura-2-loaded cells. Thioridazine was added at 25 s. The concentration of thioridazine was indicated. The experiments were performed in Ca2+-containing medium. (c) Effect of thioridazine on [Ca2+]i in Ca2+-free medium. Thioridazine was added at 25 s in Ca2+-free medium. (d) Concentration-response plots of thioridazine-induced [Ca2+]i rises. Y-axis is the percentage of the net (baseline subtracted) area under the curve (25–250 s) of the [Ca2+]irises induced by 100 μM thioridazine in Ca2+-containing medium. Data are mean ± standard error of the mean of three independent experiments. *P < 0.05 compared to open circles.|
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|Figure 2: Effect of thioridazine on Ca2+ influx by measuring Mn2+ quenching of fura-2 fluorescence. Experiments were performed in Ca2+-containing medium. MnCl2(50 μM) was added to cells 1 min before fluorescence measurements. The y-axis is fluorescence intensity (in arbitrary units) measured at the Ca2+-insensitive excitation wavelength of 360 nm and the emission wavelength of 510 nm. Trace a: Control, without thioridazine. Trace b: Thioridazine (100 μM) was added as indicated. Data are mean ± standard error of the mean of three independent experiments.|
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Mn2+ influx evoked by thioridazine in HepG2 cells
Since thioridazine induced Ca2+ influx, as shown in [Figure 2], experiments were further performed to verify that Ca2+ influx occurred during thioridazine-evoked [Ca2+]i rises. Mn2+ is a cation very similar to Ca2+. Literature shows that Mn2+ enters cells through comparable pathways as Ca2+ but quenches fura-2 fluorescence at all excitation wavelengths. Thus, the occurrence of Ca2+ influx could be deducted by observing quenching of fura-2 fluorescence excited at the Ca2+-insensitive excitation wavelength of 360 nm by Mn2+. As shown in [Figure 3], thioridazine (100 μM) induced an immediate reduction in the 360 nm excitation signal that attained a value of 78 ± 2 arbitrary units at 60 s. The results indirectly suggest that Ca2+ influx plays a significant role in the [Ca2+]i rises induced by thioridazine.
|Figure 3: Effect of Ca2+ channel modulators on thioridazine-induced [Ca2+]i rises. In blocker- or modulator-treated groups, the reagent was added 1 min before thioridazine (100 μM). The concentration was 10 nM for phorbol 12-myristate 13-acetate, 2 μM for GF109203X, 1 μM for nifedipine, 0.5 μM for econazole, 5 μM for SKF96365. Data are expressed as the percentage of control (1st column) that is the area under the curve (25-200 s) of 100 μM thioridazine-induced [Ca2+]i rises, and are mean ± standard error of the mean of three independent experiments. *P < 0.05 compared to 1st column.|
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Mechanisms underlying thioridazine-evoked Ca2+ entry in HepG2 cells
The next set of experiments was performed to examine the Ca2+ entry mechanism of the thioridazine-caused [Ca2+]i rises. The SOCE modulators (nifedipine, 1 μM; econazole, 0.5 μM; and SKF96365, 5 μM), a protein kinase C (PKC) inhibitor (GF109203X, 2 μM), and a PKC activator phorbol 12-myristate 13 acetate (1 nM) were applied 1 min before addition of 100 μM thioridazine. All these compounds suppressed thioridazine-evoked [Ca2+]i rises by about 20% [Figure 3]. The data suggest that thioridazine-induced [Ca2+]i rises involved store-operated and PKC-modulated Ca2+ influx.
Intracellular sources of thioridazine-evoked Ca2+ release in HepG2 cells
Evidence suggests that among all the intracellular organelles, the endoplasmic reticulum plays a pivotal role in releasing stored Ca2+ in most cell types studied so far. The next set of experiments was conducted to examine the role of the endoplasmic reticulum in thioridazine-induced Ca2+ release in HepG2 cells. Ca2+-free medium was used in these experiments to avoid the interference of Ca2+ influx. [Figure 4]a shows that after thapsigargin (TG; 1 μM), an endoplasmic reticulum Ca2+ pump inhibitor, evoked [Ca2+]i rises of 19 ± 1 nM, thioridazine administered afterward at 500 s did not induce [Ca2+]i rises. [Figure 4]b depicts that the addition of TG after 100 μM thioridazine-evoked [Ca2+]i rises declined failed to induce [Ca2+]i rises. These results suggest that Ca2+ release from the endoplasmic reticulum plays a key role in thioridazine-induced [Ca2+]i rises.
|Figure 4: Effect of thapsigargin on thioridazine-induced Ca2+ release. (a and b) TG (1 μM) and thioridazine (100 μM) were added at time points indicated. Experiments were performed in Ca2+-free medium. Data are mean ± standard error of the mean of three independent experiments.|
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Store-operated calcium entry
Given thioridazine-induced Ca2+ release from the endoplasmic reticulum, the next set of experiments was performed to examine the underlying mechanism. PLC has been shown to have an important part in modulating the release of stored Ca2+. The PLC inhibitor U73122 has been widely appliedin vitro to study PLC. Thus, U73122 was used in this study to examine if this enzyme was involved in thioridazine-induced Ca2+ release. In this set of experiments, first, ATP was used to verify the activity of U73122. [Figure 5]a depicts that ATP (10 μM) evoked [Ca2+]i rises of 19 ± 2 nM. The rational of using ATP in our experiments is because ATP is a well-established PLC-dependent stimulus of [Ca2+]i rises in most cell types examined, and therefore, it was used as a tool to examine whether U73122 effectively inhibited the activity of PLC. [Figure 5]b shows that treatment with 2 μM U73122 did not alter the resting [Ca2+]i but totally inhibited ATP-evoked [Ca2+]i rises. This implicates that U73122 successfully suppressed PLC activity. The findings also show that treatment with 2 μM U73122 did not change resting [Ca2+]i but eliminated 100 μM thioridazine-evoked [Ca2+]i rises. The same experiments were repeated by using U73343, a PLC-insensitive structural analog of U73122, to substitute U73122. Our data implicate that U73343 (2 μM) did not alter ATP-evoked [Ca2+]i rises (not shown). In sum, the results suggest that the activation of PLC was involved in thioridazine-induced Ca2+ release from the endoplasmic reticulum.
|Figure 5: Effect of U73122 on thioridazine-induced Ca2+ release. Experiments were performed in Ca2+-free medium. (a) ATP (10 μM) was added as indicated. (b) First column is 100 μM thioridazine-induced [Ca2+]i rises. Second column shows that 2 μM U73122 did not alter basal [Ca2+]i. Third column shows ATP-induced [Ca2+]i rises. Fourth column shows that U73122 pretreatment for 60 s abolished ATP-induced [Ca2+]i rises (*P < 0.05 compared to 3rd column). Fifth column shows that U73122 (incubation for 60 s) and ATP (incubation for 30 s) pretreatment inhibited 100 μM thioridazine-induced [Ca2+]i rises. Data are mean ± standard error of the mean of three independent experiments. *P < 0.05 compared to first bar (control). Control is the area under the curve of 100 μM thioridazine-induced [Ca2+]i rises (25–220 s).|
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Action of thioridazine on cell viability in HepG2 cells
Medical literature suggests that Ca2+ signaling and cell viability are associated under certain circumstances; thus, the following experiments were performed to explore the action of thioridazine on the viability of HepG2 cells. Cells attached on dishes were incubated with 0-80 μM thioridazine for 24 h, and the tetrazolium assay was subsequently conducted. The data show that thioridazine at concentrations between 30 and 80 μM concentration-dependently decreased cell viability [Figure 6]. The next experiment was conducted to examine if thioridazine-induced cell death was induced by accompanying [Ca2+]i rises. The intracellular Ca2+ chelator BAPTA/AM was applied to suppress [Ca2+]i rises during thioridazine pretreatment. After treatment with 5 μM BAPTA/AM, 100 μM thioridazine did not induce [Ca2+]i rises (data not shown). This implicates that BAPTA/AM efficiently chelated cytosolic Ca2+. [Figure 1] further shows that 5 μM BAPTA/AM incubation did not change the control value of cell viability. Furthermore, BAPTA/AM incubation did not reverse thioridazine-evoked cell death in the presence of 30-80 μM thioridazine. The results implicate that Ca2+ was not involved in thioridazine-evoked reduce in cell viability.
|Figure 6: Effect of thioridazine on cell viability. Cells were treated with 0–80 μM thioridazine for 24 h, and the cell viability assay was performed. Data are mean ± standard error of the mean of three independent experiments. Each treatment had six replicates (wells). Data are expressed as percentage of control that is the increase in cell numbers in thioridazine-free groups. Control had 10,111 ± 170 cells/well before experiments and had 13,888 ± 145 cells/well after incubation for 24 h. In each group, the Ca2+ chelator BAPTA/AM (5 μM) was added to cells followed by treatment with thioridazine in Ca2+-containing medium. Cell viability assay was subsequently performed. *P < 0.05 compared with control.|
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| Discussion|| |
This study has investigated the action of thioridazine on [Ca2+]i and viability in human hepatoma cells. It has been shown that thioridazine between 25 and 100 μM induced concentration-dependent [Ca2+]i rises. Thioridazine induced [Ca2+]i rises by depleting intracellular Ca2+ stores and causing Ca2+ influx from extracellular medium because removing extracellular Ca2+ reduced thioridazine-induced [Ca2+]i rises by 20%. Since the removal of extracellular Ca2+ decreased thioridazine-induced response throughout the measurement period of 225 s, it suggests that Ca2+ influx occurred during the whole stimulation period.
Thioridazine appears to cause Ca2+ entry via stimulating SOCE which is induced by the depletion of intracellular Ca2+ stores, based on the inhibition of thioridazine-induced [Ca2+]i influx by nifedipine, econazole, and SKF96365. Studies have shown that these three chemicals have an inhibitory effect on SOCE in various cells., Our data show that all of these modulators inhibited thioridazine-induced [Ca2+]i rises. Thus, it suggests that thioridazine-induced Ca2+ entry involved store-operated Ca2+ pathway. Nifedipine is a L-type voltage-gated Ca2+ channel inhibitor and has some minor effects on voltage-dependent Ca2+ channels and SOCE. Furthermore, a previous study has shown that nifedipine inhibited nonselective store-operated cation conductance in choroidal arteriolar smooth muscle. Although nifedipine may affect SOCE through the mechanism independent of L-type Ca2+ channels, it is not sure how nifedipine affects HepG2 cells. Furthermore, regarding econazole or SKF96365, although previous studies have demonstrated the multiple effects of econazole or SKF96365 on SOCE activation on other cell types,, econazole or SKF96365 itself is not commonly used as a specific SOCE inhibitor. Therefore, besides the store-operated and PKC-regulated Ca2+ entries, L-type voltage-gated Ca2+ channel may also be involved in thioridazine-induced [Ca2+]i influx.
Our results suggest that thioridazine-induced Ca2+ influx was inhibited by enhancing or inhibiting PKC activity. This implies that a normally controlled PKC level is required for thioridazine to cause a full Ca2+ response. The relationship between PKC and Ca2+ homeostasis has been well established. A study has shown that Ca2+-dependent PKC is not required for posttetanic potentiation at the hippocampal CA3 to CA1 synapse. It was also shown that protein kinases, as mediators of fluid shear stress, stimulated signal transduction in endothelial cells involving Ca2+-dependent and Ca2+-independent events activated by the flow. Therefore, the pathways underlying thioridazine-induced Ca2+ entry in HepG2 cells deserve further assessment. Our findings suggest that thioridazine-induced Ca2+ influx appears to be contributed by PKC-regulated SOCE.
The TG, a specific and potent inhibitor of endoplasmic reticulum Ca-ATPases which enhances the passive Ca2+ leak from the endoplasmic reticulum, is used to increase cytosolic [Ca2+] in a wide range of models. Our data suggest that the TG-sensitive endoplasmic reticulum stores appeared to be the major Ca2+ store contributing to thioridazine-induced Ca2+ release because thioridazine-induced Ca2+ release was abolished by TG pretreatment. One possible pathway was that thioridazine acts similarly to TG by inhibiting the endoplasmic reticulum Ca2+-ATP pump. Furthermore, the data show that after TG induced [Ca2+]i rises of 19 ± 1 nM, thioridazine added at 500 s failed to induce [Ca2+]i rises. Addition of TG after 100 μM thioridazine-induced [Ca2+]i rises failed to induce [Ca2+]i rises at 500 s. It appears that at this experimental condition (500 s), TG or thioridazine could completely inhibit the endoplasmic reticulum Ca2+-ATP pump and release Ca2+. Therefore, this time course is feasible in our study. Our findings also show that the thioridazine-induced Ca2+ release was through a PLC-associated pathway because the release was completely suppressed when PLC activity was inhibited. The relationship between PLC activation and Ca2+ signalling is well established. For instance, calretinin was shown to participate in regulating steroidogenesis by PLC-Ca2+-PKC pathway in Leydig cells.
This study shows that thioridazine was cytotoxic to HepG2 cells. Ca2+ overloading is known to initiate processes leading to an alteration in cell viability. In many cell types, Ca2+ mobilization may cause Ca2+ influx across the plasma membrane via the process of SOCE. Cell viability could be altered in a Ca2+-dependent or independent manner. In a Ca2+-dependent manner, a previous study has shown that rapamycin inhibited B-cell activating factor (BAFF)-stimulated cell proliferation and survival by suppressing Ca2+-calmodulin dependent protein kinase II (CaMKII)-dependent phosphatase and tensin homolog (PTEN)/Akt-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells. Furthermore, trigonelline, a naturally occurring alkaloidal agent, protects ultraviolet-B irradiation-induced apoptotic cell death in human skin fibroblasts via attenuation of oxidative stress, restoration of cellular Ca2+ homeostasis, and prevention of endoplasmic reticulum stress. Conversely, Ca2+-independent phospholipase A2 via arachidonic acid mobilization is involved in Caco-2 human colorectal carcinoma cell growth. This study showed that thioridazine evoked cell death in a concentration-dependent manner. However, because BAPTA/AM pretreatment abolished thioridazine-evoked [Ca2+]i rises without preventing cytotoxicity, these data suggest that this decrease in viability was not associated with preceding [Ca2+]i rises.
Several studies were performed to explore the plasma level of thioridazine in adults. The plasma level of thioridazine may reach 20–40 μM., This level may go much higher in patients with liver disorders or taking higher doses. Previous studies suggested that because thioridazine in the blood requires active transport into the liver, it is less metabolized by the cytochrome P450 family., Therefore, thioridazine might have been metabolized through the kidney in humans. Our data show that thioridazine at a concentration of 30 μM induced 10%–15% cell death. Therefore, our data may be relevant to in vivo cases.
In sum, using HepG2 cells as a model for human hepatoma research in this study, it was found that thioridazine induced [Ca2+]i rises in a concentration-dependent fashion. The Ca2+ signal was composed of Ca2+ influx from extracellular solution and also intracellular Ca2+ release from the endoplasmic reticulum. The data show that thioridazine caused Ca2+ influx from extracellular solution through PKC-dependent SOCE pathways and also intracellular Ca2+ release from the endoplasmic reticulum in a PLC-associated manner. In addition, thioridazine evoked Ca2+-independent, concentration-dependent cytotoxicity. Our data should be considered in other in vitro studies on thioridazine, given the importance of Ca2+ movement and cytotoxicity in cell physiology.
Financial support and sponsorship
This work was supported by Kaohsiung Veterans General Hospital (VGHKS108-197) to I-Shu Chen.
Conflicts of interest
There are no conflicts of interest.
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