Action of chlorzoxazone on Ca2+movement and viability in human oral cancer cells

Ti Lu; Wei-Zhe Liang; Lyh-Jyh Hao; Chun-Chi Kuo; Pochuen Shieh; Chiang-Ting Chou; Chung-Ren Jan

doi:10.4103/CJP.CJP_20_19

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ORIGINAL ARTICLE

Year : 2019 | Volume : 62 | Issue : 3 | Page : 123-130

Action of chlorzoxazone on Ca²⁺movement and viability in human oral cancer cells

Ti Lu¹, Wei-Zhe Liang², Lyh-Jyh Hao³, Chun-Chi Kuo⁴, Pochuen Shieh⁵, Chiang-Ting Chou⁶, Chung-Ren Jan⁷
¹ Department of Psychiatry, 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 Metabolism, Kaohsiung Veterans General Hospital Tainan Branch, Tainan, Taiwan
⁴ Department of Nursing, Tzu Hui Institute of Technology, Pingtung, Taiwan
⁵ Department of Pharmacy, Tajen University, Pingtung, Taiwan
⁶ Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chia-Yi, Taiwan
⁷ Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

Date of Submission	23-Feb-2019
Date of Decision	21-May-2019
Date of Acceptance	03-Jun-2019
Date of Web Publication	25-Jun-2019

Correspondence Address:
Dr. Chiang-Ting Chou
Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chia-Yi 61363
Taiwan
Dr. Chung-Ren Jan
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362
Taiwan
Dr. Pochuen Shieh
Department of Pharmacy, Tajen University, Pingtung 90741
Taiwan

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/CJP.CJP_20_19

Abstract

Chlorzoxazone is a skeletal muscle relaxant. However, the effect of chlorzoxazone on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in oral cancer cells is unclear. This study examined whether chlorzoxazone altered Ca²⁺ signaling and cell viability in OC2 human oral cancer cells. [Ca²⁺]_iin suspended cells was measured using the fluorescent Ca²⁺-sensitive dye fura-2. Cell viability was examined by water-soluble tetrazolium-1 assay. Chlorzoxazone (250–1000 μM) induced [Ca²⁺]_irises in a concentration-dependent manner. Ca²⁺ removal reduced the signal by approximately 50%. Mn²⁺ has been shown to enter cells through similar mechanisms as Ca²⁺ but quenches fura-2 fluorescence at all excitation wavelengths. Chlorzoxazone (1000 μM) induced Mn²⁺ influx, suggesting that Ca²⁺ entry occurred. Chlorzoxazone-induced Ca²⁺ entry was inhibited by 20% by inhibitors of store-operated Ca²⁺ channels and protein kinase C (PKC) modulators. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) inhibited chlorzoxazone-evoked [Ca²⁺]_irises by 88%. Conversely, treatment with chlorzoxazone-suppressed TG-evoked [Ca²⁺]_irises 75%. Chlorzoxazone induced [Ca²⁺]_irises by exclusively releasing Ca²⁺ from the endoplasmic reticulum. Inhibition of phospholipase C (PLC) with U73122 did not alter chlorzoxazone-induced [Ca²⁺]_irises. PLC activity was not involved in chlorzoxazone-evoked [Ca²⁺]_irises. Chlorzoxazone at 200–700 μM decreased cell viability, which was not reversed by pretreatment with Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl. In sum, in OC2 cells, chlorzoxazone induced [Ca²⁺]_irises by evoking PLC-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Chlorzoxazone also caused Ca²⁺-independent cell death. Since [Ca²⁺]_irises play a triggering or modulatory role in numerous cellular phenomena, the effect of chlorzoxazone on [Ca²⁺]_iand cell viability should be taken into account in other in vitro studies.

Keywords: Ca²⁺, cell viability, chlorzoxazone, endoplasmic reticulum, oral cancer cells

How to cite this article:
Lu T, Liang WZ, Hao LJ, Kuo CC, Shieh P, Chou CT, Jan CR. Action of chlorzoxazone on Ca²⁺movement and viability in human oral cancer cells. Chin J Physiol 2019;62:123-30

How to cite this URL:
Lu T, Liang WZ, Hao LJ, Kuo CC, Shieh P, Chou CT, Jan CR. Action of chlorzoxazone on Ca²⁺movement and viability in human oral cancer cells. Chin J Physiol [serial online] 2019 [cited 2023 Sep 28];62:123-30. Available from: https://www.cjphysiology.org/text.asp?2019/62/3/123/261310

Ti Lu, Wei-Zhe Liang, and Lyh-Jyh Hao contributed equally to this work.

Introduction

Chlorzoxazone, a centrally acting skeletal muscle relaxant,^[1] is metabolized to 6-hydroxychlorzoxazone (6-OH-CHZ) by cytochrome P450 2E1. Clinically, chlorzoxazone is used to treat cerebellar ataxia and nystagmus^[2] and downbeat nystagmus.^[3] Furthermore, chlorzoxazone is commonly used as a probe drug to monitor CPY2E2 and CPY2E1 enzymes.^[4],[5],[6] In ion signaling, chlorzoxazone was shown to activate SK-type K⁺ channel in rats,^[7] activate large conductance Ca²⁺-activated K⁺ channel in pituitary GH3 cells,^[8] and modulate recombinant small-conductance Ca²⁺-activated K⁺ channels in HEK293 mammalian cells.^[9] In addition, chlorzoxazone was shown to stimulate Cl⁻ secretion in the human bronchial epithelium.^[10] However, the effect of chlorzoxazone on Ca²⁺ signal transduction and cell viability is unknown in oral cancer cells.

A transient, regulated rise of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) can be evoked by various stimuli such as chemicals and hormones. This signal serves many cellular functions, such as fertilization, channel and receptor gating, gene expression, protein folding, secretion, contraction, motility, growth, proliferation, and apoptosis.^[11] Therefore, it is noteworthy to explore the mechanisms that mediate the [Ca²⁺]_i rises.^[12] Ca²⁺ can enter cells through various channels and receptors or be released from organelles such as the endoplasmic reticulum and mitochondria.^[13] Depletion of the endoplasmic reticulum Ca²⁺ store usually activates store-operated Ca²⁺ influx.^[14] Agonists can stimulate G-protein on the plasma membrane and activate phospholipase C (PLC), which subsequently increases cytosolic levels of IP₃.^[15] IP₃ can then release Ca²⁺ from the endoplasmic reticulum. A Ca²⁺ signal can be modulated by many molecules such as adenosine cyclic 3′,5′-phosphate, guanosine cyclic 3′,5′-phosphate, and protein kinase C (PKC).^[16]

Susceptibility to cancer may be associated with a large variability in cytochrome P450 2E1 activity. A previously observed correlation between the rate of 6-hydroxylation of chlorzoxazone and cytochrome P450 2E1 activity in vitro led to the postulation that this drug may be used as a noninvasive probe for P450 2E1 activity in vivo.^[17] Furthermore, a previous study has shown that cigarette smoking accelerates chlorzoxazone metabolism, most likely reflecting induction of cytochrome P450 2E1 activity, in humans. Induction of cytochrome P450 2E1 activity by cigarette smoking could contribute to tobacco-induced oral cancer.^[18] This study was aimed to explore the effect of chlorzoxazone on Ca²⁺ homeostasis and cell viability and to also explore their relationship. The human OC2 oral cancer cell was used in this study because it produces measurable [Ca²⁺]_i rises upon pharmacological stimulation. It has been shown that in this cell, [Ca²⁺]_i rises and death can be evoked by stimulation with chemicals such as methoxychlor,^[19] thimerosal,^[20] and fendiline,^[21] via causing Ca²⁺ entry and Ca²⁺ release. In our study, fura-2 was used as a Ca²⁺-sensitive dye to measure [Ca²⁺]_i. The [Ca²⁺]_i rises were characterized, the concentration–response plots were established, and the mechanisms underlying chlorzoxazone-evoked Ca²⁺ entry and Ca²⁺ release were examined. The effect of chlorzoxazone on cell viability was also explored.

Materials and Methods

Chemicals

The chemicals used for cell culture were purchased from Gibco^® (Gaithersburg, MD, USA). Aminopolycarboxylic acid/acetoxy methyl (fura-2/AM) and 1,2-bis(2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/AM (BAPTA/AM) were purchased from Molecular Probes^® (Eugene, OR, USA). Chlorzoxazone and the other reagents were purchased from Sigma-Aldrich^® (St. Louis, MO, USA) unless otherwise indicated.

Cell culture

OC2 is a cell line derived from an oral squamous cell carcinoma specimen of buccal mucosa squamous carcinoma from a Chinese man. This cell line was originated from Veterans General Hospital and Yang Ming Medical School, Taipei, Taiwan.^[22] Cells were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Experimental solutions for intracellular Ca²⁺ measurements

Ca²⁺-containing medium (pH 7.4) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 5 mM glucose. Ca²⁺-free medium contained similar chemicals as Ca²⁺-containing medium, except that CaCl₂ was substituted with 0.3 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM MgCl₂. Chlorzoxazone was dissolved in absolute ethanol as a 0.1 M stock solution. The other chemicals were dissolved in water, ethanol, or dimethyl sulfoxide. The concentration of organic solvents in the experimental solutions was <0.1% and did not change viability or basal [Ca²⁺]_i.

Intracellular Ca²⁺ assays

[Ca²⁺]_i was determined as described previously.^{[19],[20],[21]} Confluent cells grown on 6-cm dishes were trypsinized and made into a suspension in culture medium at a concentration of 10⁶ cell/ml. Cell viability was assessed by trypan blue exclusion. The viability was >95% after treatment. Cells were subsequently loaded with 2 μM fura-2/AM for 30 min at 25°C in the same medium. Cells were subsequently washed with Ca²⁺-containing medium twice and were made into a suspension in Ca²⁺-containing medium at a concentration of 10⁷ cell/ml. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25°C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was measured with the Shimadzu RF-5301PC spectrofluorophotometer immediately after 0.1 ml cell suspension was added to 0.9 ml Ca²⁺-containing or Ca²⁺-free medium, by recording excitation signals at 340 nm and 380 nm and emission signals at 510 nm at 1-s intervals. During the recording, reagents were added to the cuvette by pausing the recording for 2 s to open and close the cuvette-containing chamber. To calibrate [Ca²⁺]_i, at the end of the experiments, the detergent Triton X-100 (0.1%) and CaCl₂(5 mM) were added to the cuvette to get the maximal fura-2 fluorescence. The Ca²⁺ chelator EGTA (10 mM) was subsequently added to chelate Ca²⁺ in the cuvette to get the minimal fura-2 fluorescence. Control experiments showed that cells bathed in a cuvette had a viability of 95% after 20 min of fluorescence measurements. [Ca²⁺]_i was calculated as previously described.^[21] Mn²⁺ quenching of fura-2 fluorescence was conducted in Ca²⁺-containing medium containing 50 μM MnCl₂. MnCl₂ was added to cell suspension in the cuvette 30 s before the fluorescence recording was started. Data were recorded at excitation signal at 360 nm (Ca²⁺-insensitive) and emission signal at 510 nm at 1-s interval as described previously.^[23]

Cell viability analyses

Cell viability was determined as previously described.^{[19],[20],[21]} The measurement of viability was based on the ability of cells to cleave tetrazolium salts by dehydrogenases. An increase in the density of color correlated proportionally with the number of live cells. Assays were performed according to the manufacturer's manuals (Roche Molecular Biochemical, Indianapolis, IN, USA). Cells were seeded in 96-well plates at a concentration of 10⁴ cell/well in a culture medium for 24 h in the presence of chlorzoxazone. The fluorescent cell viability detecting reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (water-soluble tetrazolium-1; 10 μl pure solution) was added to the cells after chlorzoxazone treatment, and cells were incubated for 30 min in a humidified atmosphere. The cells were incubated with/without chlorzoxazone for 24 h. The absorbance of samples (A₄₅₀) was assessed using an enzyme-linked immunosorbent assay (ELISA) reader. In experiments using BAPTA/AM to chelate cytosolic Ca²⁺, cells were treated with 5 μM BAPTA/AM for 1 h before incubation with chlorzoxazone. The cells were washed once with Ca²⁺-containing medium and incubated with/without chlorzoxazone for 24 h. The absorbance of samples (A₄₅₀) was assayed using an ELISA reader. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value.

Statistical analyses

Data are presented as mean ± standard error of the mean of three experiments. Data were analyzed by one-way analysis of variances using the Statistical Analysis System (SAS^®, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between-group means were performed by post hoc analysis using the Tukey's honestly significantly difference procedure. A P < 0.05 was considered statistically significant.

Results

Action of chlorzoxazone on intracellular Ca²⁺ in OC2 cells

[Figure 1]a shows that the resting [Ca²⁺]_i concentration was 50 ± 1 nM. At concentrations of 250–1000 μM, chlorzoxazone evoked concentration-dependent rises in [Ca²⁺]_i. At a concentration of 1000 μM, chlorzoxazone caused [Ca²⁺]_i rises of 110 ± 2 nM. This signal was followed by a slow decay within 200 s. The Ca²⁺ signal saturated at 1000 μM chlorzoxazone because 2000 μM chlorzoxazone failed to evoke greater responses. In Ca²⁺-free medium, chlorzoxazone also evoked concentration-dependent rises in [Ca²⁺]_i at concentrations of 250–1000 μM. At a concentration of 1000 μM, chlorzoxazone caused rises in [Ca²⁺]_i of 62 ± 2 nM [Figure 1]b. [Figure 1]c depicts the concentration–response relationship. Ca²⁺ removal reduced the [Ca²⁺]_i rises by approximately 50%. The effective concentration₅₀ value was 491 ± 2 in Ca²⁺-containing or 510 ± 3 in Ca²⁺-free medium, respectively, by fitting to a Hill equation (P < 0.05).

Figure 1: Effect of chlorzoxazone on intracellular Ca²⁺ in fura-2-loaded OC2 cells. (a) Chlorzoxazone was added at 30 s. The concentration of chlorzoxazone was indicated. The experiments were performed in Ca²⁺-containing medium. Y axis is the intracellular Ca²⁺ induced by chlorzoxazone in Ca²⁺-containing medium. (b) Effect of chlorzoxazone on intracellular Ca²⁺ in the absence of extracellular Ca²⁺. Chlorzoxazone was added at 30 s in Ca²⁺-free medium. Y axis is the intracellular Ca²⁺ rises induced by chlorzoxazone in Ca²⁺-free medium. (c) Concentration–response plots of chlorzoxazone-induced intracellular Ca²⁺ rises in the presence or absence of extracellular Ca²⁺. Y axis is the percentage of the net (baseline subtracted) area under the curve (25–250 s) of the intracellular Ca²⁺ rises induced by 1000 μM chlorzoxazone in Ca²⁺-containing medium. Data are mean ± standard error of the mean of three experiments. *P < 0.05 compared to open circles

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Chlorzoxazone evoked Mn²⁺ influx-induced fluorescence quenching in OC2 cells

[Figure 1] describes that chlorzoxazone-evoked Ca²⁺ response saturated at 1000 μM; thus, in the following experiments, the response induced by 1000 μM chlorzoxazone was used as a control. The next sets of experiments were conducted to confirm that Ca²⁺ entry participated in chlorzoxazone-induced [Ca²⁺]_i rises. Store-operated Ca²⁺ entry (SOCE) pathway plays an important role in many cellular processes, which is largely studied using fluorescent Ca²⁺ indicator, fura-2.^[24] Extracellular Mn²⁺ is able to cross the plasma membrane through SOCE and quenches the fluorescence signals from fura-2. Thus, the fluorescence quenching rate by Mn²⁺ represents a convenient assay to monitor the extent of SOCE.^[24] Because Mn²⁺ and Ca²⁺ enter cells via the same mechanisms,^[25] quenching of fura-2 fluorescence excited at the Ca²⁺-insensitive excitation wavelength of 360 nm by Mn²⁺ indirectly implicates that chlorzoxazone evokes Ca²⁺ entry. [Figure 2] shows that 1000 μM chlorzoxazone evoked an immediate decrease in the 360 nm excitation signal that attained a value of 119 ± 3 arbitrary units at 200 s. This suggests that Ca²⁺ entry participated in chlorzoxazone-induced [Ca²⁺]_i rises.

Figure 2: Effect of chlorzoxazone 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 chlorzoxazone. Trace b: Chlorzoxazone (1000 μM) was added as indicated. Data are mean ± standard error of the mean of three separate experiments

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Modulation of chlorzoxazone-evoked intracellular Ca²⁺ rises in OC2 cells

Phorbol 12-myristate 13 acetate (1 nM; a PKC activator), GF109203X (2 μM; a PKC inhibitor), econazole (0.5 μM; nonspecific calcium channel blocker), nifedipine (1 μM; L-type calcium channel blocker), or SKF96365 (5 μM; SOCE inhibitor)^{[26],[27],[28]} was applied 1 min before chlorzoxazone (1000 μM) in Ca²⁺-containing medium before [Ca²⁺]_i changes were measured. All these five chemicals suppressed chlorzoxazone-evoked [Ca²⁺]_i rises by approximately 20% [Figure 3]. This suggests that SOCE and PKC-regulated Ca²⁺ entry were involved in chlorzoxazone-induced [Ca²⁺]_i rises.

Figure 3: Effect of Ca²⁺ channel modulators on chlorzoxazone-induced intracellular Ca²⁺ rises. In modulator-treated group, the modulator was added 1 min before chlorzoxazone (1000 μM). The concentration was 2 μM for GF109203X, 10 nM for phorbol 12-myristate 13 acetate, 0.5 μM for econazole, 1 μM for nifedipine, and 5 μM for SKF96365. Data are expressed as the percentage of control (1^st column) that is the area under the curve (25–200 s) of 1000 μM chlorzoxazone-induced intracellular Ca²⁺ rises in Ca²⁺-containing medium, and are mean ± standard error of the mean of three separate experiments. *P < 0.05 compared to 1^st column

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Chlorzoxazone-evoked Ca²⁺ release from the endoplasmic reticulum in OC2 cells

In most cell types including OC2 cells, the endoplasmic reticulum has been shown to be the main Ca²⁺ store.^[13] Thus, the role of the endoplasmic reticulum in chlorzoxazone-evoked Ca²⁺ release in OC2 cells was explored. The experiments were conducted in Ca²⁺-free medium to exclude the involvement of Ca²⁺ influx. [Figure 4]a shows that addition of 1 μM thapsigargin (TG), an endoplasmic reticulum Ca²⁺ pump inhibitor,^[29] induced [Ca²⁺]_i rises of 89 ± 2 nM. Chlorzoxazone (1000 μM) added afterward at 500 s induced [Ca²⁺]_i rises of 10 ± 2 nM. Conversely, [Figure 4]b shows that after 1000 μM chlorzoxazone-induced [Ca²⁺]_i rises of 82 ± 2 nM, addition of 1 μM TG at 500 s induced [Ca²⁺]_i rises of 22 ± 2 nM. TG inhibited chlorzoxazone-induced [Ca²⁺]_i rises by 88%, whereas chlorzoxazone inhibited TG-induced [Ca²⁺]_i rises by 75%. This suggests that chlorzoxazone induced [Ca²⁺]_i rises by dominantly releasing Ca²⁺ from the endoplasmic reticulum.

Figure 4: Effect of thapsigargin on chlorzoxazone-induced Ca²⁺ release. (a and b) thapsigargin (1 μM) and chlorzoxazone (1000 μM) were added at time points indicated. Experiments were performed in Ca²⁺-free medium. Data are mean ± standard error of the mean of three separate experiments

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Phospholipase C did not affect chlorzoxazone-evoked intracellular Ca²⁺ rises in OC2 cells

PLC is one of the pivotal proteins that regulate the release of Ca²⁺ from the endoplasmic reticulum.^[11] Because chlorzoxazone released Ca²⁺ from the endoplasmic reticulum, the role of PLC in this process was explored. U73122,^[30] a PLC inhibitor, was applied to examine if the activation of PLC was needed for chlorzoxazone-evoked Ca²⁺ release. [Figure 5]a shows that adenosine triphosphate (ATP) (10 μM) induced [Ca²⁺]_i rises of 25 ± 2 nM. ATP is a PLC-dependent agonist of [Ca²⁺]_i rises in most cell types.^[31] [Figure 5]b shows that incubation with 2 μM U73122 did not change basal [Ca²⁺]_i but abolished ATP-induced [Ca²⁺]_i rises. This suggests that U73122 effectively suppressed PLC activity. Conversely, the data show that incubation with 2 μM U73122 and 10 μM ATP did not inhibit 1000 μM chlorzoxazone-induced [Ca²⁺]_i rises. U73343 is a PLC-insensitive structural analog of U73122 and is often used as a control for U73122 activity. Our data show that U73343 (2 μM) did not alter ATP-evoked [Ca²⁺]_i rises (not shown). This implicates that PLC activity was not involved in chlorzoxazone-evoked [Ca²⁺]_i rises.

Figure 5: Effect of U73122 on chlorzoxazone-induced Ca²⁺ release. Experiments were performed in Ca²⁺-free medium. (a) Adenosine triphosphate (10 μM) was added at 25 s, (b) First column is 1000 μM chlorzoxazone-induced intracellular Ca²⁺ rises. Second column shows that 2 μM U73122 did not alter basal intracellular Ca²⁺. Third column shows adenosine triphosphate-induced intracellular Ca²⁺ rises. Fourth column shows that U73122 pretreatment for 30 s abolished adenosine triphosphate-induced intracellular Ca²⁺ rises (*P < 0.05 compared to 1^st column). Fifth column shows that U73122 (incubation for 30 s) and adenosine triphosphate (incubation for 30 s) pretreatment failed to inhibit 1000 μM chlorzoxazone-induced intracellular Ca²⁺ rises. Data are mean ± standard error of the mean of three experiments

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The cytotoxic effect of chlorzoxazone on OC2 cells

Because acute incubation with chlorzoxazone induced substantial [Ca²⁺]_i rises, and that unregulated [Ca²⁺]_i rises may change cell viability,^[11] experiments were performed to examine the effect of chlorzoxazone on viability of cells. Cells were treated with 0–700 μM chlorzoxazone for 24 h, and the tetrazolium assay was conducted. In the presence of 200–700 μM chlorzoxazone, cell viability decreased in a concentration-dependent manner [Figure 6]. The [Ca²⁺]_i chelator BAPTA/AM^[32] was applied to abolish [Ca²⁺]_i rises during chlorzoxazone incubation, to examine the role of Ca²⁺ in chlorzoxazone-evoked cell death. [Figure 6] also shows that 5 μM BAPTA/AM loading did not change the control value of cell viability. Chlorzoxazone (1000 μM) did not evoke [Ca²⁺]_i rises in BAPTA/AM-treated cells in Ca²⁺-containing solution (data not shown). This suggests that BAPTA loading effectively chelated cytosolic Ca²⁺. In the presence of 200–700 μM chlorzoxazone, BAPTA loading failed to reverse chlorzoxazone-evoked cell death. Therefore, the data suggest that chlorzoxazone-induced cell death was not caused by preceding rises in [Ca²⁺]_i.

Figure 6: Effect of chlorzoxazone on cell viability. Cells were treated with 0–700 μM chlorzoxazone for 24 h, and the cell viability assay was performed. Data are mean ± standard error of the mean of three separate experiments. Each treatment had six replicates (wells). Data are expressed as percentage of control response that is the increase in cell numbers in chlorzoxazone-free groups. Control had 10,123 ± 111 cells/well before experiments and had 13,987 ± 255 cells/well after incubation for 24 h. *P < 0.05 compared to control. In each group, the Ca²⁺ chelator 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (5 μM) was added to fura-2-loaded cells followed by treatment with chlorzoxazone in Ca²⁺-containing medium

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Discussion

Despite variousin vivo and in vitro actions of chlorzoxazone, its effect on Ca²⁺ signal regulation and viability in oral cancer cells is unknown. This study explored the effect of chlorzoxazone on Ca²⁺ signaling and viability in OC2 human oral cancer cells. Our study shows that chlorzoxazone increased [Ca²⁺]_i in OC2 cells. The Ca²⁺ signal was composed of Ca²⁺ entry and Ca²⁺ release because the signal was reduced by half by removing extracellular Ca²⁺. The Mn²⁺ quenching data also suggest that Ca²⁺ influx occurred during chlorzoxazone incubation.

The mechanism of chlorzoxazone-induced Ca²⁺ influx and release was explored. It has been shown that the dominant Ca²⁺ entry pathway is store-operated Ca²⁺ channels in OC2 cells.^[16],[33] Our findings show that chlorzoxazone-evoked [Ca²⁺]_i rises were inhibited by approximately 20% by econazole, nifedipine, and SKF96365. These three compounds have been used to inhibit SOCE although there are so far no selective inhibitors for this entry.^{[26],[27],[28]} Therefore, chlorzoxazone appears to cause Ca²⁺ entry via SOCE which is induced by depletion of [Ca²⁺]_i stores,^[14] based on the inhibition of chlorzoxazone-induced [Ca²⁺]_i rises by nifedipine, econazole, and SKF96365. Because Ca²⁺ entry contributed to 50% of chlorzoxazone-induced Ca²⁺ signal, whereas SOCE only accounted for 20% of the entry, the other Ca²⁺ entry pathways may involve the recently reported store-independent Ca²⁺ entry in oral cancer cells.^[28]

The activity of many protein kinases is known to associate with Ca²⁺ homeostasis.^[34],[35] Our data show that chlorzoxazone-evoked [Ca²⁺]_i rises were inhibited by 20% by enhancing or inhibiting PKC activity. This suggests that a normally maintained PKC level is needed for chlorzoxazone to induce a full Ca²⁺ response. PKC has been shown to interact with store-operated Ca²⁺ channels.^[36],[37] Because 20% of chlorzoxazone-induced [Ca²⁺]_i rises were via Ca²⁺ influx, this influx appears to involve PKC-regulated SOCE. Nifedipine is an L-type voltage-gated Ca²⁺ channel inhibitor. Although nifedipine may affect SOCE through the mechanism independent of L-type Ca²⁺ channels in rabbit arteriolar smooth muscle,^[27] it is not sure how nifedipine affects OC2 cells. Therefore, besides the SOCE and PKC-regulated Ca²⁺ entry, L-type voltage-gated Ca²⁺ channel may also be involved in chlorzoxazone-induced [Ca²⁺]_i influx.

Regarding the Ca²⁺ stores involved in chlorzoxazone-evoked Ca²⁺ release, the TG-sensitive endoplasmic reticulum store seemed to be the dominant one. Because TG did not abolish chlorzoxazone-induced Ca²⁺ release and vice versa and chlorzoxazone did not abolish TG-induced Ca²⁺ release, chlorzoxazone might release Ca²⁺ from other stores such as mitochondria and Golgi bodies.^[11] One possible mechanism for chlorzoxazone to release the endoplasmic reticulum Ca²⁺ is that chlorzoxazone acts similarly to TG by inhibiting the endoplasmic reticulum Ca²⁺-ATP pump.^[29] The data further show that PLC-dependent pathway failed to contribute to the Ca²⁺ release. The PLC-independent release could be due to other mechanisms such as nicotinic acid adenine dinucleotide phosphate-induced Ca²⁺ release^[38] and cyclic ADP-ribose-dependent Ca²⁺ release.^[39]

Cell viability could be altered in a Ca²⁺-dependent or -independent manner.^[40],[41] Our data show that chlorzoxazone induced Ca²⁺-independent cell death in a concentration-dependent manner. Although chlorzoxazone-associated Ca²⁺ signal did not induce cell death, it might affect numerous Ca²⁺-sensitive responses in the physiology of OC2 cells.^[13] Previous studies have explored the plasma concentration of chlorzoxazone after oral administration.^[42],[43] No bioresponse chlorzoxazone-related adverse effects were reported at 400 mg. The plasma level of chlorzoxazonein vivo experiments was found to reach 10–20 μM.^[42],[43] This concentration may go much higher in patients with liver or kidney dysfunction.^[42],[43] Our data show that chlorzoxazone at concentrations of and above 200 μM caused cell death. Thus, our data may have clinical significance.

[Ca²⁺]_i assays and viability assays were different methodologies in this study. [Ca²⁺]_i assays were performed online and terminated within 10 min, and trypan blue exclusion showed that after treatment with chlorzoxazone for this period of time, the cell viability was >95%. However, in viability assays, cells were incubated with chlorzoxazone for 24 h to gain significant changes in viability. This explains that 500 μM chlorzoxazone decreased cell viability by approximately 95% while 500 μM chlorzoxazone did not alter viability in [Ca²⁺]_i assays.

Together, the results show that chlorzoxazone evoked Ca²⁺ influx via PKC-sensitive SOCE and/or L-type Ca²⁺ channels and also Ca²⁺ release from the endoplasmic reticulum and/or other minor stores in a PLC-independent manner. Chlorzoxazone also caused Ca²⁺-independent cell death. Because rises in [Ca²⁺]_i can alter diverse cellular processes, caution should be taken in using chlorzoxazone for otherin vitro studies, and it should be noted that chlorzoxazone at concentrations above 200 μM may be cytotoxic to oral cancer cells.

Financial support and sponsorship

This work was supported by Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan (VGHKS106-133, 107-154).

Conflicts of interest

There are no conflicts of interest.

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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

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