ToxSci Advance Access originally published online on August 13, 2007
Toxicological Sciences 2007 100(1):109-117; doi:10.1093/toxsci/kfm205
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Thimerosal-Induced Apoptosis in Human SCM1 Gastric Cancer Cells: Activation of p38 MAP Kinase and Caspase-3 Pathways without Involvement of [Ca2+]i Elevation
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* Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813
Department of Nursery, Tzu Hui Institute of Technology, Pingtung, Taiwan 926
Department of Psychiatry, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 807
Department of Psychiatry, Tian-Sheng Memorial Hospital, Ping-Tong, Taiwan 900
¶ Laboratory Medicine Division, Zuoying Armed Forces General Hospital, Kaohsiung, Taiwan 813
|| Department of Surgery, Ping Tung Christian Hospital, Ping Tung, Taiwan 900
||| Department of Rehabilitation, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813
|||| Department of Orthopaedic Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813
# Department of Otolaryngology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813
** Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813
1 To whom correspondence should be addressed. Fax: 886-7-3468056. E-mail: crjan{at}isca.vghks.gov.tw.
Received May 15, 2007; accepted July 20, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Thimerosal is a mercury-containing preservative in some vaccines. The effect of thimerosal on human gastric cancer cells is unknown. This study shows that in cultured human gastric cancer cells (SCM1), thimerosal reduced cell viability in a concentration- and time-dependent manner. Thimerosal caused apoptosis as assessed by propidium iodide–stained cells and caspase-3 activation. Although immunoblotting data revealed that thimerosal could activate the phosphorylation of extracellular signal–regulated kinase, c-Jun NH2-terminal protein kinase, and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Thimerosal also induced [Ca2+]i increases via Ca2+ influx from the extracellular space. However, pretreatment with (bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate)/AM, a Ca2+ chelator, to prevent thimerosal-induced [Ca2+]i increases did not protect cells from death. The results suggest that in SCM1 cells, thimerosal caused Ca2+-independent apoptosis via phosphorylating p38 MAPK resulting in caspase-3 activation.
Key Words: apoptosis; caspase-3; gastric cancer cells; MAPKs; thimerosal.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Thimerosal is a mercurial compound used to preserve some vaccines and consumer products (Ueha-Ishibashi et al., 2005
). Thimerosal could also induce Ca2+ release from microsomal stores (Madi, 2005). Thimerosal has been shown to induce apoptosis and G(2)/M phase arrest in human leukemia cells (Woo et al., 2006), to induce apoptosis in a neuroblastoma cell model via the c-Jun N-terminal kinase pathway (Herdman et al., 2006), and to induce neuronal cell apoptosis by causing cytochrome c and apoptosis-inducing factor release from mitochondria (Yel et al., 2005). Thimerosal also evokes cytosolic Ca2+ elevation and subsequent cell death in human osteosarcoma cells (Chang et al., 2005) and, to induce DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons, fibroblasts (Baskin et al., 2003), and neuroblastoma cells (Parran et al., 2005). Mitochondria were thought to mediate thimerosal-induced apoptosis in neuroblastoma cells (Humphrey et al., 2005). Additionally, thimerosal was shown to stimulate ryanodine receptors from excitable cells (Hidalgo et al., 2000), to decrease transient receptor potential V1 activity by oxidation of extracellular sulfhydryl residues (Jin et al., 2004), to stimulate Ca2+ flux through inositol 1,4,5-trisphosphate (IP3) receptors (Bultynck et al., 2004), and to increase intracellular Ca2+ concentration ([Ca2+]i) of rat cerebellar neurons (Ueha-Ishibashi et al., 2004) and renal tubular cells (Jan et al., 2003). Thimerosal also inhibits sodium channels in rat sensory neurons (Song et al., 2000). In vivo immunosuppressive and autoimmune effects of thimerosal in mice were noticed (Havarinasab et al., 2005).
Apoptosis is a form of cell death that permits the removal of damaged, senescent, or unwanted cells in multicellular organisms, without damage to the cellular microenvironment. Defective apoptosis represents a major causative factor in the development and progression of cancer (Russo et al., 2006). All the criteria used to describe apoptotic cells are morphological and include several morphological changes, the presence of typical DNA fragments, caspases activation, and a phosphatidylserine shift toward the outer leaflet of the cell membrane (Hail et al., 2006). Caspases are responsible for crucial aspects of inflammation and immune-cell death that are disrupted in many genetic autoimmune and autoinflammatory diseases. The caspase family of proteases can be divided into proapoptotic and proinflammatory members based on their substrate specificity and role in separate signaling cascades (Siegel, 2006). Among the at least 11 known members of the caspase family, caspase-3 is an executioner in caspases cascades and is a main player in apoptosis (Lavrik et al., 2005).
Within cells, Ca2+, the most prominent of all intracellular messengers, mediates diverse forms of cell death with actions modulated by many proteins, including IP3 receptors, calcineurin, calpain, and cytochrome c (Hanson et al., 2004). Ca2+ plays a central role in physiological cell death such as terminal differentiation and apoptosis. Cell injury occurs when intracellular Ca2+ homeostasis is disturbed, which subsequently may lead to cell death. Ca2+ in Ca2+-dependent enzymes, transglutaminases, various proteases, phosphorylases, and kinases is involved in the process of cell death (Annunziato et al., 2003; Gupta and Pushkala, 1999).
The mitogen-activated protein kinase (MAPK) pathway is an attractive target for therapeutic intervention in different cancers. The MAPK pathway plays an integral role in the regulation of proliferation, invasiveness, and survival. The study of this pathway is made easier by the recent availability of pharmaceutical agents that inhibit the various kinases and GTPases that comprise the pathway (Panka et al., 2006). They include extracellular signal–regulated kinases (ERKs), stress-activated protein kinases (or c-Jun-N-terminal protein kinases; JNKs) and p38 MAPK. Signal transduction involves sequential phosphorylation of a tripartite kinase module, culminating in activated MAPKs. Activated MAPKs might stay in the cytoplasm to phosphorylate structural proteins or translocate to the nucleus, where it could activate transcription factors involved in DNA synthesis and cell division (Schaeffer and Weber, 1999). MAPKs are thought to play an important role in apoptosis (Cross et al., 2000; Reddy et al., 2003).
Gastritis was noted in humans ingesting 83 mg/kg thimerosal (Pfab et al., 1996). Thimerosal (0.3, 1.0, and 3.0 mg/kg) significantly and dose dependently reduced gastric secretion and acidity in rats (Postius, 1984). However, the effect of thimerosal on gastric cells has not been explored. The aim of the present study was to investigate the molecular mechanisms underlying the apoptotic pathway induced by thimerosal in human gastric cancer cells by using SCM1 cells. The SCM1 cell line has been used as a model for gastric cancer cell research (Sheu et al., 1998). The role of apoptosis, [Ca2+]i, and MAPKs in the physiology of SCM1 cells is unclear. In this study, we have explored the cytotoxic effect of thimerosal on SCM1 cells and have investigated whether apoptotic markers (such as caspase-3 activation and increase in subdiploidy nuclei), MAPKs, and Ca2+ signaling were interacting in this event.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Materials.
The reagents for cell culture were from Gibco (Gaithersburg, MD). Fura-2/AM and (bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate) (BAPTA)/AM were from Molecular Probes (Eugene, OR). Thimerosal, propidium iodide (PI), dimethyl sulfoxide (DMSO), and other reagents were from Sigma-Aldrich (St Louis, MO).
Cell culture.
SCM1 cells were obtained from American Type Culture Collection and were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were kept at 37°C in 5% CO2-containing humidified air.
Solutions.
Ca2+-containing medium (pH 7.4) had the following (in mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid 10, and glucose 5. Ca2+-free medium contained similar components as Ca2+-containing medium except that CaCl2 was substituted with 0.1mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid (EGTA). Agents were dissolved in water, ethanol, or DMSO as concentrated stocks. The concentrations of ethanol and DMSO in the final solution did not exceed 0.1% and did not alter cell viability and basal [Ca2+]i. Thimerosal was dissolved in DMSO as 1mM stock and was stored at –20°C before experiments. Thimerosal is ethylmercury thiosalicylate. Thiosalicylate at similar concentrations used for thimerosal had no effect on viability and basal [Ca2+]i (n = 3; not shown).
[Ca2+]i measurements.
Trypsinized cells (106/ml) were allowed to recover in serum-free culture medium for 1 h before being loaded with 2µM fura-2/AM for 30 min at 25°C in the same medium. Cells were washed and resuspended in Ca2+-containing medium. 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 monitored with a Shimadzu RF-5301PC spectrofluorophotometer (Shimadzu, Kyoto, Japan) by recording excitation signals at 340 and 380 nm and emission signal at 510 nm at 1-s intervals. Maximum and minimum fluorescence values were obtained by adding 0.1% Triton X-100 (plus 5mM CaCl2) and 10mM EGTA sequentially at the end of each experiment. [Ca2+]i was calculated as described previously assuming a Kd of 155nM (Grynkiewicz et al., 1985). For the experiments shown in Figure 7A, the cells were loaded with 20µM BAPTA/AM for 1 h at 37°C, followed by a washout with Ca2+-containing medium. Then cells were incubated in a 37°C incubator for 24 h. Fura-2/AM (2µM) was subsequently added to cell suspensions for 30 min at 25°C, followed by a washout with saline. The cells were ready for [Ca2+]i measurements.
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Cell viability assays.
The measurement of viability was based on the ability of viable cells to cleave tetrazolium salts by mitochondrial dehydrogenases. Augmentation in the amount of developed color directly correlated with the number of metabolically active cells. Assays were performed according to manufacturer’s instructions (Roche Molecular Biochemical, Indianapolis, IN). Cells were seeded in 96-well plates at a density of 50,000 cells/well in culture medium for 4 h to allow attachment. Then the culture medium was added with 10 µl of serum-free medium containing different concentrations of treatments. The cell viability detecting reagent WST-1 (4-[3-[4-iodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (10 µl pure solution) was added to each sample 24 h after treatments with various concentrations of thimerosal, and cells were incubated for additional 120 min in a humidified atmosphere (37°C). In experiments using BAPTA/AM to chelate intracellular Ca2+, cells were treated with 20µM BAPTA/AM for 1 h. The cells were washed once with Ca2+-containing medium and incubated with or without 5µM thimerosal for 24 h. The absorbance of samples (A450) was determined using a scanning multiwell spectrophotometer. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and was expressed as a percentage of the control value. Experiments were repeated five times in six replicates (wells).
Assessment of MAPKs and caspase-3 activation by Western immunoblotting.
Assessment of the phosphorylation of MAPKs was accomplished by immunoblotting. Cells were seeded onto 60-mm culture dishes at a density of 3 x 106 cells/dish. After 2 h of incubation, the culture medium was replaced by serum-free medium supplemented with 1 mg/ml bovine serum albumin (USB, Cleveland, OH). Serum starvation was continued for 4 h, followed by the addition of various treatments. The treatments were terminated after indicated time intervals by aspirating the supernatant and washing the dishes with physiological saline. After washing, the cells were lysed on ice for 5 min with 70 µl of lysis buffer (20mM Tris, pH 7.5, 150mM NaCl, 1mM ethylenediaminetetraacetic acid, 1mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1mM ß-glycerophosphate, 1mM Na3VO4, 1 µg/ml leupeptin, and 1mM phenylmethylsulfonyl fluoride). The lysed cells were scraped off the dish using a rubber policeman, transferred to microcentrifuge tubes, and vortexed for 10 s. The cell lysates were then centrifuged to remove insoluble materials, and the protein concentration of each sample was measured by using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Approximately 50 µg of supernatant protein from each sample was used for gel electrophoresis analysis on a 10% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, the fractionated proteins on gel were transferred to polyvinylidene difluoride membranes (NEN Life2 Science Products, Inc., Boston, MA). For immunoblotting, the membranes were blocked with 5% nonfat milk in Tris-Buffered Saline Tween-20 (TBST) (25mM Tris, pH 7.5, 150mM NaCl, and 0.1% [v/v] Tween 20) and incubated overnight with the primary antibody (rabbit antihuman phospho-ERK antibody, rabbit antihuman ERK antibody, rabbit antihuman phospho-JNK antibody, rabbit antihuman JNK antibody, rabbit antihuman phospho-p38 MAPK antibody, rabbit antihuman p38 MAPK antibody, rabbit antihuman cleaved caspase-3, or rabbit anti-human ß tubulin; all from Cell Signaling Technology, Beverly, MA). Then the membranes were extensively washed with TBST and incubated for 60 min with the secondary antibody (goat anti-rabbit antibody; Transduction Laboratories, Lexington, KY). After extensive washing with TBST, the immune complexes were detected by chemiluminescence using the Renaissance Western Blot Chemiluminescence Reagent Plus kit (NEN Life Science Products). All the Western immunoblotting data were normalized intensities of the bands of cleaved caspase-3 against the bands of ß tubulin or the bands of phospho-MAPKs against the bands of MAPKs by using NIH image 1.61.
Measurements of subdiploidy nuclei by flow cytometry.
Cells were collected from the media, washed with ice-cold physiological saline twice, and resuspended in 3 ml of 70% ethanol. The ethanol-suspended cells were centrifuged for 5 min at 200 x g. Ethanol was decanted thoroughly, and the cell pellet was washed with ice-cold saline twice and was suspended in 1 ml PI solution (1% Triton X-100, 20 µg PI and 0.1 mg/ml RNAse). The cell pellet was incubated in the dark for 30 min at room temperature. Cell fluorescence was measured in the FACScan flow cytometer (Becton Dickinson immunocytometry systems, San Jose, CA). The MODFIT software was applied to analyze the data. Data of 10,000 cells were collected. Electronic gates were set for viable and apoptotic cells with 2–4 N DNA and subnormal DNA contents, respectively, and for exclusion of debris. The percentage of apoptosis was calculated as: (number of apoptotic cells/number of total cells) x 100.
Statistics.
Data are reported as means ± SEM of five experiments. Data were analyzed by two-way ANOVA using the Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC). Multiple comparisons between group means were performed by post hoc analysis using the Tukey’s honestly significant difference procedure. A p-value less than 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Effect of Thimerosal on the Survival of SCM1 Cells
To examine the cytotoxicity of thimerosal in gastric cancer cells, SCM1 cells were cultured in the presence of various concentrations of thimerosal and cell viability assays were performed. Figure 1A shows that while 0.5µM and 1µM thimerosal had no effect on viability, 5µM thimerosal significantly reduced WST-1 metabolism to 75 ± 2% as compared with control (p < 0.05; n = 5). The data further show that approximately 5% of cells survived the incubation with 10–100µM thimerosal.
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Efforts were made to explore the time-dependent effect of thimerosal on cell viability. Figure 1B shows that 5µM thimerosal induced cell death in a time-dependent manner. When cells were treated with 5µM thimerosal for 24 h, cell viability decreased to 74 ± 2% (p < 0.05; n = 5).
Induction of Apoptosis by Thimerosal in SCM1 Cells
To examine the characteristics of cell death observed in SCM1 cells, we explored whether the apoptotic features such as subdiploid peak and caspase-3 activation were induced by thimerosal. Furthermore, the proportion of cells that underwent apoptosis was analyzed by flow cytometry after cells were treated with various concentrations of thimerosal for 18 h. As shown in Figure 2A, the marked increase in subdiploidy nuclei appeared in cells treated with 5–100µM thimerosal (p < 0.05; n = 5). Caspase-3 is thought to function as an executioner of apoptosis and its activation could reflect the percentage of apoptotic cells. Caspase-3 activation was determined by immunoblotting, and it was found that the expression of active caspase-3 was increased in the presence of 5µM thimerosal (Figs. 2B and 2C). In Figure 2C, the effect of thimerosal on the activation of caspase-3 in SCM1 cells was time dependent.
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Involvement of MAPKs in Thimerosal-induced Apoptosis
Previous studies have shown that activation of MAPKs is related to apoptosis (Suh, 2002
; Wada and Penninger, 2004). MAPKs are activated by phosphorylation of specific tyrosine and threonine residues, and the relative levels of phosphorylated MAPKs in total MAPKs represent the degree of MAPK activation. Figures 3A and 3B show that the level of phosphorylated ERK1 (phospho-ERK1) increased between 1 and 5 min after thimerosal addition, but decreased at 30, 60 and 180 min. Exposure to thimerosal seemed to increase the intensity of phosphorylated JNK2 (phospho-JNK2) at 10, 15, 120, and 180 min (Figs. 3A and 3C), and the intensity of phosphorylated p38 MAPK (phospho-p38) was increased between 3 and 120 min (Figs. 3A and 3D).
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To further examine whether MAPKs activation was involved in thimerosal-induced cell death, PD98059 (an inhibitor of ERK), SP600125 (an inhibitor of JNK), and SB203580 (an inhibitor of p38 MAPK) were administered 2 h prior to the addition of 5µM thimerosal. WST-1 metabolism was measured 24 h after treatment. While PD98059 (10–50µM) and SP600125 (10–50µM) did not inhibit thimerosal-induced cell death (Figs. 4A and 4B), pretreatment with 20µM SB203580 rescued cells from thimerosal-induced cell death by 13 ± 1% (Fig. 4C; p < 0.05; n = 5). At concentrations of 5 or 10µM, SB1203580 had no protective effect. Furthermore, pretreatment with 20µM SB203580 inhibited thimerosal-induced phosphorylation of p38 MAPK by 8 ± 1 fold compared with treatment with thimerosal alone (Figs. 4D and 4E). To further understand whether MAPKs activation was also involved in thimerosal-induced apoptosis, SB203580 was administered 2 h prior to the addition of thimerosal. Figure 4F shows that a marked increase in subdiploidy nuclei was detected 18 h after thimerosal treatment. Pretreatment with SB203580 rescued cells from thimerosal-induced apoptosis by 15 ± 1% (p < 0.05; n = 5).
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Regulation of Caspase-3 Activation by p38 MAPK
Based on the above results, it appears that activation of p38 MAPK could regulate thimerosal-induced apoptosis of SCM1 cells. But the relationship between p38 MAPK activation and activation of caspase-3 was unclear. To understand whether activation of p38 MAPK could regulate caspase-3 activation, SB203580 was pretreated for 2 h and then thimerosal was added for 24 h. Figures 5A and 5B show that pretreatment with SB203580 (the band density was 20.9 ± 1.8 fold compared with control) partially reduced the activation of caspase-3 (by 29 ± 1 fold compared with treatment with thimerosal alone, which had a band density of 49.2 ± 2.4 fold compared with control; n = 5; p < 0.05).
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Effect of Thimerosal on [Ca2+]i
Because a [Ca2+]i increase plays an important role in apoptosis in various cell types (Saris and Carafoli, 2005
; Waring, 2005), efforts were made to explore whether thimerosal could alter [Ca2+]i in SCM1 cells. In Ca2+-containing medium, the baseline [Ca2+]i was 50 ± 2nM (n = 5). Addition of thimerosal caused an immediate increase in [Ca2+]i, which lasts for at least 220 s after the addition of thimerosal (Fig. 6A). Thimerosal at concentrations of 0.5–100µM increased [Ca2+]i in a concentration-dependent manner. Five micromolar of thimerosal-induced [Ca2+]i increase attained to 119 ± 2nM over baseline (n = 5), followed by a gradual decline and a plateau. To examine the contribution of extracellular Ca2+ and mobilization of Ca2+ from the intracellular stores in the thimerosal-induced Ca2+ signal, the effect of thimerosal on [Ca2+]i was measured in the absence of extracellular Ca2+ (Fig. 6B). The baseline was 50 ± 2nM (n = 5). Thimerosal (5–100µM) failed to cause a [Ca2+]i increase (n = 5). Figure 6B shows the result of application of 5µM thimerosal. Thus, it appears that thimerosal induced only Ca2+ influx.
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No Effect of Chelating Ca2+ with BAPTA on Thimerosal-induced Cell Death
Because thimerosal induced both cell death and a [Ca2+]i increase and that a Ca2+ signal is known to trigger cell death, the next question was to see whether the apoptosis was induced by a preceding increase in [Ca2+]i. Figure 7A shows that, in BAPTA-treated cells, 5µM thimerosal added at 30s barely induce a [Ca2+]i increase (trace b) as compared with control (trace a). This experiment demonstrated that chelation of cytosolic Ca2+ with BAPTA under our circumstances could inhibit most of thimerosal-induced [Ca2+]i increase. Thimerosal (5µM) was subsequently added for 24 h before WST-1 metabolism was measured. Figure 7B show that BAPTA pretreatment fails to rescue cells from thimerosal-induced cell death (n = 5; p > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Despite the toxic effect of thimerosal observed in humans and rat (Pfab et al., 1996
; Postius, 1984), no effort was made to explore the underlying molecular mechanisms. The major novel finding of the present study is that thimerosal causes apoptosis of human SCM1 gastric cancer cells in a Ca2+-independent manner. In other cell types, thimerosal has been shown to be apoptotic (Baskin et al., 2003; Herdman et al., 2006; Parran et al., 2005; Woo et al., 2006; Yel et al., 2005). Although caspase-independent apoptosis may exist, our data show that thimerosal induces caspase-dependent apoptosis in SCM1 cells.
To further investigate the mechanisms and the signaling transduction pathways in thimerosal-induced apoptosis of SCM1 cells, immunoblotting was used to explore the alteration of phosphorylation of three members of the MAPKs family (ERK, JNK, and p38 MAPK) because recent research suggests that phosphorylation of MAPKs plays an important role in the process of apoptosis (Cross et al., 2000; Nusuetrong et al., 2005). MAPKs are activated by many stimuli, and one of their major functions is to connect cell surface receptors to transcription factors in the nucleus, which consequently triggers long-term cellular responses (Bost et al., 2005). Although thimerosal altered the phosphorylation of ERK, JNK, and p38 MAPK in SCM1 cells, only the activation of p38 MAPK phosphorylation could trigger caspase-3 activation. In contrast, thimerosal was shown to induce apoptosis in a neuroblastoma cell model via the JNK pathway (Herdman et al., 2006). The involvement of p38 MAPK in apoptosis has been reported in other cells stimulated with different ligands. For instance, resveratrol was shown to induce apoptosis of human malignant B cells by activation of caspase-3 and p38 MAPK pathways (Shimizu et al., 2006). In endothelial cells, dimethylarginine induced apoptosis via similar pathways (Jiang et al., 2006). Triptolide was shown to evoke apoptosis of dendritic cells through sequential p38 MAPK phosphorylation and caspase-3 activation (Liu et al., 2004). We are the first to show that thimerosal could utilize p38 MAPK pathway to induce apoptosis.
Another important issue was whether thimerosal-induced apoptosis required a preceding rise in [Ca2+]i. Ca2+ plays a key role in both apoptotic and necrotic cell death. Emptying of intracellular Ca2+ stores and/or influx of extracellular Ca2+ can modulate cell death in many cell types (Saris and Carafoli, 2005; Waring, 2005). However, Ca2+-independent apoptosis could be found in some cell types such as thymic lymphoma cells (Matuszyk et al., 1998), neutrophils (Das et al., 1999), pancreatic islet cells (Barbosa et al., 2002), etc. It was found that even though thimerosal-induced [Ca2+]i increases by causing extracellular Ca2+ influx, it appears that thimerosal-induced apoptosis was via a Ca2+-independent pathway because suppression of [Ca2+]i increases by BAPTA failed to prevent apoptosis.
Together, we have demonstrated that in human SCM1 gastric cancer cells, thimerosal induced cell death via a Ca2+-independent, p38 MAPK-dependent, apoptotic pathway. Although thimerosal also altered ERK and JNK phosphorylation, those effects did not participate in this apoptotic pathway.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Grants Veterans General Hospital-Kaohsiung (VGHKS)-94G-11, VGHKS94-054, VGHKS95-037, and NSC94-2320-B-075B-006 to C.-R.J. and VGHKS94-026 and VGHKS95-019 to S.-I.L.
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