OR Oncology Reports 1021-335X 1791-2431 D.A. Spandidos 10.3892/or.2018.6913 or-41-02-1324 Articles Caspase-dependent apoptotic death by gadolinium chloride (GdCl3) via reactive oxygen species production and MAPK signaling in rat C6 glioma cells TsaiYuh-Feng 1 2 ChenYuh-Fung 3 HsiaoChen-Yu 1 HuangChing-Wen 1 LuChi-Cheng 4 TsaiShih-Chang 5 YangJai-Sing 6 Department of Diagnostic Radiology, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan, R.O.C. School of Medicine, Fu-Jen Catholic University, New Taipei 202, Taiwan, R.O.C. Department of Pharmacology, School of Medicine, China Medical University, Taichung 404, Taiwan, R.O.C. Department of Sport Performance, National Taiwan University of Sport, Taichung 404, Taiwan, R.O.C. Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan, R.O.C. Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan, R.O.C. Correspondence to: Dr Jai-Sing Yang, Department of Medical Research, China Medical University Hospital, China Medical University, 2 Yude Road, Taichung 40447, Taiwan, R.O.C., E-mail: jaisingyang@gmail.com Dr Shih-Chang Tsai, Department of Biological Science and Technology, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan, R.O.C., E-mail: sctsai@mail.cmu.edu.tw 022019 07122018 41 2 1324 1332 16072018 21112018 Copyright © 2019, Spandidos Publications 2019

Gadolinium (Gd) compounds serve as magnetic resonance imaging contrast agents and exert certain anticancer activities. Yet, the molecular signaling underlying the antitumor effect of Gd chloride (GdCl3) on glioma remains unclear. In the present study, we aimed to ascertain the apoptotic mechanisms of GdCl3 on rat glioma C6 cells. Our results demonstrated that GdCl3 significantly reduced cell viability and shrunk cell morphology of C6 cells in a concentration-dependent manner. GdCl3 led to apoptotic C6 cell death as detected by TUNEL staining. An increase in cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 occurred in GdCl3-treated C6 cells as detected by immunoblotting analysis. The activities of caspase-3, caspase-8 and caspase-9 were increased, and the specific inhibitors of caspase-3/-8/-9 individually reversed cell viability, which caused apoptotic death in C6 cells prior to GdCl3 exposure. GdCl3 also caused an elevation in the cytoplasmic Ca2+ level and reactive oxygen species (ROS) production, as well as the loss of mitochondrial membrane potential (ΔΨm) as shown by flow cytometric analysis in C6 cells. The results from the immunoblotting analysis demonstrated that there were upregulated protein levels of cytochrome c and Bax but a downregulated protein level of Bcl-2 in C6 cells after GdCl3 treatment. Additionally, GdCl3 decreased the protein levels of phosphorylated-extracellular signal-regulated kinases, phosphorylated-c-Jun N-terminal kinase and phosphorylated-p38 mitogen-activated protein kinases in C6 cells. In conclusion, ROS production and MAPKs signaling pathways contribute to GdCl3-induced caspase cascade-mediated apoptosis in C6 cells. Our findings provide a better understanding of the molecular mechanisms underlying the role of GdCl3 in rat glioma C6 cells.

 gadolinium chloride GdCl3 apoptosis reactive oxygen species production MAPK pathway caspase cascade Introduction

Glioma is characterized as a malignant brain tumor with a high recurrence rate, and surgery is utilized to remove the tumor (1,2). However, patients with glioma have a high mortality rate and an extremely poor prognosis (2). Currently, chemotherapy is the main method for prolonging survival or reducing symptoms (3,4). It is known that the existence of the blood-tumor barrier (BTB) in tumor tissue limits the efficacy and the delivery of therapeutic agents to brain tumor tissue (1,3). Therefore, the development of therapeutic agents to pass through the BTB is increasingly needed to effectively limit the rapid and invasive growth of malignant gliomas (5,6).

Lanthanide (Ln) ions have the chemical properties of both a high coordination number and a charge density, and they have been previously applied in agriculture and medicine (7–12). Gadolinium (Gd), a member of the Ln series, exerts a magneto-caloric effect (8). Gd compounds including Gd chloride (GdCl3) have been applied as magnetic resonance imaging (MRI) contrast agents and may be promising anticancer drugs (8–12). GdCl3 is widely used for in-activating tumor-associated macrophages (9,13). In addition, GdCl3 was found to inhibit cell proliferation and induce apoptosis in human hepatoma HepG2 cells through a mitochondria-dependent pathway (11). Recently, we reported that GdCl3 triggers apoptotic death in human osteosarcoma U-2 OS cells through the death receptor, mitochondria-dependent and ER stress pathways (8). The exact molecular mechanism of GdCl3 that underlie the inhibition of the viability of rat C6 glioma cells remains undefined. Therefore, the present study aimed to investigate the effect of GdCl3 on rat C6 glioma cells and the possible mechanism of GdCl3-induced apoptosis.

 Materials and methods <sec> <title>Chemicals and reagents

Fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, trypsin-EDTA, and Ham's Nutrient Mixture F12 medium were obtained from HyClone; GE Healthcare Life Sciences (Logan, UT, USA). GdCl3, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), and In Situ Cell Death Detection kit, Fluorescein were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Z-VAD-FMK (a pan-caspase inhibitor), Z-DEVD-FMK (a caspase-3 inhibitor), Z-IETD-FMK (a caspase-8 inhibitor), Z-LEHD-FMK (a caspase-9 inhibitor) and Caspase-3/-8/-9 Colorimetric Assay kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). All primary antibodies against caspase-3 (cat. no. GTX110543), caspase-8 (cat. no. GTX110723), caspase-9 (cat. no. GTX112888), cytochrome c (cat. no. GTX108585), Bax (cat. no. GTX109683), Bcl-2 (cat. no. GTX100064), phosphorylated-extracellular signal-regulated kinase (p-ERK) (cat no. GTX59568), ERK (cat no. GTX59618), phosphorylated-c-Jun N-terminal kinase (p-JNK) (cat no. GTX52326), JNK (cat no. GTX52360), phosphorylated-p38 (p-p38) (cat no. GTX24822), p38 (cat no. GTX110720), β-actin (cat. no. GTX109639), as well as anti-mouse (cat. no. GTX213111-01) and anti-rabbit (cat. no. GTX213110-01) immunoglobulin (Ig) G horseradish peroxidase (HRP)-linked secondary antibodies were purchased from GeneTex International Corporation (Hsinchu, Taiwan). Dihydrorhodamine 123, 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)] and Fluo-3/AM were sourced from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

 Cell culture

Rat glial cell tumor C6 cell line was obtained from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan) and cultured in 75-cm2 flasks with Ham's Nutrient Mixture F12 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Human fetal glial SVG p12 cell line was purchased from BCRC and cultured in minimum essential Eagle's medium containing 10% FBS, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were maintained in a humidified incubator at 37°C with 5% CO2.

 Cell viability assay and morphological observation

Cell viability was determined by a colorimetric MTT assay, as previously described (14). Briefly, C6 and SVG p12 cells (1×104 cells/100 µl) in a 96-well plate were incubated at 37°C with or without GdCl3 at a final concentration of 50, 100, 150 and 200 µM for 24 h. After incubation, the medium was removed, and the cells were mixed with 0.5 mg/ml MTT solution for 4 h. The insoluble formazan crystals were dissolved in 0.1 ml dimethyl sulfoxide (DMSO) for 30 min at 37°C. The absorbance was spectrophotometrically measured with an ELISA reader at a wavelength of 570 nm. The result was calculated as a ratio of sample absorbance to the control (vehicle) absorbance, considered as 100%. Additionally, the GdCl3-treated C6 cells were measured for apoptotic characteristics and photographed via a phase-contrast microscope, as previously described (14).

 DNA breaks for apoptosis

C6 cells (1×105 cells/ml) were exposed to 50, 100, and 150 µM of GdCl3 for 24 h. The cells were subsequently collected and stained with the In Situ Cell Death Detection kit, Fluorescein (Sigma-Aldrich; Merck KGaA), following the manufacturer's instructions. The terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL)-positive cells were detected by flow cytometry (BD FACSCalibur flow cytometer; BD Biosciences, Franklin Lakes, San Jose, CA, USA), as previously described (14).

 Comet assay and DAPI staining

C6 cells (2×105 cells/well) were treated with 0, 50, 100, 150 and 200 µM of GdCl3 for 24 h. Comet assay was applied according to the vendor's instructions and a previous study (15). Additionally, chromatin undergoes a phase change from loose to condensed during apoptosis. DAPI dye was used to observe condensed chromatin, as previously described (16).

 Immunoblotting analysis

C6 cells (5×106 cells/75T flask) were incubated with the presence and absence of 0, 50, 100 and 200 µM of GdCl3 for 24 h. After cells were lysed, total protein (40 µg) was separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P Transfer membrane; Merck Millipore, Darmstadt, Germany). As previously described (17,18), the membranes were probed with primary antibodies against caspase-3, caspase-8, caspase-9, cytochrome c, Bax, Bcl-2, p-ERK, ERK, p-JNK, JNK, p-p38, p38, (all 1:1,000 dilution) and β-actin (1:5,000 dilution). The membranes were then incubated with goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP linked antibodies (1:10,000 dilution) for 1 h at room temperature. All immunoblots were hybridized using an Enhanced Chemiluminescence system kit (Immobilon Western Chemiluminescent HRP substrate; Merck Millipore). The intensity of relative protein expression was normalized to β-actin signal and quantified with the NIH ImageJ program (version 1.47; National Institutes of Health, Bethesda, MD, USA).

 Assays for caspase-3/-8/-9 activities

C6 cells (5×106 cells/75T flask) were exposed to 0, 50, 100, and 200 µM of GdCl3 for 24 h. The cell lysates were then harvested and incubated with a commercial lysis buffer according to the instructions of the manufacturer (Caspase-3,-8 and −9 Colorimetric Assay kits; R&D Systems).

 Determinations of reactive oxygen species (ROS), the mitochondrial membrane potential (ΔΨm) and intracellular Ca<sup>2+</sup> levels via flow cytometry

C6 cells (2×105 cells/well) were incubated with or without 50, 100, 150 and 200 µM of GdCl3 for 6 h. After treatment, the medium was replaced with 5 µM dihydrorhodamine 123 (an ROS indicator), 4 nM DiOC6(3) (a ΔΨm fluorescent probe) and 3 µg/ml Fluo-3/AM (cytoplasmic Ca2+ dye), respectively, and the plates were protected from light for 30 min at 37°C. The mean fluorescence intensity (MFI) was measured via flow cytometry (Muse Cell Analyzer; Merck Millipore) and expressed as % of the control (vehicle), as previously described (14,19).

 Statistical analysis

All data are represented as the mean ± standard deviation (SD) from at least three independent experiments. Statistical calculations of the data were carried out via one-way analysis of variance (ANOVA) followed by Dunnett's test using SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>GdCl<sub>3</sub> inhibits viability of rat C6 glioma cells

The MTT assay was used to evaluate the impact of GdCl3 on C6 cells. The cells were treated with GdCl3 at the different concentrations (0, 50, 100, 150, and 200 µM) for 24 h. The cell viability was significantly decreased with the treatment of >100 µM GdCl3 in a concentration-dependent manner (Fig. 1A). The half-maximal inhibitory concentration (IC50) of GdCl3 for C6 cells was 152.36±2.45 µM. GdCl3 at the concentration of 150 µM markedly caused apoptotic cell shrinkage and the morphological changes, but the untreated control was well spread in C6 cells (Fig. 1B). Our results suggested that GdCl3 possessed a cytotoxic effect on C6 cells. However, no effect of viability (Fig. 1C) and morphological change (data not shown) was observed on normal SVG p12 cells after GdCl3 treatment (0, 50, 100, 150, and 200 µM). Therefore, GdCl3 may exhibit lower toxicity in normal glial SVG p12 cells.

 GdCl<sub>3</sub> induces apoptosis in C6 cells

To explore whether GdCl3 induces C6 cell death through an apoptotic mechanism, a TUNEL assay was used to detect DNA breaks in apoptotic cells. The percentage of TUNEL-positive cells (cells undergoing apoptosis) was markedly increased in GdCl3-treated C6 cells compared to that noted in the control cells, and the effect was concentration-dependent (Fig. 2A). We also measured DNA damage using comet assay. GdCl3 triggered DNA damage through the production of a longer comet tail (Fig. 2B, upper panel) after DNA was stained with PI in C6 cells. Moreover, chromatin and apoptotic DNA condensation was observed in C6 cells after GdCl3 exposure using DAPI staining (Fig. 2B, bottom panel). Our findings indicate that GdCl3 provoked an apoptotic response in C6 cells.

 GdCl3 elevates the activities of caspase-3, caspase-8 and caspase-9 in C6 cells

To characterize the mechanism of GdCl3-induced apoptosis, we explored the expression levels of caspase-3, caspase-8 and caspase-9 using immunoblotting analysis. GdCl3 increased cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 levels in C6 cells (Fig. 3A). We also examined the activities of caspase-3, caspase-8 and caspase-9 in GdCl3-treated C6 cells. The activities of caspase-3 (Fig. 3B), caspase-8 (Fig. 3C) and caspase-9 (Fig. 3D) were elevated after GdCl3 challenge, and the effects were concentration-dependent. Furthermore, the cells were pre-treated with a pan-caspase inhibitor (Z-VAD-FMK) (Fig. 4A), and selective inhibitors to caspase-3, caspase-8 and caspase-9 (Fig. 4B) to confirm the specificities of caspases. Data showed that specific inhibitors to caspase-3 (Z-DEVD-FMK), caspase-8 (Z-IETD-FMK) and caspase-9 (Z-LEHD-FMK) significantly prevented the GdCl3-reduced cell viability (Fig. 4B). These data suggest that the caspase cascade-mediated the signaling results in GdCl3-induced apoptosis in C6 cells.

 GdCl<sub>3</sub> enhances the ROS production and loss of ΔΨm level in C6 cells

To detect the effect of GdCl3 on ROS production in C6 cells, we measured intracellular ROS levels by dihydrorhodamine 123 fluorescent dye, and the stained cells were analyzed by flow cytometry. Intracellular ROS levels were concentration-dependently increased in C6 cells following GdCl3 exposure (Fig. 5A). To further test the effect of GdCl3 on ΔΨm levels, we used the specific fluorescent probe DiOC6(3) to detect the levels of ΔΨm. GdCl3 disrupted the ΔΨm level in C6 cells, and this impact was in a concentration-dependent manner (Fig. 5B). These results indicate that GdCl3 triggered apoptosis via ROS production and mitochondrial dysfunction in C6 cells.

 GdCl<sub>3</sub> prompts cytoplasmic Ca<sup>2+</sup> level in C6 cells

To further elucidate whether the Ca2+ level is involved in GdCl3-induced apoptosis and the possible signaling in C6 cells, we detected intracellular Ca2+ levels by flow cytometric analysis. The cells were treated with 50, 100, and 150 µM of GdCl3 for 24 h. GdCl3 significantly increased the intracellular Ca2+ level (Fig. 5C). These results revealed that cytoplasmic Ca2+ signaling and ER stress-mediated pathway may contribute to GdCl3-induced apoptotic machinery in C6 cells.

 GdCl<sub>3</sub> alters the levels of apoptosis- and mitogen-activated protein kinase (MAPK)-related protein molecules in C6 cells

To understand the molecular mechanisms of the apoptotic pathway, we further explored the levels of apoptosis-related protein signals (cytochrome c, Bax and Bcl-2) using immunoblotting analysis. The levels of cytochrome c and Bax were increased. By contrast, the level of anti-apoptotic protein Bcl-2 was decreased in GdCl3-treated cells (Fig. 5D). In addition, we also investigated the levels of MAPK-related pathway proteins (p-ERK, ERK, p-JNK, JNK, p-p38 MAPK and p38 MAPK) also using immunoblotting analysis. GdCl3 downregulated the levels of the p-ERK, p-JNK and p-p38 MAPK pathway in C6 cells (Fig. 6). These data demonstrated that GdCl3-induced apoptotic cell death may result from mitochondria-dependent and MAPK-mediated signaling in C6 cells.

 Discussion

Malignant glioma is an invasive intracranial tumor that grows rapidly and has extremely high recurrence (1,20). At present, postoperative chemotherapy has become a powerful strategy for the treatment of adult glioma patients (1,2,20). However, treatment has been particularly inefficient for glioma patients administered chemotherapeutic agents due to the existence of the BTB. The permeability of the BTB and blood brain barrier (BBB) impedes the accumulation and uptake rate of potential drugs to brain tumor tissues (1,2,20). Liu et al (21) showed that the efficacy of killing brain tumor cells increased by 10-fold when the therapeutic agent was permeabilized into the area of the brain tumor tissue. Hopefully, a therapeutic agent can be found that can pass through the BTB and BBB, and with that the efficacy of chemotherapy in treating glioma would be markedly improved. Gd-based contrast agents (GBCAs) are applied as pharmaceuticals and have been approved for 30 years and are used daily in millions of patients worldwide (22). In recent years, numerous retrospective clinical studies have reported the unexpected long-term presence of Gd in the brain after receiving radiology practice (22,23). Previous studies have reported that the efficacy of chemotherapy in treating glioma would be markedly improved if the therapeutic agent can be found that can pass through the BTB and BBB (23). These results suggested that GdCl3 can accumulate and be uptaken by tumors in the brain through favorable permeability of the BTB and BBB. In addition, Gd compounds possess diverse anticancer activities (8,11). Previous studies have shown that after treatment with a relatively low concentration of GdCl3, cell cycle progression and cell growth were promoted, whereas human hepatoma HepG2 and osteosarcoma U-2 OS cells exposed to high concentrations of GdCl3 exhibited apoptosis and suppressed cell proliferation (8,11). It is suggested that GdCl3-induced apoptosis is specific to HepG2 cells (11), U-2 OS cells (8) and rat C6 glioma cells. In this study, we demonstrated that GdCl3 caused anti-proliferative effects on rat C6 glioma cells in a concentration-dependent manner (Fig. 1A). Our data demonstrated that the half-maximal inhibitory concentration (IC50) values for a 24-h treatment of GdCl3 in U-2 OS and rat C6 glioma cells were 198.26±1.69 and 152.36±2.45 µM, respectively. In addition, Shen et al (24) and Fu et al (25) demonstrated that Gd promoted cell proliferation in mouse embryo fibroblast NIH3T3 cells. Ferreira et al (26) indicated that no effect was noted in regards to the viability of Kupffer cells after GdCl3 exposure. Our data also revealed that there was no viability impact (Fig. 1C) and morphological change (data not shown) on GdCl3-treated normal SVG p12 cells, suggesting that GdCl3 has an extremely low toxicity in normal glial cells.

Apoptosis (type I programmed cell death) is a vital mechanism in antitumor drugs and cancer therapies (27,28). It is a promising approach to induce apoptosis in glioma. Induction of tumor cell apoptosis is one of the best strategies for treating glioma and multiple types of cancers (27,28). Apoptotic death can be activated by three main pathways (17,29). The extrinsic pathway is triggered through binding of extrinsic signals to death receptors, which leads to activation of caspase-8 (29,30). The mitochondrial (intrinsic signaling) pathway is activated upon cellular stresses such as ROS production and ΔΨm disruption, and this results in activation of caspase-9. Both the extrinsic and intrinsic pathways can activate caspase-3 signaling and the major executioner caspase cascade (16,29,31). Therefore, induction of apoptotic pathways by a novel agent is a potentially powerful approach to fighting cancer cells (32,33).

Our results indicate that a high concentration of GdCl3 caused the apoptosis of C6 cells (Fig. 1). This is the first study to report that GdCl3 could be successfully applied to promote cell death in rat C6 glioma cells. In the present study, an increase of DNA damage and fragmentation of cells (an apoptotic characteristic) was demonstrated after exposure to GdCl3 (Fig. 2). Previous studies have shown that Gd3+ caused distinct effects depending on the type of target cells (8,11). GdCl3 was found to exert a proliferation-promoting ability and to activatee ERK and phosphoinositide 3-kinase (PI3K) signaling pathways in NIH 3T3 cells (24). Gd triggered cell apoptosis through the mitochondrial pathway in human osteosarcoma U-2 OS cells (8). Therefore, we sought to investigate the exact effect of GdCl3 on cell proliferation and cell apoptosis in rat C6 glioma cells.

Apoptosis can be induced though activation of a cascade of caspases (29,31). Ye et al (11) showed that GdCl3 triggered HepG2 cell apoptosis through death receptor-dependent and mitochondrial pathways. Our findings previously reported that GdCl3-induced apoptosis may be mediated through the extrinsic pathway, the intrinsic pathway, and the ER stress pathway in human osteosarcoma U-2 OS cells (8). Caspase-3 (a 35-kDa protein), a critical executioner of apoptosis, is either partially or totally responsible for the proteolytic cleavage by nuclear enzyme poly (ADP-ribose) polymerase (PARP) (34). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 fragments. Caspase-8 is a 55-kDa protein and is an inactive pro-enzyme. Activation of caspase-8 involves a two-step proteolysis: i) the cleavage of caspase-8 to generate a 43- and a 12-kDa fragment which is further processed to 10 kDa; and ii) p43 is then cleaved to yield p26 and the release of the active site containing p18 (35–37). Caspase-9 is an important family protein in the intrinsic apoptotic pathway (38). Upon apoptotic stimulation, cytochrome c released from mitochondria associates with the 47-kDa procaspase-9/Apaf-1. Apaf-1-mediated activation of caspase-9 involves intrinsic proteolytic processing resulting in cleavage on Asp315 and producing a p35 subunit. Another cleavage occurs on Asp330 producing a p37 subunit that can serve to amplify the apoptotic response (39–41). In this study, GdCl3 increased the enzymatic activities of caspase-3, caspase-8, and caspase-9, as well as cleaved-caspase-3 (p19), cleaved-caspase-8 (p18), and cleaved-caspase-9 (p35) protein levels in vitro (Fig. 3). Our results suggested that GdCl3-triggered apoptosis of C6 cells resulted from caspase-dependent signaling. Furthermore, GdCl3 elevated the ROS production and decreased the levels of ΔΨm (Fig. 5). The expression level of the anti-apoptotic protein Bcl-2 was decreased, while the expression levels of cytochrome c and pro-apoptotic protein Bax were increased after GdCl3 exposure in C6 cells (Fig. 5D). Our results found that GdCl3-triggered apoptosis of C6 cells was mitochondria-dependent.

The influx of Ca2+ is involved in several biological functions including cell proliferation, apoptosis and differentiation (42,43). Ca2+ rapidly flows into the cytoplasm and into the mitochondria, leading to cell apoptosis (29,31). Xia et al (44) demonstrated that Gd caused oxidative stress in rat cortical neurons. Feng et al (45) indicated that Gd triggered ER stress and unfolded protein responses in primary cultured rat cortical astrocytes through an increase in the influx of extracellular Ca2+ level. However, no report regarding GdCl3-induced ER stress in rat C6 glioma cells has been conducted. The present study revealed that GdCl3 caused the release of Ca2+, which led to apoptosis in C6 cells (Fig. 5C). Thus, our results suggest that GdCl3-induced cell death may be mediated via the apoptotic mechanism in C6 cells.

MAPKs are divided into three main subfamilies: ERK, JNK, and p38 MAPKs. Increasing evidence has shown that MAPKs play crucial roles and exhibit cell functions in cell survival, cell proliferation, cell cycle regulation, and apoptotic death (46,47). ERK is involved in cell survival, and JNK and p38 MAPK are thought to mainly promote cell apoptosis (48,49). Activation of ERK1/2 has been demonstrated to inhibit apoptosis in response to tumor necrosis factor (TNF), Fas ligand, radiation, stress, hypoxia, and chemotherapeutic agent stimulation (50). Subramanian et al (51) suggested that estrogen-induced increase in Ca2+ leads to ERK phosphorylation and consequently CREB phosphorylation, resulting in an increase in the anti-apoptotic Bcl-2 protein level. The inactivation of pro-apoptotic Bcl-2 family member BAD is mediated through phosphorylation on Ser112 by ERK activated p90 ribosomal S6 kinase (RSK). Inhibition of JNK2 activity can also suppress tumorigenesis by promoting apoptosis (52). Yu et al (53) demonstrated that one of the molecular mechanisms by which JNK suppresses apoptosis is through phosphorylation of BAD on Thr201, thereby inhibiting its pro-apoptotic activity. p38 plays a role in cell survival supported by increased levels of Bcl-2 and Bcl-xL in response to DNA damage and stress (54,55). Furthermore, the chemical inhibition of p38 has been strongly associated with increased chemosensitivity in tumor cells (56,57). Intriguingly, in this study, the expression of p-ERK, p-JNK, and p-p38 MAPKs were downregulated after GdCl3 exposure (Fig. 6). MAPKs were crucial for GdCl3-induced apoptosis in C6 cells. We suggest that the phosphorylation of MAPKs was involved in Bcl-2 modulation in GdCl3-induced apoptosis of C6 cells. Wang et al (58) demonstrated that GdCl3 inhibits PC3 cell migration by the inactivation of both ERK and p38 MAPK pathways. Our results are in accordance with that study (58), which indicated that GdCl3 suppressed ERK and p38 MAPKs and triggered apoptosis in C6 glioma cells.

In conclusion, GdCl3 provoked apoptosis in C6 cells through upregulation of cytochrome c and Bax, downregulation of Bcl-2, and activation of caspase-3, caspase-8, and caspase-9 signaling. GdCl3 triggered C6 cell apoptosis via mitochondria-dependent pathway. GdCl3 may be a promising therapy for human glioma and an adjunct to other chemotherapies. Our findings provide a new molecular mechanism underlying the action of GdCl3 for the chemotherapy of glioma.

 Acknowledgements

Not applicable.

 Funding

The present study was supported by the Shin-Kong Wu Ho-Su Memorial Hospital (Taipei, Taiwan (grant no. SKH-8302-106-DR-26) and in part by China Medical University Hospital (Taichung, Taiwan) (grant no. DMR-107-137).

 Availability of data and materials

The datasets generated during the study are available from the corresponding author on reasonable request.

 Authors' contributions

YFT, SCT and JSY conceived and designed the experiments. YFC, CYH, CWH and CCL performed the experiments. CCL, SCT and JSY analyzed the data. YFT, SCT and JSY wrote and modified the study. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the study are appropriately investigated and resolved.

 Ethics approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References MirandaABlanco-PrietoMSousaJPaisAVitorinoCBreaching barriers in glioblastoma. Part I: Molecular pathways and novel treatment approachesInt J Pharm531372388201710.1016/j.ijpharm.2017.07.05628755993 PerryAWesselingPHistologic classification of gliomasHandb Clin Neurol1347195201610.1016/B978-0-12-802997-8.00005-026948349 SanaiNBergerMSSurgical oncology for gliomas: The state of the artNat Rev Clin Oncol15112125201810.1038/nrclinonc.2017.17129158591 NamJYde GrootJFTreatment of GlioblastomaJ Oncol Pract13629638201710.1200/JOP.2017.02553629020535 DréanAGoldwirtLVerreaultMCanneyMSchmittCGuehennecJDelattreJYCarpentierAIdbaihABlood-brain barrier, cytotoxic chemotherapies and glioblastomaExpert Rev Neurother1612851300201610.1080/14737175.2016.120276127310463 ZhanCLuWThe blood-brain/tumor barriers: Challenges and chances for malignant gliomas targeted drug deliveryCurr Pharm Biotechnol1323802387201210.2174/13892011280334179823016643 ZhaoYYYangRXiaoMGuanMJZhaoNZengTKupffer cells activation promoted binge drinking-induced fatty liver by activating lipolysis in white adipose tissuesToxicology3905360201710.1016/j.tox.2017.09.00128882574 TsaiYFHuangCWChiangJHTsaiFJHsuYMLuCCHsiaoCYYangJSGadolinium chloride elicits apoptosis in human osteosarcoma U-2 OS cells through extrinsic signaling, intrinsic pathway and endoplasmic reticulum stressOncol Rep3634213426201610.3892/or.2016.517427748868 WuYWangYLiMYangXGongJZhangWGadolinium chloride suppresses acute rejection and induces tolerance following rat liver transplantation by inhibiting Kupffer-cell activationExp Ther Med817771782201410.3892/etm.2014.2015253717324217766 HouCCFengMWangKYangXGLanthanides inhibit adipogenesis with promotion of cell proliferation in 3T3-L1 preadipocytesMetallomics5715722201310.1039/c3mt00020f23612852 YeLShiZLiuHYangXWangKGdCl3 induced Hep G2 cell death through mitochondrial and external death pathways without significant elevation of ROS generationBiol Trace Elem Res151148155201310.1007/s12011-012-9538-y23129526 YanLZhuTBWangLSPanSYTaoZXYangZCaoKHuangJInhibitory effect of hepatocyte growth factor on cardiomyocytes apoptosis is partly related to reduced calcium sensing receptor expression during a model of simulated ischemia/reperfusionMol Biol Rep3826952701201110.1007/s11033-010-0412-821088907 YangKDuCChengYLiYGongJLiuZAugmenter of liver regeneration promotes hepatic regeneration depending on the integrity of Kupffer cell in rat small-for-size liver transplantationJ Surg Res183922928201310.1016/j.jss.2013.02.03323522454 LuCCYangJSChiangJHHourMJLinKLLeeTHChungJGCell death caused by quinazolinone HMJ-38 challenge in oral carcinoma CAL 27 cells: dissections of endoplasmic reticulum stress, mitochondrial dysfunction and tumor xenograftsBiochim Biophys Acta184023102320201410.1016/j.bbagen.2014.02.02224594224 ChiangJHYangJSLuCCHourMJLiuKCLinJHLeeTHChungJGEffect of DNA damage response by quinazolinone analogue HMJ-38 on human umbilical vein endothelial cells: evidence for γH2A.X and DNA-PK-dependent pathwayHum Exp Toxicol33590601201410.1177/096032711350479124064905 ChangCHLeeCYLuCCTsaiFJHsuYMTsaoJWJuanYNChiuHYYangJSWangCCResveratrol-induced autophagy and apoptosis in cisplatin-resistant human oral cancer CAR cells: A key role of AMPK and Akt/mTOR signalingInt J Oncol50873882201710.3892/ijo.2017.386628197628 ChangHPLuCCChiangJHTsaiFJJuanYNTsaoJWChiuHYYangJSPterostilbene modulates the suppression of multidrug resistance protein 1 and triggers autophagic and apoptotic mechanisms in cisplatin-resistant human oral cancer CAR cells via AKT signalingInt J OncolMar22018(Epub ahead of print)10.3892/ijo.2018.42985873834 ChiuYJHourMJJinYALuCCTsaiFJChenTLMaHJuanYNYangJSDisruption of IGF-1R signaling by a novel quinazoline derivative, HMJ-30, inhibits invasiveness and reverses epithelial-mesenchymal transition in osteosarcoma U-2 OS cellsInt J OncolMar162018(Epub ahead of print) LuCCYangJSHuangACHsiaTCChouSTKuoCLLuHFLeeTHWoodWGChungJGChrysophanol induces necrosis through the production of ROS and alteration of ATP levels in J5 human liver cancer cellsMol Nutr Food Res54967976201010.1002/mnfr.200900265201695803031088 JensenRLBrain tumor hypoxia: Tumorigenesis, angiogenesis, imaging, pseudoprogression, and as a therapeutic targetJ Neurooncol92317335200910.1007/s11060-009-9827-219357959 LiuYHashizumeKSamotoKSugitaMNingarajNAsotraKBlackKLRepeated, short-term ischemia augments bradykinin-mediated opening of the blood-tumor barrier in rats with RG2 gliomaNeurol Res23631640200110.1179/01616410110119892911547933 RobertPFrenzelTFactorCJostGRasschaertMSchuetzGFretellierNBoykenJIdéeJMPietschHMethodological aspects for preclinical evaluation of gadolinium presence in brain tissue: critical appraisal and suggestions for harmonization-a joint initiativeInvest Radiol53499517201810.1097/RLI.0000000000000467296593816092104 FitzgeraldRTAgarwalVHoangJKGaillardFDixonAKanalEThe impact of gadolinium deposition on radiology practice: an international survey of radiologistsCurr Probl Diagn RadiolS0363-0188(17)30296-7201810.1067/j.cpradiol.2018.02.00329530452 ShenLYangAYaoPSunXChenCMoCShiLChenYLiuQGadolinium promoted proliferation in mouse embryo fibroblast NIH3T3 cells through Rac and PI3K/Akt signaling pathwaysBiometals27753762201410.1007/s10534-014-9769-925037060 FuLJLiJXYangXGWangKGadolinium-promoted cell cycle progression with enhanced S-phase entry via activation of both ERK and PI3K signaling pathways in NIH 3T3 cellsJ Biol Inorg Chem14219227200910.1007/s00775-008-0442-z18953580 FerreiraJTapiaGVidelaLAEffects of the Kupffer cell inactivator gadolinium chloride on rat liver oxygen uptake and content of mitochondrial cytochromesFEBS Lett426263265199810.1016/S0014-5793(98)00356-19599021 BöglerOWellerMApoptosis in gliomas, and its role in their current and future treatmentFront Biosci7e339e353200210.2741/bogler12133814 SteinbachJPWellerMApoptosis in gliomas: Molecular mechanisms and therapeutic implicationsJ Neurooncol70245254200410.1007/s11060-004-2753-415674481 YangJSLuCCKuoSCHsuYMTsaiSCChenSYChenYTLinYJHuangYCChenCJAutophagy and its link to type II diabetes mellitusBiomedicine (Taipei)78201710.1051/bmdcn/2017070201286127065479440 YuanCHHorngCTLeeCFChiangNNTsaiFJLuCCChiangJHHsuYMYangJSChenFAEpigallocatechin gallate sensitizes cisplatin-resistant oral cancer CAR cell apoptosis and autophagy through stimulating AKT/STAT3 pathway and suppressing multidrug resistance 1 signalingEnviron Toxicol32845855201710.1002/tox.2228427200496 PfefferCMSinghATKApoptosis: A Target for anticancer therapyInt J Mol Sci19192018 LinCTsaiSCTsengMTPengSFKuoSCLinMWHsuYMLeeMRAmagayaSHuangWWAKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cellsInt J Oncol429931000201310.3892/ijo.2013.179123354080 TsaiSCYangJSPengSFLuCCChiangJHChungJGLinMWLinJKAmagayaSWai-Shan ChungCBufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cellsInt J Oncol4114311442201210.3892/ijo.2012.157922858649 Fernandes-AlnemriTLitwackGAlnemriESCPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzymeJ Biol Chem269307613076419947983002 MuzioMChinnaiyanAMKischkelFCO'RourkeKShevchenkoANiJScaffidiCBretzJDZhangMGentzRFLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complexCell85817827199610.1016/S0092-8674(00)81266-08681377 BoldinMPGoncharovTMGoltsevYVWallachDInvolvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell deathCell85803815199610.1016/S0092-8674(00)81265-98681376 Fernandes-AlnemriTArmstrongRCKrebsJSrinivasulaSMWangLBullrichFFritzLCTrapaniJATomaselliKJLitwackGIn vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domainsProc Natl Acad Sci USA9374647469199610.1073/pnas.93.15.74648755496 DuanHOrthKChinnaiyanAMPoirierGGFroelichCJHeWWDixitVMICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme BJ Biol Chem2711672016724199610.1074/jbc.271.28.167208663294 LiuXKimCNYangJJemmersonRWangXInduction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome cCell86147157199610.1016/S0092-8674(00)80085-98689682 LiPNijhawanDBudihardjoISrinivasulaSMAhmadMAlnemriESWangXCytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascadeCell91479489199710.1016/S0092-8674(00)80434-19390557 ZouHLiYLiuXWangXAn APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9J Biol Chem2741154911556199910.1074/jbc.274.17.1154910206961 BhatTAChaudharyAKKumarSO'MalleyJInigoJRKumarRYadavNChandraDEndoplasmic reticulum-mediated unfolded protein response and mitochondrial apoptosis in cancerBiochim Biophys Acta Rev Cancer18675866201710.1016/j.bbcan.2016.12.00227988298 BaharEKimHYoonHER Stress-mediated signaling: action potential and Ca (2+) as key playersInt J Mol Sci1717201610.3390/ijms17091558 XiaQFengXHuangHDuLYangXWangKGadolinium-induced oxidative stress triggers endoplasmic reticulum stress in rat cortical neuronsJ Neurochem1173847201110.1111/j.1471-4159.2010.07162.x21198628 FengXXiaQYuanLYangXWangKImpaired mitochondrial function and oxidative stress in rat cortical neurons: implications for gadolinium-induced neurotoxicityNeurotoxicology31391398201010.1016/j.neuro.2010.04.00320398695 EblenSTExtracellular-regulated kinases: signaling from Ras to ERK substrates to control biological outcomesAdv Cancer Res13899142201810.1016/bs.acr.2018.02.004295511316007982 FaghfuriENikfarSNiazKFaramarziMAAbdollahiMMitogen-activated protein kinase (MEK) inhibitors to treat melanoma alone or in combination with other kinase inhibitorsExpert Opin Drug Metab Toxicol14317330201810.1080/17425255.2018.143259329363351 GkouverisINikitakisNGRole of JNK signaling in oral cancer: A mini reviewTumour Biol391010428317711659201710.1177/101042831771165928639904 PelusoIYarlaNSAmbraRPastoreGPerryGMAPK signalling pathway in cancers: Olive products as cancer preventive and therapeutic agentsSemin Cancer BiolSep112017(Epub ahead of print)10.1016/j.semcancer.2017.09.00228912082 LuZXuSERK1/2 MAP kinases in cell survival and apoptosisIUBMB Life58621631200610.1080/1521654060095743817085381 SubramanianMShahaCUp-regulation of Bcl-2 through ERK phosphorylation is associated with human macrophage survival in an estrogen microenvironmentJ Immunol17923302338200710.4049/jimmunol.179.4.233017675494 PotapovaOAnisimovSVGorospeMDoughertyRHGaardeWABohelerKRHolbrookNJTargets of c-Jun NH (2)-terminal kinase 2-mediated tumor growth regulation revealed by serial analysis of gene expressionCancer Res6232573263200212036942 YuCMinemotoYZhangJLiuJTangFBuiTNXiangJLinAJNK suppresses apoptosis via phosphorylation of the proapoptotic Bcl-2 family protein BADMol Cell13329340200410.1016/S1097-2765(04)00028-014967141 FlackeJPKumarSKostinSReuschHPLadilovYAcidic preconditioning protects endothelial cells against apoptosis through p38- and Akt-dependent Bcl-xL overexpressionApoptosis149096200910.1007/s10495-008-0287-519082728 KimMJChoiSYParkICHwangSGKimCChoiYHKimHLeeKHLeeSJOpposing roles of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase in the cellular response to ionizing radiation in human cervical cancer cellsMol Cancer Res617181731200810.1158/1541-7786.MCR-08-003219010820 HamanoueMSatoKTakamatsuKInhibition of p38 mitogen-activated protein kinase-induced apoptosis in cultured mature oligodendrocytes using SB202190 and SB203580Neurochem Int511624200710.1016/j.neuint.2007.03.00517459526 LimSJLeeYJLeeEp38MAPK inhibitor SB203580 sensitizes human SNU-C4 colon cancer cells to exisulind-induced apoptosisOncol Rep1611311135200617016604 WangPZouXMHuangJZhangTLWangKGadolinium inhibits prostate cancer PC3 cell migration and suppresses osteoclast differentiation in vitroCell Biol Int3511591167201110.1042/CBI2010087021605080

Effects of GdCl3 on the cell viability of rat glioma C6 and human normal fetal glial SVG p12 cells. (A) Viability of C6 cells treated with GdCl3 at the indicated concentrations for 24 h via an MTT assay. Data are expressed as the mean ± SD (n=3). *P<0.05 compared to the control. (B) Morphological changes of C6 cells treated with GdCl3 at 50, 100, and 150 µM of GdCl3 for 24 h. Images were taken from one of three independent experiments; (magnification, ×200). (C) SVG p12 cells were exposed to GdCl3 (0, 50, 100, 150, and 200 µM) for 24 h. Cell viability was determined via an MTT method. GdCl3, gadolinium chloride; SD, standard deviation.

Effects of GdCl3 on the apoptosis of rat glioma C6 cells. (A) Cells were treated with GdCl3 at indicated concentrations for 24 h. Apoptotic cells were determined by TUNEL assay kit, as described in Materials and methods. Data are presented as the mean ± SD (n=3). *P<0.05 compared to the control. (B) Fluorescence staining of apoptotic cells in GdCl3-treated cells were observed. PI was used to stain the nuclei via comet assay (upper panel), and the yellow arrows indicate the nuclei and DNA damage of apoptotic cells. DAPI dye was applied to probe the nuclei condensation (bottom panel); ×400 magnification. Representative images are from one of three independent experiments. GdCl3, gadolinium chloride; PI, propidium iodide.

Effects of GdCl3 on caspase cascade protein expression and activities in rat glioma C6 cells. Cells were treated with 50, 100, and 150 µM of GdCl3 for 24 h. The cell lysates were harvested to assess (A) the expression levels of caspase-3, caspase-8, and caspase-9 using immunoblotting analysis. β-actin was used as a control to ensure an equal loading amount. Representative chemiluminescent images were collected from one of independent duplicate experiments. The activities of (B) caspase-3, (C) caspase-8, and (D) caspase-9 were monitored, as described in Materials and methods. Data are presented as the mean ± SD (n=3). *P<0.05 compared to the control. GdCl3, gadolinium chloride; SD, standard deviation.

Effects of caspase inhibitors to reverse GdCl3-reduced viability of rat glioma C6 cells. Cells were treated with 150 µM GdCl3 for 24 h after being pre-incubated with or without (A) 15 µM Z-VAD-FMK (a pan-caspase inhibitor) and (B) 15 µM Z-DEVD-FMK (a caspase-3 inhibitor), Z-IETD-FMK (a caspase-8 inhibitor), and Z-LEHD-FMK (a caspase-9 inhibitor) for 2 h, respectively. Cell viability was determined by the MTT assay. Data are expressed as the mean ± SD (n=3). *P<0.05 compared to the control. GdCl3, gadolinium chloride; SD, standard deviation.

Effects of GdCl3 on ROS production, ΔΨm, and cytoplasmic Ca2+ level of rat glioma C6 cells. Cells were treated with 50, 100, and 150 µM of GdCl3 for 24 h. Cells were then mixed with (A) 5 µM dihydrorhodamine 123 for detecting ROS levels, (B) 4 nM fluorochrome DiOC6(3) for detecting the disruption of ΔΨm, and (C) 3 µg/ml Fluo-3/AM for detecting intracellular Ca2+ levels. MFI was measured by flow cytometry. Data are expressed as the mean ± SD (n=3). *P<0.05 compared to the control. (D) Immunoblotting analysis against cytochrome c, Bax, and Bcl-2 signals after GdCl3 challenge for 24 h. β-actin was used as a loading control. Representative chemiluminescent images were collected from one of independent duplicate experiments. ROS, reactive oxygen species; GdCl3, gadolinium chloride; ΔΨm, mitochondrial membrane potential; MFI, mean fluorescence intensity; SD, standard deviation.

Effect of GdCl3 on the levels of phosphorylation of MAPK signaling molecules in rat glioma C6 cells. Cells were exposed to 0, 50, 100, and 150 µM of GdCl3 for 24 h. Cell fractions were extracted and subjected to immunoblotting analysis using the specific antibodies (p-ERK, ERK, p-JNK, JNK, p-p38 MAPK, and p38 MAPK). β-Actin was used as a loading control. Representative chemiluminescent images were collected from one of independent duplicate experiments. GdCl3, gadolinium chloride; p-ERK, phosphorylated-extracellular signal-regulated kinase; p-JNK, phosphorylated-c-Jun N-terminal kinase; p-p38, phosphorylated-p38.