Gadolinium chloride elicits apoptosis in human osteosarcoma U-2 OS cells through extrinsic signaling, intrinsic pathway and endoplasmic reticulum stress
- Authors:
- Published online on: October 14, 2016 https://doi.org/10.3892/or.2016.5174
- Pages: 3421-3426
Abstract
Introduction
Osteosarcoma is the most common type of malignant bone tumor in children and teenagers (1,2). Unfortunately, less than 50% of patients living beyond 10 years results from the response to the current preferred treatment of preoperative adjuvant chemotherapy followed by surgery (3). Furthermore, no reliable predictors are applied to guide the choice or the intensity of the chemotherapy (4). Currently, there is still a limited role in radiotherapy which should be reserved for inoperable situations. Doxorubicin, cisplatin, ifosfamide and high-dose methotrexate with leucovorin rescue are recognized as the most effective agents against osteosarcoma, but the ideal combination remains to be defined and needs to further well investigated (5). Targeting apoptosis (programmed cell death type I) is a promising approach in the fight against osteosarcoma (6). Therefore, the induction of cell apoptosis is one of best strategies for treating osteosarcoma and multiple cancers (7,8).
Apoptotic function is associated with several diseases, including cancer genesis, and diabetes, and this process is considered a critical element of cancer prevention and therapy (6,9). The three main pathways (death receptor signal, mitochondrial regulation and endoplasmic reticulum stress) contribute to apoptotic signaling (10–12). The death receptor signal is induced by the binding of extrinsic signals to surface receptors, resulting in activation of caspase-8 followed by the activation of caspase-3/−7 (10,11). The mitochondrial pathway is regulated by endoplasmic reticulum (ER) various damage stimuli, which increase reactive oxygen species (ROS) and subsequent damage DNA. These stresses disrupt mitochondrial membrane potential (ΔΨm) to cause cells to undergo apoptosis cascade (11,13). Accumulation of unfolded/misfolded proteins elicits ER-specific pathway aggregating in ER by excessive protein traffic (12). The hallmarks of ER stress are known to stimulate the protein level of growth arrest- and DNA damage-inducible gene 153 (GADD153), glucose-regulated protein 78 (GRP78), GRP94 and activating transcription factor 4 (ATF-4), which can induce intracellular Ca2+ level to activate calpain and caspase-12 and/or caspase-4 molecules (13,14). Therefore, induction of apoptosis by a novel target in these pathways is an attractive approach to fight the tumor cell system.
The rare earth metals of Lanthanides (Lns) exhibit a variety of physical or chemical properties and have been applied in agriculture and medicine for a long time (15,16). Gadolinium (Gd), a member of Lns family, has multi-biological effects on organisms (17), and its compounds have important applications in magnetic resonance imaging (MRI) as contrast medium and are potential anticancer agents (18,19). In clinic, chelates of Gd such as gadobenate dimeglumine (MultiHance) are used as contrast agent in MRI (20). Gadolinium texaphyrin complex (MGd) was evaluated in phase III clinical trials for treating brain metastases of non-small cell lung cancer (21). The Kupffer cell inhibitor gadolinium chloride (GdCl3) has been demonstrated to inhibit human hepatoma HepG2 cell proliferation (22,23). It was also shown that GdCl3 induces HepG2 cell apoptosis through mitochondria-dependent pathway (22). Hence, the present study investigated the effects of GdCl3-induced apoptosis and the underlying mechanisms on human osteosarcoma U-2 OS cells. The experimental data indicated that GdCl3 triggered U-2 OS cell apoptosis through death receptor-, mitochondria- and ER stress-dependent pathways.
Materials and methods
Chemicals and reagents
Gadolinium chloride (GdCl3), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). McCoys 5A (modified) medium, fetal bovine serum (FBS), trypsin-EDTA, penicillin/streptomycin, Fluo-3/AM, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) and 3,3-dihexyloxacarbocyanine iodide [DiOC6(3)] were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Pan-caspase inhibitor (Z-VAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK), caspase-8 inhibitor (Z-IETD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) were purchased from R&D Systems (Minneapolis, MN, USA). Primary antibodies (anti-Fas/CD95, anti-FasL, anti-cytochrome c, anti-Apaf-1, anti-GADD153 and anti-GRP78 and anti-β-actin) and second antibodies [goat anti-rabbit IgG-horseradish peroxidase (HRP) and goat anti-mouse IgG-HRP] were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture
Human osteogenic sarcoma U-2 OS cell line was obtained from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan) and cultured in 75-cm2 tissue culture flasks with McCoys 5A (modified) medium with 10% FBS, 100 Units/ml penicillin and 100 µg/ml streptomycin at 37°C with 5% CO2.
Cell viability and morphological observation
U-2 OS cells (1×104 cells/well) in a 96-well plate were individually pretreated with or without 15 µM specific caspase inhibitors (Z-VAD-FMK, Z-IETD-FMK, Z-LEHD-FMK and Z-DEVD-FMK) for 2 h and then exposed to 0, 50, 100, 150 and 200 µM of GdCl3 for 24 h. After challenge, cell viability was assessed by an MTT assay, as previously reported by Lu et al (24). The GdCl3-treated cells were examined for apoptosis and photographed utilizing a phase-contrast microscope, as previously described (25).
Apoptosis by TUNEL and DAPI staining
U-2 OS cells (2×105 cells/well) in 24-well plates were treated with 0, 50, 100, 150 and 200 µM of GdCl3 for 24 h. Apoptotic DNA breaks were labeled with the In Situ Cell Death Detection kit, Fluorescein (Roche Diagnostics GmbH, Mannheim, Germany) according to the vendors protocol, and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) positive cells were monitored using a BD FACSCalibur flow cytometry (BD Biosciences, San Jose, CA, USA) and BD CellQuest Pro Software (BD Biosciences), as previously described (26). DAPI dye was used to countstain condensation nuclei (a characteristic of apoptosis) in U-2 OS cells with or without 200 µM GdCl3 for 24 h, as detailed by previous studies (27,28).
Assays for caspase-8/−9/−3/−4 activities
U-2 OS cells (5×106 cells per 75T flask) were exposed to 0, 50, 100, 150 and 200 µM of GdCl3 for 24 h. The cell lysates were harvested and assessed subsequently in accordance with the manufacturers instruction [caspase-8, caspase-9 and caspase-3 colorimetric assay kits (R&D System Inc.) and caspase-4 colorimetric assay kit (BioVision Incorporated, Milpitas, CA, USA)].
Determinations of intracellular Ca2+, ROS and ΔΨm levels by flow cytometry
U-2 OS cells (2×105 cells/well) were incubated with or without 50, 100, 150 and 200 µM of GdCl3 for 6 h. Cells were harvested and individually stained with 3 µg/ml Fluo-3/AM for cytoplasmic Ca2+, ROS indicator (5 µM H2DCFDA) and ΔΨm probe [4 nM DiOC6(3)] at 37°C for 30 min. The mean fluorescence intensity (MFI) was quantified by BD CellQuest Pro software (BD Biosciences) before analysis by flow cytometry, as previously described (13,26).
Western blot analysis
U-2 OS cells (5×106 cells/75T flask) were treated in the presence and absence of 0, 50, 100, 150 and 200 µM of GdCl3 for 24 h. Cell lysates were collected, and protein was fractionated on SDS-polyacrylamide gel electrophoresis (PAGE) after being mixed with protein loading dye and boiled, as previously described (14,29). The membrane (iBlot Transfer Stack, PVDF regular; Thermo Fisher Scientific Inc.) was probed with antibodies against Fas, FasL, cytochrome c, Apaf-1, GADD153 and GRP78, respectively. All blots were normalized to β-actin signal, and each signal was quantified with NIH ImageJ program for Windows (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis
Statistical calculations of the data for comparisons of two mean values were obtained using Students t-test. Statistical significance was set at P<0.05. All data were expressed as mean ± standard deviation (SD) of three independent experiments.
Results
GdCl3 reduces cell viability of human osteosarcoma U-2 OS cells
Cells were treated with GdCl3 at the different concentrations from 0 to 200 µM for 24 h. The number of viable cells was assessed by the MTT method. The cell viability was markedly decreased in GdCl3-treated U-2 OS cells in concentration-dependent manner (Fig. 1A). The inhibitory concentration 50% (IC50) for U-2 OS cells was 198.26±1.69 µM. GdCl3 at 200 µM led to the morphological changes and apoptotic cell shrinkage in U-2 OS cells. Also, GdCl3-treated cells were detached from the surface, and some cell debris was observed, whereas the untreated control cells were well spread (Fig. 1B). These findings suggested that GdCl3 possessed cytotoxic effect on human osteosarcoma U-2 OS cells.
GdCl3 provokes U-2 OS cell apoptosis
To explore whether GdCl3 induces U-2 OS cell death through an apoptotic mechanism, TUNEL assay was used for detecting DNA fragmentation in apoptotic cells. GdCl3 markedly stimulated cell apoptosis (TUNEL positive cells) in comparison with control cells (Fig. 2A), and this effect was concentration-dependent (Fig. 2B). Furthermore, apoptotic DNA condensation by DAPI stain was shown in GdCl3-treated U-2 OS cells (Fig. 2A). Our results indicated that GdCl3 provoked apoptotic cell death in U-2 OS cells.
GdCl3 stimulates the activities of caspase-3, caspase-8, and caspase-9 in U-2 OS cells
To clarify the mechanism of GdCl3-induced apoptosis, we further individually investigated the activities of caspase-3, caspase-8, and caspase-9 in U-2 OS cells prior to GdCl3 challenge. The activities of caspase-3 (Fig. 3A), caspase-8 (Fig. 3B) and caspase-9 (Fig. 3C) were increased after GdCl3 exposure in a concentration-dependent manner. Pretreatment of cells with pan-caspase inhibitor (Z-VAD-FMK) (Fig. 4A), specific inhibitors to caspase-3, caspase-8, and caspase-9 (Fig. 4B) significantly prevented the GdCl3-induced cell apoptosis. Our results suggest that the caspase cascade-dependent pathway is important in GdCl3-induced apoptotic death in U-2 OS cells.
GdCl3 increases cytoplasmic Ca2+ level and stimulated the activity of caspase-4 in U-2 OS cells
To further elucidate the possible signaling of GdCl3-induced apoptosis in U-2 OS cells, we determined intracellular Ca2+ levels and caspase-4 activity by flow cytometric analysis and caspase activity assay, respectively. Cells were treated with 50, 100, 150 and 200 µM of GdCl3 for 24 h, and our data demonstrated that GdCl3 significantly increased intracellular Ca2+ levels (Fig. 5A). In addition, caspase-4 activity was concentration-dependently increased in U-2 OS cells after GdCl3 treatment (Fig. 5B). These results showed that GdCl3 induced apoptotic response through cytoplasmic Ca2+ level and ER stress-mediated pathway.
GdCl3 stimulates the ROS production and loss of ΔΨm level in U-2 OS cells
To examine the effect of GdCl3 on ROS production and ΔΨm levels in U-2 OS cells, the specific fluorescence probes were individually used to detect the levels of ROS and ΔΨm. GdCl3 concentration-dependently increased intracellular ROS level in U-2 OS cells (Fig. 6A). GdCl3 disrupted ΔΨm level in U-2 OS cells in a concentration-dependent manner (Fig. 6B). These results suggested that GdCl3 triggered apoptosis in U-2 OS cells by way of the ROS and mitochondria-dependent signaling.
GdCl3 alters the levels of apoptosis-related proteins in U-2 OS cells
In more detail, in the molecular mechanisms of apoptotic pathway, we investigated these related protein levels by western blotting. The levels of Fas, FasL, cytochrome c, Apaf-1, GADD153 and GRP78 were increased in GdCl3-treated cells (Fig. 7). These results suggested that GdCl3-induced apoptotic action might be involved in death receptor-mediated signaling, mitochondria-dependent pathway and ER stress in U-2 OS cells.
Discussion
Lanthanide (Ln) compounds have been shown to possess contrary effects on anticancer activities such as promotion of cell cycle progression and cell growth by lower concentration treatment but induction of apoptosis and suppression of cell proliferation at higher dosing (16,18,19). Our results indicated that high concentration of GdCl3 caused apoptotic U-2 OS cell death (Fig. 1). This is first to report that the MRI contrast agent GdCl3 can be successfully applied to induce apoptosis of human osteosarcoma cells. Moreover, an increase of TUNEL positive cells (Fig. 2), activation of caspase-3, caspase-8, caspase-9 (Fig. 3) and caspase-4 (Fig. 5B) was found to suggest that GdCl3 caused apoptosis of U-2 OS through multiple pathways. It is well know that Gd3+, like other Ln ions, caused different effects depending on the type of target cells (30,31). In NIH 3T3 cells, GdCl3 at the concentrations of <100 µM exerts proliferation-promoting effect and activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling pathways (31). Gadolinium provokes cell apoptosis through mitochondrial pathway and oxidative stress in L-02 normal liver cells (32).
Ye et al (22) reported that GdCl3 induces HepG2 cell apoptosis through mitochondrial and death receptor-dependent pathways. Similarly, our results also demonstrated that GdCl3 induced apoptosis through death receptor and mitochondria-dependent pathways in U-2 OS cells. In the present study, we found that GdCl3 increased the ROS production and decreased the levels of ΔΨm (Fig. 6), and western blot analysis showed that GdCl3 treatment resulted in an increase of Fas/CD95, FasL, cytochrome c and Apaf-1 protein expression in U-2 OS cells (Fig. 7). Thus, we suggest that GdCl3-induced cell death may be mediated via extrinsic and intrinsic apoptotic pathway in U-2 OS cells.
Calcium (Ca2+) regulates cell proliferation, apoptosis or differentiation and plays a pivotal role in carcinogenesis and progression (33). Xia et al (34) has indicated that gadolinium-induced oxidative stress causes ER stress in rat cortical neurons. Feng et al (35) also showed that gadolinium triggers unfolded protein responses (UPRs) in primary cultured rat cortical astrocytes through increasing an influx of extracellular Ca2+ level. However, there is no available report regarding GdCl3-induced ER stress in U-2 OS cells. In the present study, we investigated the GdCl3-induced ER stress in apoptotic mechanism of U-2 OS cells. Our findings demonstrated that the increased levels of GADD153 and GRP78 were followed by releasing Ca2+ from ER and activating caspase-4 activity (Fig. 5), finally leading to ER stress and cell apoptosis. Our results revealed that the activation of ER stress signaling contribute to GdCl3-induced apoptosis in U-2 OS cells.
In conclusion, we found that GdCl3 exhibits direct anticancer activity and triggers suppression of tumor cell proliferation in human osteosarcoma cells. GdCl3 provoked U-2 OS cell apoptosis by the way of the death receptor, mitochondria-dependent and ER stress pathways. The proposed pathway of GdCl3-induced apoptosis of human osteosarcoma U-2 OS cells is showing in Fig. 8. Taken together, our findings provide important possible molecular mechanism against osteosarcoma of GdCl3, showing that GdClv may be a promising anti-osteosarcoma drug.
Acknowledgements
The present study was financially supported by a research grant (no. SKH-TMU-104-01) from the Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
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