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NFAT-1 hyper-activation by methionine enkephalin (MENK) significantly induces cell apoptosis of rats C6 glioma in vivo and in vitro
International Immunopharmacology 56 (2018) 1–8
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NFAT-1 hyper-activation by methionine enkephalin (MENK) significantly induces cell apoptosis of rats C6 glioma in vivo and in vitro ⁎
Wei-cheng Lua,1, Hui Xieb,1, Xin-xin Tiea, Ruizhe Wangd, An-hua Wua, , Feng-ping Shanc,
T
⁎⁎
a
Department of Neurosurgery, First Affiliated Hospital of China Medical University, Shenyang 110001, PR China Department of Histology and Embryology, College of Basic Medicine, Shenyang Medical College, Shenyang 110034, PR China c Department of Immunology, School of Basic Medical Science, China Medical University, Shenyang 110122, PR China d Department of Gynecology, First Affiliated Hospital of China Medical University, Shenyang 110001, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Methionine enkephalin Rat Glioma Apoptosis Nuclear factor of activated T cells-1
The aim of the work was to investigate the effect and possible mechanism of MENK on the growth of rat C6 glioma in vivo or in vitro. Our findings showed that MENK could inhibit the growth of rat C6 glioma, prolong median survival times in tumor-bearing rats, and induce glioma cell apoptosis. Moreover, MENK could increase the activities of caspase-3, caspase-8 and caspase-9. It also increased the expression of Fas, FasL, Bax, while decreased the expression of Bcl-2. We further confirmed that MENK could increase opioid receptors MOR and DOR expressions, Ca2 + influx into the cytoplasm, and a substantial increase of NFAT1accumulation in the nuclei in C6 glioma cell. When we specifically knocked down NFAT1, there was no effect of MENK on the cell viability and FasL up-regulation in NFAT1 knocked-down cell. These results demonstrate that MENK could bind to opioid receptors MOR and DOR on C6 glioma cells and trigger a Ca2 + influx into the cytoplasm, resulting in translocation of NFAT1 into the nucleus. The hyper-activation of NFAT1 may regulate transcription of downstream gene, such as FasL, and induce apoptosis of rat C6 glioma cells.
1. Introduction Brain glioma is the most common primary intracranial tumor with high recurrence after surgery, and post-operative chemotherapy is currently the main method for prolonging the lifespan of patients [1]. However, the existence of blood-tumor barrier (BTB) in tumor tissue limits the delivery of anti-cancer drugs to brain tumor tissue [2]. Therefore, it is urgent to develop agents, which could pass through BTB, while exerting anti-tumor effect so that the chemotherapy efficacy of glioma will be greatly improved. Methionine enkephalin (MENK), an endogenous opioid penta-peptide composed of Tyr-Ala-Ala-Phe-Met, is derived from pre-enkephalin and can pass through BTB [3]. It is suggested to be an important mediator between the immune and neuroendocrine systems [4]. Various types of opioid receptors have been described and two of the most studied ones are μ (MOR) and δ (DOR) [5,6]. These receptors are expressed on immune cells and various tumor cells [5–7]. Research of Molin et al. confirms the expressions of MOR and DOR receptors in
human gliomas [7]. The study of Lord et al. reports that MENK interacts with DOR receptor or MOR receptor [8], and all of the effects of MENK could be inhibited by naltrexone, an opioid receptor antagonist [9]. Our research group has previously reported that MENK could inhibit the tumor growth through regulating the activity of immune cells [9,10]. Meanwhile, during the process of our preliminary test we found that MENK could directly inhibit the growth of rat C6 glioma in vitro and in vivo. However, the exact molecular mechanism underlying this effect remains unclear. Apoptosis, or programmed cell death, is an important mechanism of antitumor drug in inducing cell killing and susceptibility to apoptosis of tumor cells is an important parameter of chemotherapy efficacy [11]. It is mediated by a family of caspases, with two pathways leading to caspase activation: the death receptor pathway and the mitochondrial pathway [12]. The study of Wang et al. has shown that MENK may inhibit growth and induce apoptosis of A375 melanoma cells [13]. At present, whether MENK could inhibit rat C6 glioma growth by inducing cell apoptosis has not been reported.
Abbreviations: BTB, blood-tumor barrier; MENK, methionine enkephalin; NFAT, nuclear factor of activated T cells; BBB, blood-brain barrier; TNF-R, tumor necrosis factor receptor; FasL, fas ligand ⁎ Corresponding author. ⁎⁎ Correspondence to: Feng-ping Shan, Department of Immunology, School of Basic Medical Science, China Medical University, Shenyang, 110001, PR China. E-mail addresses: [email protected] (A.-h. Wu), [email protected] (F.-p. Shan). 1 The first two authors contributed equally to this work. https://doi.org/10.1016/j.intimp.2018.01.005 Received 19 August 2017; Received in revised form 16 December 2017; Accepted 3 January 2018 1567-5769/ © 2018 Published by Elsevier B.V.
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manufacturer's protocol (Annexin-V-FITC kit, Sigma). The C6 glioma cells were exposed to 10 mg/ml MENK or vehicle for 48 h and then harvested and resuspended in Annexin-V binding buffer. Then 5 ml of Annexin V-FITC and 10 ml of PI were added, and the cells were incubated for 10 min at room temperature in the dark. The cells were analyzed immediately after staining by using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, U.S.A.) and ModFit LT software (Verity Software, Topsham, ME, U.S.A.). For each measurement, at least 20,000 cells were counted.
Nuclear factor of activated T cells (NFAT)-1 is initially identified as a transcriptional regulator with profound effects on the proliferation and migration of T cells [14]. In the steady state, NFAT1 is a heavily phosphorylated protein that is dephosphorylated and activated by the phosphatase calcineurin. De-phosphorylation of NFAT1 results in nuclear translocation, and the subsequent initiation of specific transcriptional programs [9]. It is overexpressed in glioma cells, and has been associated with tumor cell survival, apoptosis, migration and invasion [15–17]. The study of Li et al. has indicated that MENK could increase the Ca2 + influx into the cytoplasm in CD8 + T cells [11] and Ca2 + influx into the cytoplasm could also results in the translocation of NFAT1 into the nucleus, and subsequently could regulate downstream gene expression [18,19]. Thus, we speculate that MENK-induced cell apoptosis maybe associated with a significant up-regulation of Ca2 + influx into the cytoplasm and the translocation of NFAT1 into nucleus in rat C6 glioma cells. Therefore, we conducted the following work to investigate the effect and the possible mechanism of MENK on growth of C6 glioma in vitro and in vivo.
2.6. Detection of caspases-3-8 and -9 activity The activities of caspase-3, -8 and -9 were measured using the caspase activity kit (Beyotime, China) according to the manufacture's recommendation. Cells (1 × 106 cells/well) were treated with 10 mg/ ml MENK or vehicle for 48 h. The standard curve was determined by detecting the absorbance of samples at 490 nm. The cells were collected and lysed in caspase assay buffer and supernatant was collected. The activities of caspase-3, -8 and -9 were read as optical density at 405 nm with microplate reader.
2. Materials and methods 2.7. Immunofluorescence assays 2.1. Reagents The C6 cells grown on glass coverslips were exposed to 10 mg/ml MENK or vehicle for 48 h, and then cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. After blocking with 2% bovine serum albumin in PBS, cells were incubated with primary antibodies against anti-MOR Ab (1:200, Cell Signaling Technology), anti-DOR Ab (1:200, Santa Cruz Biotechnology) and antiNFAT1 Ab (1:200, Cell Signaling Technology) to assess the expressions and distributions of mu receptor, delta receptor and NFAT1. The glass slides were analyzed using immunofluorescence microscopy (Olympus, Tokyo, Japan).
MENK was provided by America Peptide Inc. (> 99% in purity). The mAbs used in this study were purchased from eBioscience, BD Pharmingen, Cell Signaling Technology, Invitrogen Life Technology and Santa Cruz Biotechnology. Rat C6 glioma cells were obtained from cell resource center of Shanghai institute of life science, Chinese Academy of Science. Other chemicals frequently used in our laboratory were products from Sigma-Aldrich, BD Pharmingen or Takara. 2.2. Rat C6 cells culture and optimal conc. of MENK in vitro The cells were cultured in high glucose Dulbecco's modified Eagle medium (DMEM; Sigma Aldrich) with 10% fetal bovine serum (Gibco) at 37 °C and 5% CO2. A cell counting kit-8 (CCK-8, Dojindo, Japan) was utilized to evaluate cell viability. The cells growth was tested with a range of concentrations of MENK (2.5, 5, 10, 20 mg/ml) for 48 h, respectively. The absorbance of each well at 450 nm was measured on microplate reader to represent cell viability. 10 mg/ml was selected as the optimum concentration of MENK for the following tests in vitro.
2.8. NFAT1 gene expression knockdown and cell viability assay NFAT1 small hairpin RNA (shRNA) plasmid and control shRNA plasmid (Santa Cruz Biotechnology) were transfected into C6 cells according to the manufacturer's protocol. C6 cells were seeded in a six well plate and grown to 50–70% confluency in antibiotic-free DMEM supplemented with 10% FBS. The cells were washed twice with 2 ml of shRNA Transfection Medium (Santa Cruz Biotechnology) and then 0.8 ml of shRNA Plasmid Transfection Medium was added. After adding 200 ml shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Santa Cruz Biotechnology), the cells were incubated for 8 h at 37 °C with 5% CO2. One milliliter of DMEM with 20% FBS was added. Forty-eight hours post-transfection, the medium was replaced with fresh medium containing 5 mg/ml puromycin for selection of stably transfected cells. Medium was changed every 2 days. Four days later, NFAT1 gene expression was monitored using Western blot analysis. NFAT1 shRNA cells and control shRNA cells were exposed to 10 mg/ml MENK or vehicle for 48 h, and CCK-8 assay was utilized to evaluate the cell viability.
2.3. Measurement of Ca2 + influx into C6 cells C6 glioma cells were treated with 10 mg/ml MENK or vehicle for 48 h, and then 2 × 106 C6 cells were collected and loaded with 5 μM Fluo3 (FITC, Sigma) for 30 min at 4 °C, and finally analyzed by flow cytometry. 2.4. Hoechst DNA staining Nuclear changes of cell apoptosis were visualized by Hoechst DNA staining. Briefly, the C6 glioma cells were seeded at 2 × 105 cells/well into 6-well plate containing sterile coverslips for 12 h culture to allow the cells to adhere to the coverslips, and then exposed to 10 mg/ml MENK or vehicle for 48 h, respectively. Coverslips were washed with PBS containing 5% heat-inactivated FBS and incubated with 100 mg/ml Hoechst 33258 tri-hydrochloride dye in PBS for 10 min at room temperature in the dark. Finally, coverslips were washed with PBS containing 5% FBS, and then photographed by using a fluorescence microscope (Olympus, Japan).
2.9. Establishment of rat brain C6 glioma model and effect of MENK on brain tumor volume and mean survival time in vivo The adult Wistar rats (180–200 g) were purchased from the Center for Experimental Animals of China Medical University. The rats were kept under conventional controlled conditions (22 °C, 55% humidity, and day–night rhythm) and had free access to a standard diet and tap water. All experimental animal procedures were in accordance with the European Communities Council Directive (86/609/EEC) for the Care and Use of Laboratory Animals. Rat C6 glioma cells were harvested at log phase by centrifugation. The rats were anesthetized with chloral hydrate (350 mg/kg, i.p.), and
2.5. Annexin-V-FITC/Propidium iodide double staining assay Annexin-V-FITC/PI staining was carried out according to the 2
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then received an intracerebral injection of 1 × 106 C6 cells in 10 μl of medium with 1.2% methylcellulose using a Hamilton syringe and stereotaxic apparatus. The coordinates were 3 mm lateral to the bregma and 4.5 mm deep to the basal ganglia in the right brain. At 24 h after tumor cell injection, the rats were divided into 5 groups and treated with various concentrations (5, 10, 20 and 50 mg/ kg) of MENK by daily i.v. injection for successive 14 days, respectively. Control group was treated with the same amount of vehicle. The rats were sacrificed at 14 days and Tumor volume (V) was calculated as follows: V = L × W2 × 0.5, where L is the length and W is the width. After this assay, 20 mg/kg was selected as the optimum concentration of MENK in the following experiments in vivo. Furthermore, rats were treated with 20 mg/kg MENK or vehicle for successive 14 days, and then these rats were observed daily and the mean survival time was assessed. 2.10. HE staining and TUNEL assay After treatment with 20 mg/kg MENK or vehicle for successive 14 days, the rats were fixed by cardiac ventricle perfusion with heparinized saline, followed by perfusion with 4% paraformaldehyde. Brain tissues were excised, fixed in formalin and sliced into a series of 2mm-thick blocks. Each block was processed and embedded in paraffin. A series of adjacent 6-um-thick coronal sections were cut with a microtome from each block. The sections were stained with hematoxylin and eosin (H&E). Moreover, to identify cellular apoptosis, TUNEL assay was performed according to the manufacture's recommendation using an ApopTag peroxidase in situ apoptosis detection Kit (Millipore, Billerica, MA).
Fig. 1. Effect of MENK on the viability of C6 glioma cells. The cell samples were exposed to various concentrations (2.5, 5, 10, 20 mg/ml) of MENK for 48 h. Control group was treated with the same amount of vehicle. The CCK-8 assay was used to determine cell viability. The data were presented as the mean ± SD of three independent experiments. **p < 0.01 compared with the control group.
concentrations (5, 10, 20 and 50 mg/kg) of MENK for successive 14 days in rats, the assay results indicated that 20 mg/kg MENK significantly inhibited intracranial tumor growth compared with control group (p < 0.01, Fig. 2A1–A2). However, there were no effect of 5, 10, and 50 mg/kg MENK on intracranial tumor growth. Moreover, HE staining was used to present the brain tumor region of control and MENK group (20 mg/kg), and the brain tumor volume of MENK group was significantly decreased compared to control group (p < 0.01, Fig. 2B1–B2). Therefore, 20 mg/kg was selected as the optimum concentration of MENK in the following experiments in vivo. Furthermore, the mean survival time was assessed after treated with 20 mg/kg MENK for successive 14 days in rats, we observed that the MENK group exhibited significantly longer median survival times (17.0 ± 1.9 days vs. 21.0 ± 0.9 days for the control group), resulting in significantly different survival curves (log-rank tests, p < 0.05, Fig. 2C).
2.11. Western blotting assay After treatment with MENK or vehicle in vitro and in vivo, cells and brain glioma tissues were collected. Total protein from tumor tissue and cells were extracted in lysis buffer (Pierce, Rockford, IL, USA) and quantified by using the BCA method. Equal amounts of protein (20–40 mg) were separated by SDS-PAGE and processed for immunoblotting with antibodies for Fas, FasL, Bax, Bcl-2, caspase-3, MOR and DOR (diluted 1:500; 1:500; 1:300; 1:300; 1:500; 1:400; 1:400, respectively). All the protein bands were scanned and integrated density values (IDVs) were quantified by Fluor Chen 2.0 software and normalized to that of β-actin.
3.2. Effects of MENK on the apoptotic proportion and morphological change in C6 glioma cells and brain tumor of rats
2.12. Statistics analysis
The Annexin-V-FITC/PI double staining assay was used to test the apoptotic cell proportion. The result showed that MENK treatment increased the apoptotic number of C6 cells compared to the control group (p < 0.01, Fig. 3A). To further observe the morphologic characteristics of apoptosis the rat C6 cells were stained with Hoechst 33258. The apoptotic cells increased significantly and displayed typical changes, including bright stained and condensed or fragmented nuclei in MENK group (p < 0.01, Fig. 3B1–B2), while the cells in control group showed an even distribution of the stain and round homogeneous nuclei. Moreover, the brain tumor tissues were removed from rats for TUNEL assay after MENK administration for successive 14 days. TUNEL staining result showed the apoptotic cells with typical dark brown, rounded or oval apoptotic bodies. The number of apoptotic cells increased significantly in MENK group compared to that in control group (p < 0.01, Fig. 3C1–C2).
All data are expressed as the mean ± standard deviation. A Student's t-test was performed to determine the significant difference between two groups. One-way ANOVA and post hoc comparisons (Bonferroni test) were used for multiple comparisons. p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of MENK on cell viability of rats C6 glioma cells and brain tumor growth of rats The CCK-8 assay was used to evaluate the impact of MENK on rats C6 glioma cell viability.C6 cells were treated for 48 h with MENK diluted to concentrations of 2.5, 5, 10, 20 mg/ml, the assay results indicated that relative to the control group, the cell viability was decreased significantly with the treatments of 10 mg/ml MENK (p < 0.01). However, there were no effect of 2.5, 5 and 20 mg/ml MENK on C6 cells viability (Fig. 1). Therefore, 10 mg/ml was selected as the optimum concentration of MENK in the following experiments in vitro. Brain tumor volumes were determined after treated with various
3.3. The expressions of opioid receptors on C6 glioma cells and brain tumor tissue of rats Expressions of MOR and DOR were examined by Western blot and immunofluorescence. Protein expressions of MOR and DOR were first examined by Western blots. Both MOR and DOR were present in C6 3
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Fig. 2. Effects of MENK on the brain tumor growth and mean survival time of rats. At 24 h after tumor cell injection, the rats were treated with 5, 10, 20 and 50 mg/kg MENK by daily i.v. injection for successive 14 days respectively. Control group was treated with the same amount of vehicle. (A1 and A2) The brain coronary section was shown, and the brain tumor volume was calculated. (B1 and B2) The HE staining and brain tumor volume of control and 20 mg/kg MENK groups were shown. (C) The mean survival time of control and 20 mg/kg MENK groups were calculated. The data were presented as the mean ± SD of three independent experiments. **p < 0.01 versus control group. Scale bar = 2 mm.
dyes that bind to Ca2 +. The mean fluorescence intensity of Ca2 + in the MENK group was increased significantly compared to control group (p < 0.01, Fig. 5A1–A2).
glioma cells and brain tumor tissue of rats, and the expressions of MOR and DOR in MENK group were increased compared to control group (Fig. 4A1–A2, p < 0.05). Analogously, immunofluorescence staining showed that there were higher expressions of MOR and DOR on C6 glioma cells both protein levels increased markedly post-MENK treatment in vitro (Fig. 4B1–B2, p < 0.05).
3.5. Effect of MENK on the localization of NFAT1 in C6 glioma cells To examine the effect of MENK on NFAT1 in C6 glioma cells, we measured the nuclear accumulation of NFAT1 in C6 glioma cells by immunofluorescence. After treatment with MENK in vitro, there was a substantial increase of NFAT1 in the nuclei (Fig. 5B).
3.4. Effect of MENK on Ca2 + influx into C6 glioma cells As a possible initial signaling event after MENK interacts with cell surface opioid receptors on C6 glioma cells, we examined whether Ca2 + influx into the cytoplasm was increased. C6 glioma cells in control and MENK groups were stained with FITC-fura3 or fura2-AM, two
Fig. 3. Effect of MENK on the apoptosis, morphological change of C6 glioma cells and brain tumor of rats. The cell samples were exposed to 10 mg/ml MENK or vehicle for 48 h respectively. (A) Apoptosis analysis in rat C6 glioma cells was assessed by Annexin V/PI double staining. (B1 and B2) Cell nuclear change with characteristic of apoptosis was visualized by Hoechst DNA staining in vitro (original magnification, × 400). Furthermore, the rats were treated with 20 mg/kg MENK for 14 days, brain tissues were excised and sliced into sections. (C1 and C2) The sections were subject to TUNEL assay (original magnification, × 400). The data were presented as the mean ± SD of three independent experiments. **p < 0.01 versus control group.
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Fig. 4. Expressions of MOR and DOR after MENK treatment in vitro and in vivo. The cell samples were exposed to 10 mg/ml MENK or vehicle for 48 h, and the rats were treated with 20 mg/kg MENK for 14 days. Subsequently the cells and brain glioma tissues were collected for Western bolt. (A1) Protein expressions of MOR and DOR in vivo and in vitro were examined by Western blots. (A2) The IDVs of MOR and DOR were shown. (B) The C6 cells grown on glass coverslips were exposed to 10 mg/ml MENK for 48 h and MOR and DOR proteins in C6 glioma cells were examined by immunofluorescence staining (original magnification, × 400). The data were presented as the mean ± SD of three independent experiments. *p < 0.05 versus control group.
data indicated that MENK could trigger apoptosis of C6 glioma cells and brain tumor of rats.
3.6. Effect of NFAT1 inhibition on the cell viability in C6 glioma cells To evaluate the effect of NFAT1 on MENK-induced cell apoptosis, NFAT1 was stably knocked down in C6 glioma cells by transfection of NFAT1-specific shRNA plasmid. The loss of NFAT1 expression was confirmed by Western blot analysis with anti-NFAT1 (Fig. 5C). NFAT1 -shRNA cells and control-shRNA cells were exposed to 10− 5 M MENK or vehicle for 48 h, and CCK-8 assay was performed to evaluate the cell viability. The results showed that MENK treatment decreased the cell viability in control-shRNA cell compared to that in control group. However, there was no effect of MENK on the cell viability in NFAT1shRNA cell (Fig. 5D).
4. Discussion Malignant glioma is the most common and deadliest form of intracranial tumor with extremely high recurrence. At present, post-operative chemotherapy of glioma becomes a promising way of treatment. Although the permeability of BTB is slightly higher than that of (bloodbrain barrier) BBB [19], it still restricts access of most chemotherapeutic drugs to brain tumor tissue. Some studies showed that if the local drug concentration was elevated by two fold, the efficacy to kill the brain tumor cells could be increased by ten folds [20]. Therefore, if an anti-tumor drug could pass through the BTB, the chemotherapy efficacy of glioma would be markedly improved. MENK, a penta-peptide is considered as being involved in the regulatory feedback loop between the immune and neuroendocrine systems, with marked modulation of various functions of human immune cells [4]. The published data demonstrated that MENK could pass through BBB and BTB [3]. Our research group previously reported that MENK inhibited tumor growth through regulating the activity of immune cells [9,10]. In the present study, we found that MENK could
3.7. Effects of MENK on the activities of caspase-3, caspase-8 and caspase9 and protein expressions of Fas, FasL, Bax, Bcl-2 and caspase-3 in C6 glioma cells and brain tumor of rats When compared with control group, the activities of caspase-3, caspase-8 and caspase-9 significantly increased in MENK group in vitro, respectively (p < 0.01, Fig. 6A). After treatment with MENK, the expression levels of Fas, FasL, Bax and caspase-3 significantly increased while the expression of Bcl-2 decreased (p < 0.05, Fig. 6B1–B2). The 5
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Fig. 5. Effect of MENK on influx of Ca2 +, NFAT1 expression as well as cell viability assay after NFAT1 Gene Knockdown in C6 glioma. The cell samples were exposed to 10 mg/ml MENK for 48 h, and influx of Ca2 +, NFAT1 expression were determined. (A1) Cells were loaded with 5 μM Fluo3, the histograms of fluorescent intensity of Fluo-3 on C6 cells were determined by flow cytometry analysis. (A2) The bar graphs represent average ± SD of Ca2 + fluorescent intensity of C6 cells from three independent experiments. (B) NFAT1 expression in C6 glioma cells were examined by immunofluorescence staining after MENK treatment (original magnification, × 100). (C) In order to knockdown NFAT1 gene expression, NFAT1 small hairpin RNA (shRNA) plasmid and control shRNA plasmid were transfected into C6 cells, respectively. NFAT1 gene expression in C6, control shRNA cells and NFAT1 shRNA cells was monitored using Western blot analysis.(D) CCK-8 assay was utilized to evaluate the cell viability in control shRNA cells and NFAT1 shRNA cells. The data were presented as the mean ± SD of three independent experiments. **p < 0.01 versus control group.
death activators though the activation of a cascade of proteolytic enzymes termed caspases. Two main apoptotic pathways have been mentioned as follows: the intrinsic mitochondrion pathway and the extrinsic death receptor pathway [21]. The intrinsic apoptotic pathway involves the release from the mitochondria into the cytosol of cytochrome c and other gene oriented apoptotic proteins [22]. This
directly inhibit the growth of rat C6 glioma in vitro and in vivo, and prolong the survival times in tumor-bearing rats, at the optimum concentration of 20 mg/kg in vivo and 10 mg/ml in vitro. Moreover, the study of Wang et al. has shown that MENK may inhibit growth and induce apoptosis of A375 melanoma cells [13]. Apoptosis can be triggered via signals within the cell or by extrinsic
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Fig. 6. Effect of MENK on the activities of caspase-3, caspase-8 and caspase-9 in vitro and apoptosis associated protein expressions in vitro and in vivo. (A) The cell samples were exposed to 10 mg/ml MENK or vehicle for 48 h and the activities of caspase-3, caspase-8 and caspase-9 were detected by colorimetric assay as described. (B1) The cell samples were exposed to 10 mg/ml MENK or vehicle for 48 h and the rats were treated with 20 mg/kg MENK or vehicle for 14 days. Subsequently, the cells and brain glioma tissues were collected and processed for Western bolt. Expressions of Fas, FasL, Bax, Bcl-2 and caspase-3 in vitro and in vivo were determined. (B2) The IDVs above proteins were shown. The data were presented as the mean ± SD of three independent experiments. *p < 0.05 and *p < 0.01 versus the control group.
receptors MOR and DOR on C6 glioma cells and triggered a Ca2 + influx into the cytoplasm, resulting in translocation of NFAT1 into the nucleus. The hyper-activation of NFAT1 may regulate transcription of downstream gene, such as FasL, and finally induced apoptosis in C6 glioma cells.
apoptotic pathway is regulated by the Bcl-2 family of proteins integrated by anti-apoptotic (Bcl-2, Bcl-xL, etc.) and pro-apoptotic (BH3 only family and Bax family) members [23]. The extrinsic apoptotic pathway is mediated by the tumor necrosis factor receptor (TNF-R) superfamily of death receptors. Fas (CD95 or APO-1) is a prototypical member of this family, whose binding with cytokines belonging to the TNF family, such as Fas ligand (FasL), induces cell death through caspase activation [24]. In this study, our results showed that MENK could induce rat C6 glioma cell apoptosis in vitro and in vivo, and MENK could increase the activities of caspase-3, caspase-8 and caspase-9 in vitro. Moreover, the expressions of Fas, FasL, Bax and caspase-3were increased after MENK treatment. However, the expression of Bcl-2 was decreased after MENK exposure in vitro and in vivo. These results proved that MENK inhibited growth and induced apoptosis of rats C6 glioma cells in vitro and in vivo. The induction of apoptosis appeared to be related with the upregulation of Fas/FasL, Bax, activation of caspase-3, caspase-8, caspase-9 and downregulation of Bcl-2, triggering major apoptotic cascades. It should be further noted that the study has also delved into the mechanisms through which MENK induced the apoptosis of rat C6 glioma. First of all, we confirmed the expressions of opioid receptors MOR and DOR on C6 glioma cells and also found that treatment with MENK increased the expression of these receptors. We further explored some aspects of the signaling events triggered by the interaction of MENK with opioid receptors on C6 glioma cells. The influx of Ca2 + could play a key role in a plethora of signaling pathways. Ca2 + influx into the cytoplasm has been shown to activate CaM/calcineurin protein phosphatase complexes, leading to de-phosphorylation of cytoplasmic NFAT-1 [25,26], which in turn translocates this factors into the nucleus, and binds to its target promotor elements and regulates the transcription of related genes, such as FasL [18,19,27]. The expression of Fas and the induced-expression of FasL is mainly responsible for the process of apoptosis [28–31]. In the present study, our results showed that MENK increased the Ca2 + influx into the cytoplasm in C6 glioma cell, and after treatment with MENK in vitro, there was a substantial increase of NFAT1 accumulation in the nuclei by immunofluorescence staining. In addition, we proceeded to explore the role of NFAT1 in MENK-induced cell apoptosis. When we specifically knocked down NFAT1 expression, there was no effect of MENK on the cell viability in NFAT1 knocked-down cell. The results of Western bolt showed that the expressions of Fas and FasL increased after MENK treatment. However, when NFAT1 was specifically knocked down, there was no effect of MENK on the FasL up-regulation. These results demonstrated that MENK could bind to opioid
5. Conclusion We believe that it is the first time that the current approach demonstrates that NFAT1 hyper-activation by MENK could significantly induce the apoptosis of rats C6 glioma cell through upregulation of Fas/ FasL and Bax, activation of caspase-3, caspase-8, caspase-9 and downregulation of Bcl-2. Clinically MENK might be with potential for the therapy of human glioma and as an adjunct to other chemotherapies. Also the present study will provide new mode of action for the chemotherapy of brain glioma. Conflict of interest The authors declare that there is no conflict of interest and we would take legal responsibility if anything would happen to this paper. Acknowledgements This work was supported financially by the grant from Science and Technology Plan Project of Educational Department of Liaoning Province (No. L2015535) and the grant from China National Funding for Natural Science (No. 31670921 to Fengping Shan). References [1] J.M. Kuijlen, E. Bremer, J.J. Mooij, W.F. den Dunnen, W. Helfrich, Review: on TRAIL for malignant glioma therapy? Neuropathol. Appl. Neurobiol. 36 (3) (2010 Apr) 168–182. [2] E.M. Kemper, W. Boogerd, I. Thuis, J.H. Beijnen, O. van Tellingen, Modulation of the blood-brain barrier in oncology: therapeutic opportunities for the treatment of brain tumours? Cancer Treat. Rev. 30 (5) (2004 Aug) 415–423. [3] S.J. Weber, T.J. Abbruscato, E.A. Brownson, A.W. Lipkowski, R. Polt, A. Misicka, R.C. Haaseth, H. Bartosz, V.J. Hruby, T.P. Davis, Assessment of an in vitro bloodbrain barrier model using several [Met5]enkephalin opioid analogs, J. Pharmacol. Exp. Ther. 266 (3) (1993 Sep) 1649–1655. [4] M. Piva, J.I. Moreno, F.S. Jenkins, J.K.A. Smith, J.L. Thomas, C. Montgomery, C.B. Wilson, R.C. Sizemore, In vitro modulation of cytokine expression by enkephalin-derived peptides, Neuroimmunomodulation 12 (6) (2005) 339–347. [5] D.M. Avella, E.T. Kimchi, R.N. Donahue, H.R. Tagaram, P.J. McLaughlin, I.S. Zagon, K.F. Staveley-O'Carroll, The opioid growth factor-opioid growth factor receptor axis regulates cell proliferation of human hepatocellular cancer, Am. J. Phys. Regul. Integr. Comp. Phys. 298 (2) (2010 Feb) R459–R466.
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NFAT-1 hyper-activation by methionine enkephalin (MENK) significantly induces cell apoptosis of rats C6 glioma in vivo and in vitro ⁎
Wei-cheng Lua,1, Hui Xieb,1, Xin-xin Tiea, Ruizhe Wangd, An-hua Wua, , Feng-ping Shanc,
T
⁎⁎
a
Department of Neurosurgery, First Affiliated Hospital of China Medical University, Shenyang 110001, PR China Department of Histology and Embryology, College of Basic Medicine, Shenyang Medical College, Shenyang 110034, PR China c Department of Immunology, School of Basic Medical Science, China Medical University, Shenyang 110122, PR China d Department of Gynecology, First Affiliated Hospital of China Medical University, Shenyang 110001, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Methionine enkephalin Rat Glioma Apoptosis Nuclear factor of activated T cells-1
The aim of the work was to investigate the effect and possible mechanism of MENK on the growth of rat C6 glioma in vivo or in vitro. Our findings showed that MENK could inhibit the growth of rat C6 glioma, prolong median survival times in tumor-bearing rats, and induce glioma cell apoptosis. Moreover, MENK could increase the activities of caspase-3, caspase-8 and caspase-9. It also increased the expression of Fas, FasL, Bax, while decreased the expression of Bcl-2. We further confirmed that MENK could increase opioid receptors MOR and DOR expressions, Ca2 + influx into the cytoplasm, and a substantial increase of NFAT1accumulation in the nuclei in C6 glioma cell. When we specifically knocked down NFAT1, there was no effect of MENK on the cell viability and FasL up-regulation in NFAT1 knocked-down cell. These results demonstrate that MENK could bind to opioid receptors MOR and DOR on C6 glioma cells and trigger a Ca2 + influx into the cytoplasm, resulting in translocation of NFAT1 into the nucleus. The hyper-activation of NFAT1 may regulate transcription of downstream gene, such as FasL, and induce apoptosis of rat C6 glioma cells.
1. Introduction Brain glioma is the most common primary intracranial tumor with high recurrence after surgery, and post-operative chemotherapy is currently the main method for prolonging the lifespan of patients [1]. However, the existence of blood-tumor barrier (BTB) in tumor tissue limits the delivery of anti-cancer drugs to brain tumor tissue [2]. Therefore, it is urgent to develop agents, which could pass through BTB, while exerting anti-tumor effect so that the chemotherapy efficacy of glioma will be greatly improved. Methionine enkephalin (MENK), an endogenous opioid penta-peptide composed of Tyr-Ala-Ala-Phe-Met, is derived from pre-enkephalin and can pass through BTB [3]. It is suggested to be an important mediator between the immune and neuroendocrine systems [4]. Various types of opioid receptors have been described and two of the most studied ones are μ (MOR) and δ (DOR) [5,6]. These receptors are expressed on immune cells and various tumor cells [5–7]. Research of Molin et al. confirms the expressions of MOR and DOR receptors in
human gliomas [7]. The study of Lord et al. reports that MENK interacts with DOR receptor or MOR receptor [8], and all of the effects of MENK could be inhibited by naltrexone, an opioid receptor antagonist [9]. Our research group has previously reported that MENK could inhibit the tumor growth through regulating the activity of immune cells [9,10]. Meanwhile, during the process of our preliminary test we found that MENK could directly inhibit the growth of rat C6 glioma in vitro and in vivo. However, the exact molecular mechanism underlying this effect remains unclear. Apoptosis, or programmed cell death, is an important mechanism of antitumor drug in inducing cell killing and susceptibility to apoptosis of tumor cells is an important parameter of chemotherapy efficacy [11]. It is mediated by a family of caspases, with two pathways leading to caspase activation: the death receptor pathway and the mitochondrial pathway [12]. The study of Wang et al. has shown that MENK may inhibit growth and induce apoptosis of A375 melanoma cells [13]. At present, whether MENK could inhibit rat C6 glioma growth by inducing cell apoptosis has not been reported.
Abbreviations: BTB, blood-tumor barrier; MENK, methionine enkephalin; NFAT, nuclear factor of activated T cells; BBB, blood-brain barrier; TNF-R, tumor necrosis factor receptor; FasL, fas ligand ⁎ Corresponding author. ⁎⁎ Correspondence to: Feng-ping Shan, Department of Immunology, School of Basic Medical Science, China Medical University, Shenyang, 110001, PR China. E-mail addresses: [email protected] (A.-h. Wu), [email protected] (F.-p. Shan). 1 The first two authors contributed equally to this work. https://doi.org/10.1016/j.intimp.2018.01.005 Received 19 August 2017; Received in revised form 16 December 2017; Accepted 3 January 2018 1567-5769/ © 2018 Published by Elsevier B.V.
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manufacturer's protocol (Annexin-V-FITC kit, Sigma). The C6 glioma cells were exposed to 10 mg/ml MENK or vehicle for 48 h and then harvested and resuspended in Annexin-V binding buffer. Then 5 ml of Annexin V-FITC and 10 ml of PI were added, and the cells were incubated for 10 min at room temperature in the dark. The cells were analyzed immediately after staining by using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, U.S.A.) and ModFit LT software (Verity Software, Topsham, ME, U.S.A.). For each measurement, at least 20,000 cells were counted.
Nuclear factor of activated T cells (NFAT)-1 is initially identified as a transcriptional regulator with profound effects on the proliferation and migration of T cells [14]. In the steady state, NFAT1 is a heavily phosphorylated protein that is dephosphorylated and activated by the phosphatase calcineurin. De-phosphorylation of NFAT1 results in nuclear translocation, and the subsequent initiation of specific transcriptional programs [9]. It is overexpressed in glioma cells, and has been associated with tumor cell survival, apoptosis, migration and invasion [15–17]. The study of Li et al. has indicated that MENK could increase the Ca2 + influx into the cytoplasm in CD8 + T cells [11] and Ca2 + influx into the cytoplasm could also results in the translocation of NFAT1 into the nucleus, and subsequently could regulate downstream gene expression [18,19]. Thus, we speculate that MENK-induced cell apoptosis maybe associated with a significant up-regulation of Ca2 + influx into the cytoplasm and the translocation of NFAT1 into nucleus in rat C6 glioma cells. Therefore, we conducted the following work to investigate the effect and the possible mechanism of MENK on growth of C6 glioma in vitro and in vivo.
2.6. Detection of caspases-3-8 and -9 activity The activities of caspase-3, -8 and -9 were measured using the caspase activity kit (Beyotime, China) according to the manufacture's recommendation. Cells (1 × 106 cells/well) were treated with 10 mg/ ml MENK or vehicle for 48 h. The standard curve was determined by detecting the absorbance of samples at 490 nm. The cells were collected and lysed in caspase assay buffer and supernatant was collected. The activities of caspase-3, -8 and -9 were read as optical density at 405 nm with microplate reader.
2. Materials and methods 2.7. Immunofluorescence assays 2.1. Reagents The C6 cells grown on glass coverslips were exposed to 10 mg/ml MENK or vehicle for 48 h, and then cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. After blocking with 2% bovine serum albumin in PBS, cells were incubated with primary antibodies against anti-MOR Ab (1:200, Cell Signaling Technology), anti-DOR Ab (1:200, Santa Cruz Biotechnology) and antiNFAT1 Ab (1:200, Cell Signaling Technology) to assess the expressions and distributions of mu receptor, delta receptor and NFAT1. The glass slides were analyzed using immunofluorescence microscopy (Olympus, Tokyo, Japan).
MENK was provided by America Peptide Inc. (> 99% in purity). The mAbs used in this study were purchased from eBioscience, BD Pharmingen, Cell Signaling Technology, Invitrogen Life Technology and Santa Cruz Biotechnology. Rat C6 glioma cells were obtained from cell resource center of Shanghai institute of life science, Chinese Academy of Science. Other chemicals frequently used in our laboratory were products from Sigma-Aldrich, BD Pharmingen or Takara. 2.2. Rat C6 cells culture and optimal conc. of MENK in vitro The cells were cultured in high glucose Dulbecco's modified Eagle medium (DMEM; Sigma Aldrich) with 10% fetal bovine serum (Gibco) at 37 °C and 5% CO2. A cell counting kit-8 (CCK-8, Dojindo, Japan) was utilized to evaluate cell viability. The cells growth was tested with a range of concentrations of MENK (2.5, 5, 10, 20 mg/ml) for 48 h, respectively. The absorbance of each well at 450 nm was measured on microplate reader to represent cell viability. 10 mg/ml was selected as the optimum concentration of MENK for the following tests in vitro.
2.8. NFAT1 gene expression knockdown and cell viability assay NFAT1 small hairpin RNA (shRNA) plasmid and control shRNA plasmid (Santa Cruz Biotechnology) were transfected into C6 cells according to the manufacturer's protocol. C6 cells were seeded in a six well plate and grown to 50–70% confluency in antibiotic-free DMEM supplemented with 10% FBS. The cells were washed twice with 2 ml of shRNA Transfection Medium (Santa Cruz Biotechnology) and then 0.8 ml of shRNA Plasmid Transfection Medium was added. After adding 200 ml shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Santa Cruz Biotechnology), the cells were incubated for 8 h at 37 °C with 5% CO2. One milliliter of DMEM with 20% FBS was added. Forty-eight hours post-transfection, the medium was replaced with fresh medium containing 5 mg/ml puromycin for selection of stably transfected cells. Medium was changed every 2 days. Four days later, NFAT1 gene expression was monitored using Western blot analysis. NFAT1 shRNA cells and control shRNA cells were exposed to 10 mg/ml MENK or vehicle for 48 h, and CCK-8 assay was utilized to evaluate the cell viability.
2.3. Measurement of Ca2 + influx into C6 cells C6 glioma cells were treated with 10 mg/ml MENK or vehicle for 48 h, and then 2 × 106 C6 cells were collected and loaded with 5 μM Fluo3 (FITC, Sigma) for 30 min at 4 °C, and finally analyzed by flow cytometry. 2.4. Hoechst DNA staining Nuclear changes of cell apoptosis were visualized by Hoechst DNA staining. Briefly, the C6 glioma cells were seeded at 2 × 105 cells/well into 6-well plate containing sterile coverslips for 12 h culture to allow the cells to adhere to the coverslips, and then exposed to 10 mg/ml MENK or vehicle for 48 h, respectively. Coverslips were washed with PBS containing 5% heat-inactivated FBS and incubated with 100 mg/ml Hoechst 33258 tri-hydrochloride dye in PBS for 10 min at room temperature in the dark. Finally, coverslips were washed with PBS containing 5% FBS, and then photographed by using a fluorescence microscope (Olympus, Japan).
2.9. Establishment of rat brain C6 glioma model and effect of MENK on brain tumor volume and mean survival time in vivo The adult Wistar rats (180–200 g) were purchased from the Center for Experimental Animals of China Medical University. The rats were kept under conventional controlled conditions (22 °C, 55% humidity, and day–night rhythm) and had free access to a standard diet and tap water. All experimental animal procedures were in accordance with the European Communities Council Directive (86/609/EEC) for the Care and Use of Laboratory Animals. Rat C6 glioma cells were harvested at log phase by centrifugation. The rats were anesthetized with chloral hydrate (350 mg/kg, i.p.), and
2.5. Annexin-V-FITC/Propidium iodide double staining assay Annexin-V-FITC/PI staining was carried out according to the 2
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then received an intracerebral injection of 1 × 106 C6 cells in 10 μl of medium with 1.2% methylcellulose using a Hamilton syringe and stereotaxic apparatus. The coordinates were 3 mm lateral to the bregma and 4.5 mm deep to the basal ganglia in the right brain. At 24 h after tumor cell injection, the rats were divided into 5 groups and treated with various concentrations (5, 10, 20 and 50 mg/ kg) of MENK by daily i.v. injection for successive 14 days, respectively. Control group was treated with the same amount of vehicle. The rats were sacrificed at 14 days and Tumor volume (V) was calculated as follows: V = L × W2 × 0.5, where L is the length and W is the width. After this assay, 20 mg/kg was selected as the optimum concentration of MENK in the following experiments in vivo. Furthermore, rats were treated with 20 mg/kg MENK or vehicle for successive 14 days, and then these rats were observed daily and the mean survival time was assessed. 2.10. HE staining and TUNEL assay After treatment with 20 mg/kg MENK or vehicle for successive 14 days, the rats were fixed by cardiac ventricle perfusion with heparinized saline, followed by perfusion with 4% paraformaldehyde. Brain tissues were excised, fixed in formalin and sliced into a series of 2mm-thick blocks. Each block was processed and embedded in paraffin. A series of adjacent 6-um-thick coronal sections were cut with a microtome from each block. The sections were stained with hematoxylin and eosin (H&E). Moreover, to identify cellular apoptosis, TUNEL assay was performed according to the manufacture's recommendation using an ApopTag peroxidase in situ apoptosis detection Kit (Millipore, Billerica, MA).
Fig. 1. Effect of MENK on the viability of C6 glioma cells. The cell samples were exposed to various concentrations (2.5, 5, 10, 20 mg/ml) of MENK for 48 h. Control group was treated with the same amount of vehicle. The CCK-8 assay was used to determine cell viability. The data were presented as the mean ± SD of three independent experiments. **p < 0.01 compared with the control group.
concentrations (5, 10, 20 and 50 mg/kg) of MENK for successive 14 days in rats, the assay results indicated that 20 mg/kg MENK significantly inhibited intracranial tumor growth compared with control group (p < 0.01, Fig. 2A1–A2). However, there were no effect of 5, 10, and 50 mg/kg MENK on intracranial tumor growth. Moreover, HE staining was used to present the brain tumor region of control and MENK group (20 mg/kg), and the brain tumor volume of MENK group was significantly decreased compared to control group (p < 0.01, Fig. 2B1–B2). Therefore, 20 mg/kg was selected as the optimum concentration of MENK in the following experiments in vivo. Furthermore, the mean survival time was assessed after treated with 20 mg/kg MENK for successive 14 days in rats, we observed that the MENK group exhibited significantly longer median survival times (17.0 ± 1.9 days vs. 21.0 ± 0.9 days for the control group), resulting in significantly different survival curves (log-rank tests, p < 0.05, Fig. 2C).
2.11. Western blotting assay After treatment with MENK or vehicle in vitro and in vivo, cells and brain glioma tissues were collected. Total protein from tumor tissue and cells were extracted in lysis buffer (Pierce, Rockford, IL, USA) and quantified by using the BCA method. Equal amounts of protein (20–40 mg) were separated by SDS-PAGE and processed for immunoblotting with antibodies for Fas, FasL, Bax, Bcl-2, caspase-3, MOR and DOR (diluted 1:500; 1:500; 1:300; 1:300; 1:500; 1:400; 1:400, respectively). All the protein bands were scanned and integrated density values (IDVs) were quantified by Fluor Chen 2.0 software and normalized to that of β-actin.
3.2. Effects of MENK on the apoptotic proportion and morphological change in C6 glioma cells and brain tumor of rats
2.12. Statistics analysis
The Annexin-V-FITC/PI double staining assay was used to test the apoptotic cell proportion. The result showed that MENK treatment increased the apoptotic number of C6 cells compared to the control group (p < 0.01, Fig. 3A). To further observe the morphologic characteristics of apoptosis the rat C6 cells were stained with Hoechst 33258. The apoptotic cells increased significantly and displayed typical changes, including bright stained and condensed or fragmented nuclei in MENK group (p < 0.01, Fig. 3B1–B2), while the cells in control group showed an even distribution of the stain and round homogeneous nuclei. Moreover, the brain tumor tissues were removed from rats for TUNEL assay after MENK administration for successive 14 days. TUNEL staining result showed the apoptotic cells with typical dark brown, rounded or oval apoptotic bodies. The number of apoptotic cells increased significantly in MENK group compared to that in control group (p < 0.01, Fig. 3C1–C2).
All data are expressed as the mean ± standard deviation. A Student's t-test was performed to determine the significant difference between two groups. One-way ANOVA and post hoc comparisons (Bonferroni test) were used for multiple comparisons. p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of MENK on cell viability of rats C6 glioma cells and brain tumor growth of rats The CCK-8 assay was used to evaluate the impact of MENK on rats C6 glioma cell viability.C6 cells were treated for 48 h with MENK diluted to concentrations of 2.5, 5, 10, 20 mg/ml, the assay results indicated that relative to the control group, the cell viability was decreased significantly with the treatments of 10 mg/ml MENK (p < 0.01). However, there were no effect of 2.5, 5 and 20 mg/ml MENK on C6 cells viability (Fig. 1). Therefore, 10 mg/ml was selected as the optimum concentration of MENK in the following experiments in vitro. Brain tumor volumes were determined after treated with various
3.3. The expressions of opioid receptors on C6 glioma cells and brain tumor tissue of rats Expressions of MOR and DOR were examined by Western blot and immunofluorescence. Protein expressions of MOR and DOR were first examined by Western blots. Both MOR and DOR were present in C6 3
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Fig. 2. Effects of MENK on the brain tumor growth and mean survival time of rats. At 24 h after tumor cell injection, the rats were treated with 5, 10, 20 and 50 mg/kg MENK by daily i.v. injection for successive 14 days respectively. Control group was treated with the same amount of vehicle. (A1 and A2) The brain coronary section was shown, and the brain tumor volume was calculated. (B1 and B2) The HE staining and brain tumor volume of control and 20 mg/kg MENK groups were shown. (C) The mean survival time of control and 20 mg/kg MENK groups were calculated. The data were presented as the mean ± SD of three independent experiments. **p < 0.01 versus control group. Scale bar = 2 mm.
dyes that bind to Ca2 +. The mean fluorescence intensity of Ca2 + in the MENK group was increased significantly compared to control group (p < 0.01, Fig. 5A1–A2).
glioma cells and brain tumor tissue of rats, and the expressions of MOR and DOR in MENK group were increased compared to control group (Fig. 4A1–A2, p < 0.05). Analogously, immunofluorescence staining showed that there were higher expressions of MOR and DOR on C6 glioma cells both protein levels increased markedly post-MENK treatment in vitro (Fig. 4B1–B2, p < 0.05).
3.5. Effect of MENK on the localization of NFAT1 in C6 glioma cells To examine the effect of MENK on NFAT1 in C6 glioma cells, we measured the nuclear accumulation of NFAT1 in C6 glioma cells by immunofluorescence. After treatment with MENK in vitro, there was a substantial increase of NFAT1 in the nuclei (Fig. 5B).
3.4. Effect of MENK on Ca2 + influx into C6 glioma cells As a possible initial signaling event after MENK interacts with cell surface opioid receptors on C6 glioma cells, we examined whether Ca2 + influx into the cytoplasm was increased. C6 glioma cells in control and MENK groups were stained with FITC-fura3 or fura2-AM, two
Fig. 3. Effect of MENK on the apoptosis, morphological change of C6 glioma cells and brain tumor of rats. The cell samples were exposed to 10 mg/ml MENK or vehicle for 48 h respectively. (A) Apoptosis analysis in rat C6 glioma cells was assessed by Annexin V/PI double staining. (B1 and B2) Cell nuclear change with characteristic of apoptosis was visualized by Hoechst DNA staining in vitro (original magnification, × 400). Furthermore, the rats were treated with 20 mg/kg MENK for 14 days, brain tissues were excised and sliced into sections. (C1 and C2) The sections were subject to TUNEL assay (original magnification, × 400). The data were presented as the mean ± SD of three independent experiments. **p < 0.01 versus control group.
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Fig. 4. Expressions of MOR and DOR after MENK treatment in vitro and in vivo. The cell samples were exposed to 10 mg/ml MENK or vehicle for 48 h, and the rats were treated with 20 mg/kg MENK for 14 days. Subsequently the cells and brain glioma tissues were collected for Western bolt. (A1) Protein expressions of MOR and DOR in vivo and in vitro were examined by Western blots. (A2) The IDVs of MOR and DOR were shown. (B) The C6 cells grown on glass coverslips were exposed to 10 mg/ml MENK for 48 h and MOR and DOR proteins in C6 glioma cells were examined by immunofluorescence staining (original magnification, × 400). The data were presented as the mean ± SD of three independent experiments. *p < 0.05 versus control group.
data indicated that MENK could trigger apoptosis of C6 glioma cells and brain tumor of rats.
3.6. Effect of NFAT1 inhibition on the cell viability in C6 glioma cells To evaluate the effect of NFAT1 on MENK-induced cell apoptosis, NFAT1 was stably knocked down in C6 glioma cells by transfection of NFAT1-specific shRNA plasmid. The loss of NFAT1 expression was confirmed by Western blot analysis with anti-NFAT1 (Fig. 5C). NFAT1 -shRNA cells and control-shRNA cells were exposed to 10− 5 M MENK or vehicle for 48 h, and CCK-8 assay was performed to evaluate the cell viability. The results showed that MENK treatment decreased the cell viability in control-shRNA cell compared to that in control group. However, there was no effect of MENK on the cell viability in NFAT1shRNA cell (Fig. 5D).
4. Discussion Malignant glioma is the most common and deadliest form of intracranial tumor with extremely high recurrence. At present, post-operative chemotherapy of glioma becomes a promising way of treatment. Although the permeability of BTB is slightly higher than that of (bloodbrain barrier) BBB [19], it still restricts access of most chemotherapeutic drugs to brain tumor tissue. Some studies showed that if the local drug concentration was elevated by two fold, the efficacy to kill the brain tumor cells could be increased by ten folds [20]. Therefore, if an anti-tumor drug could pass through the BTB, the chemotherapy efficacy of glioma would be markedly improved. MENK, a penta-peptide is considered as being involved in the regulatory feedback loop between the immune and neuroendocrine systems, with marked modulation of various functions of human immune cells [4]. The published data demonstrated that MENK could pass through BBB and BTB [3]. Our research group previously reported that MENK inhibited tumor growth through regulating the activity of immune cells [9,10]. In the present study, we found that MENK could
3.7. Effects of MENK on the activities of caspase-3, caspase-8 and caspase9 and protein expressions of Fas, FasL, Bax, Bcl-2 and caspase-3 in C6 glioma cells and brain tumor of rats When compared with control group, the activities of caspase-3, caspase-8 and caspase-9 significantly increased in MENK group in vitro, respectively (p < 0.01, Fig. 6A). After treatment with MENK, the expression levels of Fas, FasL, Bax and caspase-3 significantly increased while the expression of Bcl-2 decreased (p < 0.05, Fig. 6B1–B2). The 5
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Fig. 5. Effect of MENK on influx of Ca2 +, NFAT1 expression as well as cell viability assay after NFAT1 Gene Knockdown in C6 glioma. The cell samples were exposed to 10 mg/ml MENK for 48 h, and influx of Ca2 +, NFAT1 expression were determined. (A1) Cells were loaded with 5 μM Fluo3, the histograms of fluorescent intensity of Fluo-3 on C6 cells were determined by flow cytometry analysis. (A2) The bar graphs represent average ± SD of Ca2 + fluorescent intensity of C6 cells from three independent experiments. (B) NFAT1 expression in C6 glioma cells were examined by immunofluorescence staining after MENK treatment (original magnification, × 100). (C) In order to knockdown NFAT1 gene expression, NFAT1 small hairpin RNA (shRNA) plasmid and control shRNA plasmid were transfected into C6 cells, respectively. NFAT1 gene expression in C6, control shRNA cells and NFAT1 shRNA cells was monitored using Western blot analysis.(D) CCK-8 assay was utilized to evaluate the cell viability in control shRNA cells and NFAT1 shRNA cells. The data were presented as the mean ± SD of three independent experiments. **p < 0.01 versus control group.
death activators though the activation of a cascade of proteolytic enzymes termed caspases. Two main apoptotic pathways have been mentioned as follows: the intrinsic mitochondrion pathway and the extrinsic death receptor pathway [21]. The intrinsic apoptotic pathway involves the release from the mitochondria into the cytosol of cytochrome c and other gene oriented apoptotic proteins [22]. This
directly inhibit the growth of rat C6 glioma in vitro and in vivo, and prolong the survival times in tumor-bearing rats, at the optimum concentration of 20 mg/kg in vivo and 10 mg/ml in vitro. Moreover, the study of Wang et al. has shown that MENK may inhibit growth and induce apoptosis of A375 melanoma cells [13]. Apoptosis can be triggered via signals within the cell or by extrinsic
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Fig. 6. Effect of MENK on the activities of caspase-3, caspase-8 and caspase-9 in vitro and apoptosis associated protein expressions in vitro and in vivo. (A) The cell samples were exposed to 10 mg/ml MENK or vehicle for 48 h and the activities of caspase-3, caspase-8 and caspase-9 were detected by colorimetric assay as described. (B1) The cell samples were exposed to 10 mg/ml MENK or vehicle for 48 h and the rats were treated with 20 mg/kg MENK or vehicle for 14 days. Subsequently, the cells and brain glioma tissues were collected and processed for Western bolt. Expressions of Fas, FasL, Bax, Bcl-2 and caspase-3 in vitro and in vivo were determined. (B2) The IDVs above proteins were shown. The data were presented as the mean ± SD of three independent experiments. *p < 0.05 and *p < 0.01 versus the control group.
receptors MOR and DOR on C6 glioma cells and triggered a Ca2 + influx into the cytoplasm, resulting in translocation of NFAT1 into the nucleus. The hyper-activation of NFAT1 may regulate transcription of downstream gene, such as FasL, and finally induced apoptosis in C6 glioma cells.
apoptotic pathway is regulated by the Bcl-2 family of proteins integrated by anti-apoptotic (Bcl-2, Bcl-xL, etc.) and pro-apoptotic (BH3 only family and Bax family) members [23]. The extrinsic apoptotic pathway is mediated by the tumor necrosis factor receptor (TNF-R) superfamily of death receptors. Fas (CD95 or APO-1) is a prototypical member of this family, whose binding with cytokines belonging to the TNF family, such as Fas ligand (FasL), induces cell death through caspase activation [24]. In this study, our results showed that MENK could induce rat C6 glioma cell apoptosis in vitro and in vivo, and MENK could increase the activities of caspase-3, caspase-8 and caspase-9 in vitro. Moreover, the expressions of Fas, FasL, Bax and caspase-3were increased after MENK treatment. However, the expression of Bcl-2 was decreased after MENK exposure in vitro and in vivo. These results proved that MENK inhibited growth and induced apoptosis of rats C6 glioma cells in vitro and in vivo. The induction of apoptosis appeared to be related with the upregulation of Fas/FasL, Bax, activation of caspase-3, caspase-8, caspase-9 and downregulation of Bcl-2, triggering major apoptotic cascades. It should be further noted that the study has also delved into the mechanisms through which MENK induced the apoptosis of rat C6 glioma. First of all, we confirmed the expressions of opioid receptors MOR and DOR on C6 glioma cells and also found that treatment with MENK increased the expression of these receptors. We further explored some aspects of the signaling events triggered by the interaction of MENK with opioid receptors on C6 glioma cells. The influx of Ca2 + could play a key role in a plethora of signaling pathways. Ca2 + influx into the cytoplasm has been shown to activate CaM/calcineurin protein phosphatase complexes, leading to de-phosphorylation of cytoplasmic NFAT-1 [25,26], which in turn translocates this factors into the nucleus, and binds to its target promotor elements and regulates the transcription of related genes, such as FasL [18,19,27]. The expression of Fas and the induced-expression of FasL is mainly responsible for the process of apoptosis [28–31]. In the present study, our results showed that MENK increased the Ca2 + influx into the cytoplasm in C6 glioma cell, and after treatment with MENK in vitro, there was a substantial increase of NFAT1 accumulation in the nuclei by immunofluorescence staining. In addition, we proceeded to explore the role of NFAT1 in MENK-induced cell apoptosis. When we specifically knocked down NFAT1 expression, there was no effect of MENK on the cell viability in NFAT1 knocked-down cell. The results of Western bolt showed that the expressions of Fas and FasL increased after MENK treatment. However, when NFAT1 was specifically knocked down, there was no effect of MENK on the FasL up-regulation. These results demonstrated that MENK could bind to opioid
5. Conclusion We believe that it is the first time that the current approach demonstrates that NFAT1 hyper-activation by MENK could significantly induce the apoptosis of rats C6 glioma cell through upregulation of Fas/ FasL and Bax, activation of caspase-3, caspase-8, caspase-9 and downregulation of Bcl-2. Clinically MENK might be with potential for the therapy of human glioma and as an adjunct to other chemotherapies. Also the present study will provide new mode of action for the chemotherapy of brain glioma. Conflict of interest The authors declare that there is no conflict of interest and we would take legal responsibility if anything would happen to this paper. Acknowledgements This work was supported financially by the grant from Science and Technology Plan Project of Educational Department of Liaoning Province (No. L2015535) and the grant from China National Funding for Natural Science (No. 31670921 to Fengping Shan). References [1] J.M. Kuijlen, E. Bremer, J.J. Mooij, W.F. den Dunnen, W. Helfrich, Review: on TRAIL for malignant glioma therapy? Neuropathol. Appl. Neurobiol. 36 (3) (2010 Apr) 168–182. [2] E.M. Kemper, W. Boogerd, I. Thuis, J.H. Beijnen, O. van Tellingen, Modulation of the blood-brain barrier in oncology: therapeutic opportunities for the treatment of brain tumours? Cancer Treat. Rev. 30 (5) (2004 Aug) 415–423. [3] S.J. Weber, T.J. Abbruscato, E.A. Brownson, A.W. Lipkowski, R. Polt, A. Misicka, R.C. Haaseth, H. Bartosz, V.J. Hruby, T.P. Davis, Assessment of an in vitro bloodbrain barrier model using several [Met5]enkephalin opioid analogs, J. Pharmacol. Exp. Ther. 266 (3) (1993 Sep) 1649–1655. [4] M. Piva, J.I. Moreno, F.S. Jenkins, J.K.A. Smith, J.L. Thomas, C. Montgomery, C.B. Wilson, R.C. Sizemore, In vitro modulation of cytokine expression by enkephalin-derived peptides, Neuroimmunomodulation 12 (6) (2005) 339–347. [5] D.M. Avella, E.T. Kimchi, R.N. Donahue, H.R. Tagaram, P.J. McLaughlin, I.S. Zagon, K.F. Staveley-O'Carroll, The opioid growth factor-opioid growth factor receptor axis regulates cell proliferation of human hepatocellular cancer, Am. J. Phys. Regul. Integr. Comp. Phys. 298 (2) (2010 Feb) R459–R466.
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