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Ginsenoside F1 suppresses astrocytic senescence-associated secretory phenotype
Chemico-Biological Interactions 283 (2018) 75–83
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Ginsenoside F1 suppresses astrocytic senescence-associated secretory phenotype
T
Jingang Houa, Changhao Cuia, Sunchang Kima,b,∗, Changkeun Sungc, Chulhee Choid a
Intelligent Synthetic Biology Center, Daejeon 34141, Republic of Korea Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea c Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University, Daejon 305764, Republic of Korea d Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Keywords: Astrocytic senescent Ginsenoside F1 p38MAPK NF-κB SASP Glioblastoma
Senescence is one of the hallmarks of aging and identified as a potential therapeutic target in the treatment of aging and aging-related diseases. Senescent cells accumulate with age in a variety of human tissues where they develop a complex senescence-associated secretory phenotype (SASP). SASP in brain could contribute to agerelated inflammation and chronic neurodegenerative diseases. We confirmed that senescent astrocytes express a characteristic of SASP in vitro by human cytokine antibody array. Ginsenoside F1 suppresses the SASP from astrocytes induced by D-galactose via suppressing p38MAPK-dependent NF-κB activity. A specific inhibitor of p38MAPK, SB203580 significantly decreased the secretion of IL-6 and IL-8, the major components of SASPs. Additionally, treatment of senescent astrocytes with NF-κB inhibitor, BAY 11–7092, also suppressed the secretion of IL-6 and IL-8, suggesting NF-κB was required for SASP. Importantly, conditioned media from senescent astrocytes promoted the migration of glioblastoma cells, such as U373-MG, U251-MG and U87-MG assessed by scratch wound healing. This migration was significantly decreased by F1 treatment in senescent astrocytes. Interestingly, IL-8, the main mediator regulating glioblastoma cell invasion, was suppressed in both transcriptional and protein level. Herein, we propose ginsenoside F1 as a potential therapeutic strategy for reducing the deleterious contribution of senescent astrocytes in aged brain and related diseases.
1. Introduction
and reactive astroglial cells contribute to aging of brain [15]. Interestingly, astrocytes undergo functional recession with age and partially lose their neuroprotective ability as well as exacerbating neuronal injury in age-related neurodegenerative processes [16,17]. Additionally, astrocytes in the aging brain express characteristics of senescence-associated secretory phenotype, giving rise to the decline in functional capacity of the brain [18]. Important evidence has been demonstrated that senescent astrocytes accumulate in age brain, and further, in brain from patient of Alzheimer's disease [19]. Ginsenosides has long been a popularity of great interest in a wide range of field. Up to date, great challenges by ginsenosides are antiaging and neuroprotective functions [20,21]. Furthermore, since most of senescent astrocytes express SASP and SASPs reprogram neighboring microenvironment [22]; we examined whether ginsenoside regulates SASP in astrocytes in the present study. We found that ginsenoside F1 significantly inhibited the expression and secretion of IL-6 and IL-8, the major SASP components claimed in aged brain [23]. Accordingly, we propose that ginsenoside F1 maybe one candidate for reducing the contribution of senescence in aging brain and related pathologies.
Aging is defined as a progressive loss of tissue and organ function over time [1,2]. Although aging itself is not a kind of disease, it is the largest risk factor for host of age-related diseases [3–6]. Nine tentative candidate hallmarks were recently enumerated as potential pharmaceutical targets to improve human health during aging, with minimal side effects [7]. Among these hallmarks is cellular senescence. Cellular senescence is one of the two independent stress-response mechanisms that is able to initially maintain the tissue integrity when organism repair (DNA) falls short [8,9]. Cellular senescence causes a permanent cell growth arrest of proliferative cells and develop senescence-associated secretory phenotype (SASP) that includes cytokines, chemokines, growth factors [10]. This emerging evidence suggest that cellular senescence is a potent anticancer mechanism, but also has been indicating as a driver for aging and aging related diseases [11,12]. Senescent cells are lurking in the heart, liver, kidney and even brain tissues [13,14]. Astrocytes are fundamental for homoeostasis, defense and regeneration of central nervous system. Loss of astroglial function
∗
Corresponding author. 291 Daehak-ro, Yuseong-gu, Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea. E-mail address: [email protected] (S. Kim).
https://doi.org/10.1016/j.cbi.2018.02.002 Received 17 October 2017; Received in revised form 31 December 2017; Accepted 1 February 2018 Available online 03 February 2018 0009-2797/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods 2.1. Cells and cell culture Astrocytic CRT and U373-MG cells were maintained in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS,G), 100U of penicillin/mL, and 100 μg of streptomycin/mL as previously described [24]. U251-MG-EGFP cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100U of penicillin/mL, and 100 μg of streptomycin/mL. Primary rat and human astrocytes were maintained in 10% FBS-DMEM containing 1% nonessential amino acids (Gibco-BRL, Grand Island, NY, USA). Stable cell line CRT-MG/IL-8p-d2EGFP cells were prepared and maintained as previously described [25]. Hippocampi were aseptically dissected out from embryonic Day 18 Sprague-Dawley rat embryos acquired from OrientBio, Inc. (Seongnam, Korea). After trituration and trypsinization, hippocampal cells were resuspended in plating medium (86.55% of MEM Eagle's with Earle's BSS, 10% re-filtered and heat inactivated FBS, 0.45% of glucose, 100 μM of sodium pyruvate, 200 μM glutamine and 100 mg/L streptomycin, 100 U/mL penicillin). The single-cell suspension was seeded in 100 mm petri-dishes containing poly-L-lysine coated coverslips at a density of 5 × 105/mL. After 4 h, cells were maintained in Neurobasal medium supplemented with 1% B27, 200 μM glutamine, 100U of penicillin/mL, and 100 μg of streptomycin/mL in a humidified atmosphere of 5% CO2 at 37 °C. Cerebral cortices were trypsinized and resuspended in plating medium DMEM20S (DMEM, 4 mM L-glutamine, 1 mM sodium pyruvate, 20% FBS, 50 U/mL penicillin and 50 mg/L streptomycin). The cell suspension was seeded in a T75 poly-D-lysine-coated flask (approximately 10 million cells), and fed every 2–3 days with 10 ml DMED20S for 10 days. Then the flask was pre-shaken on the shaker for 1 h at 200 r.p.m. at 37 °C to remove microglial cells. After a shake at 200 r.p.m. overnight, the medium were collected and incubated in untreated petri-dish to remove microglia and astrocytes. Subsequently, cells were pelleted and plated onto poly-D, L-ornithine-coated petridishes to achieve a density of 1 × 104 per cm2 with oligodendrocyte progenitor cell medium (containing 10 ng/mL PDGF-AA and 10 ng/mL bFGF) in a humidified atmosphere of 5% CO2 at 37 °C. 2.2. Reagents Fig. 1. Selected ginsenosides suppresses IL-6 secretion. A. Senescent astrocytic CRT cells were prepared with 20 g/L D-galactose for 14 days. Ginsenoside F1, Rh1, Rg3, Rh2 and CK were used with 20 μg/mL to treat senescent astrocytic CRT cells at the 12th day, after which conditioned medium (CM) were collected and analyzed with Ray Biotech IL-6 ELISA kit. Cells treated with DMSO (vehicle) as negative control and 100 nM Corticosterone as positive control. B. Senescent astrocytic cells were treated with a serial of ginsenoside F1. CM were collected and analyzed for IL-6 secretion. * indicates p < 0.05 versus DMSO, ** indicates p < 0.01 versus DMSO.
Ginsenosides Rg1, Re, F1, Rh1, Rg2, PPT, Rb1, Rd, Gyp75, F2, Rg3, Rh2, CK and PPD, with a purity of more than 98%, were prepared with High Performance Liquid Chromatography (HPLC). Each ginsenoside was dissolved in dimethyl sulfoxide (DMSO) as 10 mg/mL solution. Corticosterone, BAY11-7082, SB203580 and D-galactose were purchased from Sigma (St. Louis, MO). 2.3. Senescence induction and assessment
www.cellprofiler.org). SA-β-gal staining was quantified by light microscopy and a researcher that was blinded to the treatments. Specific inhibitor of p38MAPK, SB203580 and NF-κB inhibitor, BAY 11–7092 were added at 12th day in case of SASP assessment.
Astrocytic CRT cells were induced to senescence by exposure to 20 g/L D-galactose supplemented in complete culture medium. Briefly, proliferating cells were treated with above indicated concentration of Dgalactose for consecutive 14 days. Later, cells were scored for senescence markers, including SA-β-gal activity and the presence of persistent DNA-damage foci. Senescence-associated β-galactosidase (SA-βgal) staining was performed using a SA-β-gal kit (#9860, Cell Signaling Technology, Inc. MA) in accordance with the manufacturer's protocol. The cells were fixed for 10–15 min at room temperature, then rinsed twice with PBS and stained with staining solution at a final pH of 6.0 for at least overnight. The SA-β-gal positive cells develop blue color and were counted under a phase-contrast microscope. DNA-damage foci were assessed by immunostaining for 53BP1. For DNA-damage foci and SA-β-gal positivity, random fields were shown. Fluorescent images were quantified using CellProfiler, an open source software program (http://
2.4. Cell proliferation assay Cell viability was evaluated by the WST-1 assay, which is based on the enzymatic cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenase present in viable cells. In brief, after 24 h treatment, 20 μl of WST-1 was added to each well and the plates were incubated at 37 °C for 2 h. The plates were then centrifuged and 100 μl of the medium was withdrawn to be determined by measuring the absorbance value at a wavelength of 450 nm using microplate reader. 76
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Fig. 2. F1 suppresses the secretion of other SASP factors. A. CM from non-senescent or senescent cells treated with DMSO or F1 for 14 days were analyzed by antibody arrays. The average signal from non-senescent cells were used as the baseline. B. Senescent astrocytic CRT cells with construct IL-8p-d2EGFP were treated with DMSO or F1 for 14 days and fluorescence density were analyzed. C. The secretion of IL-8 was analyzed by Ray Biotech ELISA kit. * indicates p < 0.05 versus DMSO.
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equivalent to 1 × 106 cells per 1.5 ml, and applied to antibody array (Ray Biotech). The signals were detected with Kodak chemiluminescent imaging system. Signals were averaged and expressed as described in figure legend. 2.7. Quantification of IL-6 and IL-8 in conditioned media The conditioned medium were collected from both presenescent and senescent cells after 48 h in serum-free medium as described above. IL-6 and IL-8 levels in conditioned media were quantified using Human IL-6 and IL-8 ELISA Ray Biotech protocol respectively. 2.8. Scratch wound healing assay U373-MG cells were scraped off from the bottom of a culture plate using a pipette tip to produce a cell-free area. U373-MG cells were washed with RPMI to remove cell debris and then incubated with conditioned media prepared from senescent astrocytic CRT cells treated without or with p38 inhibitor SB203580, ginsenoside F1 (30 μg/mL) for 24 h in 3% FBS culture media. The wound areas were imaged at 0 h and 24 h and quantified using CellProfiler Software (2.2.0). 2.9. Western blotting Cell lysate was prepared from various indicated treatments. The samples were loaded into a 10% SDS-PAGE and separated by electrophoresis for 2 h at 100 V. Protein were transferred polyvinylidene difluoride membrane for 1 h at 110 V. The following primary antibodies were used: antibody p38, phosphorylated-p38, NF-κBp65 (rabbit polyclonal, 1:2000, CST, USA); GAPDH and lamin B1 as loading control (rabbit polyclonal, 1:4000, Santa Cruz, USA; rabbit monoclonal, 1:3000, Cell signaling Technology). The bands from the Western blots were densitometrically visualized and the signals quantified using ImageJ software. For immunofluorescence assay, the cells were first washed twice with PBS, then cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with buffer (0.15% Triton X-100 in PBS) for 20 min at room temperature. Cells were blocked with 3% BSA for 30min and then incubated with primary antibody against 53BP1 (rabbit polyclonal, Abcam, 1:200) overnight at 4 °C. Following washing three times for 5 min each with PBS contain 0.05% Triton X-100. Cells were incubated with appropriate Alexa 488 fluorogenic secondary antibody (Invitrogen) to detect the signal at room temperature for 1.5 h. After another set of washing, cell were mounted with DAPI and images were captured with Zeiss inverted epifluorescence microscope.
Fig. 3. F1 suppresses both establishment and maintenance of SASP. A. Effects of two different F1 regimens on IL-6 secretion by senescent astrocytic CRT cells, one treated immediately after induction (day 0) and continued for 14 days, the other treated 10 days after induction and continued for 4 days. B. On day 14, senescent astrocytic CRT cells were washed and incubated with serum-free medium. Samples for subsequent time points were similar washed and replaced with serum-free medium 24 h before collection. CM from all the time points were analyzed at day 24.
2.5. Human MAPK phosphorylation array Presenescent and senescent astrocytic CRT cells were treated with 20 g/L D-galactose for 14 days, and then lysed with array lysis buffer. Quantitation of protein concentration was performed by BCA assay. Cell lysates (500 μg) were applied to the array assay according to the manufacturer's protocol (Ray Biotech). Briefly, the membranes were blocked for 30 min on a rocking platform shaker. Cell lysates were diluted in array buffer and incubated overnight at 4 °C. Then the arrays were incubated with detection antibody cocktail for 1.5 h at RT, followed by streptavidin-HRP for 1 h.The arrays were imaged with chemiluminescent imaging system (Kokda, Japan). The MAPK phosphorylation activity were determined by densitometric analysis.
2.10. Statistical analyses All data that show error bars are presented as mean ± s.e.m. The significance of difference in the mean was determined using Student's ttest and one-way ANOVA unless otherwise mentioned. P < 0.05 was considered significant. All calculations were performed using GraphPad Prism software. 3. Results
2.6. Antibody array
3.1. Selected ginsenosides decrease SASP
The conditioned medium for anti-body array analyses were prepared by washing approximately 3–4 × 106 presenescent and senescent cells 3 times with PBS, and incubating them with serum-free medium for 48 h. The conditioned medium were collected and the remained cells were counted to normalize conditioned medium volumes for cell number. Then they were centrifuged for 20 min at 5000 rpm, filtered through 0.22 μm bottle-top filters (Sartorius Stedim Biotech, Göttingen, Germany) diluted with serum-free medium to a concentration
On screening the ginsenoside compounds for ability to regulate the secretion of IL-6, a major SASP component in mouse and human cells [26], we identified selected ginsenosides F1, Rh1, Rg3, Rh2 and CK as significantly more competent than the vehicle (DMSO) control (Fig. 1A). These selected ginsenosides reduced IL-6 secretion by both astrocytic CRT and primary rat astrocytes. However, the following studies used F1 as only ginsenoside F1 was detected in brain after intravenous injection. Additionally, apart from F1, above mentioned 78
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Fig. 4. F1 does not act on DNA-damage response signaling. Astrocytic CRT cells were treated with DMSO or F1 for indicated durations and immunostained for DNA-SCAR marker 53BP1. The number of 53BP1 foci was determined using CellProfiler. Shown is the percentage of cells with > 2 53BP1 foci.
days after consecutive D-galactose treatment; Fig. 3A). Intriguingly, we also examined the recovery of IL-6 secretion after F1 treatment. The senescent astrocytic CRT cells were treated with F1 or DMSO for consecutive 14 days, then cells were replaced with fresh normal medium. The following 10 days, conditioned media were collected and IL-6 secretion was quantified (Fig. 3B). F1 delayed the recovery for IL-6 levels for 4 days. Thus, F1 suppressed both establishment and maintenance of the SASP.
ginsenosides started to show cytotoxicity to normal neurons and oligodendrocyte progenitor cells (not published data). F1 decreased IL-6 secretion in a dose-dependent manner, reaching maximal suppression of about 57% at 30 μg/mL (Fig. 1B). We used 30 μg/ml F1 for subsequent experiments. To test the hypothesis whether F1 decreased the secretion of other SASP factors, we measured the level of 42 secreted proteins using antibody arrays (Fig. 2A). Of the factors detected by arrays and secreted at significantly higher level (GRO, IL-6, IL-8, MCP-1 and OSM) by senescent astrocytic cells, F1 reduced the secretion of IL-6 and IL-8. Notably, these two F1-sensitive SASP factors were previously proposed as major components as well as the targets of NF-κB transcription factor [27]. To address the possibility that F1 affects IL-8 expression at the transcriptional level, IL-8 promoter activity was examined. Astrocytic CRT cells were stably transfected with construct IL-8p-d2EGFP, which is a destabilized enhanced green fluorescent protein (EGFP)-expressing plasmid under the control of the IL-8 promoter. As we postulated, F1 reduced the IL-8 EGFP reporter activity by 3.5 fold (Fig. 2B). Additionally, we next determined IL-8 protein expression by ELISA. The IL-8 protein expression was normalized to the cell number (Fig. 2C). Collectively, F1 leads to the reduction of IL-8 mRNA and protein expression and this regulation is mediated at the transcriptional level. In culture, the SASP normally requires 5–7 days to develop once cells exposed to senescence-inducing stimulus [28]. Ginsenoside F1 reduced IL-6 expression not only when supplemented immediately after senescence stimulus but also after SASP fully developed (for example, 7
3.2. F1 does not interfere with DNA-damage response signaling SASP maintenance requires persistent DNA-damage response signaling (DDR), comprising stable nuclear foci termed DNA-SCARS (DNA segments with chromatin alterations). To test the possibility that F1 inhibits the SASP by interfering with DDR signaling, we determined the alterations of 53BP1, a typical feature of DNA-SCARS [29]. Ginsenoside F1 did not statistically alter the number of 53BP1 foci in senescent astrocytic CRT cells, indicating that F1 acted downstream of DDR signaling or other parallel pathways (Fig. 4). 3.3. F1 decreases NF-κB activity by decreasing p38 MAPK activation In fibroblasts model, NF-κB signaling controls the appearance of SASP and regulates the transcript of SASP genes [30]. In addition, as astrocyte senescence is strongly connected to p38MAPK activation confirmed by human MAPK phosphokinase signaling arrays (Fig. 5A), 79
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Fig. 5. F1 decreases p38MAPK-dependent NF-κB activity. A. Non-senescent cell or senescent cells treated with DMSO or F1 for 14 days were collected and analyzed by MAPK antibody arrays. The average signal from non-senescent cells were used as the baseline. B. Extracts from non-senescent or senescent astrocytic CRT cells, treated with SB203580 or F1 were analyzed for the indicated proteins by western blotting, GAPDH and lamin B1 served as the loading control. C. Specific inhibitor of p38MAPK, SB203580 and F1 suppressed the secretion of IL-6. ** indicates p < 0.01 versus DMSO. D. NF-κB inhibitor, BAY11-7082, decreased the secretion of IL-6 and nuclear translocation of NF- κB in senescent astrocytic CRT cells. * indicates p < 0.05 versus DMSO, ** indicates p < 0.01 versus DMSO.
Ginsenoside F1 significantly decreases the expression levels of p38MAPK and NF-κB from nuclear extract lysates (Fig. 5B) and the secretion of IL-6 (Fig. 5C). Furthermore, NF-κB inhibition (BAY117082) also significantly decrease the secretion of IL-6 and the nuclear
we asked whether ginsenoside F1 decreases SASP by p38MAPK-dependent NF-κB activation. Therefore, we examined the contribution of p38MAPK on NF-κB activation in senescent astrocytic CRT cells using ginsenoside F1 and specific inhibitor for p38MAPK SB203580 80
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Fig. 6. F1 suppresses SASP from senescent astrocytes induced migration of glioblastoma cell but does not affect the viability of senescent cell and normal cells in brain. A. Representative Images of the scratch wound healing assay in U373-MG glioblastoma cells treated with CM from non-senescent astrocytic CRT cells, or senescent cells treated with DMSO, SB203580 or F1 for 48 h. Migration activity was determined with CellProfiler. Data are presented as the fold induction compared with non-senescent cells. ** indicates p < 0.01 versus non-treated cells; ## indicates p < 0.01 versus DMSO. B. Senescent astrocytic CRT cells were treated with DMSO, 1 μM staurosporin and 30 μg/mL F1 for 48 h, then cells were stained with caspase3/7 fluorescent probe. Positive cells were imaged and calculated. ** indicates p < 0.01 versus non-senescent group. C. 30 μg/mL F1 were used to treat primary rat astrocytes, primary rat neuron and rat oligodendrocyte progenitor cells for 7 days, and cell viability were assayed compared with non-treated groups.
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translocation of NF-κB analyzed by fluorescence microscopy (Fig. 5D), suggesting that NF-κB is required for the SASP.
Additionally, since p38MAPK regulates both SASP and NF-κB activity, we asked whether NF-κB is required for SASP. We used the NF-κB inhibitor BAY 11–7092 to treat senescent astrocytes. NF-κB inhibition significantly decreased the secretion of IL-6 and IL-8. Collectively, these demonstrated that IL-6 and IL-8 secretion induced by p38MAPK activation were NF-κB dependent. Thus, p38MAPK acts primarily through NF-κB to induce the SASP. A possible explanation for F1's suppression on p38MAPK activity might be consistent with a computer docking and molecular modeling study that F1 can act like drug and interact with p38MAPK active sites but does not affect the enzyme's structural stability [37]. Interleukin-8 (IL-8, CXCL8), a CXC chemokines, was crucial mediator of glioblastoma multiforme (GBM) malignancy and pathogenesis [38,39]. Previous studies indicated that IL-8 secretion was in response to various stimuli, such as astrocytes and glioblastoma cell induced by LPS, TNF-α and hypoxia [40–42]. Our results observed that senescent astrocytes increased IL-8 in both transcriptional and protein level. This secretion significantly promoted the GBM (U373-MG, U251-MG and U87-MG) migration. While F1 suppressed IL-8 secretion from senescent astrocytes, suggesting that F1 may be a potential candidate for mitigation on brain cancer development or invasion in the aged. Additionally, normal counterparts such as neuron and oligodendrocyte progenitor cells were not affected by the required dose of F1, indicating a safe therapeutic intervention in potential senescence-associated diseases in brain.
3.4. F1 inhibits the migration of glioblastoma induced by SASP SASP is reported to disrupt normal tissue structure, promote malignant phenotype and even promote tumor growth [31]. To test the idea that SASP from senescent astrocytes promotes migration of glioblastoma and confirm the effects of F1 on the SASP, we assessed the cell migration of U373-MG treated with SASP from astrocytic CRT cells with a scratch would healing assay. The migration was greatly increased by SASP from astrocytic CRT cells treated with DMSO, but significantly retained by that of SASP treated with 30 μg/mL of F1 or p38MAPK specific inhibitor SB203580 (Fig. 6A). Similar effects were also observed in U251-MG and U87-MG cells (Data not shown). The results suggested that astrocytic SASP promote the migration of glioblastoma. To test whether F1 has deleterious effects on non-senescent counterparts, we asked if it induces apoptosis or inhibits cell proliferation. Primary rat neurons, primary rat and human astrocytes, and oligodendrocyte progenitor cells were treated with DMSO or 30 μg/ml F1 and analyze apoptosis by activated caspase-3/7 activity in above mentioned cell types. F1 failed to induce cell death in any of these cell types (Fig. 6B). In addition, 30 μg/ml F1 does not inhibit the cell proliferation in all cell types after 7 days treatment (Fig. 6C). 4. Discussion
5. Conclusions The principles of the present study have demonstrated that ginsenoside F1 decreased the SASP of astrocytes through regulation of IL-6, and the suppression on the secretion of IL-6 was modulated by p38MAPK and NF-κB activation. Furthermore, F1 also lead to the decrease of IL-8 on both transcriptional and protein levels. Importantly, conditioned media from senescent astrocytes greatly promoted the migration of U373-MG, U251-MG and U87-MG cells, while this deleterious effect was abrogated by that of F1 treatment. Judith Campisi and her research team first revealed that senescent cells are not merely defined as irreversible cell-growth arrest, they secret a myriad of factors associated with inflammation and oncogenesis. They termed this cellular state as the senescence-associated with secretory phenotype (SASP). SASP mainly include interleukins and chemokines, such as, IL-6, IL-8, MCP-1, growth factors, such as EGF and VEGF, and several matrix metalloproteinases [32]. SASP contributes to ‘inflammaging (low level and chronic inflammation in brain)’, which is thought to cause to most, if not all, major pathologies associated with aging [33]. Interestingly, in the case of Alzheimer's disease (AD), senescent astrocytes were reported in human brain tissue and expressed SASP factors especially higher in aged patients. It is also confirmed by Amyloid-β peptide treated astrocytes in vitro [19]. DNA damage is the common contributor inducing cell cycle arrest and cellular senescence associated with accumulation of chromatin changes, such as DNASCARS [34]. It is well demonstrated that DNA damage can stimulate a robust secretion of inflammatory factors and a unanimous model in SASP studies [35]. To test the idea that F1 might decrease SASP by preventing DNA damage, we examined the typical marker 53BP1 foci and found no statistical differences. F1 did not significantly alter the number of 53BP1 in senescent astrocytes, indicating that F1 may act in the downstream or parallel pathways. Mammalian p38 mitogen-activated protein kinase (p38MAPK) is the signaling target of a wide variety of cellular stresses, especially DNA damage. In HCA2 cell model, p38MAPK activity is sufficient to induce an SASP and demonstrated that the IL-6, IL-8 and GM-CSF secretion through activated NF-κB was controlled by p38MAPK [36]. We therefore asked whether NF-κB activity increases during astrocytic senescence, and whether the increase is p38MAPK dependent. Interestingly, p38MAPK inhibition (SB203580) significantly decreased the NF-κB nuclear translocation (P < 0.05). We conclude that NF-κB is regulated by p38MAPK during senescence.
In summary, our findings propose a potential mechanism for suppression of the SASP by which ginsenoside F1 might suppress brain agerelated pathologies, including brain cancer. Importantly, understanding how F1 control the SASP of senescent astrocytes could provide a potentially therapeutic approach in development antiaging drug. Conflicts of interest The authors declare no conflict of interest. Acknowledgement This work was supported by the Intelligent Synthetic Biology Center of the Global Frontier Project, funded by the Ministry of Education, Science and Technology, Republic of Korea. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2018.02.002. References [1] J.A. Buckwalter, S.L. Woo, V.M. Goldberg, E.C. Hadley, F. Booth, T.R. Oegema, D.R. Eyre, Soft-tissue aging and musculoskeletal function, J. Bone Joint Surg. Am. 75 (1993) 1533–1548. [2] F.B. Johnson, D.A. Sinclair, L. Guarente, Molecular biology of aging, Cell 96 (1999) 291–302. [3] D.G. Nicholls, Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease, Int. J. Biochem. Cell Biol. 34 (2002) 1372–1381. [4] L.J. Yan, Positive oxidative stress in aging and aging-related disease tolerance, Redox Biol. 2 (2014) 165–169. [5] T. Fulop, A. Larbi, J.M. Witkowski, J. McElhaney, M. Loeb, A. Mitnitski, G. Pawelec, Aging, frailty and age-related diseases, Biogerontology 11 (2010) 547–563. [6] J. Neves, M. Demaria, J. Campisi, H. Jasper, Of flies, mice, and men: evolutionarily conserved tissue damage responses and aging, Dev. Cell 32 (2015) 9–18. [7] C. Lopez-Otin, M.A. Blasco, L. Partridge, M. Serrano, G. Kroemer, The hallmarks of aging, Cell 153 (2013) 1194–1217. [8] T. Von Zglinicki, A. Bürkle, T.B. Kirkwood, Stress, DNA damage and ageing—an integrative approach, Exp. Gerontol. 36 (2001) 1049–1062. [9] F.d.A. Di Fagagna, Living on a break: cellular senescence as a DNA-damage response, Nat. Rev. Canc. 8 (2008) 512–522.
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Ginsenoside F1 suppresses astrocytic senescence-associated secretory phenotype
T
Jingang Houa, Changhao Cuia, Sunchang Kima,b,∗, Changkeun Sungc, Chulhee Choid a
Intelligent Synthetic Biology Center, Daejeon 34141, Republic of Korea Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea c Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University, Daejon 305764, Republic of Korea d Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Keywords: Astrocytic senescent Ginsenoside F1 p38MAPK NF-κB SASP Glioblastoma
Senescence is one of the hallmarks of aging and identified as a potential therapeutic target in the treatment of aging and aging-related diseases. Senescent cells accumulate with age in a variety of human tissues where they develop a complex senescence-associated secretory phenotype (SASP). SASP in brain could contribute to agerelated inflammation and chronic neurodegenerative diseases. We confirmed that senescent astrocytes express a characteristic of SASP in vitro by human cytokine antibody array. Ginsenoside F1 suppresses the SASP from astrocytes induced by D-galactose via suppressing p38MAPK-dependent NF-κB activity. A specific inhibitor of p38MAPK, SB203580 significantly decreased the secretion of IL-6 and IL-8, the major components of SASPs. Additionally, treatment of senescent astrocytes with NF-κB inhibitor, BAY 11–7092, also suppressed the secretion of IL-6 and IL-8, suggesting NF-κB was required for SASP. Importantly, conditioned media from senescent astrocytes promoted the migration of glioblastoma cells, such as U373-MG, U251-MG and U87-MG assessed by scratch wound healing. This migration was significantly decreased by F1 treatment in senescent astrocytes. Interestingly, IL-8, the main mediator regulating glioblastoma cell invasion, was suppressed in both transcriptional and protein level. Herein, we propose ginsenoside F1 as a potential therapeutic strategy for reducing the deleterious contribution of senescent astrocytes in aged brain and related diseases.
1. Introduction
and reactive astroglial cells contribute to aging of brain [15]. Interestingly, astrocytes undergo functional recession with age and partially lose their neuroprotective ability as well as exacerbating neuronal injury in age-related neurodegenerative processes [16,17]. Additionally, astrocytes in the aging brain express characteristics of senescence-associated secretory phenotype, giving rise to the decline in functional capacity of the brain [18]. Important evidence has been demonstrated that senescent astrocytes accumulate in age brain, and further, in brain from patient of Alzheimer's disease [19]. Ginsenosides has long been a popularity of great interest in a wide range of field. Up to date, great challenges by ginsenosides are antiaging and neuroprotective functions [20,21]. Furthermore, since most of senescent astrocytes express SASP and SASPs reprogram neighboring microenvironment [22]; we examined whether ginsenoside regulates SASP in astrocytes in the present study. We found that ginsenoside F1 significantly inhibited the expression and secretion of IL-6 and IL-8, the major SASP components claimed in aged brain [23]. Accordingly, we propose that ginsenoside F1 maybe one candidate for reducing the contribution of senescence in aging brain and related pathologies.
Aging is defined as a progressive loss of tissue and organ function over time [1,2]. Although aging itself is not a kind of disease, it is the largest risk factor for host of age-related diseases [3–6]. Nine tentative candidate hallmarks were recently enumerated as potential pharmaceutical targets to improve human health during aging, with minimal side effects [7]. Among these hallmarks is cellular senescence. Cellular senescence is one of the two independent stress-response mechanisms that is able to initially maintain the tissue integrity when organism repair (DNA) falls short [8,9]. Cellular senescence causes a permanent cell growth arrest of proliferative cells and develop senescence-associated secretory phenotype (SASP) that includes cytokines, chemokines, growth factors [10]. This emerging evidence suggest that cellular senescence is a potent anticancer mechanism, but also has been indicating as a driver for aging and aging related diseases [11,12]. Senescent cells are lurking in the heart, liver, kidney and even brain tissues [13,14]. Astrocytes are fundamental for homoeostasis, defense and regeneration of central nervous system. Loss of astroglial function
∗
Corresponding author. 291 Daehak-ro, Yuseong-gu, Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea. E-mail address: [email protected] (S. Kim).
https://doi.org/10.1016/j.cbi.2018.02.002 Received 17 October 2017; Received in revised form 31 December 2017; Accepted 1 February 2018 Available online 03 February 2018 0009-2797/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods 2.1. Cells and cell culture Astrocytic CRT and U373-MG cells were maintained in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS,G), 100U of penicillin/mL, and 100 μg of streptomycin/mL as previously described [24]. U251-MG-EGFP cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100U of penicillin/mL, and 100 μg of streptomycin/mL. Primary rat and human astrocytes were maintained in 10% FBS-DMEM containing 1% nonessential amino acids (Gibco-BRL, Grand Island, NY, USA). Stable cell line CRT-MG/IL-8p-d2EGFP cells were prepared and maintained as previously described [25]. Hippocampi were aseptically dissected out from embryonic Day 18 Sprague-Dawley rat embryos acquired from OrientBio, Inc. (Seongnam, Korea). After trituration and trypsinization, hippocampal cells were resuspended in plating medium (86.55% of MEM Eagle's with Earle's BSS, 10% re-filtered and heat inactivated FBS, 0.45% of glucose, 100 μM of sodium pyruvate, 200 μM glutamine and 100 mg/L streptomycin, 100 U/mL penicillin). The single-cell suspension was seeded in 100 mm petri-dishes containing poly-L-lysine coated coverslips at a density of 5 × 105/mL. After 4 h, cells were maintained in Neurobasal medium supplemented with 1% B27, 200 μM glutamine, 100U of penicillin/mL, and 100 μg of streptomycin/mL in a humidified atmosphere of 5% CO2 at 37 °C. Cerebral cortices were trypsinized and resuspended in plating medium DMEM20S (DMEM, 4 mM L-glutamine, 1 mM sodium pyruvate, 20% FBS, 50 U/mL penicillin and 50 mg/L streptomycin). The cell suspension was seeded in a T75 poly-D-lysine-coated flask (approximately 10 million cells), and fed every 2–3 days with 10 ml DMED20S for 10 days. Then the flask was pre-shaken on the shaker for 1 h at 200 r.p.m. at 37 °C to remove microglial cells. After a shake at 200 r.p.m. overnight, the medium were collected and incubated in untreated petri-dish to remove microglia and astrocytes. Subsequently, cells were pelleted and plated onto poly-D, L-ornithine-coated petridishes to achieve a density of 1 × 104 per cm2 with oligodendrocyte progenitor cell medium (containing 10 ng/mL PDGF-AA and 10 ng/mL bFGF) in a humidified atmosphere of 5% CO2 at 37 °C. 2.2. Reagents Fig. 1. Selected ginsenosides suppresses IL-6 secretion. A. Senescent astrocytic CRT cells were prepared with 20 g/L D-galactose for 14 days. Ginsenoside F1, Rh1, Rg3, Rh2 and CK were used with 20 μg/mL to treat senescent astrocytic CRT cells at the 12th day, after which conditioned medium (CM) were collected and analyzed with Ray Biotech IL-6 ELISA kit. Cells treated with DMSO (vehicle) as negative control and 100 nM Corticosterone as positive control. B. Senescent astrocytic cells were treated with a serial of ginsenoside F1. CM were collected and analyzed for IL-6 secretion. * indicates p < 0.05 versus DMSO, ** indicates p < 0.01 versus DMSO.
Ginsenosides Rg1, Re, F1, Rh1, Rg2, PPT, Rb1, Rd, Gyp75, F2, Rg3, Rh2, CK and PPD, with a purity of more than 98%, were prepared with High Performance Liquid Chromatography (HPLC). Each ginsenoside was dissolved in dimethyl sulfoxide (DMSO) as 10 mg/mL solution. Corticosterone, BAY11-7082, SB203580 and D-galactose were purchased from Sigma (St. Louis, MO). 2.3. Senescence induction and assessment
www.cellprofiler.org). SA-β-gal staining was quantified by light microscopy and a researcher that was blinded to the treatments. Specific inhibitor of p38MAPK, SB203580 and NF-κB inhibitor, BAY 11–7092 were added at 12th day in case of SASP assessment.
Astrocytic CRT cells were induced to senescence by exposure to 20 g/L D-galactose supplemented in complete culture medium. Briefly, proliferating cells were treated with above indicated concentration of Dgalactose for consecutive 14 days. Later, cells were scored for senescence markers, including SA-β-gal activity and the presence of persistent DNA-damage foci. Senescence-associated β-galactosidase (SA-βgal) staining was performed using a SA-β-gal kit (#9860, Cell Signaling Technology, Inc. MA) in accordance with the manufacturer's protocol. The cells were fixed for 10–15 min at room temperature, then rinsed twice with PBS and stained with staining solution at a final pH of 6.0 for at least overnight. The SA-β-gal positive cells develop blue color and were counted under a phase-contrast microscope. DNA-damage foci were assessed by immunostaining for 53BP1. For DNA-damage foci and SA-β-gal positivity, random fields were shown. Fluorescent images were quantified using CellProfiler, an open source software program (http://
2.4. Cell proliferation assay Cell viability was evaluated by the WST-1 assay, which is based on the enzymatic cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenase present in viable cells. In brief, after 24 h treatment, 20 μl of WST-1 was added to each well and the plates were incubated at 37 °C for 2 h. The plates were then centrifuged and 100 μl of the medium was withdrawn to be determined by measuring the absorbance value at a wavelength of 450 nm using microplate reader. 76
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Fig. 2. F1 suppresses the secretion of other SASP factors. A. CM from non-senescent or senescent cells treated with DMSO or F1 for 14 days were analyzed by antibody arrays. The average signal from non-senescent cells were used as the baseline. B. Senescent astrocytic CRT cells with construct IL-8p-d2EGFP were treated with DMSO or F1 for 14 days and fluorescence density were analyzed. C. The secretion of IL-8 was analyzed by Ray Biotech ELISA kit. * indicates p < 0.05 versus DMSO.
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equivalent to 1 × 106 cells per 1.5 ml, and applied to antibody array (Ray Biotech). The signals were detected with Kodak chemiluminescent imaging system. Signals were averaged and expressed as described in figure legend. 2.7. Quantification of IL-6 and IL-8 in conditioned media The conditioned medium were collected from both presenescent and senescent cells after 48 h in serum-free medium as described above. IL-6 and IL-8 levels in conditioned media were quantified using Human IL-6 and IL-8 ELISA Ray Biotech protocol respectively. 2.8. Scratch wound healing assay U373-MG cells were scraped off from the bottom of a culture plate using a pipette tip to produce a cell-free area. U373-MG cells were washed with RPMI to remove cell debris and then incubated with conditioned media prepared from senescent astrocytic CRT cells treated without or with p38 inhibitor SB203580, ginsenoside F1 (30 μg/mL) for 24 h in 3% FBS culture media. The wound areas were imaged at 0 h and 24 h and quantified using CellProfiler Software (2.2.0). 2.9. Western blotting Cell lysate was prepared from various indicated treatments. The samples were loaded into a 10% SDS-PAGE and separated by electrophoresis for 2 h at 100 V. Protein were transferred polyvinylidene difluoride membrane for 1 h at 110 V. The following primary antibodies were used: antibody p38, phosphorylated-p38, NF-κBp65 (rabbit polyclonal, 1:2000, CST, USA); GAPDH and lamin B1 as loading control (rabbit polyclonal, 1:4000, Santa Cruz, USA; rabbit monoclonal, 1:3000, Cell signaling Technology). The bands from the Western blots were densitometrically visualized and the signals quantified using ImageJ software. For immunofluorescence assay, the cells were first washed twice with PBS, then cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with buffer (0.15% Triton X-100 in PBS) for 20 min at room temperature. Cells were blocked with 3% BSA for 30min and then incubated with primary antibody against 53BP1 (rabbit polyclonal, Abcam, 1:200) overnight at 4 °C. Following washing three times for 5 min each with PBS contain 0.05% Triton X-100. Cells were incubated with appropriate Alexa 488 fluorogenic secondary antibody (Invitrogen) to detect the signal at room temperature for 1.5 h. After another set of washing, cell were mounted with DAPI and images were captured with Zeiss inverted epifluorescence microscope.
Fig. 3. F1 suppresses both establishment and maintenance of SASP. A. Effects of two different F1 regimens on IL-6 secretion by senescent astrocytic CRT cells, one treated immediately after induction (day 0) and continued for 14 days, the other treated 10 days after induction and continued for 4 days. B. On day 14, senescent astrocytic CRT cells were washed and incubated with serum-free medium. Samples for subsequent time points were similar washed and replaced with serum-free medium 24 h before collection. CM from all the time points were analyzed at day 24.
2.5. Human MAPK phosphorylation array Presenescent and senescent astrocytic CRT cells were treated with 20 g/L D-galactose for 14 days, and then lysed with array lysis buffer. Quantitation of protein concentration was performed by BCA assay. Cell lysates (500 μg) were applied to the array assay according to the manufacturer's protocol (Ray Biotech). Briefly, the membranes were blocked for 30 min on a rocking platform shaker. Cell lysates were diluted in array buffer and incubated overnight at 4 °C. Then the arrays were incubated with detection antibody cocktail for 1.5 h at RT, followed by streptavidin-HRP for 1 h.The arrays were imaged with chemiluminescent imaging system (Kokda, Japan). The MAPK phosphorylation activity were determined by densitometric analysis.
2.10. Statistical analyses All data that show error bars are presented as mean ± s.e.m. The significance of difference in the mean was determined using Student's ttest and one-way ANOVA unless otherwise mentioned. P < 0.05 was considered significant. All calculations were performed using GraphPad Prism software. 3. Results
2.6. Antibody array
3.1. Selected ginsenosides decrease SASP
The conditioned medium for anti-body array analyses were prepared by washing approximately 3–4 × 106 presenescent and senescent cells 3 times with PBS, and incubating them with serum-free medium for 48 h. The conditioned medium were collected and the remained cells were counted to normalize conditioned medium volumes for cell number. Then they were centrifuged for 20 min at 5000 rpm, filtered through 0.22 μm bottle-top filters (Sartorius Stedim Biotech, Göttingen, Germany) diluted with serum-free medium to a concentration
On screening the ginsenoside compounds for ability to regulate the secretion of IL-6, a major SASP component in mouse and human cells [26], we identified selected ginsenosides F1, Rh1, Rg3, Rh2 and CK as significantly more competent than the vehicle (DMSO) control (Fig. 1A). These selected ginsenosides reduced IL-6 secretion by both astrocytic CRT and primary rat astrocytes. However, the following studies used F1 as only ginsenoside F1 was detected in brain after intravenous injection. Additionally, apart from F1, above mentioned 78
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Fig. 4. F1 does not act on DNA-damage response signaling. Astrocytic CRT cells were treated with DMSO or F1 for indicated durations and immunostained for DNA-SCAR marker 53BP1. The number of 53BP1 foci was determined using CellProfiler. Shown is the percentage of cells with > 2 53BP1 foci.
days after consecutive D-galactose treatment; Fig. 3A). Intriguingly, we also examined the recovery of IL-6 secretion after F1 treatment. The senescent astrocytic CRT cells were treated with F1 or DMSO for consecutive 14 days, then cells were replaced with fresh normal medium. The following 10 days, conditioned media were collected and IL-6 secretion was quantified (Fig. 3B). F1 delayed the recovery for IL-6 levels for 4 days. Thus, F1 suppressed both establishment and maintenance of the SASP.
ginsenosides started to show cytotoxicity to normal neurons and oligodendrocyte progenitor cells (not published data). F1 decreased IL-6 secretion in a dose-dependent manner, reaching maximal suppression of about 57% at 30 μg/mL (Fig. 1B). We used 30 μg/ml F1 for subsequent experiments. To test the hypothesis whether F1 decreased the secretion of other SASP factors, we measured the level of 42 secreted proteins using antibody arrays (Fig. 2A). Of the factors detected by arrays and secreted at significantly higher level (GRO, IL-6, IL-8, MCP-1 and OSM) by senescent astrocytic cells, F1 reduced the secretion of IL-6 and IL-8. Notably, these two F1-sensitive SASP factors were previously proposed as major components as well as the targets of NF-κB transcription factor [27]. To address the possibility that F1 affects IL-8 expression at the transcriptional level, IL-8 promoter activity was examined. Astrocytic CRT cells were stably transfected with construct IL-8p-d2EGFP, which is a destabilized enhanced green fluorescent protein (EGFP)-expressing plasmid under the control of the IL-8 promoter. As we postulated, F1 reduced the IL-8 EGFP reporter activity by 3.5 fold (Fig. 2B). Additionally, we next determined IL-8 protein expression by ELISA. The IL-8 protein expression was normalized to the cell number (Fig. 2C). Collectively, F1 leads to the reduction of IL-8 mRNA and protein expression and this regulation is mediated at the transcriptional level. In culture, the SASP normally requires 5–7 days to develop once cells exposed to senescence-inducing stimulus [28]. Ginsenoside F1 reduced IL-6 expression not only when supplemented immediately after senescence stimulus but also after SASP fully developed (for example, 7
3.2. F1 does not interfere with DNA-damage response signaling SASP maintenance requires persistent DNA-damage response signaling (DDR), comprising stable nuclear foci termed DNA-SCARS (DNA segments with chromatin alterations). To test the possibility that F1 inhibits the SASP by interfering with DDR signaling, we determined the alterations of 53BP1, a typical feature of DNA-SCARS [29]. Ginsenoside F1 did not statistically alter the number of 53BP1 foci in senescent astrocytic CRT cells, indicating that F1 acted downstream of DDR signaling or other parallel pathways (Fig. 4). 3.3. F1 decreases NF-κB activity by decreasing p38 MAPK activation In fibroblasts model, NF-κB signaling controls the appearance of SASP and regulates the transcript of SASP genes [30]. In addition, as astrocyte senescence is strongly connected to p38MAPK activation confirmed by human MAPK phosphokinase signaling arrays (Fig. 5A), 79
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Fig. 5. F1 decreases p38MAPK-dependent NF-κB activity. A. Non-senescent cell or senescent cells treated with DMSO or F1 for 14 days were collected and analyzed by MAPK antibody arrays. The average signal from non-senescent cells were used as the baseline. B. Extracts from non-senescent or senescent astrocytic CRT cells, treated with SB203580 or F1 were analyzed for the indicated proteins by western blotting, GAPDH and lamin B1 served as the loading control. C. Specific inhibitor of p38MAPK, SB203580 and F1 suppressed the secretion of IL-6. ** indicates p < 0.01 versus DMSO. D. NF-κB inhibitor, BAY11-7082, decreased the secretion of IL-6 and nuclear translocation of NF- κB in senescent astrocytic CRT cells. * indicates p < 0.05 versus DMSO, ** indicates p < 0.01 versus DMSO.
Ginsenoside F1 significantly decreases the expression levels of p38MAPK and NF-κB from nuclear extract lysates (Fig. 5B) and the secretion of IL-6 (Fig. 5C). Furthermore, NF-κB inhibition (BAY117082) also significantly decrease the secretion of IL-6 and the nuclear
we asked whether ginsenoside F1 decreases SASP by p38MAPK-dependent NF-κB activation. Therefore, we examined the contribution of p38MAPK on NF-κB activation in senescent astrocytic CRT cells using ginsenoside F1 and specific inhibitor for p38MAPK SB203580 80
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Fig. 6. F1 suppresses SASP from senescent astrocytes induced migration of glioblastoma cell but does not affect the viability of senescent cell and normal cells in brain. A. Representative Images of the scratch wound healing assay in U373-MG glioblastoma cells treated with CM from non-senescent astrocytic CRT cells, or senescent cells treated with DMSO, SB203580 or F1 for 48 h. Migration activity was determined with CellProfiler. Data are presented as the fold induction compared with non-senescent cells. ** indicates p < 0.01 versus non-treated cells; ## indicates p < 0.01 versus DMSO. B. Senescent astrocytic CRT cells were treated with DMSO, 1 μM staurosporin and 30 μg/mL F1 for 48 h, then cells were stained with caspase3/7 fluorescent probe. Positive cells were imaged and calculated. ** indicates p < 0.01 versus non-senescent group. C. 30 μg/mL F1 were used to treat primary rat astrocytes, primary rat neuron and rat oligodendrocyte progenitor cells for 7 days, and cell viability were assayed compared with non-treated groups.
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translocation of NF-κB analyzed by fluorescence microscopy (Fig. 5D), suggesting that NF-κB is required for the SASP.
Additionally, since p38MAPK regulates both SASP and NF-κB activity, we asked whether NF-κB is required for SASP. We used the NF-κB inhibitor BAY 11–7092 to treat senescent astrocytes. NF-κB inhibition significantly decreased the secretion of IL-6 and IL-8. Collectively, these demonstrated that IL-6 and IL-8 secretion induced by p38MAPK activation were NF-κB dependent. Thus, p38MAPK acts primarily through NF-κB to induce the SASP. A possible explanation for F1's suppression on p38MAPK activity might be consistent with a computer docking and molecular modeling study that F1 can act like drug and interact with p38MAPK active sites but does not affect the enzyme's structural stability [37]. Interleukin-8 (IL-8, CXCL8), a CXC chemokines, was crucial mediator of glioblastoma multiforme (GBM) malignancy and pathogenesis [38,39]. Previous studies indicated that IL-8 secretion was in response to various stimuli, such as astrocytes and glioblastoma cell induced by LPS, TNF-α and hypoxia [40–42]. Our results observed that senescent astrocytes increased IL-8 in both transcriptional and protein level. This secretion significantly promoted the GBM (U373-MG, U251-MG and U87-MG) migration. While F1 suppressed IL-8 secretion from senescent astrocytes, suggesting that F1 may be a potential candidate for mitigation on brain cancer development or invasion in the aged. Additionally, normal counterparts such as neuron and oligodendrocyte progenitor cells were not affected by the required dose of F1, indicating a safe therapeutic intervention in potential senescence-associated diseases in brain.
3.4. F1 inhibits the migration of glioblastoma induced by SASP SASP is reported to disrupt normal tissue structure, promote malignant phenotype and even promote tumor growth [31]. To test the idea that SASP from senescent astrocytes promotes migration of glioblastoma and confirm the effects of F1 on the SASP, we assessed the cell migration of U373-MG treated with SASP from astrocytic CRT cells with a scratch would healing assay. The migration was greatly increased by SASP from astrocytic CRT cells treated with DMSO, but significantly retained by that of SASP treated with 30 μg/mL of F1 or p38MAPK specific inhibitor SB203580 (Fig. 6A). Similar effects were also observed in U251-MG and U87-MG cells (Data not shown). The results suggested that astrocytic SASP promote the migration of glioblastoma. To test whether F1 has deleterious effects on non-senescent counterparts, we asked if it induces apoptosis or inhibits cell proliferation. Primary rat neurons, primary rat and human astrocytes, and oligodendrocyte progenitor cells were treated with DMSO or 30 μg/ml F1 and analyze apoptosis by activated caspase-3/7 activity in above mentioned cell types. F1 failed to induce cell death in any of these cell types (Fig. 6B). In addition, 30 μg/ml F1 does not inhibit the cell proliferation in all cell types after 7 days treatment (Fig. 6C). 4. Discussion
5. Conclusions The principles of the present study have demonstrated that ginsenoside F1 decreased the SASP of astrocytes through regulation of IL-6, and the suppression on the secretion of IL-6 was modulated by p38MAPK and NF-κB activation. Furthermore, F1 also lead to the decrease of IL-8 on both transcriptional and protein levels. Importantly, conditioned media from senescent astrocytes greatly promoted the migration of U373-MG, U251-MG and U87-MG cells, while this deleterious effect was abrogated by that of F1 treatment. Judith Campisi and her research team first revealed that senescent cells are not merely defined as irreversible cell-growth arrest, they secret a myriad of factors associated with inflammation and oncogenesis. They termed this cellular state as the senescence-associated with secretory phenotype (SASP). SASP mainly include interleukins and chemokines, such as, IL-6, IL-8, MCP-1, growth factors, such as EGF and VEGF, and several matrix metalloproteinases [32]. SASP contributes to ‘inflammaging (low level and chronic inflammation in brain)’, which is thought to cause to most, if not all, major pathologies associated with aging [33]. Interestingly, in the case of Alzheimer's disease (AD), senescent astrocytes were reported in human brain tissue and expressed SASP factors especially higher in aged patients. It is also confirmed by Amyloid-β peptide treated astrocytes in vitro [19]. DNA damage is the common contributor inducing cell cycle arrest and cellular senescence associated with accumulation of chromatin changes, such as DNASCARS [34]. It is well demonstrated that DNA damage can stimulate a robust secretion of inflammatory factors and a unanimous model in SASP studies [35]. To test the idea that F1 might decrease SASP by preventing DNA damage, we examined the typical marker 53BP1 foci and found no statistical differences. F1 did not significantly alter the number of 53BP1 in senescent astrocytes, indicating that F1 may act in the downstream or parallel pathways. Mammalian p38 mitogen-activated protein kinase (p38MAPK) is the signaling target of a wide variety of cellular stresses, especially DNA damage. In HCA2 cell model, p38MAPK activity is sufficient to induce an SASP and demonstrated that the IL-6, IL-8 and GM-CSF secretion through activated NF-κB was controlled by p38MAPK [36]. We therefore asked whether NF-κB activity increases during astrocytic senescence, and whether the increase is p38MAPK dependent. Interestingly, p38MAPK inhibition (SB203580) significantly decreased the NF-κB nuclear translocation (P < 0.05). We conclude that NF-κB is regulated by p38MAPK during senescence.
In summary, our findings propose a potential mechanism for suppression of the SASP by which ginsenoside F1 might suppress brain agerelated pathologies, including brain cancer. Importantly, understanding how F1 control the SASP of senescent astrocytes could provide a potentially therapeutic approach in development antiaging drug. Conflicts of interest The authors declare no conflict of interest. Acknowledgement This work was supported by the Intelligent Synthetic Biology Center of the Global Frontier Project, funded by the Ministry of Education, Science and Technology, Republic of Korea. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2018.02.002. References [1] J.A. Buckwalter, S.L. Woo, V.M. Goldberg, E.C. Hadley, F. Booth, T.R. Oegema, D.R. Eyre, Soft-tissue aging and musculoskeletal function, J. Bone Joint Surg. Am. 75 (1993) 1533–1548. [2] F.B. Johnson, D.A. Sinclair, L. Guarente, Molecular biology of aging, Cell 96 (1999) 291–302. [3] D.G. Nicholls, Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease, Int. J. Biochem. Cell Biol. 34 (2002) 1372–1381. [4] L.J. Yan, Positive oxidative stress in aging and aging-related disease tolerance, Redox Biol. 2 (2014) 165–169. [5] T. Fulop, A. Larbi, J.M. Witkowski, J. McElhaney, M. Loeb, A. Mitnitski, G. Pawelec, Aging, frailty and age-related diseases, Biogerontology 11 (2010) 547–563. [6] J. Neves, M. Demaria, J. Campisi, H. Jasper, Of flies, mice, and men: evolutionarily conserved tissue damage responses and aging, Dev. Cell 32 (2015) 9–18. [7] C. Lopez-Otin, M.A. Blasco, L. Partridge, M. Serrano, G. Kroemer, The hallmarks of aging, Cell 153 (2013) 1194–1217. [8] T. Von Zglinicki, A. Bürkle, T.B. Kirkwood, Stress, DNA damage and ageing—an integrative approach, Exp. Gerontol. 36 (2001) 1049–1062. [9] F.d.A. Di Fagagna, Living on a break: cellular senescence as a DNA-damage response, Nat. Rev. Canc. 8 (2008) 512–522.
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