D-galactose and Bay 11–7082 were purchased from Sigma-Aldrich (Merck KGaA). Astrocytic CRT and U373-MG Uppsala cells (kindly provided by Professor Chul-hee Choi, Korea Advanced Institute of Science and Technology, South Korea) were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 µg/ml of streptomycin (Thermo Fisher Scientific, Inc.) as previously described (18). N2a cells (ATCC^® CCL-131™) were grown in Dulbecco's Modified Eagles medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 1 or 10% heat-inactivated FBS (Thermo Fisher Scientific, Inc.), 100 U/ml of penicillin and 100 µg/ml of streptomycin. Primary rat astrocytes were maintained in 10% FBS-DMEM containing 1% nonessential amino acids (Gibco; Thermo Fisher Scientific, Inc.). Stable cell line CRT-MG/IL-8p-d2EGFP cells were prepared and maintained as previously described (19). All the cells were incubated at 37°C in a 5% CO₂ atmosphere.

Human astrocytic CRT cells and rat primary astrocytes were treated with varying doses of D-galactose resolved in culture medium (0–60 g/l) at 37°C and the cell viability after 72 h exposure was determined. The half-maximal inhibitory concentration, 50 g/l, of D-galactose was used for the subsequent experiments. SA-β-gal staining was performed using an SA-β-gal kit (cat. no. 9860, Cell Signaling Technology, Inc.) in accordance with the manufacturer's protocols to confirm cellular senescence. 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 overnight. The SA-β-gal-positive cells were seen as blue, and were counted under a phase-contrast microscope (magnification, ×100; 5 fields per view analyzed). The experiment was repeated three times in each group.

Cell viability was evaluated by a 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 following treatment, 20 µl of WST-1 was added to each well (12-well plate with 1×10⁵ cells per well) and the plates were incubated at 37°C for 2 h. The plates were employed for centrifugation (300 × g) at room temperature for 3 min and 100 µl of the medium was withdrawn and analyzed by measuring the absorbance at a wavelength of 450 nm using microplate reader (Tecan Group, Inc.).

Astrocytic CRT cells (8×10⁵ cells in 90 mm petri dish) were exposed to D-galactose (50 g/l) at 37°C for 10 days to induce premature secretory senescence. Then, 8 days following senescence induction, cells were cultured with complete culture medium (without 10% FBS) for the collection of CM. The CM was collected from both pre-senescent (untreated cells) and senescent cells after 48 h. CM were centrifuged for 20 min at 800 × g at 4°C, filtered through 0.22 µm bottle-top filters (Sartorius Stedim Biotech) and used for subsequent experiments. CM was used to treat U373-MG and N2a cells with different percentages (25, 50, and 100%) at 37°C for 24 h. Cells were then exposed with or without 0.5 mM Temozolomide (TMZ, Sigma-Aldrich; Merck KGaA) at 37°C for 48 h followed by cell viability tests using the WST-1 method and observations of morphological changes by microscopy (magnification, ×100; 6 fields per view analyzed).

Interleukin (IL)-6 and IL-8 levels in the CM were quantified using the Human IL-6 and IL-8 ELISA kits (cat. nos. D6050 and D8000C, R&D Systems, Inc.) according to the manufacturer's protocols. Briefly, 100 µl of culture media normalized by cell number was added in the wells with five replicates and incubated for 2.5 h at room temperature with gentle agitation. Then, the medium was discarded and wells were washed with washing buffer for four times. After that, 100 µl of human IL-6 or IL-8 conjugate were added to each well and plates was incubated for 2 h at room temperature with gentle agitation. Following washing with washing buffer, 100 µl of substrate solution was added to each well and incubated for 20 min at room temperature. Lastly, 50 µl of stop solution was added and wells were assayed with a microplate reader set to 450 nm within 30 min.

Whole cell lysate (20 mg) was prepared from various treatments. The samples (30 µg determined by Bradford Assay) were loaded onto 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 p16ink4a, p53, p21, PARP, Cleaved caspase 3, c-Myc and Tju1 (rabbit polyclonal, 1:2,000, Abcam, cat. nos. ab51243; ab32389; ab109520; ab74290; ab2302; ab39688 and ab18207, respectively); senescence marker protein 30 (SMP30; mouse monoclonal, 1:1,000, Santa Cruz Biotechnology, Inc., cat. no. sc-390098), GAPDH as a loading control (mouse monoclonal, 1:4,000, Santa Cruz Biotechnology, Inc., cat. no. sc-47724). Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; Cell Signaling Technology, Inc., cat. nos. sc-2370 and sc-2380). The bands from the western blots were densitometrically visualized by ECL detection and the signals quantified using ImageJ software (version 1.5.1, National Institutes of Health).

For the immunofluorescence assay, the cells were first washed twice with PBS, then 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% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 30 min and then incubated with primary antibodies against NF-κB p65 (rabbit polyclonal, 1:200, Santa Cruz Biotechnology, Inc., cat.no. sc-8008), and IL-6 (mouse monoclonal, 1:1,000, Abcam) overnight at 4°C. Following washing three times for 5 min each with PBS and 0.05% Triton X-100. Cells were incubated with appropriate Alexa Fluor^® secondary antibodies 488 or 594 (Invitrogen; Thermo Fisher Scientific, Inc., cat. nos. A-11008; A21209) to detect the signal at room temperature for 1.5 h. After another set of washing, images were captured with a Nikon i2 U fluorescence microscope (magnification, ×100; Nikon Corporation).

Optical density analysis was conducted to quantify the fluorescence signal and was performed by using CellProfiler Software (2.2.0, http://cellprofiler.org). Each evaluation was conducted on five fields randomly selected for each of the target proteins.

All data were expressed as the mean ± standard deviation except unless otherwise indicated. P-values were generate using a Student's t-test or one-way analysis of variance; P<0.05 was considered to indicate a statistically significant difference. A protected Fisher's Least Significant Difference post hoc test was used for multiple comparisons. All calculations were performed using GraphPad Prism software (v7.0, GraphPad Software, Inc.).

To determine whether D-galactose can induce the senescence of astrocytic cells, we first cultured astrocytic CRT cells and rat primary astrocytes in the absence or presence of varying doses of D-galactose (0–60 g/l) and observed the cell viability after 72 h treatment (Fig. 1A). Treatment with D-galactose significantly suppressed cellular viability in a dose-dependent manner from 40 g/l of D-galactose upwards. Importantly, a higher dose of D-galactose (60 g/l) induced a considerable cell death (profound apoptotic bodies) as observed under microscopy. Then, we investigated further the classic markers of the senescence-related phenotype. The astrocytic CRT and rat primary cells exhibited flattened morphology and significantly increased levels of SA-β-gal activity compared with the control (Fig. 1B). Additionally, the expression of hallmark regulatory proteins, such as p16, p53, p21 and SMP30 were markedly elevated after D-galactose exposure (Fig. 1C). Taken together, these observations demonstrated that, in a relative long-term of 3 days with D-galactose, astrocytic cells were susceptible to the stress, thereby inducing a characteristic senescent phenotype.

Since the treatment of D-galactose induced typical the senescent phenotype in CRT cells, we next examined at the intracellular level the expression of proinflammatory cytokines IL-6 and IL-8, the major SASP components in rodent and human cells (20). As expected, D-galactose significantly increased IL-6 expression by astrocytic CRT-cells compared with the control (Fig. 2A). In addition, significantly increased IL-8 expression at the transcriptional level was also exhibited by the stable cell line CRT-MG/IL-8p-d2EGFP cells compared with the control (Fig. 2B). To explore the molecular mechanism, we examined the expression of NF-κB by immunofluorescence, which has been revealed to stimulate the transcription of many SASP genes (10), and the results indicated a significant increase in the nuclear translocation after D-galactose exposure compared with the control (Fig. 2C). Our findings suggested that D-galactose increased the expression of SASP-related proteins through activating the NF-κB pathway as early as 6 days after treatment as determined by elevated secretions of IL-6 and IL-8, which was abrogated by a NF-κB pathway inhibitor, Bay 11–7082 (Fig. 2D), suggesting that it may act as a potential target for inhibition on development of SASP in astrocytes.

To explore the role of CM in the process of cellular senescence, we used two different brain tumor cell lines: U373-MG and N2a. CM was used to treat U373-MG and N2a cells at various concentrations (25–100% of culture medium; Fig. 3A and D). Treatment of U373-MG and N2a cells (with 10% FBS) promoted cell viability, suggesting a pro-tumoral action. Furthermore, U373 cells treated with different concentrations of CM were exposed to 0.5 mM temozolomide, and strong chemotherapy resistance was observed with sustained cell viability compared with the control treated with temozolomide (Fig. 3B). This resistance was further confirmed by the decreased levels of activated apoptosis-associated molecules cleaved caspase-3 and PARP (Fig. 3C). Intriguingly, N2a cell-derived neurons (with 1% FBS) were observed as tumor-like neuroblasts after treatment with CM. The results herein showed that neuron-specific class III β-tubulin expression levels were markedly decreased, and tumor-like alterations were observed with reduced dendritic extensions. In addition, we also observed the increased expression of c-Myc protein (Fig. 3E), which is important for cancer cell proliferation (21). Therefore, these studies suggest a potential key role of CM derived from senescent astrocytic cells in human brain tumor progression.