Fresh human tissue samples were obtained with the required Institutional Review Board (IRB) approval from Seoul National University Hospital (IRB number: 0106-080-001).

Two types of primary glioblastoma cells (GBL-13 and GBL-15) and two types of glioblastoma cell lines (U87MG and U373MG) were used for this study. The U87MG (human glioblastoma) cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The U373 (human glioblastoma) cell line was acquired from the Korean Cell Line Bank (KCLB: Seoul, Korea). GBL-13 and GBL-15 were obtained during brain tumor lobectomy. After pathological analysis, brain tumor was diagnosed as glioblastoma. The tumor mass was processed to a single cell by papain digestion. These primary cells were cultured with Dulbecco Modified Eagled Medium (DMEM, WelGENE, LM001-05, Korea) containing 10% fetal bovine serum (FBS, Gibco Corp.,16000, Grand Island, NY, USA) and 100 U/ml penicillin/streptomycin (Gibco Corp.,15140-122, Grand Island, NY, USA) in a humidified incubator with 5% CO2 at 37℃ and were passaged by trypsinization using TrypLE™ (Gibco Corp.,12604-013, Grand Island, NY, USA) every 7~8 days. U87MG and U373MG were cultured with 10% FBS (FBS, Gibco Corp.,16000, Grand Island, NY, USA), supplemented Eagle's Minimum Essential Media (MEM, WelGENE., LM001-05, Korea) containing 100 U/ml penicillin/streptomycin (Gibco Corp.,15140-122, Grand Island, NY, USA) in 5% CO2 incubator at 37℃ and were passaged by TrypLE™ (GibcoCorp.,12604-013, Grand Island, NY, USA) every 3~4 days.

Cells were plated at a density of 3×10³ cells per each well into 96-multiwell plate for 24 h (Falcon, 353072, Franklin Lakes, USA). A day after the initial plating, cells were treated and cultured in the same ethanol diluted growth media containing six different dilution concentrations of ethanol (EtOH, Absolute Ethanol 99.9%. Duksan Pure Chemicals Co., Gyeonggi, Korea). Ethanol concentrations were as follows: 0%, 0.1%, 0.5%, 1%, 3% and 5% v/v. Ethanol containing media were changed after 24 h, 48 h and 72 h from the initial planting. The cells were cultured in ethanol diluted growth media with 5% CO2 incubation at 37℃ during the whole test periods.

The MTT assay was applied to evaluate the ethanol effects in proliferation and viability of glioblastoma cells. MTT salt is cleaved by mitochondrial dehydrogenase in the metabolic active cells and is reduced to an insoluble formazan crystal which displays a purple color. The color was detected by a Multi-scan MS spectrophotometer (Labsystems, Stockholm, Sweden). The In vitro Cell Proliferation KIT 1 (Roche, 11465007001, Mannheim, Germany) was used. U87MG, U373MG, GBL-13 and GBL-15 were seeded at a density of 3×10³ cells/well in 96-well plate. After ethanol treatment, 10 µl of MTT (5 µg/ml) were added to every single well in the plate which was incubated for 4 h at 37℃. Then 100 µl of solubilization buffer (SDS 10% in 0.01N HCl) were added to the wells in the plate which was incubated at 37℃ overnight. After the incubation, the absorbance was measured at 570 nm in a Multiscan MS spectrophotometer (Labsystems, Stockholm, Sweden).

After the treatment of 5% ethanol for 24 h, morphological changes of the cells were observed by phase contrast microscopy (Nikon, TS100, Japan).

The cells were plated in 96-well plate. After ethanol treatments, cells attached on the 96-well plate were washed using phosphate-buffered saline (PBS, pH 7.4) once, trypsinized for 1 min with TrypLE™ (GIBCO Corp.,12604-013) at 37℃ incubator and then neutralized with fetal bovine serum (FBS) supplemented growth media. The cells were stained using 4% trypan-blue to determine live cells. Cell count was performed manually with a hemocytometer. Counting was at minimum triplicated samples, and experiments were performed at least three times.

The CytoTox-Glo cytotoxicity assay (Promega Corp., G9292, WI, U.S.A.) was employed to evaluate the cytotoxicity effects of ethanol on glioblastoma cells. The CytoTox-Glo cytotoxicity assay distinctively quantifies the extracellular activity of an intracellular protease (dead-cell protease). Intracellular protease is associated with cytotoxicity and reacts with a luminogenic peptide substrate (alanyl-alanylphenylalanyl-aminoluciferin). The degree of protease reaction can measure dead-cell protease activity. As a control, we treated 1% TritonX-100 to exclude the background value of media color. 5 µl of luminogenic peptide substrate was added to each well to measure the luminescence to determine the number of dead cells. The peptide substrate was incubated for 15 min at 37℃. The luminescence was measured in a 1420 Luminescence Counter (Perkin Elmer, Waltham, MA). To determine the total number of cells, we measured luminescence by adding 50 µg of lysis reagent to each well of the plates. The luminescence was incubated for 15 min at 37℃. To calculate viability, luminescence was measured before and after lysis. The calculating formula used is described below. The luminescence value before lysis is the experimental dead cell luminescence and after lysis is the total luminescence (Viable cell luminescence=Total luminescence-Experimental dead cell luminescence).

Lactate dehydrogenase (LDH) was applied to evaluate the quantification of plasma membrane damage and cytotoxicity. We conducted LDH assay with the LDH-Cytotoxicity Assay Kit (LDH, BioVision Inc., K311-400, SF, USA) adhering to the manufacturer's protocol. In brief, 1×10⁴ cells were seeded in 96-well plate with 1% assay medium and were exposed to ethanol for 24 h in an incubator (5% CO2, 90% relative humidity, 37℃). Then the 96-well plate was centrifuged at 250 g for 10 min. 100 µl of supernatant was transferred into a new 96-well plate. Then, 100 µl reaction was added and incubated for 30 min at room temperature. After incubation, the absorbance of the solution was measured at 492 nm using a Multi-scan MS spectrophotometer (Labsystems, Stockholm, Sweden). LDH levels in the media were quantified and compared to control values according to the kit instructions.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Inc. Valencia, CA, U.S.A.) at the 24 h ethanol treatment. Total RNA was reverse transcribed using the Reverse Transcription System (Promega, A3500, WI, USA) as per instructions. The primers used were as follows: Bax, (forward: 5'-AGGATGCGTCCACCAAGAAG-3') and (reverse: 5'-GCTCCCGGAGGAAGTCCAAT-3'), Bcl-2, (forward: 5'-GACTTCGCCGAGATGTCCAG-3') and (reverse: 5'-CATCCCAGCCTCCGTTATCC-3'), Fas, (forward: 5'-GGACCCTCCTACCTCTGGTTCTT-3') and (reverse: 5'-CAGTCCCTAGCTTTCCTTTCACC-3'), and GAPDH, (forward: 5'-CGTGGAAGGACTCATGAC-3') and (reverse: 5'-CAAATTCGTTGTCATACCAG-3'). 30 cycles polymerase chain reaction (PCR) was performed for amplification of these products with each cycle, which consists of denaturation at 94℃ for 30 s, annealing at Tm (melting temperature) for 30 s and primer extension at 72℃ for 1 min. PCR products were separated on 1.5% agarose gel (Bioline, BIO-41025, London, U.K.) and were visualized by RAS-3000 (FUJI, RAS-3000, Japan).

After 24 h of ethanol treatment, four types of cells were collected and the proteins were isolated. The proteins were extracted using PRO-PREP, (iNtRON Biotechnology Inc., 17081, Sungnam, Korea). 10 µg proteins were electrophoresed on 10% SDS-PAGE and were transferred to a polyvinylidene fluoride (PVDF) membrane. (GE Healthcare, RPN 2020F, Buckinghamshire, UK). In the case of cleaved form such as caspase-3 and caspase-9, 30 µg proteins were used and 0.2 µm pore size PVDF membrane (Millipore Corporation, ISEQ00010, Billerica, MA, USA) was applied. After blocking the membrane with 5% (w/v) skim milk (BD Biosciences, 232100, CA, USA) and washing with Tris-buffered saline-Tween solution (TBST), membranes were incubated with anti-BCL-2 (1:1000), anti-Bax (1:1000), anti-caspase-3 (1:1000), anti-caspase-9 (1:1000) (Cell Signaling Technology, Inc., MA, USA), and β-actin (1:500) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), for 12 h at 4℃, respectively. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000, Vector labs,. PI-1000) or goat anti-mouse IgG (1:1000, Vector labs, PI-2000, CA, USA) or rabbit anti-goat IgG secondary antibodies (1:1000, Vector labs, PI-9500, CA, USA) for 1 h~2 h at room temperature. After washing with TBST, signals were detected using enhanced chemiluminescence, ECL Western Blot Detection Kit (BIOSESANG, W6001, Seoul, Korea) on the membrane.

Nicotinamide nucleotide is a main indicator of the redox state in cells. Intracellular nucleotides such as NAD and NADH ratios were detected by the NAD+/NADH Quantification kit (BioVision, Inc. K347-100, SF, USA). This assay was performed adhering to the manufacturer's protocol. In short, U87MG, U373MG, GBL-13 and GBL-15 were treated with ethanol at specific concentrations of 0%, 1%, and 5% and incubated for 24 h (5% CO2, 90% relative humidity, 37℃). Then, the 2×10⁵ cell pellet was collected (2000 rpm, 5 min) for the NAD+/NADH measurement per each assay. The cell pellet was extracted by Extraction Buffer through the freeze/thaw cycle method. Then, the extraction sample was briefly vortexed and spun down at 14,000 rpm for 5 min. Only supernatant was applied in an e-tube. To detect the total NAD/NADH (NADt), 50 µl of the extracted supernatant was placed into 96-well plate. Heat was applied to the supernatant sample at 60℃ for 30 min in a water bath to detect NADH only. Throughout this process, all NAD was decomposed while the NADH remained intact. The samples were then cooled on ice. 50 µl of NADH samples were then placed in 96-well plate. 100 µl of Cycling Mix was also added in each well. Afterwards, 5 min plate incubation was applied at room temperature, and 10 µl of developer was continuously added. The plates were incubated for 1 to 4 hours and 10 µl of stop solution were added. Finally, the plates were read at 450 nm by a Multiscan MS spectrophotometer (Labsystems, Stockholm, Sweden). And finally, the amount of NADt and NADH (pmol) and NAD/NADH Ratio was calculated (NAD/NADH Ratio = (NADt - NADH)/NADH).

The CCK-8 assay (WST-8, Dojindi labs, Kumamoto, Japan) was applied to evaluate the effects of ethanol for proliferation and viability of the cells used. CCK-8 assay uses water soluble tetrazolium salt that is called WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]. WST-8 produces a water soluble formazan dye according to the dehydrogenase activity in the presence of an electro mediator. Through this process, colorless WST-8 becomes WST-8 formazan with an orange color. CCK-8 assay was used by following the manufacturer's instructions. U87MG, U373MG, GBL-13 and GBL-15 were seeded at a density of 3000 cell/well in 96-well plate and were treated with ethanol at the concentrations of 0, 0.1, 0.5, 1, 3 and 5%. And then the plates were incubated for 24 h, 48 h and 72 h at 5% CO2, 90% relative humidity, and 37℃. After incubation, 10 µl of CCK-8 solution was added to each well of the 96-well plate and the wells were incubated for 1~4 hours. Finally, the absorbance at 450 nm was evaluated using a Multiscan MS spectrophotometer (Labsystems, Stockholm, Sweden).

All data are expressed as means±standard deviation (SD). Statistical significance between different test conditions was determined by using the Mann-Whitney U test. The differences between groups were considered significant at p<0.05. Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Prism, San Diego, California, U.S.A.).

Cells were examined by MTT-assay to determine the ethanol-induced cytotoxicity. Ethanol exposure showed that cytotoxicity for two glioblastoma cell lines (U87MG and U373MG) and two primary glioblastoma cells (GBL-13 and GBL 15) was increased in a time- and ethanol concentration-dependent manner (Fig. 1). The viabilities of U87MG and U373MG were reduced as the ethanol concentration was increased (Fig. 1A, 1C). The viability of U87MG was significantly decreased to 82%, 66%, and 42% at ethanol concentrations of 1%, 3% and 5%, respectively, at 72 h. In the case of U373MG, cell viability was also dramatically decreased to 38% in the 5% ethanol-treated group at 72 h (Fig. 1A). However, different patterns were observed for the primary glioblastoma cells in the MTT assay (Fig. 1A). These cells - GBL-13 and GBL-15 - seemed to be affected by the treatments with low dose ethanol concentration, in a similar pattern as the two glioblastoma cell lines (Fig. 1A). However, intriguingly, when the higher dose of ethanol was treated, the MTT assay revealed significantly increased cell viabilities in primary glioblastoma cells, especially at the 5% ethanol concentration (Fig. 1A).

Cells were counted by trypan blue exclusion assay to determine the effect of actual ethanol-induced cytotoxicity on glioblastoma cells (Fig. 1C). The experimental condition was the same as the MTT assay described previously; 0%, 0.5%, 1%, 3%, and 5% ethanol treated media were used. The number of viable cell for U87MG and U373MG was found to be gradually decreased as the ethanol concentration was increased (Fig. 1C). The number of viable cells of the cell line was reduced to almost less than 5% at 72 h of the 5% ethanol treatment. Likewise, primary glioblastoma cells -GBL-13 and GBL-15 - exhibited decreased number of viable cell in an ethanol concentration-dependent manner (Fig. 1C). When the 5% ethanol was treated, the viability of the primary glioblastoma cells was found to be significantly dropped to less than 20% at 72 h.

As expected, every type of cell used in this study underwent morphological changes when ethanol was treated compared to the control (Fig. 1B). Cell density was decreased and the size of the most cells in the 5% ethanol-treated group was shrunk compared with the control one. Cell pictures of the 5% ethanol treatment are only shown because the MTT results of the 5% ethanol treatment were not found to match with the results obtained from the trypan blue exclusion assay and the microscopic images of the cells treated with ethanol. (Nikon, ECLIPSS, TS100, Japan).

CytoTox-Glo cytotoxicity assay (Promega Corp., G9292, WI, U.S.A.) was used to examine dead-cell protease activity which measured the ethanol-induced cytotoxicity on glioblastoma cell lines and primary cells (Fig. 2A). Accordingly, cell viability was calculated using the formula: Viable cell luminescence=Total luminescence-Experimental dead cell luminescence. Through CytoTox-Glo cytotoxicity assay, it was shown that the viability of U87MG and U373MG apparently declined to 43% and 30%, respectively, at a 5% ethanol concentration at 72 h. Likewise, the viability of primary glioblastoma cells - GBL-13 and GBL-15 - was decreased to 39% and 50%, respectively, at a 5% ethanol concentration at 72 h. Through this assay, it was found that the cell viabilities of U87MG and U373MG were reduced in an ethanol concentration-dependent manner, and that a very similar pattern was also observed for the primary glioblastoma cells. These results clearly suggest that ethanol induced the cytotoxicity not only on the glioblastoma cell lines but also on the primary glioblastoma cells.

The generation of reactive oxygen species (ROS) and oxidative stress leads to DNA damage and ultimately cellular apoptosis. Markers of apoptosis were detected by RT-PCR to confirm the effects of ethanol. BAX and FAS are well-known pro-apoptotic markers, whose expression levels were found to be increased in U87MG, U373MG, GBL-13 and GBL-15 in an ethanol concentration-dependent manner (Fig. 3A, 4A). BCL-2 is a well-known anti-apoptotic marker whose expression level was found to be decreased in every type of cells that were treated with ethanol (Fig. 3A, 4A). Additionally, ethanol-induced apoptosis was checked at protein level using Western Blot Analysis. The expressions of Bcl-2, full length caspase-3 and full length caspase-9 were decreased as the ethanol concentration was increased (Fig. 3B, 4B). In contrast, the expressions of Bax, cleaved caspase-3 and cleaved caspase-9 were increased in in an ethanol dose-dependent manner (Fig. 3B, 4B). In short, it was found that when exposed to 5% ethanol for 24 h, two primary glioblastoma cells and immortalized glioblastoma cell lines exhibited increased expression levels of the pro-apoptotic proteins and decreased expression levels of anti-apoptotic proteins in an ethanol dose-dependent manner. These expression patterns indicate that ethanol treatment caused an apoptotic event in all 4 types of cells tested in our study.

LDH are well known tests for the measurement of membrane integrity and cell toxicity. LDH leakage of primary glioblastoma cells was found to be gradually increased in an ethanol dose-dependent manner (Fig. 2B). LDH leakage of GBL-13 was observed to be gradually increased up to 151 degrees at a concentration of 5% ethanol at 24 h when compared with the control group (no ethanol-treated, LDH leakage level=100). LDH leakage of GBL-15 was also gradually increased up to 136 degrees at 24 h. However, immortalized glioblastoma cell lines showed a different pattern of LDH leakage (Fig. 2B). LDH leakage of U87MG increased only up to 111 degrees at a 5% ethanol concentration at 24 h. And strikingly, LDH leakage of U373MG was slightly reduced to 92 degrees at a 5% ethanol concentration at 24 h. These results suggest that LDH leakage of the primary glioblastoma cells due to ethanol treatment more easily occurred than that of immortalized cell lines.

Ethanol treatments at concentrations of 0%, 1%, and 5% were applied to examine the intracellular reduction system and especially to determine the effects of the nicotinamide nucleotide. Four types of cells were treated with indicated dose of ethanol and were measured for NAD+/NADH ratio (Fig. 5). NAD+/NADH ratio of U87MG was found to be decreased by approximately 14% compared with the control group at a 5% ethanol concentration at 24 h. NAD+/NADH ratio of U373MG was also decreased by roughly 39% compared with the control group at 24 h. In addition, NAD+/NADH ratio of primary glioblastoma cells was decreased by approximately 20% compared with the control group at a 5% ethanol concentration at 24 h. These results indicate that ethanol exposure enhances the intracellular NADH concentration in both primary and immortalized glioblastoma cells.

In order to examine the effect of ethanol-induced cytotoxicity, viabilities of primary and immortalized glioblastoma cells were measured by CCK-8 assay using water-soluble tetrazolium salt called WST-8 (Fig. 6). Ethanol at concentrations of 0%, 0.1%, 0.5%, 1%, 3%, and 5% was treated on two glioblastoma immortalized cell lines (U87MG and U373MG) and two primary glioblastoma cells (GBL-13 and GBL-15) for 24 h, 48 h, and 72 h. Viabilities of U87MG and U373MG were found to be reduced as ethanol concentration was increased. Specifically, viability of U87MG was significantly decreased to 32% at a 5% ethanol treatment at 72 h. In the case of U373MG, the cell viability was apparently decreased to 7% in the same condition. Furthermore, the primary glioblastoma cells showed the exact same patterns as the immortalized cell lines. At 72 h, viability of GBL-15 was dramatically decreased to 28% at a 5% ethanol concentration. And similarly, viability of GBL-13 was found to be decreased to 43% in the same condition as GBL-15. These results clearly show that not only the immortalized cell lines, but also the primary glioblastoma cells were affected by the ethanol treatments at various concentrations.