Essay: Culturing of SK-N-SH cells

SK-N-SH human neuroblastoma cell lines obtained from National Center for Cell Science (NCCS) Pune, India were cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) (Himedia), 2 mM glutamine and penicillin/streptomycin (100 U/ml) in an incubator humidified with 95 % air and 5 % CO2. The medium was changed for every 2 days. Cells were maintained at 37 °C in CO2 incubator in a saturated humidity atmosphere containing 95% air and 5% CO2. Rotenone and GE were made fresh in DMSO (0.05 %), prior to each experiment. GE was added 4 h prior to rotenone treatment.
2.3. Measurement of cell viability (Dose fixation study)
Cell viability was measured by the MTT method [26, 27]. SK-N-SH (1×103cells/well) was pre-treated with GE (7.5, 15, 30 and 60 nM of medium). After 4 h incubation, 100 nM of rotenone was added to the respective groups. 0.5 ml of MTT reagent was added to each well after 24 h. Then, the cells were centrifuged for 10 min and the supernatant was removed. 200 μl of DMSO was added into each well to dissolve the formazan crystals and absorbance was measured in a microplate reader at 560 nm. Cell viability was expressed as a percentage of the control culture value.
2.4. Measurement of intracellular ATP Levels
Experimentally treated cells were collected via centrifugation and intracellular ATP was measured with a illuminometer, using an ATP Bioluminescence Assay Kit HS II (Roche Molecular Biochemicals; catalogue no: 11699709001) following the manufacturer’s instructions.
2.5. Determination of intracellular ROS generation
Intracellular ROS levels were measured by using a non-fluorescent probe 2, 7-diacetyl dichlorofluorescein diacetate (DCFH-DA) [28]. Upon interaction with intracellular H2O2, DCFH gives rise to green fluorescence. The percentage of ROS was estimated in the control and experimental SK-N-SH cells. Briefly, aliquots of the isolated cells 3 x 106 cells/ml were made up to a final volume of 2 ml in normal PBS (pH 7.4). After pre-treatment with GE (60 nM) for 2 h, the cells were incubated with rotenone (100 nM) for 24 h. Thereafter, they were treated with
100 µl DCFH-DA for 30 min at 37 °C, washed twice with PBS and visualized using a fluorescent microscope. Fluorescent measurements were made with excitation and emission filters set at 485 ± 10 nm and 530 ± 12.5 nm, respectively (Shimadzu RF-5301 PC spectroflurometer). The images were captured using fluorescence microscope (450 – 490 nm; blue filter).
2.6. Estimation of Oxidant and Antioxidant Indices
Following the incubation with GE and/rotenone, SK-N-SH neuroblastoma cells (5×103 cells/well in a six well plate) were suspended in 130 mM KCl and 50 mM PBS containing 0.1 ml of 0.1 M dithiothreitol and centrifuged at 20,000 g for 15 min (4 °C). The supernatant was taken for the following biochemical analysis.
2.6.1. TBARS assay: The level of lipid peroxidation was determined by analysing TBARS, as described earlier [29]. Briefly, 0.2 mL of sample was diluted with double distilled water (0.2 mL) and mixed well, and then, 2.0 mL of TBA-TCA-HCl reagent was added.
The mixture was kept in a boiling water bath for 15 min. After cooling, the tubes were centrifuged at 1,000g for 10 min and the supernatant was estimated at 535 nm. The pink coloured chromogen formed by the reaction of 2-TBA with breakdown products of lipid peroxidation was measured.
2.6.2. Assay of Superoxide dismutaseactivity: Superoxide dismutase (SOD) activity was examined in the supernatants according to the method described by Kakkar et al. [30]. This assay is based on the ability of SOD to scavenge superoxide anion radical (O2), which decreases the overall rate of pyrogallol autoxidation. In brief, 1 mL of 0.05 mol/L Tris-HCl buffer (pH 8.2) containing 1 mmol/L DTPA was added to 200 μl of sample. The reaction was initiated by the addition of 0.2 mmol/L pyrogallol, and the change in optical density at 420 nm was recorded for 3 min.
2.6.3. Catalase activity assay: Intracellular catalase activity was assayed by the decomposition of hydrogen peroxide (H2O2) as described earlier [31]. Decrease in absorbance due to H2O2 degradation was monitored at 240 nm for 1 min. The reaction mixture contained 50 mM phosphate buffer (7.0), 40 mM of H2O2 and 200 μl of cell lysate [32]. 1 U of catalase is defined as the amount of enzyme that transforms 1 μmol of H2O2 per minute at 25 °C.
2.6.4. Glutathione peroxidase activity assay: The activity of glutathione peroxidase (GPx) was measured by the method described by Rotruck et al. [33]. The reaction mixture contained 2.0 ml of 0.4 M Tris-HCl buffer, pH 7.0, 0.01 ml of 10 mM sodium azide, 200 μl of cell lysate, 0.2 ml of 10 mM glutathione and 0.5 ml of 0.2 mM. H2O2. The contents were incubated at 37 °C for 10 min followed by the termination of the reaction by the addition of 0.4 ml 10 % (v/v) TCA, centrifuged at 5,000 rpm for 5 min. The absorbance of the product was read at 430 nm and calculated as nmol of NADPH consumed/min/mg protein and expressed as mU/mg protein.
2.6.5. Estimation of glutathione: Glutathione (GSH) was determined by the method of
Ellman et al. [34]. Briefly, to 200 μl of cell lysate, 0.5 ml of Ellmans reagent (19.8 mg of 5, 50 -dithiobisnitro benzoic acid (DTNB) in 100 ml of 0.1 % sodium nitrate), 3.0 ml of phosphate buffer (0.2 M, pH 8.0) and 0.4 ml of distilled water was added. The mixture was thoroughly mixed and the absorbance was read at 412 nm, expressed as nmol/mg protein.
2.7. Measurement of nitrite concentrations
Nitric oxide (NO) production was quantified by measuring nitrite, a stable oxidation end product of NO. Briefly, 50 ml of culture medium was mixed with an equal volume of Griess reagent (1.5% sulfanilamide in 1N HCl and 0.15% N (1-naphtyl)-ethylenediamine dihydrochloride in distilled water, v/v). After 10 min of incubation at room temperature, the absorbance at 540 nm was determined in a Bio-Rad microplate reader. Sodium nitrite was used as a standard.
2.8. Mitochondrial complex I estimation
2.8.1. Isolation of mitochondria
Mitochondrial fractions were prepared by the method described by Vander Heiden et al.. [35]. Briefly, 2 × 107 SK-N-SH cells were trypsinized and resuspended in 0.8 ml of ice-cold buffer A (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and protease inhibitors cocktail). The suspended cells were then passed through an ice-cold glass Dounce homogenizer with 40 strokes of the B pestle. Unlysed cells, cell debris and nuclei were pelleted by centrifugation (1,000 × g force) for 10mins. The supernatant fluids were then centrifuged at 10,000 × g for 25 min. The pellets were resuspended in buffer A and represented the mitochondrial fractions.
2.8.2. Mitochondrial complex I activity
The mitochondrial complex I buffer containing 25 mM KH2PO4 (pH 7.2), 5.0 mM MgCl2, 3mM potassium cyanide and 2.5 mg/ml BSA in the presence of 125 μM of NADH and CoQ10. Briefly the mitochondria fractions (80 μg) were incubated in the assay buffer (100 μl) for 15 min. To initiate the reaction, 100 μl of NADH and coenzyme Q10 solutions were added. The plate was read for 5 min in a microplate reader and OD was recorded after a 1-min interval [35]. The change in optical density at 550 nm was recorded using BIO-RAD microplate reader and the activity was expressed as percentage of control [36].
2.8.3. Changes in mitochondrial transmembrane potential (Δψm)
The uptake of rhodamine123 (Rh123) into mitochondria is an indicator of the Δψm. The alteration of mitochondrial membrane potential was estimated in the control, rotenone treated, GE and rotenone treated SK-N-SH cells. Briefly, cells were pre-treated with GE (60 nM) and then the cells were incubated with rotenone (100 nM) for 24 h at 37 °C. After 24 h, 1 µl of fluorescent dye Rh-123 (5 mmol/l) was added and kept in incubator for 15 min. Then, the cells were washed with PBS and observed under fluorescence microscope using blue filter (450-490 nm) (Shimadzu RF-5301 PC spectroflurometer) [37].
2.9. Detection of apoptotic morphological changes by EtBr/AO staining
Acridine orange (AO) is a DNA selective and membrane permeable fluorescent cationic dye. It can freely enter the normal cell nuclei and emits green fluorescence under 525 nm [38]. The control and experimental neuroblastoma cells were seeded in a 6-well plate (3×103 cells/well) and incubated in CO2 incubator for 24 h. The cells were fixed in methanol: glacial acetic acid (3:1) for 30 mins at room temperature. The cells were washed in PBS and stained with 1:1 ratio of ethidium bromide/acridine orange (EtBr/AO). Stained cells were immediately washed again with PBS and viewed under a fluorescence microscope (450-490 nm; blue filter; Nikon, Eclipse TS 100, Japan) with a magnification of 40X. The number of cells showing features of apoptosis was counted as a function of the total number of cells present in the field.
2.10. Detection of cytoplasmic autophagic vacuoles
To detect the presence of cytoplasmic autophagic vacuoles (AVOs) of the cells were stained with the vital dye acridine orange (1µg/ml) [39, 40] Cells were seeded in 6 well plates at a density of 3×103 cells/well in complete medium and incubated overnight. Cells were then experimentally treated with rotenone and GE for 48 h. The AVOs were counted using fluorescent microscope in at least 100 cells for each group.
2.11. Transmission electron microscope
Treated SK-N-SH cells were fixed in 0.1 mol/L sodium cacodylate buffered (pH 7.4) 2.5% glutaraldehyde solution for 2 h, then rinsed (3610 minutes) in 0.1 mol/L sodium cacodylate-buffered (pH 7.4) 7.5% saccharose and postfixed in 1% OsO4 solution for 1 hour. After dehydration in an ethanol gradient (70% ethanol for 20 min, 96% ethanol for 20 min, 100% ethanol for 2620 min), samples were embedded in Durcupan ACM and then were stained with uranylacetate and lead citrate. The sections were examined using Olympus electron microscope and the degenerating cell bodies were captured digitally (Olympus Soft Imaging Solutions GmbH, Germany).
2.12. Immuno blotting
Briefly, cells seeded in 6-well plates were harvested and washed with PBS. Cells were lysed in 100𝜇L lysis buffer containing 20 mM Tris-Hcl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 30𝜇g/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride and subjected to 12.5% polyacrylamide gel electrophoresis. A total volume of 40 𝜇g of protein was loaded per lane. The separated proteins were blotted onto a PVDF membrane by semidry transfer (Bio-Rad). After blocking with 5% nonfat milk in TBS, the membranes were then incubated with various antibodies: P13K, mTOR, AKT, LC3-I, LC3-II, Atg-5, -12, P62, Ub-protein, Ub- E1 and
β-actin. The following dilutions were used for P13K (1:400), mTOR (1:500), AKT (1:1000), LC3-I (1:200), LC3-II (1:200), Atg-5, -12 (1:500), P62 (1:800), Ub-protein, Ub- E1 (1:500) and β-actin (1:1500). After primary antibody incubation, the membranes were incubated with secondary antibody at a concentration of 1: 2000. Then, the membranes were washed with Tris-buffered saline and 0.05% Tween 20 thrice for 10 min interval; after extensive washes in TBST, the bands were visualised by treating the membranes with 3, 3’-diaminobenzidine tetrahydrochloride (western blot detection reagent, Sigma, USA). Densitometry was performed using “Image J” analysis software.
2.13. Data Analysis
Statistical analysis was performed using one-way analysis of variance followed by Dun-can’s multiple range test (DMRT) using Statistical Package for the Social Science (SPSS) soft-ware package version 12.0. Results were expressed as mean±SD for four experiments in each group. 𝑃 values < 0.05 were considered significant.
3. Result
3.1. Cytoprotective effect of GE on rotenone induced cytotoxicity by MTT assay
MTT assay was used to find out the LD50 of rotenone toxicity and optimum dose of GE treatment. Rotenone treatment (0, 0.5, 5, 50, 100 and 200 nM for 24 h) to SK-N-SH cells induced dose-dependent cytotoxicity, with approximately 50% of cell viability at 100 nM (LD50) (Fig. 1a and 1b). 100 nM of rotenone fixed as optimum dose and used to carry out for further in vitro studies in SK-N-SH cells. Pretreatment with GE dose-dependently (7.5, 15, 30 and 60 nM) protected cell death against the cytotoxicity induced by rotenone (Fig. 1a and 1b). The optimum protection for rotenone induced cell death was observed at 60 nM GE pretreatment. GE dose-dependently (7.5, 15, 30 and 60 nM) protected the GE against cell death induced by 100 nM rotenone (Fig. 1b), with about 80% protection at 60 nM of GE. A further increase in GE concentration (120 nM) decreased the cell viability against death induced by 100 nM rotenone.
3.2. Effect of GE on rotenone depleted intracellular ATP stores
Treatment with rotenone significantly depleted intracellular ATP stores (~20% at 100 nm; Fig. 2) as compared with control group. Pre-treatment with GE significantly improved intra cellu-lar ATP stores compared to rotenone alone treated cells. No significant changes were detected between control and GE alone treated SK-N-SH cells.
3.3. Effect of GE on rotenone-induced ROS and nitrite generation in SK-N-SH cells
Intracellular ROS was measured in terms of fluorescence by DCFDA (Fig. 3 a and 3 b). Addition of rotenone (100 nM) to cells caused a significant increase in DCFDA fluorescence. Pre-treatment of the cells with GE (60 nM) lowered rotenone induced free radical release as compared to rotenone alone treated group. No significant changes in ROS formation were detected in SK-N-SH cells treated only with GE.
Nitrite levels in medium from rotenone and GE treated cell cultures were measured to confirm the possible involvement of NO in rotenone induced cytotoxicity. No difference was observed in the production of nitrite between rotenone and GE treated cells when compared with controls. (data not shown).
3.4. Effect of GE on rotenone induced oxidative stress formation.
Figures 4 indicates the levels of TBARS and GSH in rotenone-treated SK-N-SH cells incubated with and without GE. Rotenone treatment (100 nM) significantly increased the levels of TBARS parallel to the decreased levels of GSH in SK-N-SH cells compared with non-treated cells. Pretreatment with GE (60nM) to rotenone treated cells significantly decreased the levels of TBARS and increased GSH levels significantly, compared to cells treated with rotenone alone. Compared with GE untreated cells, rotenone (100 nM) treatment increased SOD, catalase and GPx activities in SK-N-SH cells. Pretreatment with GE significantly decreased the activities of SOD, catalase and GPx, compared to cells treated with rotenone alone.
3.5. Effect of GE on rotenone induced Mitochondrial ETC-1 activity
Mitochondrial ETC-I activity was measured in mitochondrial enriched fractions from SK-N-SH cells as shown in fig 5. Rotenone treated cells showed that reduced ETC-I activity as compared to control cells. Pre-treatment with GE to rotenone significantly restored ETC-I activi-ty in mitochondria compared to rotenone alone treated cells. No changes observed between con-trol and GE alone treated cells.
3.6. Effect of GE on rotenone-induced Δψm alteration in SK-N-SH cells
Figure 6 (a and b) show changes in ΔΨm (mitochondrial membrane potential), which was measured by determining the red/green fluorescence ratio of rhodamine-123 in control and experimental groups. Treatment of cells with 100 nM rotenone for 24 h resulted in significant dissipation of ΔΨm. The average red fluorescence ratio was decreased in rotenone alone treated SK-N-SH cells as compared to the control group. Cells treated with rotenone displayed higher green fluorescence indicating a polarized state, whereas cells pre-treated with GE displayed significant decrease in green fluorescence. GE alone treated cells showed no significant effect on ΔΨm as compared to the controls.
3.7. Effect of GE on rotenone induced apoptotic features in SK-N-SH cells by dual staining
Figure 7 (a and b) show the apoptotic morphological changes measured in terms of fluorescence by AO and EtBr. Our results indicated that the control cells had intact nuclei and the rotenone (100 nM) treated cells exhibited significant nuclear fragmentation and destruction, characteristic of apoptosis (bright orange color) and necrosis (red color), respectively. However, the amount of nuclear fragmentation and destruction of rotenone treated cells were dramatically reduced, when the cells were pretreated with GE (60 nM).
3.8. GE decreased autophagic signaling molecules in rotenone treated SK-N-SH cells
The immunoblotting protein expression levels of P13K, AKT, mTOR (a negative regulator of autophagy) and autophagy complex signaling proteins such as Atg-5, Atg-7 and Atg-12 were significantly increased in rotenone alone treated cells after 24 h (Figure 8 (a and b) and 10 (a and b)). In contrast, pretreatment with GE to rotenone treated cells caused a significant decrease in P13K/AKT, mTOR, autophagy complex signaling proteins such as Atg-5, -7 and -12 significantly (p < 0.05).
In contrast, P62, a positive regulator of autophagy, increased significantly. These results suggested the GE critically regulated autophagy in SK-N-SH cells through P13K/AKT mediated pathway, by maintaining mTOR and P62 ratio (Fig. 11).
3.9. GE decelerated autophagic markers expression in rotenone treated SK-N-SH cells
The immunoblotting study showed that rotenone treatment significantly enhanced the expression of autophagic markers such as LC3-I and LC3-II respectively (Figure 9; a and b). Pretreatment with GE decelerated autophagic markers via enhanced the expression of a light chain of the microtubule-associated protein as compared to rotenone alone treated cells. No significant changes in LC3-I and LC3-II expression levels were detected in SK-N-SH cells treated with GE alone. These data clearly demonstrates that GE has the ability to regulate rotenone induced autophagic flux significantly.
3.10. GE improved intracellular Ub protein and Ub-E1 in rotenone treated SK-N-SH cells
To study the effects GE on rotenone induced intracellular Ub protein and Ub-E1 in SK-N-SH cells as showed in figure 11 (a and b). Rotenone treated cells show decreased levels of Ub protein and Ub-E1 expressions were observed as compared to control mice. However, pretreatment with GE to rotenone treated cells showed a significant increase in the expression of Ub protein and Ub-E1 (p< 0.05), no differences were observed between control and GE alone treated cells.
3.11. GE reduced AVOs formation in rotenone treated SK-N-SH cells
Formation of AVOs in cytoplasm is one of the characteristic features of autophagy process. This can be detected and measured by vital staining with acridine orange as showed in figure 12 (a and b). The number of AVOs significantly increased in rotenone alone treated cells when compared with the control cells. Formation of AVOs was significantly reduced by pretreatment with GE to rotenone as compared with rotenone alone treated cells. No further significant changes were observed between control and GE alone treated cells.
3.12. Effect of GE on rotenone induced ultrastructural manifestation
The ultrastructural manifestation represents a “gold standard” method to detect AVOs and damaged mitochondrial state as showed in figure 13. Observed through a TEM, SK-N-SH had a high nucleo-cytoplasmic ratio, damaged nuclear membrane and an irregular appearance in the rotenone treated cells. In the absence of rotenone, there were no AVOs except normal mitochondria and rough endoplasmic reticulum, which were observed in SK-N-SH cells. In the presence of rotenone, formation of AVOs, mitochondrial swellings, mitochondrial crest fracture and damaged nuclear membrane within 24 h administration. Interestingly, an increase in mitochondrial numbers was observed.