FilipinComplex br Cell lines and maintenance of cell culture
2.2. Cell lines and maintenance of cell culture
Human breast cancer cell lines (MBA-MB-435, MDA-MB-231, T47D and MCF-7), colon cancer cell line (HCT-116), lung cancer cell line (A549) and normal breast cell line (MCF-10) used in this study were purchased from National Cancer Institute (NCI), Bethesda, USA. All cell lines were grown in tissue culture flasks containing RPMI-1640 growth media supplemented with 100 units/mL of penicillin, 100 μg/mL of streptomycin and 10% fetal bovine serum. The FilipinComplex were maintained in a CO2 incubator (New Brunswick, Galaxy 170R, Eppendorf) at 37 °C with 5% CO2 and a relative humidity of about 98%.
2.3. Collection of plant material
The plant material was collected from Pir Panjal range of Kashmir Himalayas and was identified by Centre of Plant Taxonomy, Department of Botany, University of Kashmir. A reference specimen under reference number KASHbot/Ku/AB-712-TAG has been retained in the concerned herbarium of University of Kashmir.
2.4. Preparation of WSPF
After washing with distilled water, the shade dried roots of Withania somnifera were powdered to a fine powder with a blender. For protein extraction powdered root material of 100 g, suspended in 400 mL of 0.1 M Tris buffer (pH 7.0), containing 2% w/v PVPP, 0.1 M NaCl, 1 mM DTT, 1 mM PMSF and 1 mM EDTA, was kept overnight at 4 °C with continuous stirring. Homogenate obtained was filtered through cheese cloth followed by centrifugation at 12,000 rpm for 20 min to pellet down cell debris or any other insoluble material. The resultant supernatant was further processed for downstream purification process. For this, ammonium sulphate precipitation was first carried out by adding solid ammonium sulphate to the su-pernatant kept at 4 °C to reach a 30% salt saturation, followed by 45% and 60% salt precipitation. Precipitated ammonium sulphate protein fractions i.e. (30%, 45% and 60%) were separately collected by centrifugation at 12,000 rpm for 20 min and were dissolved in 20 mM Tris buffer (pH 7.0) for dialysis.
2.5. Electrophoretic and mass spectrometric analysis
After extensive dialysis, the diluted WSPF was concentrated to 3 mg/mL by Amicon Ultra-4 (10 kDa cut-off) concentrator. Samples of
WSPF were mixed with 1× loading buffer and boiled for 5 min at 95 °C. After boiling, WSPF and the pre-stained molecular weight protein marker were loaded into wells of 12% gel and allowed to run in an elec-trophoretic assembly at 100 V for 2.5 h. After running for 2.5 h, the pro-tein bands in the gel were visualized by Coomassie brilliant blue. In addition to this, WSPF was subjected to intact protein mass spectromet-ric analysis by using UltrafleXtreme MALDI TOF/TOF (Bruker Daltonics) mass spectrometer.
2.6. Cancer cell proliferation
Effect of WSPF on cell proliferation was evaluated by MTT assay . Briefly, before WSPF treatment, the MDA-MB-231 cells at a density of (1 × 104 cells/mL) were seeded in a 96 well plate for 12 h. After 72 h of treatment with different concentrations of WSPF, 20 μL of MTT (5 mg/mL) was added to each well and incubated for 4 h at 37 °C. Fol-lowing this, the media was removed from each well by tapping it slowly
on a blotting paper and 150 μL of dimethyl sulphoxide was added to each well. After 10 min of incubation at 37 °C, absorbance at 570 nm was measured by using microplate reader (Biotech, USA).
2.7. Assessment of loss in mitochondrial membrane potential (ΔΨm)
For measuring changes in ΔΨm, MDA-MB-231 cells (1 × 105 cells/ mL) seeded in a 6-well plate containing RPMI media were allowed to at-tach for 24 h. After this, the cells were treated for 72 h with various con-centrations of WSPF in a dose-dependent manner. Subsequent to removal of the exhausted media, rhodamine 123 (Rh 123) at a concen-tration of 1 μM was added to each well containing fresh media and incu-bated for 30 min. After incubation, the unbound Rh 123 dye was removed by washing each well twice by phosphate buffer saline (PBS) and then cells were harvested by trypsinization followed by centrifuga-tion at 1200 rpm for 10 min. Cells suspended in PBS were assayed for any change in ΔΨm by flow cytometry.
2.8. Expression analysis of apoptosis associated proteins
MDA-MB-231 cells of density 1 × 105 cells/mL, seeded in a 6-well microplate containing complete media, were incubated under stan-dard culture conditions for 24 h. After this, the cells were treated with different concentrations of WSPF for 72 h in a dose-dependent fashion. Subsequent to this, the cells were harvested by centrifuga-tion at 1200 rpm for 5 min. Cells obtained were washed with chilled PBS and then lysed in RIPA buffer containing protease inhibitor cock-tail. The supernatant, obtained after centrifugation at 12,000 rpm for 20 min was quantified for total protein by using Bradford assay. Equal amount of the protein was loaded in each well of 12% SDS-PAGE and then transferred to nitrocellulose membrane after com-plete running. 5% skimmed milk was used for blocking of membrane and was probed with respective primary antibodies. After washing, secondary antibodies conjugated with horseradish peroxidase were incubated with membrane. Chemi-luminescence method was used for detection of proteins of interest on PVDF membrane. From West-ern blots, the protein band intensity was quantified by using ImageJ software.