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  • br In light of this significant efforts are


    In light of this, significant efforts are currently devoted for explora-tion of novel, less toxic and effective anti-cancerous agent(s) from me-dicinal plants. Withania somnifera, belonging to the family Solanaceae, is a well-known and widely used plant in Indian traditional medicine system. In modern medicine, the medicinal attributes of Withania somnifera have become increasingly attractive, due in part to observed anti-microbial, anti-oxidant, anti-diabetic, anti-cancerous and anti-neurodegeneration properties [13–15]. Despite the numerous studies characterizing the anti-cancerous potential of secondary metabolite constituents of this plant, studies evaluating the protein constituents of this plant for cytotoxic activity have not been executed thus far. In this context, the present study aimed to investigate the effect of WSPF, the protein fraction extracted from the roots of Withania somnifera, against a panel of cancer cell lines. Among the cell lines tested, WSPF was found to be most active against MDA-MB-231, a metastatic breast cancer cell line, and all subsequent detailed mechanistic studies were carried out against this cell line only. It was observed that WSPF was able to induce apoptotic cell death and cell cycle inhibition in a dose-
    dependent manner. Future studies for identification of the effector pro-tein molecule(s) are highly warranted.
    2. Material and methods
    2.1. Materials and chemicals
    Tris buffer, polyvinylpolypyrrolidone (PVPP), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic Midostaurin (EDTA) and Coomassie brilliant blue were purchased from Hi-media, India. Sodium dodecyl sulphate (SDS), ammonium sulphate, bis-acrylamide, ammonium persulphate, polyvinylidene difluoride (PVDF) membranes and acrylamide were purchased from Merck, Germany. Dialysis tubing (molecular weight cut off 12 kDa), penicillin, propidium iodide, streptomycin, Roswell Park Memorial Institute me-dium (RPMI), acridine orange, ethidium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Dulbecco's minimal essential medium (DMEM), 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) were procured from Sigma-Aldrich, USA. All the primary and secondary antibodies used in the study were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, USA.
    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 cells 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.