br a Department of Clinical Biochemistry University of
a Department of Clinical Biochemistry, University of Kashmir, Srinagar, Jammu and Kashmir, India
b Cancer Pharmacology Division, CSIR-IIIM, Jammu, Jammu and Kashmir, India
c Department of Biotechnology, Central University of Kashmir, Jammu and Kashmir, India
d Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi, India
Withania somnifera exhibits different pharmacological activities which mainly stem from its broad range of bio-active molecules. Majority of these bioactive molecules, fall into the groupings of alkaloids, steroidal lactones, phenolic compounds and glycoproteins. In this study, we evaluated a novel protein fraction, named here as WSPF, isolated from Withania somnifera roots for its cytotoxic properties against various human cancer cell lines. WSPF exhibited apoptotic activity for each cancer cell line tested, demonstrating significant activity against MDA-MB-231 human breast cancer cells with an IC50 value of 92 μg/mL. WSPF induced mitochondrial-mediated apoptosis of MDA-MB-231 cells via extensive reactive oxygen species generation, dysregulation of Bax/Bcl-2, loss of mitochondrial membrane potential and caspase-3 activation. Additionally, we observed G2/M-phase 11078-21-0 arrest, cleavage of nuclear lamin A/C proteins, and nuclear morphological changes. The present results highlight the anti-cancer properties of WSPF, indicating that the proteins in this fraction can be potential therapeutic agents for triple negative breast cancer treatment.
A great number of the currently available anti-cancer drugs have been produced either from natural sources or compounds derived from these natural sources [1,2]. Among these natural sources, plants have been a valuable source of approved anti-cancer drugs which mostly belong to secondary metabolites or their derivatives . Al-though these drugs are efficient anti-cancer drugs in vitro, they are often unsuccessful in the clinic due to their limited bioavailability or sig-nificant toxicity and other side effects due to non-specific target engage-ment [4,5]. This has necessitated a discovery effort in search of novel, effective and non-toxic anti-cancer agents derived from natural re-sources . To this end, a number of potent anti-cancer proteins and peptides have been extracted from traditional medicinal plants (e.g. Momordica charantia Lectin from Momordica charantia , Tricosanthin from Trichosanthes kirilowii , MAP30 from Momordica charantia  and glycoprotein from Codium decorticatum ). Moreover, a few of these plant protein-based therapies, like Bowman-Birk inhibitor for
E-mail addresses: [email protected] (F. Malik), [email protected]
prostate and oral carcinoma, Mistletoe lectin-1 for tumour inhibition, are in clinical trials [10–12]. With the exception of a few reports, the bio-active proteinaceous constituents of medicinal plants are mostly uncharacterized.
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 acid (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.