TITLE:
EWS Knockdown and Taxifolin Treatment Induced Differentiation and Removed DNA Methylation from p53 Promoter to Promote Expression of Puma and Noxa for Apoptosis in Ewing’s Sarcoma
AUTHORS:
Mohammad Motarab Hossain, Swapan Kumar Ray
KEYWORDS:
Apoptosis, Differentiation, Ewing’s Sarcoma, EWS shRNA, p53 Promoter, Taxifolin
JOURNAL NAME:
Journal of Cancer Therapy,
Vol.5 No.12,
October
28,
2014
ABSTRACT: Ewing’s sarcoma is a
pediatric tumor that mainly occurs in soft tissues and bones. Malignant
characteristics of Ewing’s sarcoma are correlated with expression of EWS
oncogene. We achieved knockdown of EWS expression using a plasmid vector
encoding EWS short hairpin RNA (shRNA) to increase anti-tumor mechanisms of
taxifolin (TFL), a new flavonoid, in human Ewing’s sarcoma cells in culture and
animal models. Immunofluorescence microscopy and flow cytometric analysis
showed high expression of EWS in human Ewing’s sarcoma SK-N-MC and RD-ES cell
lines. EWS shRNA plus TFL inhibited 80% cell viability and caused the highest
decreases in EWS expression at mRNA and protein levels in both cell lines.
Knockdown of EWS expression induced morphological features of differentiation.
EWS shRNA plus TFL caused more alterations in molecular markers of
differentiation than either agent alone. EWS shRNA plus TFL caused the highest
decreases in cell migration with inhibition of survival, angiogenic and
invasive factors. Knockdown of EWS expression was associated with removal of
DNA methylation from p53 promoter, promoting expression of p53, Puma, and Noxa.
EWS shRNA plus TFL induced the highest amounts of apoptosis with activation of
extrinsic and intrinsic pathways in both cell lines in culture. EWS shRNA plus
TFL also inhibited growth of Ewing’s sarcoma tumors in animal models due to
inhibition of differentiation inhibitors and angiogenic and invasive factors
and also induction of activation of caspase-3 for apoptosis. Collectively,
knockdown of EWS expression increased various anti-tumor mechanisms of TFL in
human Ewing’s sarcoma in cell culture and animal models.