Transcription factor Specificity protein 1 (Sp1) and its downstream target
survivin (inhibitor of apoptosis protein), play major roles in the pathogenesis of various
cancers.
Ewing Sarcoma (ES) is a common soft tissue/bone
tumor in adolescent and young adults. Overexpression of
survivin is also linked to the aggressiveness and poor prognosis of ES. Small molecule
Tolfenamic acid (TA) inhibits Sp1 and
survivin in
cancer cells. In this investigation, we demonstrate a strategy to target Sp1 and
survivin using TA and positive control
Mithramycin A (Mit) to inhibit ES cell growth. Knock down of Sp1 using
small interfering RNA (
siRNA) resulted in significant (p < 0.05) inhibition of CHLA-9 and TC-32 cell growth as assessed by CellTiter-Glo assay kit. TA or Mit treatment caused dose/time-dependent inhibition of cell viability, and this inhibition was correlated with a decrease in Sp1 and
survivin protein levels in ES cells. Quantitative PCR results showed that Mit treatment decreased the
mRNA expression of both
survivin and Sp1, whereas TA diminished only
survivin but not Sp1.
Proteasome inhibitor restored TA-induced inhibition of Sp1
protein expression suggesting that TA might cause
proteasome-dependent degradation. Gel shift assay using ES cell nuclear extract and biotinylated Sp1 consensus
oligonucleotides confirmed that both TA and Mit decreased
DNA-binding activity of Sp1. These results demonstrate that both Mit and TA reduce expression of Sp1 and
survivin, disrupt Sp1
DNA-binding and inhibit ES cell proliferation. This investigation suggests that targeting Sp1 and
survivin could be an effective strategy for inhibiting ES cell growth.