Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth

Sagar Shelake, Umesh Tanaji Sankpal, Paul Bowman, Matthew Wise, Anish Ray, Riyaz Mahammad Basha

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)158-165
Number of pages8
JournalInvestigational New Drugs
Volume35
Issue number2
DOIs
StatePublished - 1 Apr 2017

Fingerprint

Sp1 Transcription Factor
Ewing's Sarcoma
Growth
Neoplasms
Proteasome Inhibitors
tolfenamic acid
DNA
Proteasome Endopeptidase Complex
Cell Extracts
Oligonucleotides
Small Interfering RNA
Young Adult
Cell Survival
Proteins
Transcription Factors
Gels
Cell Proliferation
Bone and Bones
Polymerase Chain Reaction
Messenger RNA

Keywords

  • Ewing sarcoma
  • Mithramycin
  • Sp1
  • Survivin
  • Targeted therapy
  • Tolfenamic acid

Cite this

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title = "Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth",
abstract = "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.",
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Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth. / Shelake, Sagar; Sankpal, Umesh Tanaji; Bowman, Paul; Wise, Matthew; Ray, Anish; Basha, Riyaz Mahammad.

In: Investigational New Drugs, Vol. 35, No. 2, 01.04.2017, p. 158-165.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth

AU - Shelake, Sagar

AU - Sankpal, Umesh Tanaji

AU - Bowman, Paul

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AU - Basha, Riyaz Mahammad

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N2 - 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.

AB - 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.

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