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Targeting Canine KIT Promoter by Candidate DNA G-Quadruplex Ligands.

Eleonora Zorzan1, Silvia Da Ros1, Mery Giantin1

  • 1Department of Comparative Biomedicine and Food Science, University of Padua, Agripolis Legnaro, Padua, Italy (E.Z., M.G., L.Z.S., G.G., M.D.), and Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy (S.D.R., M.P., C.S.).

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Anthracene derivative AN6 effectively down-regulates canine KIT expression by interacting with its promoter. This G-quadruplex stabilizer shows promise for targeting canine mast cell tumors.

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Area of Science:

  • Molecular Biology
  • Oncology
  • Drug Discovery

Background:

  • G-quadruplexes (G4) are nucleic acid structures in G-rich regions like telomeres and proto-oncogene promoters.
  • Canine KIT proto-oncogene promoter contains G-rich sequences (d_kit1, d_kit2_A16) capable of forming G4 structures.

Purpose of the Study:

  • To evaluate the efficacy of G4-stabilizing ligands (anthraquinone AQ1, anthracene derivative AN6) in down-regulating canine KIT expression.
  • To assess the potential of these ligands for targeting canine mast cell tumors (MCTs).

Main Methods:

  • Cytotoxicity assessed via Alamar Blue test.
  • Gene expression (KIT, BCL2, VEGFα, VEGFR2, KRAS, TERT) quantified using qRT-PCR.
  • Ligand interaction with KIT promoter confirmed through DNA binding studies and dual-luciferase reporter assay.

Main Results:

  • Both AQ1 and AN6 reduced canine KIT mRNA and c-kit protein levels.
  • AN6 demonstrated greater efficacy than AQ1 in down-regulating KIT.
  • AN6's mechanism involves direct interaction with the canine KIT proximal promoter.

Conclusions:

  • AN6 is a promising candidate for selective targeting of canine KIT-dependent tumors.
  • Results partially differ from human cell line studies, highlighting species-specific responses to G4 ligands.