PT  - JOURNAL ARTICLE
AU  - Eleonora Zorzan
AU  - Silvia Da Ros
AU  - Mery Giantin
AU  - Lara Zorro Shahidian
AU  - Giorgia Guerra
AU  - Manlio Palumbo
AU  - Claudia Sissi
AU  - Mauro Dacasto
TI  - Targeting Canine KIT Promoter by Candidate DNA G-quadruplex Ligands
AID  - 10.1124/jpet.118.248997
DP  - 2018 Jan 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - jpet.118.248997
4099  - http://jpet.aspetjournals.org/content/early/2018/10/01/jpet.118.248997.short
4100  - http://jpet.aspetjournals.org/content/early/2018/10/01/jpet.118.248997.full
AB  - G-quadruplexes (G4) are nucleic acid secondary structures frequently assumed by G-rich sequences located mostly at telomeres and proto-oncogenes promoters. Recently, we identified, in canine KIT promoter, two G-rich sequences able to fold into G4: d_kit1 and d_kit2_A16. In this study, an anthraquinone (AQ1) and an anthracene derivative (AN6), known to stabilize the G4 structures of the corresponding human h_kit1 and h_kit2, were tested on the canine G4 and in two canine mast cell tumor (MCT) cell lines (C2 and NI-1) to verify their capability to downregulate KIT expression. The cytotoxicity of AQ1 and AN6 was determined using the Alamar Blue test; meantime, the constitutive expression of KIT and other proto-oncogenes containing G4 structures in their promoter (BCL2, VEGFα, VEGFR2, KRAS, and TERT) was assessed by quantitative real-time PCR (qPCR). Then, the time- and dose-dependent effects of both ligands on target gene expression were assessed by qPCR. All target genes were constitutively expressed up to 96 hours of culture. Both ligands decreased KIT mRNA levels and c-kit protein amount, and AN6 was comparatively fairly more effective. DNA interaction studies and a dual-luciferase gene reporter assay performed on a non-cancerous canine cell line (MDCK) proved that this downregulation was the result of the interaction of AN6 with KIT proximal promoter. Interestingly, present results only partially overlap with former ones previously obtained in human cell lines, where AQ1 was found as the most effective compound. These preliminary data might suggest AN6 as a promising candidate for the selective targeting of canine KIT-dependent tumors.