Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models

Abstract

Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an “in house” library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters.From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT–dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., α155, HMC1.2 and ROSA cells).Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations.

Targeting CanineKITPromoter by Candidate DNA G-Quadruplex Ligands

“…Moreover, and fairly similar to C2 cell line, the exposure to AQ1 subcytotoxic concentrations resulted in a significant down-regulation of KIT mRNA levels and c-kit protein. Even so, this level of inhibition in canine models is less pronounced when compared with the inhibition obtained in the human mast cell leukemia cell line HMC1.2 (i.e., 2-fold vs. 5-fold decrease in dog vs. human cell line, respectively, Zorzan et al, 2016).…”

“…Overall, only BCL2 showed a trend to mRNA down-regulation in both canine cell lines. This result was not unexpected-in fact, AQ1 causes a marked inhibition of BCL2 mRNA levels in human cell lines as well (Zorzan et al, 2016). Moreover, some anthraquinone derivatives have been shown to induce apoptosis in vitro, a phenomenon that usually implies a decrease of BCL2 mRNA/protein (Huang et al, 2007(Huang et al, , 2014Hasan et al, 2011;Dong et al, 2017).…”

“…AQ1 and AN6 were synthesized by Prof. G. Zagotto (University of Padua, Italy). Stock solutions were prepared as previously reported elsewhere (Zorzan et al, 2016).…”

“…The cell pellets were washed once with phosphatebuffered saline 1X containing 0.02% EDTA, then they were resuspended in 0.5 ml of TRIzol reagent (Thermo Fisher Scientific). Total RNA extraction, its qualitative and quantitative evaluation, and the reverse transcription into cDNA were performed according to the methods of Zorzan et al (2016).…”

“…In a previous study, we selected and tested in different human neoplastic cell lines an anthraquinone and an anthracene derivative: AQ1 and AN6, respectively. Both compounds stabilized h_kit1 and h_kit2 and led to an inhibition of cell proliferation and KIT down-regulation, with AQ1 being more effective than AN6 (Zorzan et al, 2016).…”