Abstract
A putative anticancer plant alkaloid, Chelerythrine binds to G-quadruplexes at promoters of VEGFA, BCL2 and KRAS genes and down regulates their expression. The association of Chelerythrine to G-quadruplex at the promoters of these oncogenes were monitored using UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy, CD melting, isothermal titration calorimetry, molecular dynamics simulation and quantitative RT-PCR technique. The pronounced hypochromism accompanied by red shifts in UV absorption spectroscopy in conjunction with ethidium bromide displacement assay indicates end stacking mode of interaction of Chelerythrine with the corresponding G-quadruplex structures. An increase in fluorescence anisotropy and CD melting temperature of Chelerythrine-quadruplex complex revealed the formation of stable Chelerythrine-quadruplex complex. Isothermal titration calorimetry data confirmed that Chelerythrine-quadruplex complex formation is thermodynamically favourable. Results of quantative RT-PCR experiment in combination with luciferase assay showed that Chelerythrine treatment to MCF7 breast cancer cells effectively down regulated transcript level of all three genes, suggesting that Chelerythrine efficiently binds to in cellulo quadruplex motifs. MD simulation provides the molecular picture showing interaction between Chelerythrine and G-quadruplex. Binding of Chelerythrine with BCL2, VEGFA and KRAS genes involved in evasion, angiogenesis and self sufficiency of cancer cells provides a new insight for the development of future therapeutics against cancer.
Highlights
DNA can adopt right handed double helical B-form structure[1]
In the present study we have shown that Chelerythrine binds to human telomeric DNA and RNA G-quadruplex it binds to quadruplex structures formed in the promoter region of oncogenes like B-cell CLL/lymphoma 2 (BCL2), Vascular endothelial growth factor A (VEGFA) and KRAS
Various spectroscopic techniques (UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy and CD melting) and molecular dynamics simulation fruitfully monitored the binding of Chelerythrine to these G quadruplex structures
Summary
DNA can adopt right handed double helical B-form structure[1]. Under certain conditions DNA can adopt an array of non B-form unique unusual secondary structures based on particular sequence motif[2]. Six important proceedings in the malignant process are evasion of apoptosis, insensitivity to anti‐growth signals, self‐sufficiency in growth signals, sustained angiogenesis, limitless replicative potential and tissue invasion and metastasis These are known as six hallmarks of cancer[7]. These important events are linked with G‐quadruplex forming gene promoters including c-MYC8, c-KIT9 and KRAS10 (self‐sufficiency), RB1 (insensitivity), BCL211 (evasion of apoptosis), VEGFA (angiogenesis)[12] hTERT (limitless replication)[13] and PDGFA (metastasis). Several studies suggested that formation and stabilization of G-quadruplex in the promoter region of proto oncogene like c-MYC3, c-KIT14, KRAS15, BCL216, VEGFA17 etc. In this report we have studied the binding of Chelerythrine with G-quadruplex forming promoter sequences of BCL2, VEGFA and KRAS.
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