Chelerythrine down regulates expression of VEGFA, BCL2 and KRAS by arresting G-Quadruplex structures at their promoter regions

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.

Figure 1. UV absorption spectra of Chelerythrine (10 μM) with increasing concentration of promoter sequences (BCL2, KRAS and VEGFA) showing red shift and hypochromism.

(B) BCL2 (12 nm red shift, hypochromism 61%), (C) KRAS (14 nm red shift, hypochromism 50%), (D) VEGFA (12 nm red shift, hypochromism 63%). All experiments were carried out using 10 mM potassium phosphate buffer containing 100 mM potassium chloride at pH 7.0. (A) Chemical structure of Chelerythrine.

Figure 2. Fluorescence anisotropy of Chelerythrine (CHL) (10 μM) vs incresaing concentration of promoter sequences (BCL2, KRAS and VEGFA).

(A) BCL2 (B) KRAS (C) VEGFA. All experiments were carried out using 10 mM potassium phosphate buffer containing 100 mM potassium chloride at pH 7.0.

Figure 3. Ethidium bromide (EtBr) displacement assay with increasing concentration of Chelerythrine displaying stacking mode of interaction.

(A) BCL2 (B) KRAS (C) VEGFA. All experiments were carried out using 10 mM potassium phosphate buffer containing 100 mM potassium chloride at pH 7.0.

Figure 4. Far UV CD spectra of promoter sequences (BCL2, KRAS, VEGFA) with increasing concentration of Chelerythrine.

(A) BCL2 (B) KRAS (C) VEGFA. All experiments were carried out using 10 mM potassium phosphate buffer containing 100 mM potassium chloride at pH 7.0.

Figure 5. CD melting profile of free promoter sequences (BCL2, KRAS and VEGFA) and their complex with Chelerytrine in the ratio 1:2.

(A) BCL2 (B) KRAS (C) VEGFA. All experiments were carried out using 10 mM potassium phosphate buffer containing 100 mM potassium chloride at pH 7.0.

Figure 6. Isothermal titration calorimetry (ITC) profile of Chelerythrine with promoter sequences (BCL2, KRAS and VEGFA).

(A) BCL2 (B) KRAS (C) VEGFA. The top panel display the isothermal plot of the Chelerythrine-promoter sequences complex formation, whereas lower panel represent the integrated binding isotherm generated from the integration of peak area as a function of molar ratio. The solid line represents the best fit data using ‘one site binding model’.

Figure 7

(A) Real time PCR analysis show Chelerythrine-mediated repression of VEGFA, KRAS and BCL2 genes expression in MCF7 cells. Bar diagram showing relative expression level, quantified using real time PCR analysis, of VEGFA, KRAS and BCL2 genes after treatment of cells with 15 and 150 nM (n = 3 batches of cells for each treatment) Chelerythrine for 24 h. Control received only the buffer in which Chelerytrine was suspended. Data presented as relative expression level (±Standard Error) where expression in control calculated as almost 1 (±Standard Error). Expressions in different samples normalized based on expression of GAPDH gene. *significantly changed from control, and **significantly changed from cognate 15 nM sample (indicated with dashed line). (B–G) Chelerythrine downregulates the promoter activity of BCL2, KRAS and VEGFA through targeting G-quadruplex, confirmed by luciferase assay. (B,D,F) Schematic representations of the reporter luciferase constructs are given. BCL2 G-Quadruplex (GQ), KRAS GQ, and VEGFA GQ are the quadruplex- scaffolds which are located at the upstream of hRlucCP. GQ deleted constructs are also demonstrated below. (C,E,G) Promoter activity is shown in terms of relative luciferase units, which dictates the expression of the reporter gene. Significant reduction in luciferase activity is found for the constructs which harbour the quadruplex motifs in the upstream of promoters compared to those which are devoid of quadruplex motifs. Error bars in the bar plots (E,F and G) represent means ± s.d. from three independent experiments. Asterisks (*) indicate statistical significance as determined from Student’s t-test (*indicates P < 0.05, **indicates P < 0.01, ***indicates P < 0.001), which denote significant differences in the promoter activities of BCL2, KRAS, and VEGFA, compared with values for untreated cells (control).

Figure 8

(A–D) Ensemble structure of BCL2. (A) control (B) BCL2-Chelerythrine (1:2) complex. The frames were collected from 45–50 ns of MD trajectory. (grey colour showed DNA bases present in the loop) (blue (A) and orange (B) color showed G-quartet forming bases) (C) A plot of RMSD of all atom vs time. (D) A plot of RMSF of individual residue of BCL2 control and BCL2-Chelerythrine complex. (E–H) Ensemble structure of VEGFA. (E) control (F) VEGFA-Chelerythrine (1:2) complex. The frames were collected from 45-50 ns of MD trajectory. (blue colour showed DNA bases present in the loop). (light pink (A) and (B) color showed G-quartet forming bases) (G) A plot of RMSD of all atom vs time. (H) A plot of RMSF of individual residue of BCL2 control and VEGFA-Chelerythrine complex.