Department of Biophysics, Bose Institute, Kolkata, WB, India.
Division of Plant Biology, Bose Institute, Kolkata, WB, India.
Sci Rep. 2017 Jan 19;7:40706. doi: 10.1038/srep40706.
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.
一种假定的抗癌植物生物碱，Chelerythrine 与 VEGFA、BCL2 和 KRAS 基因启动子中的 G-四链体结合，并下调它们的表达。使用紫外吸收光谱、荧光各向异性、圆二色性光谱、CD 熔融、等温滴定量热法、分子动力学模拟和定量 RT-PCR 技术监测 Chelerythrine 与这些致癌基因启动子中 G-四链体的结合。紫外吸收光谱中明显的减色伴随着红移，与溴化乙锭置换实验相结合，表明 Chelerythrine 与相应的 G-四链体结构以末端堆积模式相互作用。荧光各向异性和 Chelerythrine-四链体复合物的 CD 熔融温度的增加表明形成了稳定的 Chelerythrine-四链体复合物。等温滴定量热法数据证实 Chelerythrine-四链体复合物的形成在热力学上是有利的。定量 RT-PCR 实验与荧光素酶测定结果表明，Chelerythrine 处理 MCF7 乳腺癌细胞有效地下调了这三个基因的转录水平，表明 Chelerythrine 有效地结合了细胞内的四链体基序。MD 模拟提供了分子图像，显示了 Chelerythrine 与 G-四链体的相互作用。Chelerythrine 与 BCL2、VEGFA 和 KRAS 基因的结合，这些基因参与了癌细胞的逃逸、血管生成和自给自足，为开发针对癌症的未来治疗方法提供了新的见解。
