Paul Avijit, Bishayee Kausik, Ghosh Samrat, Mukherjee Avinaba, Sikdar Sourav, Chakraborty Debrup, Boujedaini Naoual, Khuda-Bukhsh Anisur Rahman
Department of Zoology, University of Kalyani, Kalyani, India.
Zhong Xi Yi Jie He Xue Bao. 2012 Sep;10(9):1025-38. doi: 10.3736/jcim20120912.
To evaluate the role of chelidonine isolated from ethanolic extract of Chelidonium majus in inducing apoptosis in HeLa cells and to assess the main signalling pathways involved.
Cells were initially treated with different concentrations of chelidonine for 48 h and the median lethal dose (LD50) value was selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological analysis of nuclear condensation and DNA damage and fragmentation were measured by 4',6-diamidino-2-phenylindole staining and comet assay. Further, reactive oxygen species (ROS) generation, cell cycle arrest and change in mitochondrial membrane potential were also examined and analyzed by flow cytometry. Evaluation of interaction of drug with CT DNA was investigated by circular dichroism (CD) spectral analysis to find any possible drug-CT DNA interaction. The mRNA and protein expressions of major signal proteins like p38, p53, protein kinase B (AKT), phosphatidylinositol 3-kinases (PI3K), Janus kinase 3 (JAK3), signal transducer and activator of transcription 3 (STAT3) and E6 and E7 oncoproteins as well as the pro-apoptotic genes and antiapoptotic genes were also estimated by reverse transcriptase-polymerase chain reaction and Western blotting.
Based on LD(50) value (30 μg/mL) of chelidonine, three doses were selected, namely, 22.5 μg/mL (D1), 30.0 μg/mL (D2) and 37.5 μg/mL (D3). Results showed that chelidonine inhibited proliferation and induced apoptosis in HeLa cells through generation of ROS, cell cycle arrest at sub-G1 and G0/G1 stage, change in mitochondrial membrane potential and fragmentation of DNA. Results of CD spectra showed effective interaction between chelidonine and calf thymus DNA. Studies of signalling pathway revealed that chelidonine could efficiently induce apoptosis through up-regulation of expressions of p38, p53 and other pro-apoptotic genes and down-regulation of expressions of AKT, PI3K, JAK3, STAT3, E6, E7 and other antiapoptotic genes.
Chelidonine isolated from Chelidonium majus efficiently induced apoptosis in HeLa cells through possible alteration of p38-p53 and AKT/PI3 kinase signalling pathways.
评估从白屈菜乙醇提取物中分离得到的白屈菜碱在诱导人宫颈癌HeLa细胞凋亡中的作用，并评估其涉及的主要信号通路。
首先用不同浓度的白屈菜碱处理细胞48小时，通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法确定半数致死剂量(LD50)值。通过4',6-二脒基-2-苯基吲哚染色和彗星试验对核浓缩、DNA损伤和片段化进行形态学分析。此外，还通过流式细胞术检测并分析活性氧(ROS)生成、细胞周期阻滞和线粒体膜电位变化。通过圆二色(CD)光谱分析研究药物与小牛胸腺DNA(CT DNA)的相互作用，以发现任何可能的药物-CT DNA相互作用。通过逆转录聚合酶链反应和蛋白质免疫印迹法评估主要信号蛋白如p38、p53、蛋白激酶B(AKT)、磷脂酰肌醇3激酶(PI3K)、Janus激酶3(JAK3)、信号转导子和转录激活子3(STAT3)以及E6和E7癌蛋白的mRNA和蛋白表达，以及促凋亡基因和抗凋亡基因。
根据白屈菜碱的LD(50)值(30μg/mL)，选择三个剂量，即22.5μg/mL(D1)、30.0μg/mL(D2)和37.5μg/mL(D3)。结果表明，白屈菜碱通过产生活性氧、使细胞周期阻滞在亚G1期和G0/G1期、改变线粒体膜电位和使DNA片段化，抑制HeLa细胞增殖并诱导其凋亡。CD光谱结果表明白屈菜碱与小牛胸腺DNA之间存在有效相互作用。信号通路研究表明，白屈菜碱可通过上调p38、p53和其他促凋亡基因的表达以及下调AKT、PI3K、JAK3、STAT3、E6、E7和其他抗凋亡基因的表达有效诱导凋亡。
从白屈菜中分离得到的白屈菜碱可能通过改变p38-p53和AKT/PI3激酶信号通路，有效诱导HeLa细胞凋亡。
