Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India.
Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India.
Eur J Pharmacol. 2014 Dec 5;744:132-46. doi: 10.1016/j.ejphar.2014.09.048. Epub 2014 Oct 18.
Increased evidence of chemo-resistance, toxicity and carcinogenicity necessitates search for alternative approaches for determining next generation cancer therapeutics and targets. We therefore tested the efficacy of plant alkaloid berberine on human papilloma virus (HPV) -18 positive cervical cancer cell HeLa systematically-involving certain cellular, viral and epigenetic factors. We observed disruptions of microtubule network and changes in membrane topology due to berberine influx through confocal and atomic force microscopies (AFM). We examined nuclear uptake, internucleosomal DNA damages, mitochondrial membrane potential (MMP) alterations and cell migration assays to validate possible mode of cell death events. Analytical data on interactions of berberine with pBR322 through fourier transform infrared (FTIR) and gel migration assay strengthen berberine׳s biologically significant DNA binding abilities. We measured cellular uptake, DNA ploidy and DNA strand-breaks through fluorescence activated cell sorting (FACS). To elucidate epigenetic modifications, in support of DNA binding associated processes, if any, we conducted methylation-specific restriction enzyme (RE) assay, methylation specific-PCR (MSP) and expression studies of histone proteins. We also analyzed differential interactions and localization of cellular tumor suppressor p53 and viral oncoproteins HPV-18 E6-E7 through siRNA approach. We further made in-silico approaches to determine possible binding sites of berberine on histone proteins. Overall results indicated cellular uptake of berberine through cell membrane depolarization causing disruption of microtubule networks and its biological DNA binding abilities that probably contributed to epigenetic modifications. Results of modulation in p53 and viral oncoproteins HPV-18 E6-E7 by berberine further proved its potential as a promising chemotherapeutic agent in cervical cancer.
越来越多的证据表明，化疗耐药性、毒性和致癌性需要寻找替代方法来确定下一代癌症治疗方法和靶点。因此，我们系统地测试了植物生物碱小檗碱对人乳头瘤病毒 (HPV) -18 阳性宫颈癌 HeLa 细胞的疗效，涉及某些细胞、病毒和表观遗传因素。我们通过共聚焦和原子力显微镜 (AFM) 观察到微管网络的破坏和由于小檗碱流入引起的膜拓扑结构的变化。我们检查了核摄取、核小体间 DNA 损伤、线粒体膜电位 (MMP) 改变和细胞迁移实验，以验证可能的细胞死亡方式。通过傅里叶变换红外 (FTIR) 和凝胶迁移实验分析小檗碱与 pBR322 的相互作用数据，证实了小檗碱具有重要的生物学 DNA 结合能力。我们通过荧光激活细胞分选 (FACS) 测量细胞摄取、DNA 倍性和 DNA 链断裂。为了阐明表观遗传修饰，以支持任何与 DNA 结合相关的过程，我们进行了甲基化特异性限制性内切酶 (RE) 测定、甲基化特异性聚合酶链反应 (MSP) 和组蛋白蛋白表达研究。我们还通过 siRNA 方法分析了细胞肿瘤抑制因子 p53 和病毒癌蛋白 HPV-18 E6-E7 的差异相互作用和定位。我们还进行了计算机模拟方法来确定小檗碱在组蛋白蛋白上的可能结合位点。总体结果表明，小檗碱通过细胞膜去极化进入细胞，导致微管网络的破坏及其生物学 DNA 结合能力，这可能有助于表观遗传修饰。小檗碱对 p53 和 HPV-18 E6-E7 病毒癌蛋白的调节作用进一步证明了它在宫颈癌中作为一种有前途的化疗药物的潜力。
