Abstract：
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.
摘要：
化疗耐药的证据越来越多,毒性和致癌性需要寻找确定下一代癌症治疗方法和靶点的替代方法。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阳性宫颈癌细胞HeLa的功效，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）观察到由于小檗碱流入而引起的微管网络破坏和膜拓扑变化。我们检查了核摄取，核小体间DNA损伤，线粒体膜电位（MMP）改变和细胞迁移试验，以验证细胞死亡事件的可能模式。通过傅立叶变换红外（FTIR）和凝胶迁移实验对小檗碱与pBR322相互作用的分析数据增强了小檗碱的生物学意义。我们测量了细胞摄取，通过荧光激活细胞分选(FACS)的DNA倍性和DNA链断裂。为了阐明表观遗传修饰，支持DNA结合相关过程，如果有的话,我们进行了甲基化特异性限制酶（RE）测定，甲基化特异性PCR（MSP）和组蛋白的表达研究。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-18E6-E7的差异相互作用和定位。我们进一步进行了计算机内方法来确定小檗碱在组蛋白上的可能结合位点。总体结果表明，细胞通过细胞膜去极化摄取小檗碱，导致微管网络的破坏及其生物DNA结合能力，这可能是表观遗传修饰的原因。小檗碱对p53和病毒癌蛋白HPV-18E6-E7的调节结果进一步证明了其作为宫颈癌有前途的化学治疗剂的潜力。
