title: Berberine alters epigenetic modifications, disrupts microtubule network, and modulates HPV-18 E6-E7 oncoproteins by targeting p53 in cervical cancer cell HeLa: a mechanistic study including molecular docking.
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.
authors.affiliation: Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India.
authors.forename: Santu Kumar
authors.initials: SK
authors.lastname: Saha
authors.affiliation: Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India. Electronic address: prof_arkb@yahoo.co.in.
authors.forename: Anisur Rahman
authors.initials: AR
authors.lastname: Khuda-Bukhsh
mesh_terms: D001599:Berberine / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000187:drug effects* / Q000235:genetics; D005260:Female; D006367:HeLa Cells; D052161:Human papillomavirus 18 / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008870:Microtubules / Q000187:drug effects* / Q000235:genetics; D062105:Molecular Docking Simulation / Q000379:methods; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D001599:Berberine
keywords: Berberine; Berberine (Pubchem SID_162173365); Cell signaling; Cervical cancer; Cytoskeleton structures; Epigenetic modification; Molecular docking