title: Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro.
abstract: OBJECTIVE
  Methylation-specific epigenetic process and gene expression profiles of HeLa cells treated with ultra-high dilutions (HDs) of two plant extracts, Hydrastis canadensis (HC-30) and Marsdenia condurango (Condu-30), diluted 1060 times, were analyzed against placebo 30C (Pl-30) for alterations in gene profiles linked to epigenetic modifications.
  
  
  METHODS
  Separate groups of cells were subjected to treatment of Condu-30, HC-30, and Pl-30 prepared by serial dilutions and succussions. Global microarray data recorded on Affymetrix platform, using 25-mer probes were provided by iLifeDiscoveries, India. Slides were scanned with 3000 7G microarray scanner and raw data sets were extracted from Cel (raw intensity) files. Analyses of global microarray data profile, differential gene expression, fold change and clusters were made using GeneSpring GX12.5 software and standard normalization procedure. Before microarray study, concentration of RNA (ng/μL), RIN value and rRNA ratio for all the samples were analysed by Agilant Bioanalyzer 2100. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RT-PCR were done for analyzing SMAD-4 expression. Fluorescence-activated cell sorting study was further made to elucidate fate of cells at divisional stages. Methylation-specific restriction enzyme assay was conducted for ascertaining methylation status of DNA at specific sites.
  
  
  RESULTS
  HDs of HC-30 and Condu-30 differentially altered methylation in specific regions of DNA and expression profiles of certain genes linked to carcinogenesis, as compared to Pl-30. Two separate cut sites were found in genomic DNA of untreated and placebo-treated HeLa cells when digested with McrBC, compared to a single cut observed in Condu-30-treated genomic DNA. SMAD-4 gene expression validated the expression pattern observed in microarray profile. Methylation-specific restriction enzyme assay elucidated differential epigenetic modifications in drug-treated and control cells.
  
  
  CONCLUSIONS
  HDs triggered epigenetic modifications and alterations in microarray gene expression profiles of many genes associated with carcinogenesis in HeLa cells in vitro.
authors.affiliation: Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India.
authors.forename: Santu Kumar
authors.initials: SK
authors.lastname: Saha
authors.affiliation: Department of Entomology and Institute for Integrative Genome Biology, University of California Riverside, CA 92521, USA.
authors.forename: Sourav
authors.initials: S
authors.lastname: Roy
authors.affiliation: Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India.
authors.forename: Anisur Rahman
authors.initials: AR
authors.lastname: Khuda-Bukhsh
mesh_terms: D002453:Cell Cycle; D016000:Cluster Analysis; D019175:DNA Methylation; D044127:Epigenesis, Genetic / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D039321:Hydrastis; D031101:Marsdenia; D010936:Plant Extracts / Q000494:pharmacology*; D059467:Transcriptome / Q000187:drug effects*
chemical_list: D010936:Plant Extracts