Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro  

作　　者：Santu Kumar Saha Sourav Roy Anisur Rahman Khud a-Bukhsh 

机构地区：[1]Cytogenetics and Molecular Biology Laboratory,Department of Zoology,University of Kalyani [2]Department of Entomology and Institute for Integrative Genome Biology,University of California Riverside

出　　处：《Journal of Integrative Medicine》2015年第6期400-411,共12页结合医学学报（英文版）

基　　金：financially supported by Boiron Laboratories,Lyon,France;Emeritus Fellowship of UGC granted to ARKB

摘　　要：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 106o times, were analyzed against placebo 30C （PI-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 PI- 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/IJL）, 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 PI-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. CONCLUSION： HDsOBJECTIVE： 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 106o times, were analyzed against placebo 30C （PI-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 PI- 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/IJL）, 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 PI-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. CONCLUSION： HDs

关 键 词：plant extracts HOMEOPATHY reactive oxygen species apoptosis gene expression epigenesis genetic Smad4 protein 

分 类 号：R285[医药卫生—中药学]