OBJECTIVE: Methylation-specifi c epigenetic process and gene expression profi les of He La 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 profi les linked to epigenetic modifi cations.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 i Life Discoveries, India. Slides were scanned with 3000 7G microarray scanner and raw data sets were extracted from Cel(raw intensity) fi les. Analyses of global microarray data profi le, differential gene expression, fold change and clusters were made using Gene Spring GX12.5 software and standard normalization procedure. Before microarray study, concentration of RNA(ng/μL), RIN value and r RNA ratio for all the samples were analysed by Agilant Bioanalyzer 2100. Reverse transcriptase polymerase chain reaction(RT-PCR) and quantitative RTPCR were done for analyzing SMAD-4 expression. Fluorescence-activated cell sorting study was further made to elucidate fate of cells at divisional stages. Methylation-specifi c restriction enzyme assay was conducted for ascertaining methylation status of DNA at specifi c sites.RESULTS: HDs of HC-30 and Condu-30 differentially altered methylation in specifi c regions of DNA and expression profi les 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 He La cells when digested with McrB C, compared to a single cut observed in Condu-30-treated genomic DNA. SMAD-4 gene expression validated the expression pattern observed in microarray profi le. Methylation-specifi c restriction enzyme assay elucidated differential epigenetic modifi cations in drug-treated and control cells.CONCLUSION: HDs triggered epigenetic modifi cations and alterations in microarray gene expression profi les of many genes associated with carcinogenesis in HeLa cells in vitro. OBJECTIVE: Methylation-specifi c epigenetic process and gene expression profi les of He La 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 profi les linked to epigenetic modifi cations. 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 i Life Discoveries, India. Slides were scanned with 3000 7G microarray scanner and raw data sets were extracted from Cel (raw intensity) fi les. Analyzes of global microarray data profi le, fold gene expression, fold change and cluster were made using Gene Spring GX12.5 software and standard normalization procedure. Before microarray study, concentration of RNA (ng / μL), RIN value and r RNA ratio for all the samples were analysed by Agilant Bioanalyzer 2100. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RTPCR were done for analyzing SMAD-4 expression. Fluorescence-activated cell sorting study was further made to elucidate fate of cells at divisional stages. Methylation -specifi c restriction enzyme assay was conducted for ascertaining methylation status of DNA at specifi c sites .RESULTS: HDs of HC-30 and Condu-30 differentially altered methylation in specifi c regions of DNA and expression profi les 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 He La cells when digested with McrB C, compared to a single cut observed in Condu-30-treated genomic DNA. SMAD-4 gene expression validated the expression pattern observed in microarray profi le. Methylation-specific c restriction enzyme assay elucidated differential epigenetic modifi cations in drug-treated and control ce lls.CONCLUSION: HDs triggered epigenetic modifi cations and alterations in microarray gene expression profi les of many genes associated with carcinogenesis in HeLa cells in vitro.