Project description:Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. In the present study, transcriptional profiling experiments coupled with the analysis of mutants were performed in an effort to elucidate its mechanism of action. Using Saccharomyces cerevisiae as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low oxygen conditions. Several additional lines of evidence obtained suggest that these responses could involve effects on heme. First, the hem1 deletion mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells. In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine. Furthermore, spectral analysis of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells. Transcriptional profiling experiments were also performed in C. albicans to investigate the response of a pathogenic fungal species to sampangine. Taking into account the known differences in the physiological responses of C. albicans and S. cerevisiae to low oxygen, significant correlations were observed between the two transcription profiles suggestive of heme-related defects. Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme. Experiment Overall Design: S. cerevisiae S288C cells at OD 0.2 were treated with either sampangine (SMP) at IC50 concentration (1.17 ug/ml), or solvent (0.25% DMSO), allowed to grow to OD 0.5, then harvested and frozen. Three biological replicate samples were analyzed for each treatment.

Project description:Background: Epigenetic modifications such as methylation silencing of genes with CpG-island-associated promoters is frequently observed in cancer. Studies regarding the implications of epigenetic modifications in osteosarcoma (OS) have been limited. The epigenetic drug decitabine is a potential re-activator of silenced genes through de-methylation, and is currently undergoing clinical trials for cancer treatment. No study to date has utilized decitabine to modify gene expression in OS-derived cells to identify gene-specific methylation targets that may have therapeutic importance. The objective of this study was to measure the response of the OS cell line, U-2OS, to decitabine treatment both in vitro and in vivo. Results: Genome-wide screening was used to distinguish decitabine-dependent changes in gene expression in U-2OS and to identify responsive loci with de-methylated CpG regions. U-2OS xenografts were established in the sub-renal capsule of immune-deficient mice to study the effect of decitabine in vivo on tumor growth and differentiation. Genome-wide screening of U-2OS cells in vitro revealed that decitabine treatment at low-dosage (1 ÂµM) caused a significant induction (p<0.0025) in the expression of 88 genes, 13 had a â?¥2-fold change, 11 of which had CpG-island-associated promoters. Interestingly, 6 of these 11 were pro-apoptotic genes. 1 ÂµM decitabine resulted in a significant induction of cell death in U-2OS cells in vitro (p<0.05). Decitabine treatment also reduced tumor volume size significantly (p<0.05) in U-2OS in vivo at a 2.5 mg/kg dose. Histological analysis of U-2OS xenograft sections revealed a lower mitotic activity (p<0.0001), a higher bone matrix production (p<0.0001), and higher apoptosis (p = 0.0329) after treatment. The 6 pro-apoptotic genes were also induced to â?¥2-fold in vivo. Quantitative Methylation Pyrosequencing (Pyro Q-CpG) showed that 4 pro-apoptotic genes had CpG-island DNA de-methylation as a result of treatment in vitro and in vivo. Conclusion: Our data provide new insights regarding the use of epigenetic modifiers in OS, and have important implications for therapeutic trials involving demethylation drugs. The findings of this study suggest an epigenetic mechanism, whereby OS tumor cells silence expression of pro-apoptotic genes through promoter-CpG methylation, to maintain high proliferation capacity and continuous growth. Experiment Overall Design: Total RNA was extracted using the RNeasy kit (Qiagen, Germany) from duplicate experiments of U-2OS cells at Day3 after treatment with 1 ÂµM decitabine or medium alone (control). In each experiment RNA yields were pooled from two independent cultures per treatment arm to minimize experimental noise. For each case, 10 Âµg of RNA was labeled and hybridized to the Affymetrix HG-U133A GeneChips using the manufacturerâ??s protocol (Affymetrix, Santa Clara, CA) by the Center of Applied Genomics at the Hospital for Sick Children (Toronto, Canada). Data were extracted using the Microarray Suite (MAS) version 5.0 (Affymetrix) and linearly scaled to achieve an average intensity of 150 across each chip. The candidate gene list obtained from the MAS 5.0-extracted data was selected by eliminating genes that were not present in at least one experiment. The arrays were subjected to a pair wise comparison using MAS 5.0, with signal intensities from the no-treatment cells as the baseline. The statistical significance for the change of expression for each probe set between the decitabine treated and control was calculated by the MAS5.0 software. The criteria for gene selection for real-time expression validation analysis was based on the statistically significant up-regulation (p<0.0025) and fold change of â?¥ 2 for expression after decitabine treatment. The gene list was annotated based on the NetAffx data-base (http://www.affymetrix.com/analysis /index.affx) and further verified using the Human Genome Browser data base (http://genome.ucsc.edu/)

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Transcription profiling of human Mia Paca2 cells treated with AM251

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