Investigation into the biological properties of the olive polyphenol, hydroxytyrosol: mechanistic insights by genome-wide mRNA-Seq analysis.

The medicinal properties of the leaves and fruit of Olea Europaea (olive tree) have been known since antiquity. Numerous contemporary studies have linked the Mediterranean diet with increased health. In particular, consumption of olive oil has been associated with a decreased risk of cardiovascular disease and certain cancers. Increasingly, there has been an interest in the biological properties of polyphenols, which are minor constituents of olive oil. For example, hydroxytyrosol has been shown to be a potent antioxidant and has anti-atherogenic and anti-cancer properties. The overall aim of this study was to provide insights into the molecular mechanisms of action of hydroxytyrosol using genome-wide mRNA-Seq. Initial experiments were aimed at assessing cytotoxicity, apoptosis and cell cycle effects of hydroxytyrosol in various cell lines. The findings indicated a dose-dependent reduction in cell viability in human erythroleukemic K562 and human keratinocytes. When comparing the viability in parental CEM-CCRF and R100 cells (which overexpress the P-glycoprotein pump), it was determined that the R100 cells were more resistant to effects of hydroxytyrosol suggesting efflux by the multi-drug resistance pump. By comparing the uptake of Hoechst 33342 in the two cell lines that had been pretreated with hydroxytyrosol, it was determined that the polyphenol may have P-glycoprotein-modulating activity. Further, initial studies indicated modest radioprotective effects of relatively low doses of hydroxytyrosol in human keratinocytes. Analysis of mRNA sequencing data identified that treatment of keratinocytes with 20 µM hydroxytyrosol results in the upregulation of numerous antioxidant proteins and enzymes, including heme oxygenase-1 (15.46-fold upregulation), glutaredoxin (1.65) and glutathione peroxidase (1.53). This may account for the radioprotective activity of the compound, and reduction in oxidative stress suggests a mechanism for chemoprevention of cancer by hydroxytyrosol. Alteration in the expression of transcription factors may also contribute to the anti-cancer effects described in numerous studies. These include changes in the expression of STAT3, STAT6, SMAD7 and ETS-1. The telomerase subunit TERT was also found to be downregulated in K562 cells. Overall, our findings provide insights into the mechanisms of action of hydroxytyrosol, and more generally, we identify potential gene candidates for further exploration.

γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis.

The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage.

PURPOSE: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. MATERIALS AND METHODS: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (¹³7Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. RESULTS: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 µM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 µM. CONCLUSIONS: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.

Radio- and chemo-sensitization of human erythroleukemic K562 cells by the histone deacetylase inhibitor Trichostatin A.

Histone deacetylase inhibitors are emerging therapeutic agents for cancer. In addition to effecting hyperacetylation of core histones, they have been shown to induce biologic effects such as cell cycle redistribution, cytostasis and in certain cases apoptosis in a variety of cell lines. In this study, the purpose was to investigate the effects of Trichostatin A (TSA) - the most potent histone deacetylase inhibitor identified to date - in human erythroleukemic K562 cells. Further aims were to examine the effect of TSA pre-treatment on the chemosensitivity of the cells to the anthracycline, doxorubicin, and on radiosensitivity. In all experiments the cells were treated with 0.2, 0.5 and 2 mM TSA for 24 h prior to analysis for histone acetylation status, cell growth and survival. In parallel assays, TSA treated cells were exposed to doxorubicin or g-radiation and subsequently analyzed for clonogenic survival. The findings indicated that TSA exhibits potent histone deacetylase inhibitor activity in K562 cells, resulting in hyperacetylation of histones 3 and 4 at the concentrations tested. Furthermore, treatment of cells with TSA resulted in dose-dependent inhibition of proliferation, reduction in clonogenic survival and induction of apoptosis. Moreover, the findings of clonogenic survival assays indicated that pre-treatment of K562 cells with TSA augmented the cytotoxic potency of doxorubicin. The magnitude of sensitization to 10 mM doxorubicin-mediated cell death was approximately 2-fold in cells that were treated with 0.2 mM TSA and 5-fold in cells exposed to 0.5 and 2 mM TSA, compared to cells that had not been pre-treated with the histone deacetylase inhibitor. Similarly, exposure of K562 cells to TSA prior to irradiation resulted in dose-dependent radiosensitization. The dose modification factors at D(37) were calculated to be 1.3, 1.6 and 2.5 for cells treated with 0.2, 0.5 and 2 mM TSA, respectively. These findings provide additional evidence that histone deacetylase inhibitors can increase the cytotoxic efficiency of chemotherapeutic drugs, particularly those which target DNA, and can enhance the sensitivity of cells to g-radiation. More generally, the results support the development of histone deacetylase inhibitors as potential clinical chemo- and radio-sensitizers.