Mechanisms of growth inhibition of primary prostate epithelial cells following gamma irradiation or photodynamic therapy including senscence, necrosis, and autophagy, but not apoptosis

In comparison to more differentiated cells, prostate cancer stem-like cells are radioresistant, which could explain radio-recurrent prostate cancer. Improvement of radiotherapeutic efficacy may therefore require combination therapy. We have investigated the consequences of treating primary prostate epithelial cells with gamma irradiation and photodynamic therapy (PDT), both of which act through production of reactive oxygen species (ROS). Primary prostate epithelial cells were cultured from patient samples of benign prostatic hyperplasia and prostate cancer prior to treatment with PDT or gamma irradiation. Cell viability was measured using MTT and alamar blue assay, and cell recovery by colony-forming assays. Immunofluorescence of gamma-H2AX foci was used to quantify DNA damage, and autophagy and apoptosis were assessed using Western blots. Necrosis and senescence were measured by propidium iodide staining and beta-galactosidase staining, respectively. Both PDT and gamma irradiation reduced the colony-forming ability of primary prostate epithelial cells. PDT reduced the viability of all types of cells in the cultures, including stem-like cells and more differentiated cells. PDT induced necrosis and autophagy, whereas gamma irradiation induced senescence, but neither treatment induced apoptosis. PDT and gamma irradiation therefore inhibit cell growth by different mechanisms. We suggest these treatments would be suitable for use in combination as sequential treatments against prostate cancer

At-grade stabilization structure impact on surface water quality of an agricultural watershed

Decades of farming and fertilization of farm land in the unglaciated/Driftless Area (DA) of southwestern Wisconsin have resulted in the build-up of P and to some extent, N, in soils. This build-up, combined with steep topography and upper and lower elevation farming (tiered farming), exacerbates problems associated with runoff and nutrient transport in these landscapes. Use of an at-grade stabilization structure (AGSS) as an additional conservation practice to contour strip cropping and no-tillage, proved to be successful in reducing organic and sediment bound N and P within an agricultural watershed located in the DA. The research site was designed as a paired watershed study, in which monitoring stations were installed on the perennial streams draining both control and treatment watersheds. Linear mixed effects statistics were used to determine significant changes in nutrient concentrations before and after installation of an AGSS. Results indicate a significant reduction in storm event total P (TP) concentrations (P = 0.01) within the agricultural watershed after installation of the AGSS, but not total dissolved P (P = 0.23). This indicates that the reduction in P concentration is that of the particulate form. Storm event organic N concentrations were also significantly reduced (P = 0.03) after the AGSS was installed. We conclude that AGSS was successful in reducing the organic and sediment bound N and P concentrations in runoff waters thus reducing their delivery to nearby surface waters

Innovation in regulated electricity distribution networks : a review of the effectiveness of Great Britain's low carbon network fund

Introduced in 2010, the Low Carbon Networks Fund (LCNF) was a major development in the regulatory regime for electricity distribution networks in Great Britain, yet evaluation of its design and implementation has been limited. This paper examines the type and quality of innovation arising from the LCNF. Novel frameworks for assessing innovation project activity and learning are presented and results from their application to the LCNF are discussed. Reduction of uncertainty through the production of high quality evidence is argued to be the primary purpose of innovation project funding support. The analysis of LCNF project activity finds a step change in Research Development & Demonstration (RD&D) spend and stakeholder engagement by network licensees in Britain; however, the innovation observed was considered to be conservative and incremental in nature. It was found that the LCNF lacked a strategic approach to targeted learning and the reduction of uncertainty for innovation priority areas. Project learning outputs were contradictory and inconclusive for several innovations. Strategic learning should be a core part of policy makers’ design of innovation funding mechanisms for energy technology, and a framework for shaping, capturing and assessing the learning outputs of funded innovation projects is essential

At-grade stabilization structure impact on surface water quality of an agricultural watershed

Decades of farming and fertilization of farm land in the unglaciated/Driftless Area (DA) of southwestern Wisconsin have resulted in the build-up of P and to some extent, N, in soils. This build-up, combined with steep topography and upper and lower elevation farming (tiered farming), exacerbates problems associated with runoff and nutrient transport in these landscapes. Use of an at-grade stabilization structure (AGSS) as an additional conservation practice to contour strip cropping and no-tillage, proved to be successful in reducing organic and sediment bound N and P within an agricultural watershed located in the DA. The research site was designed as a paired watershed study, in which monitoring stations were installed on the perennial streams draining both control and treatment watersheds. Linear mixed effects statistics were used to determine significant changes in nutrient concentrations before and after installation of an AGSS. Results indicate a significant reduction in storm event total P (TP) concentrations (P = 0.01) within the agricultural watershed after installation of the AGSS, but not total dissolved P (P = 0.23). This indicates that the reduction in P concentration is that of the particulate form. Storm event organic N concentrations were also significantly reduced (P = 0.03) after the AGSS was installed. We conclude that AGSS was successful in reducing the organic and sediment bound N and P concentrations in runoff waters thus reducing their delivery to nearby surface waters

Correction to: “Constraining climate forecasts: The role of prior assumptions”

N/

Differential cytotoxic activity of a novel palladium-based compound on prostate cell lines, primary prostate epithelial cells and prostate stem cells

The outcome for patients with advanced metastatic and recurrent prostate cancer is still poor. Therefore, new chemotherapeutics are required, especially for killing cancer stem cells that are thought to be responsible for disease recurrence. In this study, we screened the effect of a novel palladium-based anticancer agent (Pd complex) against six different prostate cancer cell lines, and primary cultures from seven Gleason 6/7 prostate cancer, three Gleason 8/9 prostate cancer and four benign prostate hyperplasia patient samples, as well as cancer stem cells selected from primary cultures. MTT and ATP viability assays were used to assess cell growth and flow cytometry to assess cell cycle status. In addition, immunofluorescence was used to detect γH2AX nuclear foci, indicative of DNA damage, and Western blotting to assess the induction of apoptosis and autophagy. The Pd complex showed a powerful growth-inhibitory effect against both cell lines and primary cultures. More importantly, it successfully reduced the viability of cancer stem cells as first reported in this study. The Pd complex induced DNA damage and differentially induced evidence of cell death, as well as autophagy. In conclusion, this novel agent may be promising for use against the bulk of the tumour cell population as well as the prostate cancer stem cells, which are thought to be responsible for the resistance of metastatic prostate cancer to chemotherapy. This study also indicates that the combined use of the Pd complex with an autophagy modulator may be a more promising approach to treat prostate cancer. In addition, the differential effects observed between cell lines and primary cells emphasise the importance of the model used to test novel drugs including its genetic background, and indeed the necessity of using cells cultured from patient samples

Inhibition of the PI3K/AKT/mTOR pathway activates autophagy and compensatory Ras/Raf/MEK/ERK signalling in prostate cancer

The PI3K/AKT/mTOR pathway is frequently activated in advanced prostate cancer, due to loss of the tumour suppressor PTEN, and is an important axis for drug development. We have assessed the molecular and functional consequences of pathway blockade by inhibiting AKT and mTOR kinases either in combination or as individual drug treatments. In established prostate cancer cell lines, a decrease in cell viability and in phospho-biomarker expression was observed. Although apoptosis was not induced, a G1 growth arrest was observed in PTEN null LNCaP cells, but not in BPH1 or PC3 cells. In contrast, when the AKT inhibitor AZD7328 was applied to patient-derived prostate cultures that retained expression of PTEN, activation of a compensatory Ras/MEK/ERK pathway was observed. Moreover, whilst autophagy was induced following treatment with AZD7328, cell viability was less affected in the patient-derived cultures than in cell lines. Surprisingly, treatment with a combination of both AZD7328 and two separate MEK1/2 inhibitors further enhanced phosphorylation of ERK1/2 in primary prostate cultures. However, it also induced irreversible growth arrest and senescence.Ex vivo treatment of a patient-derived xenograft (PDX) of prostate cancer with a combination of AZD7328 and the mTOR inhibitor KU-0063794, significantly reduced tumour frequency upon re-engraftment of tumour cells.The results demonstrate that single agent targeting of the PI3K/AKT/mTOR pathway triggers activation of the Ras/MEK/ERK compensatory pathway in near-patient samples. Therefore, blockade of one pathway is insufficient to treat prostate cancer in man

Regulation of the stem cell marker CD133 is independent of promoter hypermethylation in human epithelial differentiation and cancer

BackgroundEpigenetic control is essential for maintenance of tissue hierarchy and correct differentiation. In cancer, this hierarchical structure is altered and epigenetic control deregulated, but the relationship between these two phenomena is still unclear. CD133 is a marker for adult stem cells in various tissues and tumour types. Stem cell specificity is maintained by tight regulation of CD133 expression at both transcriptional and post-translational levels. In this study we investigated the role of epigenetic regulation of CD133 in epithelial differentiation and cancer.MethodsDNA methylation analysis of the CD133 promoter was done by pyrosequencing and methylation specific PCR; qRT-PCR was used to measure CD133 expression and chromatin structure was determined by ChIP. Cells were treated with DNA demethylating agents and HDAC inhibitors. All the experiments were carried out in both cell lines and primary samples.ResultsWe found that CD133 expression is repressed by DNA methylation in the majority of prostate epithelial cell lines examined, where the promoter is heavily CpG hypermethylated, whereas in primary prostate cancer and benign prostatic hyperplasia, low levels of DNA methylation, accompanied by low levels of mRNA, were found. Moreover, differential methylation of CD133 was absent from both benign or malignant CD133+/α2β1integrinhi prostate (stem) cells, when compared to CD133-/α2β1integrinhi (transit amplifying) cells or CD133-/α2β1integrinlow (basal committed) cells, selected from primary epithelial cultures. Condensed chromatin was associated with CD133 downregulation in all of the cell lines, and treatment with HDAC inhibitors resulted in CD133 re-expression in both cell lines and primary samples.ConclusionsCD133 is tightly regulated by DNA methylation only in cell lines, where promoter methylation and gene expression inversely correlate. This highlights the crucial choice of cell model systems when studying epigenetic control in cancer biology and stem cell biology. Significantly, in both benign and malignant prostate primary tissues, regulation of CD133 is independent of DNA methylation, but is under the dynamic control of chromatin condensation. This indicates that CD133 expression is not altered in prostate cancer and it is consistent with an important role for CD133 in the maintenance of the hierarchical cell differentiation patterns in cancer