Irradiation specifically sensitises solid tumour cell lines to TRAIL mediated apoptosis-3

<p><b>Copyright information:</b></p><p>Taken from "Irradiation specifically sensitises solid tumour cell lines to TRAIL mediated apoptosis"</p><p>BMC Cancer 2005;5():5-5.</p><p>Published online 14 Jan 2005</p><p>PMCID:PMC547906.</p><p>Copyright © 2005 Marini et al; licensee BioMed Central Ltd.</p>analyis using the Quantibrite™ evalution system from BD(Heidelberg, Germany) according to manufacturer's instructions. Data shown are from one representative experiment (n ≥ 3). A: Cell surface expression of R1/DR4 B: Cell surface expression R2/DR

DataSheet_1_CD9- and CD81-positive extracellular vesicles provide a marker to monitor glioblastoma cell response to photon-based and proton-based radiotherapy.docx

Glioblastoma multiforme (GBM) is the most aggressive tumor of the central nervous system with a poor prognosis. In the treatment of GBM tumors, radiotherapy plays a major role. Typically, GBM tumors cannot be cured by irradiation because of intrinsic resistance machanisms. An escalation of the irradiation dose in the GBM tumor is difficult due to the high risk of severe side effects in the brain. In the last decade, the development of new irradiation techniques, including proton-based irradiation, promised new chances in the treatment of brain tumors. In contrast to conventional radiotherapy, irradiation with protons allows a dosimetrically more confined dose deposition in the tumor while better sparing the normal tissue surrounding the tumor. A systematic comparison of both irradiation techniques on glioblastoma cells has not been performed so far. Despite the improvements in radiotherapy, it remains challenging to predict the therapeutical response of GBM tumors. Recent publications suggest extracellular vesicles (EVs) as promising markers predicting tumor response. Being part of an ancient intercellular communication system, virtually all cells release specifically composed EVs. The assembly of EVs varies between cell types and depends on environmental parameters. Here, we compared the impact of photon-based with proton-based radiotherapy on cell viability and phenotype of four different glioblastoma cell lines. Furthermore, we characterized EVs released by different glioblastoma cells and correlated released EVs with the cellular response to radiotherapy. Our results demonstrated that glioblastoma cells reacted more sensitive to irradiation with protons than photons, while radiation-induced cell death 72 h after single dose irradiation was independent of the irradiation modality. Moreover, we detected CD9 and CD81-positive EVs in the supernatant of all glioblastoma cells, although at different concentrations. The amount of released CD9 and CD81-positive EVs increased after irradiation when cells became apoptotic. Although secreted EVs of non-irradiated cells were not predictive for radiosensitivity, their increased EV release after irradiation correlated with the cytotoxic response to radiotherapy 72 h after irradiation. Thus, our data suggest a novel application of EVs in the surveillance of anti-cancer therapies.</p

Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway-3

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p>24 h with medium or 5, 10 and 50 μg/ml Ukrain. Apoptosis was then determined by flow cytometry using light scatter characteristics (left panel). Alternatively, A3, FADD-deficient and caspase-8-deficient Jurkat cells were treated for 3, 6, 12 and 24 h with medium or 10 μg/ml Ukrain. Apoptosis induction was then quantified by determination of the mitochondrial membrane potential (Δψm) (right panel). Data show induction of specific apoptosis (means ± SD; n ≥ 3). One-way ANOVA was performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, . . Jurkat A3 as well as FADD-deficient and caspase-8-deficient Jurkat cells were treated for 6, 12 and 24 h with medium or 10 μg/ml Ukrain. Caspase activation was then determined by Western blot analysis of cytosolic extracts with specific antibodies against caspase-3, cleaved caspase-3, PARP and cleaved PARP, respectively. In addition, cells were treated for 3, 6, 12 and 24 h with medium or 10 μg/ml Ukrain and Western blot analysis with specific antibodies against caspase-8 was performed. Data from one representative experiment are shown

Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway-14

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p>istics (white bars), measurement of mitochondrial membrane potential Δψm (grey bars) and the activation of caspase-3 (black bars) upon treatment of Jurkat Vector cells for 24 h with medium or 0, 50, 100, 150 and 250 μM allocryptopine. Data show means ± SD (n ≥ 3) (left panel). Fluorescence microscopy upon combined staining with Hoechst33342 and propidium iodide staining of cells treated with 250 μM allocryptopine for 24 h showed increased numbers of cells with apoptotic morphology upon treatment (right panel)

Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway-6

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p> 10 μg/ml Ukrain for 6, 12 and 24 h (left panel) or for 24 h with 5, 10 and 50 μg/ml Ukrain (right panel). Apoptosis was then measured by fluorescence microscopy upon Hoechst33342-staining. Data show means ± SD (n = 3). Paired t-test was performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, )

Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway-15

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p>topine, and extract adjusted to 1, 5 or 50 μM chelidonine. Caspase-activation was then determined by Western blot analysis with antibodies specific for PARP and cleaved PARP. A representative experiment is shown

Mock-transfected Jurkat control cells (Jurkat Vector) were treated for 24 h with medium or 10 μg/ml Ukrain and subsequently stained with Hoechst33342

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p> Apoptotic cells show characteristic chromatin condensation and nuclear fragmentation. Micrographs of a representative experiment are shown. . Jurkat Vector cells were treated for 24 h with medium or 5, 10 and 50 μg/ml Ukrain. Apoptosis was then quantified by flow cytometric analysis using light scatter characteristics (white bars), determination of the mitochondrial membrane potential Δψm upon staining with the potential sensitive dye TMRE (grey bars) and detection of caspase-activation upon staining with CaspACE(black bars). Data show specific apoptosis (% apoptosis in Ukrain-treated cells minus % apoptosis in untreated control cells) as means ± SD (n ≥ 3). . Jurkat Vector cells were treated with medium or 10 μg/ml Ukrain for 3, 6, 12 and 24 h and subsequently analysed by flow cytometry using light scatter characteristics (white bars), determination of the mitochondrial membrane potential upon staining with the potential sensitive dye TMRE (grey bars) and detection of caspase-activation upon staining with CaspACE(black bars). Data represent specific apoptosis (means ± SD, n ≥ 3). . Jurkat Vector cells were treated with medium or 10 μg/ml Ukrain for 6, 12 and 24 h. Caspase activation was determined by Western blot analysis of cytosolic extracts with specific antibodies against caspase-8 and caspase-3 and their respective cleavage products. In addition, caspase-3 activation was verified by detection of the cleavage of the caspase-3 substrate PARP. Data from one representative experiment are shown

Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway-12

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p> extract adjusted to the respective chelidonine concentration. Apoptosis induction was analysed by fluorescence microscopy upon combined staining with Hoechst33342 and propidium iodide. The percentage of cells with characteristic apoptotic nuclear morphology was determined by counting a minimum of 250 cells per data point in each of at least three independent experiments. Data represent the percentage of cells with apoptotic nuclear morphology (means ± SD, n = 3). . Jurkat Vector cells were treated for 24 h with medium supplemented with solvent or 50 μM extract adjusted to the respective chelidonine-concentration. Apoptosis induction was analysed by fluorescence microscopy upon combined staining with Hoechst33342 and propidium iodide. Characteristic micrographs of nuclear morphology are shown. . Jurkat vector cells were treated for 24 h with medium supplemented with solvent or 1, 5, 10 and 50 μM L. extract adjusted to the respective chelidonine-concentration. Induction of apoptosis was then measured using flow cytometry (cell shrinkage: white bars; breakdown of mitochondrial membrane potential: grey bars; caspase-activation: black bars). Data show means ± SD (n ≥ 3)

Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway-5

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p>medium or 10 μg/ml Ukrain, for 12 h with medium or 25 μM Etoposide and for 48 h with medium or irradiated with 10 Gy 48 h prior to determination of apoptosis. Apoptosis was quantified by fluorescence microscopy upon Hoechst33342-staining by counting cells with apoptotic nuclear morphology. Data show specific apoptosis (apoptosis rates of treated cells minus apoptosis rates of untreated cells) as means ± SD (n = 3). One-way ANOVA was performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, . . Jurkat Vector, Jurkat Bcl-2 as well as Jurkat Caspase-9 DN cells were treated for 24 h with medium or 5, 10 and 50 μg/ml Ukrain. Apoptosis induction was then quantified by determination of the mitochondrial membrane potential (Δψm) (right panel). Data show induction of specific apoptosis (means ± SD; n ≥ 3). One-way ANOVA was performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, . . Expression of Bcl-2 in Jurkat Vector and Bcl-2 overexpressing Jurkat cells (Jurkat Bcl-2) (upper panel) as well as expression of caspase-9 in Jurkat Vector and Jurkat cells expressing a dominant negative caspase-9 (Jurkat Caspase-9 DN) (lower panel) were verified by Western blot analysis of cytotsolic extracts with the respective antibodies. Jurkat Vector cells, Jurkat Bcl-2 cells as well as Jurkat Caspase-9 DN cells were treated for 0, 6, 12 and 24 h with medium or 10 μg/ml Ukrain. Caspase-activation was then determined by Western blot analysis of cytosolic extracts with antibodies against full length and active caspase-3, caspase-8 as well as PARP and cleaved PARP. Data from one representative experiment are shown

Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway-10

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p>/z 348.4; : LC-MS/MS ion chromatogram of m/z 354.1; : LC-MS/MS ion chromatogram of m/z 370.2