Effects of parthenolide on the expression of the anti-inflammatory gene HO1.

<p>LNCaP, PC3 and DU145 cells were kept in normoxia and exposed to 1% O₂ in the presence and absence of 5 µM parthenolide. mRNA levels for HO1 were analyzed by real-time PCR, normalized to the housekeeping gene 18S rRNA and expressed as fold induction with respect to the normoxic untreated control (set at 1). We show the effect of parthenolide after 4 h treatment when the drug maximally upregulated HO1 expression in normoxic cultures and after 24 h (LNCaP, DU145) and 48 h (PC3) stimulation when hypoxia exerted a maximum increase on its transcription. Mean values ± SE. * Normoxic and hypoxic parthenolide treated cells <i>vs</i> normoxic control cells. C: control normoxic untreated cells. P: normoxic parthenolide treated cells. H: hypoxic cells. HP: parthenolide treated hypoxic cells.</p

Normoxic expression levels of pro-inflammatory genes in LNCaP, DU145 and PC3 cells.

<p>Normoxic expression levels of pro-inflammatory genes in LNCaP, DU145 and PC3 cells. Values express the mRNA level of each gene quantified by real-time PCR and normalized to the housekeeping gene 18S rRNA. Mean ± SE. Asterisks indicate a significant higher value compared to LNCaP cells.</p

Hypoxic regulation of NF-kB nuclear expression in LNCaP, DU145 and PC3 cells.

<p>Cell were exposed to 1% O₂ from 0.5 up to 24 h or left untreated. After the times indicated, cells were processed and the nuclear content of p50 and p65 was detected by immunoblot analysis. β-actin was used as a loading control. A representative experiment for each gene in all the studied cell lines is shown. Mean densitometry of NF-kB p50 and p65 relative to β-actin ± SE is also evidenced and expressed as arbitrary units. * Hypoxic cells <i>vs</i> normoxic control cells.</p

Effects of parthenolide on the expression of pro-inflammatory genes.

<p>LNCaP (<b>A</b>), PC3 (<b>B</b>), DU145 (<b>C</b>) cells were kept in normoxia and exposed to 1% O₂ in the presence and absence of 5 µM parthenolide. mRNA levels for VEGF, RAGE, P2X7R, PTX3, COX2, CXCR4 and HO1 were analyzed by real-time PCR, normalized to the housekeeping gene 18S rRNA and expressed as fold induction with respect to the normoxic untreated control (set at 1). For each gene we present the effect of parthenolide at the time points where hypoxia exerted a maximum increase on its transcription. *Hypoxic parthenolide treated cells <i>vs</i> hypoxic cells. C: control normoxic untreated cells. P: normoxic parthenolide treated cells. H: hypoxic cells. HP: parthenolide treated hypoxic cells.</p

Effects of Parthenolide on HIF1α and HIF3α hypoxia dependent activation.

<p>A LNCaP, DU145 and PC3 cells were kept in normoxia and exposed to 1% O₂ for 4 h in the presence and absence of 5 µM parthenolide. HIF1α content was measured in the nuclear fraction by immunoblot analysis. β-actin was used as a loading control. For each gene a representative experiment is shown. Mean densitometry of HIF1α relative to β-actin is also evidenced and expressed as arbitrary units. <b>B</b> DU145 cells were incubated under normoxic or hypoxic conditions for 48 h in the presence and absence of 5 µM parthenolide. HIF3α content was measured in the nuclear fraction by immunoblot analysis. β-actin was used as a loading control. For each gene a representative experiment is shown. Mean densitometry of HIF3α relative to β-actin is also evidenced and expressed as arbitrary units. Mean values ± SE. C: control normoxic untreated cells. P: normoxic parthenolide treated cells. H: hypoxic cells. HP: parthenolide treated hypoxic cells. *Hypoxic parthenolide treated cells <i>vs</i> hypoxic cells.</p

mRNA expression levels of pro-inflammatory genes in hypoxic LNCaP, DU145 and PC3 cells.

<p>Cells were exposed to 1% O₂ from 2 up to 72 h. mRNA levels for VEGF, RAGE, P2X7R, PTX3, COX2, CXCR4 and HO1 were analyzed by real-time PCR and normalized to the housekeeping gene 18S rRNA. Graphs show the ratio between the expression of hypoxia treated cells with respect to normoxic control cells (set at 1). Mean values ± SE. * Hypoxic cells <i>vs</i> normoxic control cells.</p

Effects of HIF1α knockdown and of parthenolide on the normoxic and hypoxic expression of the pro-inflammatory phenotype and HO1 transcription in DU145 cells.

<p><b>A</b> HIF1α shRNA cells were exposed to 1% O₂ or left untreated from 2 up to 72 h in the presence and absence of 5 µM parthenolide. mRNA levels for VEGF, RAGE, PTX3, COX2 and CXCR4 were analyzed by real-time PCR and normalized to the housekeeping gene 18S rRNA. For each gene we present the result on the time point where hypoxia exerted a maximum increase in transcription. Graphs show the ratio between the expression of parthenolide, hypoxia, parthenolide and hypoxia treated HIF1α shRNA cells with respect to the mean normoxic control of wt cell cultures (set at 1). Mean values ± SE. ° HIF1α shRNA normoxic control cells <i>vs</i> wt normoxic control cells, * hypoxic HIF1α shRNA cells <i>vs</i> HIF1α shRNA normoxic control cells. # Parthenolide treated hypoxic HIF1α shRNA cells <i>vs</i> hypoxic HIF1α shRNA cells. <b>B</b> Effect of parthenolide on the transcription the anti-inflammatory gene HO1 in HIF1α shRNA cells in normoxia and in hypoxia after 4 h (when parthenolide maximally upregulated HO1 expression in normoxic cultures) and 24 h treatment. Graph shows the ratio between the expression of parthenolide, hypoxia, parthenolide and hypoxia treated HIF1α shRNA cells with respect to the mean normoxic control of wt cell cultures (set at 1). Mean values ± SE. * Normoxic and hypoxic parthenolide treated HIF1α shRNA cells <i>vs</i> HIF1α shRNA normoxic control cells. C: control normoxic untreated cells. P: parthenolide treated cells. H: hypoxic cells. HP: parthenolide treated hypoxic cells.</p

Parallelism between the phenotype of cells held in tumors and of CSC grown <i>in vitro</i>.

<p>In particular, CSC grown in FBS-medium aimed to reproduce the injection of 1 spheroid in mice, whereas CSC grown in CM from DU145 cells allowed us to simulate the injection of 5,000 unsorted DU145 cells.</p

Effect of CM from DU145 cells on the phenotype of CSC.

<p>(<b>A</b>) The proliferation of CSC contained in spheroids grown in FBS-containing medium or in CM from DU145 cells was evaluated at different times of culture. Data are shown as the mean ± SEM from 3 experiments. Asterisks indicate a significant inhibitory effect of CM with respect to FBS-medium (*, <i>p</i><0.05). (<b>B</b>) Phase contrast (10×) of cells after 20 days in FBS-containing medium or in CM showing the different pattern of cell growth. In the left panel, the rectangle highlights the presence of NE cells. (<b>C</b>) Phase contrast (left, 20×) and IF staining (right, 40×) of branched NE cells generated from CSC grown in FBS-medium. In particular, the right panel shows the expression of CD56 in the cytoplasm and in the long cell processes. (<b>D</b>) Representative western blots showing the changes in the expression of E-cadherin, β-catenin, and vimentin in spheroid CSC grown in the different culture conditions with respect to control spheroids and DU145 cells. The expression levels of the 3 proteins were determined by densitometric analysis of the respective bands and normalized to β-actin levels. Values reported in the histograms are mean ± SEM from 3 independent experiments.</p

Influence of culture conditions on the expression of lipocalin-2 and E-cadherin in CSC.

<p>Q-PCR showing changes in mRNA levels for lipocalin-2 and E-cadherin in DU145 cells, CSC contained in control spheroids, and CSC grown for 20 days in FBS-medium or CM from DU145 cells. Data represent mean ± SEM from 3 individual experiments. Asterisks indicate a significant difference (*, <i>p</i><0.05).</p