Reactivation of apoptosis by gene transfer in uterine sarcoma cells.

<p>Measurements of apoptosis in uterine sarcoma cells by caspase-3/-7 activation that was reinduced by gene transfer (A and C). ESS-1 (A) and MES-SA cells (C) were transfected with caspase-8 (A) or DR4 (C) expression plasmids driven by a CMV promoter, respectively, and were supplemented with or without TRAIL before caspase-3/-7 activation was measured 24 hours later. Controls were mock-transfected and treated with or without TRAIL. For comparison, cells that received 3 μM SAHA and/or TRAIL were measured. Presented is the relative caspase activation in percentage as compared to untreated control cells. Asterisks (* <i>p</i><0.05) or number signs (# <i>p</i><0.001) indicate statistically significant differences as compared to the untreated control. Western blot analyses of activated executioner caspases of ESS-1 cells (B) and MES-SA cells (D) in order to observe apoptosis reinduction upon gene rescue experiments as demonstrated in (A). Samples with 50 μg (B) or 30 μg (D) protein were immunoblotted and analyzed with antibodies against cleaved (CL.) caspases-3, -6, -7, and β-tubulin as loading control. Untreated cells were used as control. The molecular weights of presented bands are indicated in brackets.</p

Time course of combined SAHA and TRAIL-induced caspase activation in uterine sarcoma cells.

<p>Western blot analysis of ESS-1 cells (A) and MES-SA cells (B) after treatment with 3 μM SAHA and/or 100 ng/ml TRAIL for 4, 8, and 24 hours to compare the induction of apoptosis. Untreated cells were used as control. Cell extracts were prepared, then 50 μg of protein were loaded onto a SDS-PAGE (12% Bis-tris gel) of each sample, and blotted onto nitrocellulose membrane. Subsequently, the membrane was incubated with the indicated antibodies against cleaved (CL.) caspases-3, -6, -7, -8, and β-tubulin (as loading control) followed by detection with a secondary hrp-coupled antibody. The molecular weights of presented bands are indicated in brackets. Note the weak expression of caspase-8 in ESS-1 cells (C) The amount of caspase-3 and -7 activation (Caspase-Glo 3/7 Assay; upper panel) and LDH release (CytoTox-ONE Homogeneous Membrane Integrity Assay; lower panel) of both uterine sarcoma cell lines was measured 4, 8, and 24 hours after treatment with 3 μM SAHA and/or 100 ng/ml TRAIL. The results are expressed as percentage of relative caspase-3/-7 activation or LDH release as compared to the untreated control or lysis control, respectively. Cells were seeded at a density of 5×10³ cells per well. Each value represents the average of 3 independent experiments with 5 replicates each. Asterisks (* <i>p</i><0.05) or number signs (# <i>p</i><0.001) indicate statistically significant differences between the combined SAHA/TRAIL treatment and the control.</p

Reactivation of apoptosis through DNA demethylation by 5-Aza-dC in uterine sarcoma cells.

<p>Caspase-3 and -7 activation was measured in the uterine sarcoma cell lines, ESS-1 and MES-SA, upon DNA demethylation analysis (A). Therefore, treatment of cells with different concentrations (0.5, 1, 5, or 10 μM) of the DNA methylation inhibitor 5-Aza-dC was performed for 5 days with or without final addition of TRAIL (for 8 hours). Cells treated with 3 μM SAHA and/or 100 ng/ml TRAIL were included as positive controls. The cells received daily exchange of medium supplemented with fresh 5-Aza-dC whereas the control received no treatment at all. Western blot analysis of ESS-1 cells (B) and MES-SA cells (C) that were treated as in (A) for monitoring caspase activity upon DNA demethylation. Samples with 50 μg of protein were separated by SDS-PAGE (12% Bis-tris gel), transferred onto nitrocellulose membrane, and analyzed with antibodies against cleaved (CL.) caspases-3, -7, -6, -8, and β-tubulin as loading control. Untreated cells were used as control. The molecular weights of presented bands are indicated in brackets.</p

Promoter hypermethylation, and demethylation analysis of caspase-8 and DR4 genes in uterine sarcoma cells.

<p>Methylation-specific PCR (MSP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091558#pone.0091558-Kubota1" target="_blank">[36]</a> of bisulfite treated genomic DNA, isolated from untreated ESS-1 and MES-SA cells, was performed of the promoter regions of <i>caspase-8</i> and <i>DR4</i> (<i>TNSFR10A</i>) for assessing the methylation status (A). Amplification with the help of primer pairs that either bind unmethylated (U) or methylated (M) DNA was conducted before the PCR product was visualized by gel electrophoresis. Unmodified genomic DNA was used as a negative control (CO). Note that the lower band in the MSP performed for <i>DR4</i> represents primers. Following DNA standards were used: (1) Gene ruler 50 bp DNA ladder; (2) λBst91I marker. (B) Bisulfite sequencing analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091558#pone.0091558-Frommer1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091558#pone.0091558-Frhlich1" target="_blank">[38]</a> of the <i>caspase-8</i> and <i>DR4</i> promoter regions in ESS-1 and MES-SA cells for identifying individual 5-methycytosine residues in genomic DNA. Eleven CpG sites located upstream of the transcription start site, between nucleotides – 300 and -697 for <i>caspase-8,</i> or nucleotides – 27 and – 267 for <i>DR4,</i> were analyzed for DNA methylation. Bisulfite converted genomic DNA, was amplified by PCR, subcloned, and sequenced by the Sanger method. The results of eight sequenced clones are depicted schematically for each promoter region and cell line, respectively. Methylated and unmethylated CpG nucleotides are presented as black or white circles, respectively. (C) MSP analysis as in (A) upon treatment of cells for 5 days with 0.5 μM 5-Aza-dC for examining induced alterations in genomic DNA methylation pattern for the CASP8 and DR4 promoter regions. Corresponding untreated cells were used as control. (D) Expression analysis of 5 day 5-Aza-dC treated ESS-1 and MES-SA cells for relative caspase-8 and DR4 expression by qRT-PCR. Triplicate ct values of three independent experiments were normalized to cyclophillin (CYC) expression and averaged. The depicted graphs represent the relative expression values as compared to the untreated control. Asterisks (* <i>p</i><0.05) indicate statistically significant differences as compared to the untreated control.</p

Reduced expression of caspase-8 in ESS-1 cells and DR4 (TRAIL-R1) in MES-SA cells.

<p>TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5), both TRAIL decoy receptors (Dc-R1, Dc-R2), and caspase-8 (CASP8) were amplified from cDNA in order to monitor defects in gene expression (A). Therefore, RNA of untreated cells was isolated, reversely transcribed, and subjected to qRT-PCR with primers binding to exonic sequences. PCR products were run on a 1.5% agarose gel, stained with ethidium bromide, and photographed. Amplification of beta-actin with or without genomic DNA served as a positive control (ACTB) or negative control (CO), respectively. Note the weaker bands for caspase-8 and DR4 in ESS-1 and MES-SA cells, respectively. The additional high molecular weight band for Dc-R2 in ESS-1 cells represents the genomic amplicon. M1, Gene ruler 50 bp DNA ladder; M2, λ<i>Bst</i>91I marker. (B) The relative caspase-8 expression of SAHA and/or TRAIL treated ESS-1, and MES-SA cells, was quantitated by qRT-PCR. Technical triplicate cycle threshold (ct) values of three biological replicates were normalized to cyclophillin (CYC) expression. Asterisks (* <i>p</i><0.05) indicate statistically significant differences as compared to the untreated control. (C) Western blot analysis of ESS-1 and MES-SA cells treated for 8 hours with 3 μM SAHA and/or 100 ng/ml TRAIL. Untreated cells were used as control. Cell extracts were prepared, then 30 μg of protein subjected to SDS-PAGE (4 to 12% Bis-tris gel), and immunoblotted with the indicated antibodies against DR4 (TRAIL-R1), DR5 (TRAIL-R2) and β-tubulin as loading control. The molecular weights of presented bands are indicated in brackets.</p

SAHA/TRAIL treatment induces apoptosis in uterine sarcoma cells involving the mitochondrial pathway.

<p>Confocal laser scanning microscopy of ESS-1 and MES-SA cells which were stained after 8 hours of 3 μM SAHA and/or 100 ng/ml TRAIL treatment with YoPro-1/PI in order to detect apoptotic and non-apoptotic cells (A). Control cells received neither SAHA nor TRAIL treatment. Red staining (PI) represents dead or necrotic cells, green staining (YoPro-1) represents apoptotic staining, merged (yellow/orange) staining represents secondary apoptotic cells (uptake of both dyes), and no staining represents living cells. Representative images of three independent experiments that were acquired at 505 to 530 nm for the green channel and 543 nm for the red channel are shown (magnification 40 x). (B) Western blot analysis of ESS-1 and MES-SA cells treated for 8 hours with 3 μM SAHA and/or 100 ng/ml TRAIL for PARP-1 in order to demonstrate apoptotis. Untreated cells were used as control. Cell extracts were prepared, subjected to SDS-PAGE (30 μg of protein; 4-12% Bis-Tris gel), and immunoblotted with antibodies against cleaved PARP-1 (89 kDa) and β-tubulin (for loading control). The presented 89 kDa PARP-1 fragment is only processed during induction of apoptosis but not necrosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091558#pone.0091558-Oliver1" target="_blank">[41]</a>. (C) The mitochondrial membrane potential (Δψ_m) was determined in uterine sarcoma cells (1×10⁴ cells per well) by JC-1 staining for confirming involvement of the intrinsic pathway of SAHA/TRAIL-induced apoptosis. Upon collapse of the Δψ_m, JC-1 molecules can enter mitochondria where they form red J-aggregates. The red (∼590 nm; high Δψ_m) to green (∼529 nm; low Δψ_m) ratio therefore indicates the amount of apoptosis in SAHA/TRAIL-treated cells after 4, 8, and 24 hours in arbitrary units. Mitochondrial depolarization in dead cells or cells undergoing apoptosis is indicated by a decrease in the red/green fluorescence intensity ratio. Asterisks (* <i>p</i><0.05) or number signs (# <i>p</i><0.001) indicate statistically significant differences between the combined SAHA/TRAIL treatment and the untreated control.</p

Quantitative bivariate AnnV/PI cytofluorometric analysis of apoptosis in SAHA and TRAIL-induced uterine sarcoma cells.

<p>*Presented is the mean ± SD of three independent experiments. <b>^#</b> Untreated Control; ^‡ Combined S(AHA) [3 μM] and T(RAIL) [100 ng/ml] treatment.</p

Cytotoxic effects of combined SAHA and TRAIL treatment in the cell lines ESS-1 and MES-SA.

<p>Cell viability (MTS) assays were performed after treatment of ESS-1 and MES-SA endometrial stroma sarcoma cells as well as HESC control cells for 24 hours with 3 μM of the HDAC inhibitor SAHA and/or different concentrations of human recombinant TRAIL (5, 25, 50, and 100 ng/ml) to determine the most effective cytotoxic concentration (A). Cell viability of ESS-1, MES-SA, and HESC control cells was determined in a time course experiment after treatment of cells for 0, 12, 24, 36, or 48 hours with 3 μM SAHA and/or 100 ng/ml TRAIL (B). The results in (A) and (B) are expressed as percentage of relative viability as compared to the untreated control. Cells were seeded at a density of 5×10³ cells per well. Each value represents the average of 3 independent experiments with 5 replicates each. Asterisks (* <i>p</i><0.05) or number signs (# <i>p</i><0.001) indicate statistically significant differences as compared to the untreated control.</p

Mode of action of PPS in conventional cultures.

<p>Activation of caspases 3 and 7 (A and B) and release of LDH (C and D) upon exposure of EAhy 926 cells to 20 and 200 nm PPS for 4, 8, and 24 hours compared to untreated cells. Data are presented as mean ± SD. (h), hours.</p

Acute cytotoxicity of NPs exerted on EAhy 926 in different cell cultures after 24 hours.

<p>Cells in conventional cultures were treated with NPs dissolved in serum-free medium (A) as well as in medium with 10% FBS (B). Cells cultured on microcarriers were exposed to NMs dissolved in medium with 10% FBS. Data are presented as mean ± SD.</p