Cinanthrenol A, an Estrogenic Steroid Containing Phenanthrene Nucleus, from a Marine Sponge <i>Cinachyrella</i> sp.

Cinanthrenol A (<b>1</b>), a new steroid composed of a phenanthrene and a spiro­[2,4]­heptane system, was isolated from the marine sponge <i>Cinachyrella</i> sp. It is the first phenathrene-containing steroid with estrogen activity

The Detergent-Soluble Cytoplasmic Pool of Survivin Suppresses Anoikis and Its Expression Is Associated with Metastatic Disease of Human Colon Cancer

<div><p>Survivin is a component of the chromosomal passenger complex (CPC) that is essential for accurate chromosome segregation. Interfering with the function of Survivin in mitosis leads to chromosome segregation errors and defective cytokinesis. Survivin contains a Baculovirus IAP Repeat (BIR) and therefore was originally classified as inhibitor of apopotosis protein (IAP), yet its role in apoptosis after cellular stress remains largely unknown. We demonstrate here, that Survivin predominantly suppresses anoikis, a form of programmed cell death induced by loss of cellular adhesion to extracellular matrix. Interestingly, cells ectopically overexpressing EGFP-Survivin showed after loss of cell-matrix-interaction a decreased expression of IκB-α. Subsequent subcellular protein fractionation and immunoprecipitation experiments revealed that XIAP interacts with detergent-soluble Survivin which is known to cooperatively activate NF-κB signaling. Examination of the expression levels of detergent soluble Survivin in colorectal cancer cell lines and in colorectal cancerous tissues revealed that detergent soluble cytoplasmic Survivin levels correlated inversely with anoikis susceptibility in colorectal cancer. Therefore, the detergent soluble cytoplasmic Survivin might be a promising predictive biomarker for lymph node and distant metastases of colorectal cancer. We conclude that an anti-apoptotic function of detergent-soluble Survivin in interphase cells experiencing anoikis is mediated at least via XIAP/IκB-α/NF-κB signaling.</p> </div

Overexpression of Survivin does not significantly protect radiations-induced apoptosis, but does protect apoptosis under other stresses.

<p><b>A.</b> Frequency of apoptosis in CHE cells (with p53+/+ and p53−/−) transfected with pEGFP-empty and pEGFP-Survivin after treatment with X-irradiation (10 Gy) and UV-C (10 J/m²). The transfected cells were exposed to IR or UV-C at 24 h after transfection. Transfection frequencies were 80–90%, and EGFP-positive cells were counted for Annexin V-positive or -negative cells (<i>upper panel</i>) and TUNEL assay-positive or -negative cells (<i>lower panel</i>). X-irradiated CHE-p53−/− cells had significantly greater fraction of apoptosis-positive cells when compared to X-irradiated CHE-p53+/+ cells (<i>P</i><0.03 for Annexin V staining and <i>P</i><0.08 for TUNEL assay), and UV-C irradiated CHE-p53−/− cells had significantly greater fraction of apoptosis-positive cells when compared to UV-C irradiated CHE-p53+/+ cells (<i>P</i><0.001 for Annexin V staining and <i>P</i><0.02 for TUNEL assay). Apoptosis-positive cells were not significantly decreased in pEGFP-Survivin-transfected cells when compared to pEGFP-transfected cells in each case (<i>P</i>>0.4 for Annexin V staining and <i>P</i>>0.4 for TUNEL assay). Values indicate means ± S.D. (n = 3). <b>B.</b> Increase of the retention in the lung after intravenous injection of EGFP- or EGFP-Survivin-expressing cells. The number of surviving CHE-p53−/− cells in the lung was significantly increased when compared to the number of surviving control cells expressing EGFP. Values indicate means ± S.D. of six mice. *Significant difference (<i>P</i><0.005). <b>C.</b> Caspase-3 activation in CHE-p53−/− cells transfected with pEGFP-empty and pEGFP-Survivin after anoikis induction. The transfected cells were detached from extracellular matrix and simultaneously serum-starved to induce anoikis at 24 h after transfection. Cells were suspended in serum-free medium for 6–36 h, harvested, and lysed in Laemmli SDS-sample buffer for immunoblot analysis with anti-activated caspase-3 antibody. Transfection frequencies were checked by using fluorescence microscopy and confirmed to be 80–90%. <b>D.</b> Caspase-3 activation in CHE-p53−/− cells transfected with pEGFP-empty and pEGFP-Survivin after treatment with UV-C (10 J/m²). The transfected cells were exposed to UV-C at 24 h after transfection. Cells were cultured for 24 h, harvested, and lysed in Laemmli SDS-sample buffer for immunoblot analysis with anti-activated caspase-3 antibody. Transfection frequencies were checked by using fluorescence microscopy and confirmed to be 80–90%.</p

Survivin expression and anoikis susceptibility in colorectal cancer cells.

<p><b>A.</b> Subcellular fractionation and immunoblot analysis of Survivin. The cell lysate from exponentially growing cells was fractionated into the detergent-soluble cytoplasmic (<i>C</i>), and nuclear (<i>N</i>) fractions and the detergent-insoluble pellet (<i>P</i>) fraction for immunoblot analysis with anti-Survivin, anti-α-tubulin, anti-H3-histone, and anti-β-actin. The detergent-soluble cytoplasmic Survivin level was estimated by immunoblot analysis, and expressed as a percentage of the level of 8505C, which is undifferentiated thyroid carcinoma showing anoikis resistance (+++, >80%; ++, 40–80%; +, 10–40%; −, <10%). <b>B.</b> Anoikis susceptibility in in colorectal cancer cells. Cells were suspended in serum-free medium for 72 h. Cell viability was determined by WST-1 assay.</p

Anoikis suppression pathways triggered by the detergent-soluble cytoplasmic Survivin.

<p>Overexpressed Survivin, which is fractionated into the detergent-soluble cytoplasmic fraction, associates with XIPA. The Survivin-XIPA heterodimer increases NF-κB transcriptional activity due to IκB-α degradation and NF-κB nuclear translocation. Simultaneously, the Survivin-XIPA heterodimer inhibits c-Jun phosphorylation. In our proposed model, cross-talk between the NF-κB and JNK pathways relies on the Survivin-XIPA heterodimer leading to the shutdown of JNK activity and to rescue from anoikis.</p

Survivin functions during metastases.

<p>The detergent soluble cytoplasmic Survivin is imperative for survival of not only primary tumor cells but also intravasated cells.</p

EGFP-Survivin and XIPA found in the detergent-soluble fractions.

<p><b>A.</b> Subcellular fractionation and immunoblot analysis of activated caspase-3, Survivin, and XIAP. Transfection frequencies were checked by using fluorescence microscopy and confirmed to be 80–90%. Cells were kept in serum-free medium for 3–6 h, harvested, and lysed. The cell lysate was fractionated into the detergent-soluble cytoplasmic (<i>C</i>), and nuclear (<i>N</i>) fractions and the detergent-insoluble pellet (<i>P</i>) fraction for immunoblot analysis with anti-activated caspase-3, anti-Survivin, anti-GFP, anti-XIAP, anti-α-tubulin, anti-H3-histone, and anti-β-actin. <b>B.</b> Protein-protein interactions between EGFP-Survivn and XIAP. Cell lysate from the detergent-soluble cytoplasmic fraction was immunoprecipitated by anti-XIAP antibody and anti-GFP antibody and subsequently immunoblotted with anti-GFP antibody (<i>lower panel</i>) and anti-XIAP antibody (<i>upper panel</i>), respectively. EGFP-Survivin and XIAP were co-imunoprecipitated only in cell lysate form EGFP-Survivin-expressing cells (<i>lane 2</i>).</p

Overexpression of Survivin predominantly protects anoikis.

<p><b>A.</b> Frequency of anoikis in CHE-p53−/− cells transfected with pEGFP-empty and pEGFP-Survivin. The transfected cells were detached from extracellular matrix and simultaneously serum-starved to induce anoikis at 24 h after transfection. Transfection frequencies were 80–90%, and EGFP-positive cells were counted for anoikis-positive or -negative cells. Overexpression of EGFP-Survivin significantly suppressed anoikis when compared to the CHE-p53−/− control cells. Values indicate means ± S.D. (n = 3). *Significant difference (<i>P</i><0.08). **Significant difference (<i>P</i><0.04). <b>B.</b> Caspase-3 activation in EGFP- and EGFP-Survivin-expressing cells after serum starvation under attached or detached culture conditions. The experimental protocol was illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055710#pone.0055710.s001" target="_blank">Figure S1</a>. Transfection frequencies were checked by using fluorescence microscopy and confirmed to be 80–90%. Cells were kept in serum-free medium for 24–72 h, harvested, and lysed in Laemmli SDS-sample buffer for immunoblot analysis with anti-GFP, anti-Survivin, anti-activated caspase-3 antibody, anti-LC3B, and anti-α-tubulin. <b>C.</b> Survivin-induced apoptosis repression rate compared between EGFP-expressing cells and EGFP-Survivin-expressing cells. Levels of activated caspase-3 protein were estimated by immunoblot analysis. The repression rates were expressed in a ratio of activated caspase-3 protein level in EGFP-Survivin-expressing cells to the level in EGFP-expressing cells. Values indicate means ± S.D. (n = 3). *Significant difference (<i>P</i><0.04). **Significant difference (<i>P</i><0.02). ***Significant difference (<i>P</i><0.007). <b>D.</b> DNA fragmentation analysis in EGFP-expressing cells and EGFP-Survivin-expressing cells. The transfected cells were detached from extracellular matrix and simultaneously serum-starved to induce anoikis at 24 h after transfection. The cells were suspended for 24 h, and harvested. Genomic DNA was extracted and electrophoresed on agarose gels. <b>E.</b> Representative images of EGFP-expressing cells and EGFP-Survivin-expressing cells, using laser confocal microscopy. Transfected cells were suspended in serum-free medium for 24, and imaged by Nomarski differential interference contrast (<i>lane DIC</i>), by rhodamine phalloidin staining (<i>lane F-actin</i>), by EGFP fluorescence (<i>lane EGFP</i>), and by DAPI staining (<i>lane DAPI</i>). Rhodamine phalloidin staining and DAPI staining typically indicated that EGFP-expressing cells underwent anoikis, but EGFP-Survivin-expressing cells did not. <b>F.</b> Determination of cell viability by WST-1 assay. Transfected cells were suspended in serum-free medium for 24 h, and then cell viability of EGFP-expressing cells and EGFP-Survivin-expressing cells was assessed. Values indicate means ± S.D. (n = 3). *Significant difference (<i>P</i><0.04).</p

Expression and phosphorylation of signaling molecules relating to Survivin in EGFP- and EGFP-Survivin-expressing cells after serum starvation under attached or detached culture conditions.

<p>The experimental protocol was illustrated in Figure S1. Transfection frequencies were checked by using fluorescence microscopy and confirmed to be 80–90%. Cells were kept in serum-free medium for 24–72 h, harvested, and lysed in Laemmli SDS-sample buffer for immunoblot analysis with anti-β-actin, anti-Bax, anti-Smac/DIABLO, anti-XIAP, anti-IκB-α, anti-NF-κB, anti-JNK, anti-c-Jun-P(S73), anti-c-Jun, anti-FAK-P(Y397), and anti-FAK.</p

Clinicopathological significance of the detergent-soluble cytoplasmic Survivin expression in colorectal cancer.

a<p>Tumor invasion of mucosa (m), submucosa (sm), muscularis propria (mp), subserosa (ss), penetration of serosa (se), and invasion of adjacent strucures (si).</p>*<p>Significant difference.</p