Authors’ Affiliations: Cell Death Regulation Laboratory, Departments of Medicine and Cell and Molecular Biology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois and Division of Medical Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

PURPOSE: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic antibodies targeting its receptors are promising cancer therapies because of their tumor selectivity, many tumors are resistant to TRAIL-based therapies. We examined whether the nonsteroidal anti-inflammatory drug aspirin sensitized cancer cells to TRAIL agonists in vitro and in vivo and investigated the underlying mechanism. EXPERIMENTAL DESIGN: The effects of aspirin on sensitivity to TRAIL agonists and expression of apoptosis regulators was determined in human breast cancer cell lines and xenograft tumors. The specific role of survivin depletion in the TRAIL-sensitizing effects of aspirin was determined by silencing survivin. RESULTS: Aspirin sensitized human breast cancer cells, but not untransformed human mammary epithelial cells, to TRAIL-induced caspase activation and apoptosis by a cyclooxygenase-2-independent mechanism. Aspirin also sensitized breast cancer cells to apoptosis induced by a human agonistic TRAIL receptor-2 monoclonal antibody (lexatumumab). Aspirin treatment led to G(1) cell cycle arrest and a robust reduction in the levels of the antiapoptotic protein survivin by inducing its proteasomal degradation, but did not affect the levels of many other apoptosis regulators. Silencing survivin with small interfering RNAs sensitized breast cancer cells to TRAIL-induced apoptosis, underscoring the functional role of survivin depletion in the TRAIL-sensitizing actions of aspirin. Moreover, aspirin acted synergistically with TRAIL to promote apoptosis and reduce tumor burden in an orthotopic breast cancer xenograft model. CONCLUSIONS: Aspirin sensitizes transformed breast epithelial cells to TRAIL-based therapies in vitro and in vivo by a novel mechanism involving survivin depletion. These findings provide the first in vivo evidence for the therapeutic utility of this combination.

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College of Pharmacy, Pusan National University, San 30, Jangjeon-dong, Geumjung-gu, Busan 609-735, Republic of Korea.

OBJECTIVE.: Histone deacetylase (HDAC) inhibitors are promising new class of anticancer agents that act by inhibiting cell proliferation and inducing cell cycle arrest of various cancer cells. Psammaplin A (PsA) is a phenolic natural product that has been isolated from marine sponges, and has been suggested to be a promising novel HDAC inhibitor. However, the precise mechanism of PsA as a HDAC inhibitor is poorly understood. This study investigated the anti-tumor effect of PsA on endometrial human cancer cells. METHODS.: The cell proliferation, cell cycle, and apoptosis were measured in Ishikawa endometrial cancer cells after PsA treatment. RESULTS.: PsA significantly inhibited the proliferation of Ishikawa cells in a dose-dependent manner. PsA markedly induced the expression of acetylated H3 and H4 histone proteins. In addition, PsA markedly up-regulated the expression of cyclin-dependent kinase inhibitor, p21(WAF1), and down-regulated the expression of pRb, cyclins, and CDKs, which lead to induce cell cycle arrest. Cell cycle analysis indicated that PsA treatment increased the proportion of cells in the G0/G1 and G2/M phases, and decreased the ratio of cells in the S phase. CONCLUSION.: The PsA treatment resulted in the significant induction of apoptosis, which was associated with p53 independent p21(WAF1) expression. These results suggest that PsA exhibits the antiproliferative effects on endometrial cancer cells through selective induction of genes related to cell cycle arrest and apoptosis.

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The prostate androgen-regulated (PAR) gene is ubiquitously overexpressed in prostate cancer (PCa) cells and is involved in proliferation of PCa. However, the mechanism by which the modulation of PAR gene expression elicits its biological effects on PCa cells is not well documented. Here, we investigate the mechanism of PAR depletion inhibiting PCa cell growth. METHODS: PAR expression was depleted by small interfering RNA (siRNA) and its subsequent effects on proliferation of PC3 cells were determined by the trypan blue exclusion assay. Flow cytometric analysis provided the evidence for the progression of cell cycle and the induction of apoptosis which was further confirmed by the observation of cleavage of poly(ADP-ribose) polymerase. Western blot analysis was performed to investigate the involvement of critical molecular events known to regulate the cell cycle and the apoptotic machinery. RESULTS: siRNA transfection results in a dose-dependent inhibition of cell growth in PC3 cells by causing G2/M phase cell cycle arrest and apoptosis. The G2/M arrest by PAR depletion was associated with decreased levels of cyclin B1, pCdc2 (Tyr15), Cdc2 and Cdc25C. PAR depletion also was found to result in inhibition of procaspases 9, 8, 6 and 3 with significant increase in the ratio of Bax : Bcl-2. CONCLUSIONS: Our data indicate that PAR depletion induces G2/M arrest via the Cdc25C-Cdc2/cyclin B1 pathway. Furthermore, the results of the present study point toward involvement of pathways mediated by both caspase 8 and caspase 9 in apoptosis induction by PAR depletion. Copyright (c) 2007 John Wiley & Sons, Ltd.
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Lymphocyte Signal Transduction Laboratory, Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.

The fermented wheat germ extract (code name: MSC, trade name: Avemar), with standardized benzoquinone content has been shown to inhibit tumor propagation and metastases formation in vivo. The aim of this study was to understand the molecular and cellular mechanisms of the anti-tumor effect of MSC. Therefore, we have designed in vitro model experiments using T and B tumor lymphocytic cell lines. Tyrosine phosphorylation of intracellular proteins and elevation of the intracellular Ca2+ concentration were examined using immunoblotting with anti-phosphotyrosine antibody and cytofluorimetry by means of Ca2+ sensitive fluorescence dyes, Fluo-3AM and FuraRed-AM, respectively. Apoptosis was measured with cytofluorimetry by staining the DNA with propidium iodide and detecting the cell population. The level of the cell surface MHC class I molecules was analysed with indirect immunofluorescence on cytofluorimeter using a monoclonal antibody to the non-polymorphic region of the human MHC class I. MSC stimulated tyrosine phosphorylation of intracellular proteins and the influx of extracellular Ca2+ resulted in elevation of intracellular Ca2+ concentration. Prominent apoptosis of 20-40% was detected upon 24 h of MSC treatment of the cell lines. As a result of the MSC treatment, the amount of the cell surface MHC class I proteins was downregulated by 70-85% compared to the non-stimulated control. MSC did not induce a similar degree of apoptosis in healthy peripheral blood mononuclear cells. Inhibition of the cellular tyrosine phosphatase activity or Ca2+ influx resulted in the opposite effect increasing or diminishing the Avemar induced apoptosis as well as the MHC class I downregulation, respectively. A benzoquinone component (2,6-dimethoxi-p-benzoquinone) in MSC induced similar apoptosis and downregulation of the MHC class I molecules in the tumor T and B cell lines to that of MSC. These results suggest that MSC acts on lymphoid tumor cells by reducing MHC class I expression and selectively promoting apoptosis of tumor cells on a tyrosine phosphorylation and Ca2+ influx dependent way. One of the components in MSC, 2,6-dimethoxi-p-benzoquinone was shown to be an important factor in MSC mediated cell response.

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