Guy Fuhrmann

The Small α5β1 Integrin Antagonist, SJ749, Reduces Proliferation and Clonogenicity of Human Astrocytoma Cells

The potential role of α 5 β 1 integrins in cancer has recently attracted much interest. However, few α 5 β 1 -selective antagonists have been developed compared with other integrins. The most specific nonpeptidic α 5 β 1 antagonist described thus far, SJ749, inhibits angiogenesis by affecting adhesion and migration of endothelial cells. We investigated the effects of SJ749 in two human astrocytoma cell lines, A172 and U87, which express different levels of α 5 β 1 . SJ749 dose-dependently inhibited adhesion of both cell types on fibronectin. Application of SJ749 to spread cells led to formation of nonadherent spheroids for A172 cells but had no effect on U87 cell morphology. SJ749 also reduced proliferation of A172 cells due to a long lasting G 0 -G 1 arrest, whereas U87 cells were only slightly affected. However, under nonadherent culture conditions (soft agar), SJ749 significantly reduced the number of colonies formed only by U87 cells. As U87 cells express more α 5 β 1 than A172 cells, we specifically examined the effect of SJ749 on A172 cells overexpressing α 5 . Treatment of α 5 -A172 cells with SJ749 decreased colony formation similarly to that observed in U87 cells. Therefore, in nonadherent conditions, the effect of SJ749 on tumor cell growth characteristics depends on the level of α 5 β 1 expression. Our study highlights the importance of α 5 β 1 as an anticancer target and shows for the first time that a small nonpeptidic α 5 β 1 -specific antagonist affects proliferation of tumor cells. (Cancer Res 2006; 66(12): 6002-7)

Red wine polyphenols cause growth inhibition and apoptosis in acute lymphoblastic leukaemia cells by inducing a redox-sensitive up-regulation of p73 and down-regulation of UHRF1

Several epidemiological studies suggest that a diet rich in fruits and vegetables, which contain high levels of polyphenols, is associated with a reduced risk of cancer. The aim of the present study was to determine whether a red wine polyphenolic extract (RWPs, a rich source of polyphenols; 2.9g/L) affects the proliferation of human lymphoblastic leukaemia cells (Jurkat cells) and, if so, to determine the underlying mechanism. Cell proliferation and viability were determined by the MTS and trypan blue exclusion assays, respectively. Cell cycle analysis, apoptosis activity and oxidative stress levels were assessed by flow cytometry, and the expression of p73, UHRF1 and active caspase-3 by Western blot analysis. RWPs inhibited the proliferation of Jurkat cells and induced G0/G1 cell cycle arrest in a concentration-dependent manner. Moreover, RWPs triggered apoptosis, which is associated with an increased expression level of the pro-apoptotic protein p73 and the active caspase-3. RWPs induced apoptosis confirmed by DNA fragmentation analysis, and this effect was associated with down-regulation of the antiapoptotic protein UHRF1. Furthermore RWPs significantly increased the formation of reactive oxygen species (ROS). Intracellular scavengers of superoxide anions (MnTMPyP, MnTBAP, PEG-SOD) prevented the RWPs-induced formation of ROS and apoptosis, while native extracellular superoxide dismutase (SOD) was without effect. In addition, the effect of RWPs on the expression levels of p73, active caspase-3 and UHRF1 was also prevented by MnTMPyP. Thus, these findings indicate that RWPs induce apoptosis in Jurkat cells by a redox-sensitive mechanism involving the intracellular formation of superoxide anions and consequently the up-regulation of p73 and down-regulation of UHRF1.

UHRF1 recruits the histone acetyltransferase Tip60 and controls its expression and activity

Tat-interactive protein, 60kDa (Tip60) is a histone acetyltransferase with specificity toward lysine 5 of histone H2A (H2AK5) and plays multiple roles in chromatin remodeling processes. Co-immunoprecipitation experiments performed on Jurkat cells, showed that Tip60 is present in the same macro-molecular complex as UHRF1 (Ubiquitin-like containing PHD and RING domain 1), DNMT1 (DNA methyltransferase 1), and HDAC1 (histone deacetylase 1). Furthermore, immunocytochemistry experiments confirmed that Tip60 co-localizes with the UHRF1/DNMT1 complex. Although down-regulation of UHRF1 by RNA interference enhanced Tip60 expression, a significant decrease of the level of acetylated H2AK5 was observed. Consistently, we have observed that down-regulation of Tip60 and DNMT1 by RNA interference, dramatically reduced the levels of acetylated H2AK5. Altogether, these results suggest that Tip60 is a novel partner of the epigenetic integration platform interplayed by UHRF1, DNMT1 and HDAC1 involved in the epigenetic code replication.

Down-regulation of cyclic nucleotide phosphodiesterase PDE1A is the key event of p73 and UHRF1 deregulation in thymoquinone-induced acute lymphoblastic leukemia cell apoptosis.

Thymoquinone (TQ), the active principle of Nigella sativa black seeds, has anti-proliferative properties on numerous cancer cell types. Others and we have previously reported that TQ acts as agent that triggers cell cycle arrest and apoptosis through either a p53- or p73-dependent pathway. However, the immediate targets recruited upon TQ-induced cytotoxicity have not yet been clearly identified. We therefore asked whether cyclic nucleotide phosphodiesterases (PDEs) could be involved in TQ-triggered pro-apoptotic reactivity; PDEs are regulators of intracellular levels of cyclic nucleotides and therefore can modulate cAMP and cGMP-dependent cell death pathways. Our results showed that TQ specifically repressed PDE1A expression in the acute lymphoblastic leukemia Jurkat cell line. This effect is concomitant with the previously described sequential deregulation of the expression of the tumor suppressor protein p73 and the epigenetic integrator UHRF1 (Ubiquitin-like, PHD Ring Finger 1). Interestingly, RNA-interference knock-down of PDE1A expression as well as decreased PDE1A expression induced growth inhibition of Jurkat cells, cell cycle arrest and apoptosis through an activation of p73 and a repression of UHRF1. Conversely, PDE1A re-expression counteracted the cellular pro-apoptotic effects of TQ in association with a p73 repression and UHRF1 re-expression. Altogether, our results show that TQ induced an initial down-regulation of PDE1A with a subsequent down-regulation of UHRF1 via a p73-dependent mechanism. This study further proposes that PDE1A might be involved in the epigenetic code inheritance by regulating, via p73, the epigenetic integrator UHRF1. Our findings also suggest that a forced inhibition of PDE1A expression might be a new therapeutic strategy for the management of acute lymphoblastic leukemia.

Aronia melanocarpa juice induces a redox-sensitive p73-related caspase 3-dependent apoptosis in human leukemia cells.

Polyphenols are natural compounds widely present in fruits and vegetables, which have antimutagenic and anticancer properties. The aim of the present study was to determine the anticancer effect of a polyphenol-rich Aronia melanocarpa juice (AMJ) containing 7.15 g/L of polyphenols in the acute lymphoblastic leukemia Jurkat cell line, and, if so, to clarify the underlying mechanism and to identify the active polyphenols involved. AMJ inhibited cell proliferation, which was associated with cell cycle arrest in G2/M phase, and caused the induction of apoptosis. These effects were associated with an upregulation of the expression of tumor suppressor p73 and active caspase 3, and a downregulation of the expression of cyclin B1 and the epigenetic integrator UHRF1. AMJ significantly increased the formation of reactive oxygen species (ROS), decreased the mitochondrial membrane potential and caused the release of cytochrome c into the cytoplasm. Treatment with intracellular ROS scavengers prevented the AMJ-induced apoptosis and upregulation of the expression of p73 and active caspase 3. The fractionation of the AMJ and the use of identified isolated compounds indicated that the anticancer activity was associated predominantly with chlorogenic acids, some cyanidin glycosides, and derivatives of quercetin. AMJ treatment also induced apoptosis of different human lymphoblastic leukemia cells (HSB-2, Molt-4 and CCRF-CEM). In addition, AMJ exerted a strong pro-apoptotic effect in human primary lymphoblastic leukemia cells but not in human normal primary T-lymphocytes. Thus, the present findings indicate that AMJ exhibits strong anticancer activity through a redox-sensitive mechanism in the p53-deficient Jurkat cells and that this effect involves several types of polyphenols. They further suggest that AMJ has chemotherapeutic properties against acute lymphoblastic leukemia by selectively targeting lymphoblast-derived tumor cells.

UHRF1 Links the Histone Code and DNA Methylation to Ensure Faithful Epigenetic Memory Inheritance

Epigenetics is the study of the transmission of cell memory through mitosis or meiosis that is not based on the DNA sequence. At the molecular level the epigenetic memory of a cell is embedded in DNA methylation, histone post-translational modifications, RNA interference and histone isoform variation. There is a tight link between histone post-translational modifications (the histone code) and DNA methylation, as modifications of histones contribute to the establishment of DNA methylation patterns and vice versa. Interestingly, proteins have recently been identified that can simultaneously read both methylated DNA and the histone code. UHRF1 fulfills these requirements by having unique structural domains that allow concurrent recognition of histone modifications and methylated DNA. Herein, we review our current knowledge of UHRF1 and discuss how this protein ensures the link between histone marks and DNA methylation. Understanding the molecular functions of this protein may reveal the physiological relevance of the linkage between these layers of epigenetic marks.

Selective proapoptotic activity of polyphenols from red wine on teratocarcinoma cell, a model of cancer stem-like cell

SummaryCancer stem cells are expected to be responsible for tumor initiation and metastasis. These cells are therefore potential targets for innovative anticancer therapies. However, the absence of bona fide cancer stem cell lines is a real problem for the development of such approaches. Since teratocarcinoma cells are totipotent stem cells with a high degree of malignancy, we used them as a model of cancer stem cells in order to evaluate the anticancer chemopreventive activity of red wine polyphenols (RWPs) and to determine the underlying cellular and molecular mechanisms. We therefore investigated the effects of RWPs on the embryonal carcinoma (EC) cell line P19 which was grown in the same culture conditions as the most appropriate normal cell line counterpart, the pluripotent embryonic fibroblast cell line NIH/3T3. The present study indicates that RWPs selectively inhibited the proliferation of P19 EC cells and induced G1 cell cycle arrest in a dose-dependent manner. Moreover, RWPs treatment specifically triggered apoptosis of P19 EC cells in association with a dramatic upregulation of the tumor suppressor gene p53 and caspase-3 activation. Our findings suggest that the chemopreventive activity of RWPs on tumor initiation and development is related to a growth inhibition and a p53-dependent induction of apoptosis in teratocarcinoma cells. In addition, this study also shows that the EC cell line is a convenient source for studying the responses of cancer stem cells to new potential anticancer agents.

Selective anticancer effects of a synthetic flavagline on human Oct4-expressing cancer stem-like cells via a p38 MAPK-dependent caspase-3-dependent pathway

Cancer stem cells (CSCs) are considered as the initiators of the carcinogenic process and are therefore emerging targets for innovative anticancer therapies. In order to evaluate the anticancer chemopreventive activity of flavagline derivatives, we used the pluripotent teratocarcinomal cell as a model of Oct4-expressing cancer stem-like cell and determined the underlying cellular and molecular mechanisms induced by a synthetic flavagline. We precisely investigated the effects of the flavagline derivative FL3 on the human embryonal carcinoma (EC) cell line NT2/D1 and compared the responses to those of a normal more restrictive pluripotent stem cell line (i.e. BJ fibroblast cell line). FL3 selectively inhibited the proliferation of NT2/D1 cells by inducing G1 phase cell cycle arrest in a dose-dependent manner. Moreover, FL3 treatment specifically triggered apoptosis in association with an induction of the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and caspase-3 activation followed by a drastic downregulation of the master regulator of stemness Oct4. Forced inhibition of p38 MAPK activity by the specific pharmacological inhibitor SB203580 or by p38 MAPK gene knockdown using small-interfering RNA (siRNA) counteracted the effects of FL3, demonstrating that its chemopreventive action is related to growth inhibition and a p38-dependent caspase-3-dependent induction of apoptosis in Oct4-expressing CSCs. This study also shows that FL3 selectively kills poorly differentiated and highly aggressive carcinomal cells, but has little effect on normal stem-like cells. Thus FL3 offers great promise for cancer treatment since it is able to target the carcinogenic process without affecting normal cells.

Selective ROS-dependent p53-associated anticancer effects of the hypoxoside derivative rooperol on human teratocarcinomal cancer stem-like cells

Cancer stem cells (CSCs) are potential targets for innovative anticancer therapies that involve natural products with potential chemopreventive effects. We therefore analyzed the antineoplastic activity of rooperol, the aglycone of the plant-derived compound hypoxoside, on a model of Oct4-expressing cancer stem-like cell, i.e. the human embryonal carcinoma (EC) cell NT2/D1.Rooperol selectively inhibited the proliferation of NT2/D1 cells in a concentration-dependent manner and had no effect on either normal embryonic fibroblasts which are more restrictive pluripotent stem cells or on NCCIT p53-mutant EC cells. Accordingly, rooperol only eliminates colon carcinoma cells expressing p53.Rooperol treatment triggered cell death on NT2/D1 cells through the alteration of mitochondrial membrane potential and production of reactive oxygen species (ROS). Rooperol-induced apoptosis was associated with activation of p53 and concentration-dependent changes of the expression levels of both caspase 3 and poly ADP ribose polymerase type 1 cleaved subunits. These modifications were accompanied by a downregulation of Oct4 and its two partners involved in the maintenance of cell pluripotency and self-renewal, Nanog and Sox2.Treatment with intracellular membrane permeant O2− scavengers prevented rooperol-induced apoptosis and upregulation of the expression of p53 and active caspase-3. Our findings indicate that rooperol mediates its growth inhibitory effects on CSCs via a mitochondrial redox-sensitive mechanism. We propose that abrogating the expression of the stemness regulators is a prerequisite for rooperol to fully exert its pro-apoptotic properties on wild-type p53-bearing CSCs.

Combining 2D angiogenesis and 3D osteosarcoma microtissues to improve vascularization

Abstract Angiogenesis is now well known for being involved in tumor progression, aggressiveness, emergence of metastases, and also resistance to cancer therapies. In this study, to better mimic tumor angiogenesis encountered in vivo, we used 3D culture of osteosarcoma cells (MG‐63) that we deposited on 2D endothelial cells (HUVEC) grown in monolayer. We report that endothelial cells combined with tumor cells were able to form a well‐organized network, and that tubule‐like structures corresponding to new vessels infiltrate tumor spheroids. These vessels presented a lumen and expressed specific markers as CD31 and collagen IV. The combination of 2D endothelial cells and 3D microtissues of tumor cells also increased expression of angiogenic factors as VEGF, CXCR4 and ICAM1. The cell environment is the key point to develop tumor vascularization in vitro and to be closer to tumor encountered in vivo. Graphical abstract Figure. No caption available. HighlightsCombining 2D endothelial cells/3D tumor cells leads to a well‐organized network.This network consists of tubule‐like structures able to infiltrate 3D tumor.These vessels present a lumen and express specific marker as CD31 and Collagen IV.Combined 2D/3D strategy allows to an increased expression of angiogenic factors.