AS602868, a dual inhibitor of IKK2 and FLT3 to target AML cells.

Acute myeloid leukemia (AML) cells carry molecular defects that promote their leukemic proliferation, resistance to apoptosis and defect in differentiation. Pharmacological targeting of the nuclear factor kappaB (NF-kappaB) pathway has been shown to promote apoptosis of primary AML cells and to sensitize blasts to neoplastic drugs (Frelin, Blood 2005, 105, 804). The Fms-like tyrosine kinase 3 (FLT3), which sustains proliferation of normal hematopoietic progenitors is frequently overexpressed or mutated in AML patients. Using Ba/F3 murine pre-B cells transfected with various mutants of FLT3 (ITD, D835V, D835Y) and the MV4-11 human AML line, we show that normal or oncogenic stimulation of FLT3 led to activation of NF-kappaB. Pharmacological inhibition of either FLT3 with AG1296 or NF-kappaB with the small molecule inhibitor of IkappaB kinase-2 AS602868 reduced viability and triggered cell death. Moreover, AS602868 was also found to interfere directly with FLT3 kinase activation. AS602868 thus appears to target two different kinases that play a crucial role in the pathogenesis of AML, making it particularly attractive as a new therapeutical approach for AML.

The response of pre-osteoblasts and osteoclasts to gallium containing mesoporous bioactive glasses.

Mesoporous bioactive glasses (MBGs) in the system SiO2-CaO-P2O5-Ga2O3 have been synthesized by the evaporation induced self-assembly method and subsequent impregnation with Ga cations. Two different compositions have been prepared and the local environment of Ga(III) has been characterized using 29Si, 71Ga and 31P NMR analysis, demonstrating that Ga(III) is efficiently incorporated as both, network former (GaO4 units) and network modifier (GaO6 units). In vitro bioactivity tests evidenced that Ga-containing MBGs retain their capability for nucleation and growth of an apatite-like layer in contact with a simulated body fluid with ion concentrations nearly equal to those of human blood plasma. Finally, in vitro cell culture tests evidenced that Ga incorporation results in a selective effect on osteoblasts and osteoclasts. Indeed, the presence of this element enhances the early differentiation towards osteoblast phenotype while disturbing osteoclastogenesis. Considering these results, Ga-doped MBGs might be proposed as bone substitutes, especially in osteoporosis scenarios. STATEMENT OF SIGNIFCANCE: Osteoporosis is the most prevalent bone disease affecting millions of patients every year. However, there is a lack of bone grafts specifically designed for the treatment of bone defects occurred because of osteoporotic fractures. The consequence is that osteoporotic bone defects are commonly treated with the same biomaterials intended for high quality bone tissue. In this work we have prepared mesoporous bioactive glasses doped with gallium, demonstrating osteoinductive capability by promoting the differentiation of pre-osteoblast toward osteoblasts and partial inhibition of osteoclastogenesis. Through a deep study of the local environment of gallium within the mesoporous matrix, this work shows that gallium release is not required to produce this effect on osteoblasts and osteoclasts. In this sense, the presence of this element at the surface of the mesoporous bioactive glasses would be enough to locally promote bone formation while reducing bone resorption.

DOCK4 promotes loss of proliferation in glioblastoma progenitor cells through nuclear beta-catenin accumulation and subsequent miR-302-367 cluster expression.

Glioblastomas (GBM) are lethal primitive brain tumours characterized by a strong intra-tumour heterogeneity. We observed in GBM tissues the coexistence of functionally divergent micro-territories either enriched in more differentiated and non-mitotic cells or in mitotic undifferentiated OLIG2 positive cells while sharing similar genomic abnormalities. Understanding the formation of such functionally divergent micro-territories in glioblastomas (GBM) is essential to comprehend GBM biogenesis, plasticity and to develop therapies. Here we report an unexpected anti-proliferative role of beta-catenin in non-mitotic differentiated GBM cells. By cell type specific stimulation of miR-302, which directly represses cyclin D1 and stemness features, beta-catenin is capable to change its known proliferative function. Nuclear beta-catenin accumulation in non-mitotic cells is due to a feed forward mechanism between DOCK4 and beta-catenin, allowed by increased GSK3-beta activity. DOCK4 over expression suppresses selfrenewal and tumorigenicity of GBM stem-like cells. Accordingly in the frame of GBM median of survival, increased level of DOCK4 predicts improved patient survival.

The BMI1 polycomb protein represses cyclin G2-induced autophagy to support proliferation in chronic myeloid leukemia cells.

The BMI1 polycomb protein regulates self-renewal, proliferation and survival of cancer-initiating cells essentially through epigenetic repression of the CDKN2A tumor suppressor locus. We demonstrate here for the first time that BMI1 also prevents autophagy in chronic myeloid leukemia (CML) cell lines, to support their proliferation and clonogenic activity. Using chromatin immunoprecipitation, we identified CCNG2/cyclin G2 (CCNG2) as a direct BMI1 target. BMI1 downregulation in CD34+ CML cells by PTC-209 pharmacological treatment or shBMI1 transduction triggered CCNG2 expression and decreased clonogenic activity. Also, ectopic expression of CCNG2 in CD34+ CML cells strongly decreased their clonogenicity. CCNG2 was shown to act by disrupting the phosphatase 2A complex, which activates a PKCζ-AMPK-JNK-ERK pathway that engages autophagy. We observed that BMI1 and CCNG2 levels evolved inversely during the progression of CML towards an acute deadly phase, and therefore hypothesized that BMI1 could support acute transformation of CML through the silencing of a CCNG2-mediated tumor-suppressive autophagy response.

Involvement of T lymphocytes in curative effect of a new immunomodulator OM 163 on rat colon cancer metastases.

In a model of colon cancer in syngeneic rats, a new immunomodulator, OM 163, induced the complete disappearance of peritoneal carcinomatosis (nodules measuring 1-5 mm) in 41 out of 82 rats. Those results were confirmed in a survival experiment in which 3 out of 10 treated rats died free of tumour 10, 18 and 28 months after the tumour cell injection while all the untreated control rats died of their tumours within 3 months. OM 163 had a systemic effect, since injected intraperitoneally it completely inhibited the growth of lung metastases in 13 out of 20 rats. The antitumour effect of OM 163 was also observed in two rat strains on original tumours. Lymphocyte infiltration was observed in the tumours mainly constituted of CD4+ and CD8+ cells. The treatment had no effect in nude rats, confirming the involvement of T lymphocytes. Furthermore, rats cured by OM 163 were protected against a second challenge of tumour cells and in a Winn's assay, splenocytes from cured rats protected normal rats against tumour cells.

Strain dependence of rat macrophage natural tumoricidal capacity and its stimulation by endotoxins.

Without known stimulation in vivo and in vitro, resident peritoneal macrophages from 5 conventional or specific pathogen-free (SPF) rat strains [Hairless (H), BDIX, Wistar (W), Sprague-Dawley (SD) and Long-Evans (LE)] exhibited an in vitro strain-dependent cytolysis against DHD-K12/TS cancer cells. This natural cytolysis was also observed when polymyxin B was added to the culture medium. The percentage of natural cytolysis varied from one rat to another but was significantly different according to the strain. In the presence of 10 micrograms endotoxin/ml, macrophages from BDIX, W, SD and LE rats were always cytolytic, whilst those of H rats were irregularly cytolytic. Endotoxins induced or increased macrophage-mediated cytolysis from H, BDIX, W and SD rats, but they were without effect for LE rats. The endotoxin effect depended on the level of natural cytolysis. In contrast to mouse resident peritoneal macrophages, which were not naturally cytolytic and not activated in vitro by endotoxins, these results show that rat resident peritoneal macrophages can be naturally cytolytic. This cytolysis can be enhanced by endotoxins as the sole in vitro stimulus. Rat macrophage natural cytolytic activity is strain-dependent.

Effects of cyclosporin A on progressive and regressive tumors induced by two cancer lines derived from a single colon carcinoma chemically induced in the rat.

The effects of cyclosporin-induced immunosuppression were assessed in a rat model of progressive and regressive colonic tumors. Two cloned cell variants, obtained from the same chemically induced colonic carcinoma, differ in their capacity to grow when injected into the syngeneic rat. PROb cells yield progressive tumors and often metastases; in contrast, REGb cells produce tumors which regress in 3 to 6 weeks. Cyclosporin A (CsA) administered daily, 20 mg/kg subcutaneously (s.c.) for 30 days after tumor cell inoculation, drastically enhanced the local growth of PROb tumors and increased the number of metastases. It increased the local growth and prevented the regression of REGb tumors which persisted even as long as 8 weeks after the termination of CsA administration and occasionally yielded metastases. CsA prevented the accumulation of inflammatory cells with the T lymphocyte phenotype at the periphery of both PROb and REGb tumors but did not alter the tumor infiltration by macrophages and NK cells. CsA did not modify the natural cytotoxicity of peripheral blood mononuclear cells against PROb and REGb target cells. These results suggest that CsA-induced suppression of T lymphocyte activity may enhance tumor progression and suppress tumor regression in this model.

Correlation between the capacity to activate macrophages in vitro and the antitumor activity in vivo of lipopolysaccharides from different bacterial species.

The correlation between the activation of macrophages by lipopolysaccharides (LPS) from four different bacterial species and their antitumor effect in a rat model of colon cancer was investigated. The efficacy of LPS from Neisseria meningitidis (Nm), Salmonella minnesota (Sm), Escherichia coli (Ec) and Bordetella pertussis (Bp) was evaluated as the smallest concentration inducing rat peritoneal macrophages (pm psi) to produce tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and nitric oxide (NO). The cytokine production was measured in bioassays and NO production quantitatively with Griess reactant. Nm was the most effective LPS with concentrations of 1 ng/10(6) pm psi for the induction of TNF, IL-1 and IL-6 activities and 0.01 ng/10(6) pm psi for the induction of NO production. The range between efficacy of different LPS was broad from 1 to 10(4)-10(5) for TNF activity, 1 to 10(2)-10(3) for NO production and IL-6 activity and 1 to 10-10(2) for IL-1 activity. In vivo antitumor effect was evaluated on the growth of peritoneal carcinomatosis. Complete tumor regressions were observed, the LPS rating with respect to decreasing efficacy was Nm, Sm, Ec then Bp; Nm, Sm and Ec were very closed while Bp was not effective. These results show the correlation between the antitumor effect in vivo of LPS and their capacity to induce in vitro IL-1 activity, but not between their ability to induce NO production, TNF and IL-6 activities.

In vitro natural killer activity against progressive and regressive variants of a rat colon adenocarcinoma. Effect of treatments with anti-asialo GM1 plus complement.

In a previous work, a cell line (DHD/K12) was established from a colon adenocarcinoma induced in a BDIX rat by 1,2-dimethylhydrazine. From this line, two cloned sublines, PROb and REGb, were then isolated. When subcutaneously inoculated into syngeneic rats, PROb cells yield progressive tumors, whereas REGb cells yield tumors which regress. In this study, in a 16-h 51Cr release assay, natural cytotoxicity mediated by BDIX splenic nonadherent lymphoid cells (NK cells) was shown to be much higher against REGb cells than against PROb cells. Whatever the target cells, NK cytotoxicity was always higher when the effector cells were obtained from males rather than from females. Treatment of BDIX splenic lymphocytes by anti-asGM1 serum plus complement revealed that both anti-asGM1 sensitive and non-sensitive NK cells exist. The activity of anti-asGM1 non-sensitive NK cells appeared to be minor and to be detected only when the level of cytotoxicity before treatment was sufficiently high. The difference between PROb and REGb tumor growth appears to be linked, at least in part, to a higher sensitivity of REGb cells to NK cells and especially to anti-asGM1-sensitive NK cells.

Synergistic effect of liposomes and endotoxins on the activation of rat macrophage tumoricidal activity.

Macrophage biological responses to endotoxins have been extensively studied; nevertheless, the mechanisms by which endotoxins activate macrophage tumoricidal activity are not currently understood. We used liposomes to investigate the interaction of endotoxins with macrophages. In a medium containing 10 micrograms endotoxin/ml, macrophage-mediated cytolysis ranged from -7 to 36%. In all the experiments, 1mM dipalmitoyl phosphatidyl choline (DPPC) small unilamellar liposomes significantly induced or enhanced cytolysis, ranging from 30-90%. Liposomes and endotoxins had a synergistic effect on the macrophage cytolytic activity. This effect was dose-dependent on liposome concentration, ranging from 0.25-1 mM or 2 mM. Liposomes decreased the endotoxin concentration threshold necessary to induce cytolysis. They did not modify the kinetics of macrophage activation. Liposomes did not modify the binding of tumor cells to macrophages. The optimum synergistic effect was obtained when liposomes were present during the first 18 h of the mixed culture of macrophages and target cells, before adding endotoxins for the next 18 h. When cholesterol was added to DPPC (M/M), liposomes did not enhance but rather inhibited macrophage activation by endotoxins.

Interleukin-8 antitumour effect is associated with a local infiltration but not with a systemic activation of T lymphocytes.

In a model of colon cancer in rats (peritoneal carcinomatosis), IL-8 was found to have a highly reproducible antitumoural effect. During IL-8-induced tumour regression the infiltration of nodules by CD4+ T lymphocytes was enhanced. However, splenic lymphocytes did not proliferate in response to tumour cells in vitro. IL-8 antitumour effect was associated with a local but not with a systemic activation of T lymphocytes.

In vivo and in vitro effects of fish-containing diets on colon tumour cells in rats.

Polyunsaturated n-3 fatty acids, abundant in sea fish, can inhibit the growth of chemoinduced or transplanted mammary tumours in the rat. Since mammary and colonic cancers have both been linked to a high fat consumption, we studied the effect of 2 diets moderately (7% fish meal) or strongly (9% fish oil) enriched in fish fatty acids on the growth of colon cancer cells subcutaneously inoculated into syngeneic rats. The diets had no effect on the in vivo tumor growth and on the in vitro tumouricidal activity of peritoneal macrophages or splenic lymphocytes.

Involvement of effector cells in the treatment with endotoxins of peritoneal carcinomatosis induced by colon tumour cells.

Peritoneal carcinomatoses, an ordinary way for human colon carcinoma to spread, are incurable. When rat peritoneal carcinomatoses of colon origin were treated with endotoxins i.p. administered 3 days after the tumour cell injection, 70% of the BDIX rats were alive 30 weeks after the PROb tumour cell injection whereas all the untreated rats died of their tumour within 14 weeks. The study of the effector cells involved in the antitumour effect of endotoxins suggests that T lymphocytes are required for this effect. The endotoxin effect is local and is not reflected by the cytolytic activity of peritoneal cells.

Role of macrophage in the defense against intestinal cancers.

The capability of activated macrophages to kill tumor cells in vitro is now well documented. The tumoricidal activation of macrophages against intestinal tumor cells by different agents is described and the main hypothesis on the mechanisms of tumor cell killing in vitro are discussed. These in vitro results suggest that the macrophage can constitute an efficient effector cell in the defense against intestinal tumors. The distribution and ratio of macrophages in normal intestine and intestinal tumors is described. At the moment, potent activators of macrophages studied in vivo on experimental and human intestinal tumors give poor results or even enhance the growth of tumors. Macrophages may also interfere with the specific immune response in two directions by enhancing the immune response or decreasing it by elaboration of mediators such as prostaglandins.

Influence of the injection site on the tumorigenicity of a cloned colon tumor cell line in the rat.

The cloned DHD/K12/TSb line obtained from a chemically-induced rat colon carcinoma, presents tumors which always regress when injected subcutaneously to the syngeneic animal. This study reports that an intraperitoneal injection of the DHD/K12/TSb cells induces a progressive carcinomatosis in a high proportion of the syngeneic rats. This result underlines the effect that the local environment has on tumorigenicity.

Pharmacological targeting of NF-kappaB potentiates the effect of the topoisomerase inhibitor CPT-11 on colon cancer cells.

NF-kappaB interferes with the effect of most anti-cancer drugs through induction of anti-apoptotic genes. Targeting NF-kappaB is therefore expected to potentiate conventional treatments in adjuvant strategies. Here we used a pharmacological inhibitor of the IKK2 kinase (AS602868) to block NF-kappaB activation. In human colon cancer cells, inhibition of NF-kappaB using 10 microM AS602868 induced a 30-50% growth inhibitory effect and strongly enhanced the action of SN-38, the topoisomerase I inhibitor and CPT-11 active metabolite. AS602868 also potentiated the cytotoxic effect of two other antineoplasic drugs: 5-fluorouracil and etoposide. In xenografts experiments, inhibition of NF-kappaB potentiated the antitumoural effect of CPT-11 in a dose-dependent manner. Eighty-five and 75% decreases in tumour size were observed when mice were treated with, respectively, 20 or 5 mg kg(-1) AS602868 associated with 30 mg kg(-1) CPT-11 compared to 47% with CPT-11 alone. Ex vivo tumour analyses as well as in vitro studies showed that AS602868 impaired CPT-11-induced NF-kappaB activation, and enhanced tumour cell cycle arrest and apoptosis. AS602868 also enhanced the apoptotic potential of TNFalpha on HT-29 cells. This study is the first demonstration that a pharmacological inhibitor of the IKK2 kinase can potentiate the therapeutic efficiency of antineoplasic drugs on solid tumours.

Design of a digital phantom of the neonatal brain.

In this paper, we present the design and implementation of a 3D digital phantom of the neonatal brain. Commonly used digital brain phantoms (e.g. BrainWeb) are based on adults' brains. With the increasing interest in computer aided analysis of neonatal Magnetic Resonance (MR) images, it becomes necessary to create a special digital phantom for neonatal brains. This is because of the pronounced differences not only in size but more important in geometrical proportions of different brain tissues in adults and neonates and the additional need to subdivide the white matter of neonatal brains into two different types. Thus, the here created neonatal brain phantom consists of 6 different tissue types: scalp, skull, gray matter, myelinated and non-myelinated white matter and cerebrospinal fluid. Every voxel has a vector consisting of 6 probabilities of being part of one of these six tissues. The digital brain phantom will be used for simulation of tomographic images of the newborns' head and may serve as well as an evaluation data set for comparison of analysis methods for neonatal MR images, e.g. segmentation/registration algorithms, providing the possibility of controlled degradation of image data.

Human small-cell lung-cancer cells are cytokine-resistant but NK/LAK-sensitive.

We have studied the effects of 8 cytokines and their combinations on the in vitro growth of 10 human small-cell cancer lines (SCLC). Interferon-alpha and gamma (IFN-alpha and gamma) caused significant but slight growth inhibition over a 7-day incubation period. However, none of the other 6 cytokines, tumor necrosis factor (TNF), lymphotoxin (LT), interleukin-1 beta (IL-1 beta) interleukin-2 (IL-2), transforming growth factor-beta 1 (TGF-beta 1), or granulocyte colony-stimulating factor (G-CSF), modified SCLC cell proliferation. In contrast, all 10 lines were sensitive to lysis by natural killer (NK) and lymphokine-activated killer (LAK) cells. Sensitivity to LAK cells could be increased by pretreatment of SCLC cells with IFN-gamma. As resistance to the cytostatic/cytotoxic activity of some cytokines has been associated with autocrine production of cytokines, we screened the SCLC lines for cytokine mRNAs. Within the limits of detection of the assay we found no expression of TNF, TGF-beta 1, IL-1 beta or IL-6 mRNA in the 10 SCLC lines.