Fibronectin expression in glioblastomas promotes cell cohesion, collective invasion of basement membrane in vitro and orthotopic tumor growth in mice.

Glioblastoma multiforme (GBM) are highly invasive and angiogenic malignancies with a median survival time from diagnosis of <15 months. Previous work has revealed robust overexpression of fibronectin (FN) mRNA in GBM, although immunohistochemical staining of FN in these tumors is typically associated with the angiogenic vasculature. Here we sought to examine the expression of tumor cell FN and address its possible involvement in the invasive phenotype of GBM. We found that FN was expressed and assembled into fibrillar arrays in human tumors and in established GBM lines. Cultured cells spontaneously formed dense cellular networks and spheroid-like domes. Depletion of FN by targeted-short hairpin RNA expression disrupted matrix assembly and multicellular network organization by exerting profound effects on cell adhesion and motility. Although FN depletion enhanced persistent directional migration of single cells, it compromised collective invasion of spheroids through a laminin-rich matrix and sensitized cells to ionizing radiation. In orthotopic grafts, FN depletion significantly reduced tumor growth and angiogenesis. Together our results show that FN produced by the tumor cells has a role in GBM pathophysiology and they provide insights into the implications that targeting FN interactions may have for combating this dreaded disease.

Cooperative effect of roscovitine and irradiation targets angiogenesis and induces vascular destabilization in human breast carcinoma.

Angiogenesis is considered as an essential process for tumour development and invasion. Previously, we demonstrated that cyclin-dependent kinase inhibition by roscovitine induces a radiosensitization and a synergistic antitumoral effect in human carcinoma but its effect on the microenvironment and tumour angiogenesis remains unknown. Here, we investigated the effect of the combination roscovitine and ionizing radiation (IR) on normal cells in vitro and on tumour angiogenesis in MDA-MB 231 tumour xenografts. We observed that the combination roscovitine and IR induced a marked reduction of angiogenic hot spot and microvascular density in comparison with IR or roscovitine treatments alone. The Ang-2/Tie-2 ratio was increased in presence of reduced vascular endothelial growth factor level suggesting vessel destabilization. In vitro, no radiosensitization effect of roscovitine was found in endothelial, fibroblast, and keratinocyte cells. IR potentiated the antiproliferative effect of roscovitine without inducing apoptosis in endothelial cells. Roscovitine decreased IR-stimulated vascular endothelial growth factor secretion of MDA-MB 231 and endothelial cells. A reduction in the endothelial cells invasion and the capillary-like tube formation in Matrigel were observed following the combination roscovitine and IR. This combined treatment targets angiogenesis resulting in microvessel destabilization without inducing normal cell toxicity.

[New strategies to interfere with radiation response: "biomodulation" of radiation therapy]. / Les stratégies nouvelles pour moduler les effets de l'irradiation: exemples d'interactions entre biomodulateurs et radiothérapie.

The development of several new anti cancer agents has been made possible because of recent significant achievements in our global understanding of cancer biology. These new "targeted" agents selectively inhibit targets necessary for tumor cell growth and viability with little toxicity to normal cells compared to conventional cytotoxic agents. So far, the efficacy of many of these new promising agents when used alone treatment remains limited, it is likely that the optimal use of these agents could be obtained in combination with conventional agents such as radiation therapy. The potential benefit of these targeted therapies combined with irradiation seems important. They might offer the advantage of increasing the tumor response to radiation with no or little increase in normal tissue damage. Therefore, these new types of chemo-radiation approaches might respect the normal tissue versus tumor cell "therapeutic ratio". These approaches can be sub divided in three sub groups: 1) Therapeutics targeting selectively one tumor related biochemical activity such as EGFR inhibitors. These approaches are efficient but one mutation of the target might render them inefficient. 2) Therapeutics directed against a widely expressed target. This is the case for anti Insulin Growth Factor-1 (IGF1R) interventions: IGF1R inhibition seems to specifically alter tumor cell viability with a minimal effect on normal cells viability. 3) Strategies which are not targeted against the tumor but the microenvironment, especially angiogenesis. This type of approaches seems to be applicable independently of tumor intrinsic biologic related factors.

Tyrosine kinase inhibitor AG1024 exerts antileukaemic effects on STI571-resistant Bcr-Abl expressing cells and decreases AKT phosphorylation.

Chronic myelogenous leukaemia (CML) is a clonal malignancy of the pluripotent haematopoietic stem cell, characterised by an uncontrolled proliferation and expansion of myeloid progenitors expressing a fusion oncogene, BCR-ABL, the molecular counterpart of the Ph1 chromosome. The tyrosine kinase (TK) activity of BCR-ABL is known to activate several major signalling pathways in malignant cells, including Ras, JAK/STAT and PI3K/Akt with evidence of proteasome-mediated degradation of other targets such as the DNA repair protein DNA-PKcs and cyclin-dependent kinases inhibitor p27. Targeting these abnormalities by blocking TK of BCR-ABL with STI571 provided a promising approach for the therapy of CML. The recent development of resistance to STI571 illustrates, however, that the use of other TK inhibitors could be of major interest for therapeutic purposes. To this end, the TK inhibitor Tyrphostin AG1024 was used to evaluate effect on regulation of BCR-ABL expression, inhibition of cell proliferation and tumour formation in vivo in human and murine BCR-ABL expressing cell lines. Tyrphostin AG1024 was shown to downregulate expression of BCR-ABL and P-Akt, and to upregulate DNA-PKcs expression. In addition, Tyrphostin AG1024 was able to inhibit cell proliferation, and delay tumour growth in vivo. Thus, AG1024 is able to interfere with three major targets of BCR-ABL in leukaemic cells. Interestingly, Tyrphostin AG1024 was also effective against cells resistant to STI571 by distinct mechanisms including Bcr-Abl mutation. Therefore, these data suggest that Tyrphostin AG1024 could represent the basis of a novel therapy for STI571 refractory CML.

Mononuclear cell adhesion and cell adhesion molecule liberation after X-irradiation of activated endothelial cells in vitro.

PURPOSE: Increased expression of cell adhesion molecules on endothelial cells is an important early event in inflammation. Low-dose radiotherapy is very effective anti-inflammatory treatment. The hypothesis that it may act by modulation of cell adhesion molecule expression in activated endothelial cells and the subsequent adhesion of mononuclear cells onto the activated endothelial cells was tested. MATERIALS AND METHODS: EA.hy.926 endothelial cells were irradiated with 0.3-10 Gy X-rays at different times before or after stimulation with TNFalpha. ICAM-1 or E-selectin expression was measured by ELISA and FACS. Isolated peripheral blood mononuclear cells were incubated with an activated and irradiated confluent monolayer of endothelial cells 4 h, 12 h or 24 h after stimulation, and adhesion was determined in dynamic and static adhesion assays. RESULTS: In the static adhesion assay, where integrin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion of mononuclear cells onto EA.hy.926-EC in vitro by 25-40% and 15-25% of the control level 4 h and 24 h after stimulation, respectively, but increased adhesion 12 h after stimulation. In the dynamic adhesion assay, where selectin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion events by 40-50% and 30-40% of the control level 4 h and 24 h after stimulation, respectively, and again increased adhesion 12h after stimulation. X-ray doses of < or =5 Gy did not induce ICAM-1 expression, or modulate TNFalpha-induced ICAM-1 expression. E-selectin expression was, however, increased in a dose-dependent way 6 h after irradiation. In contrast, X-irradiation 2-5 h before stimulation decreased the characteristic transient expression of E-selectin after TNFalpha stimulation. CONCLUSIONS: Modulation of E-selectin liberation on activated endothelial cells may be one mechanism to decrease leukocyte adhesion after low-dose irradiation in vitro, and could be involved in the therapeutic action of anti-inflammatory radiotherapy.

Tyrphostin AG 1024 modulates radiosensitivity in human breast cancer cells.

Insulin-like growth factor-1 (IGF-1) plays an important growth-promoting effect by activating the PI3K/Akt signalling pathway, inhibiting apoptotic pathways and mediating mitogenic actions. Tyrphostin AG 1024, one selective inhibitor of IGF-1R, was used to evaluate effects on proliferation, radiosensitivity, and radiation-induced cell apoptosis in a human breast cancer cell line MCF-7. Exposure to Tyrphostin AG 1024 inhibited proliferation and induced apoptosis in a time-dependent manner, and the degree of growth inhibition for IC20 plus irradiation (4 Gy) was up to 50% compared to the control. Examination of Tyrphostin AG 1024 effects on radiation response demonstrated a marked enhancement in radiosensitivity and amplification of radiation-induced apoptosis. Western blot analysis indicated that Tyrphostin AG 1024-induced apoptosis was associated with a downregulation of expression of phospho-Akt1, increased expression of Bax, p53 and p21, and a decreased expression of bcl-2 expression, especially when combined with irradiation. To our knowledge, this is the first report showing that an IGF-1 inhibitor was able to markedly increase the response of tumour cells to ionizing radiation. These results suggest that Tyrphostin AG 1024 could be used as a potential therapeutic agent in combination with irradiation.

The olivacine S16020 enhances the antitumor effect of ionizing radiation without increasing radio-induced mucositis.

The combination of a novel topoisomerase II inhibitor, S16020, and ionizing radiation (IR) was investigated with the aim of assessing normal tissue tolerance using a mouse mucosal lip model and antitumor activity in a human carcinoma (HEP2) cell line. No increase of acute mucosal reactions was seen when combining S16020 with IR as compared with IR alone. Using clonogenic cell survival assay, a marked enhancement of HEP2 cell killing was found when S16020 was combined with irradiation. Additional in vivo combination of S16020-IR was able to increase markedly the antitumor efficacy as compared with S16020 or irradiation alone. Interestingly, the radiosensitization effect in vivo was observed at relatively low and nontoxic concentrations of S16020, and no dose-effect relationship was found beyond 30 mg/kg. In conclusion, the combination of IR and S16020 seems promising to enhance antitumor activity without increasing deleterious effect in normal tissues and to provide the basis for a new radio-chemotherapy combination.

BCR-ABL down-regulates the DNA repair protein DNA-PKcs.

This study demonstrates in both stable and inducible BCR-ABL-expressing hematopoietic cells a down-regulation of the major mammalian DNA repair protein DNA-PKcs by BCR-ABL. Similar results were found in BCR-ABL CD34(+) cells from patients with chronic myelogenous leukemia (CML). DNA-PKcs down-regulation is a proteasome-dependent degradation that requires tyrosine kinase activity and is associated with a marked DNA repair deficiency along with increased sensitivity to ionizing radiation. The conjunction of a major DNA repair deficiency and a resistance to apoptosis, both induced by BCR-ABL, provides a new mechanism to explain how secondary genetic alterations can accumulate in CML, eventually leading to blast crisis. The down-regulation of DNA-PKcs was reversible in CD34(+) CML cells suggesting that this approach might offer a novel and powerful therapeutic strategy in this disease, especially to delay the blast crisis. (Blood. 2001;97:2084-2090)