Anna Flugy

Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis

Our study is designed to assess if exosomes released from chronic myelogenous leukemia (CML) cells may modulate angiogenesis. We have isolated and characterized the exosomes generated from LAMA84 CML cells and demonstrated that addition of exosomes to human vascular endothelial cells (HUVEC) induces an increase of both ICAM‐1 and VCAM‐1 cell adhesion molecules and interleukin‐8 expression. The stimulation of cell‐cell adhesion molecules was paralleled by a dose‐dependent increase of adhesion of CML cells to a HUVEC monolayer. We further showed that the treatment with exosomes from CML cells caused an increase in endothelial cell motility accompanied by a loss of VE‐cadherin and β‐catenin from the endothelial cell surface. Functional characterization of exosomes isolated from CML patients confirmed the data obtained with exosomes derived from CML cell line. CML exosomes caused reorganization into tubes of HUVEC cells cultured on Matrigel. When added to Matrigel plugs in vivo, exosomes induced ingrowth of murine endothelial cells and vascularization of the Matrigel plugs. Our results suggest for the first time that exosomes released from CML cells directly affect endothelial cells modulating the process of neovascularization.

Exosome-mediated crosstalk between chronic myelogenous leukemia cells and human bone marrow stromal cells triggers an interleukin 8-dependent survival of leukemia cells.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by the Bcr-Abl oncoprotein with constitutive tyrosine kinase activity. Exosomes are nanovesicles released by cancer cells that are involved in cell-to-cell communication thus potentially affecting cancer progression. It is well known that bone marrow stromal microenvironment contributes to disease progression through the establishment of a bi-directional crosstalk with cancer cells. Our hypothesis is that exosomes could have a functional role in this crosstalk. Interleukin-8 (IL 8) is a proinflammatory chemokine that activates multiple signalling pathways downstream of two receptors (CXCR1 and CXCR2). We demonstrated that exosomes released from CML cells stimulate bone marrow stromal cells to produce IL 8 that, in turn, is able to modulate both in vitro and in vivo the leukemia cell malignant phenotype.

Continental and subcontinental distributions of mtDNA control region types

When the mtDNA profile of a crime scene matches that of a suspect, it is necessary to determine the probability of a chance match by consulting the frequencies of the identified allele in a “reference population”. The ceiling principle suggests that that population should be chosen in which the allele of the suspect is found at the highest frequency, in order to give the suspect the maximum benefit of doubt. Recently, we advocated the use of a worldwide mitochondrial database combined with a geographical information system to identify the regions of the world with the highest frequencies of matching mtDNA types. Here, we demonstrate that the alternative approach of defining a ceiling reference population on the basis of continent or phenotype (race) is too coarse for a non-negligible percentage of mtDNA control region types.

carboxyamidotriazole-orotate inhibits the growth of imatinib resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

The Bcr/Abl kinase has been targeted for the treatment of chronic myelogenous leukaemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML are often resistant. New therapeutic options are therefore needed for this fatal disease. Although more common in solid tumors, increased microvessel density was also reported in chronic myelogenous leukaemia and was associated with a significant increase of angiogenic factors, suggesting that vascularity in hematologic malignancies is a controlled process and may play a role in the leukaemogenic process thus representing an alternative therapeutic target. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable signal transduction inhibitor that in vitro has been shown to possess antileukaemic activities. CTO, which has a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound, was tested in this study for its ability to affect imatinib-resistant CML tumor growth in a xenograft model. The active cross talk between endothelial cells and leukemic cells in the bone marrow involving exosomes plays an important role in modulating the process of neovascularization in CML. We have thus investigated the effects of CTO on exosome-stimulated angiogenesis. Our results indicate that CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility for CTO in leukaemia patients.

E-selectin modulates the malignant properties of T84 colon carcinoma cells

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of cells in the host connective tissue. E-selectin on endothelial cells and sialyl Lewis X carbohydrate component on tumor cells are mainly involved in the adhesion of colon carcinoma cells to the endothelium of target organ. Interaction of T84 colon cancer cells to purified E-selectin in vitro caused an increase in the tyrosine phosphorylation of a number of proteins as well as the modulation of cellular properties correlated to the metastatic phenotype. Specifically, E-selectin-stimulated actin reorganization, increased collagenase secretion, and induced cell migration. Treatment of T84 cells with herbimycin A inhibited cell adhesion as well as selectin-induced increase of cell migration, and cytoskeleton assembly. Our data demonstrate that binding of cancer cells to E-selectin starts signal transduction pathways which may affect the tumor metastatic abilities.

Effects of desipramine and alprazolam in the forced swim test in rats after long-lasting termination of chronic exposure to picrotoxin and pentylenetetrazol

Rats were treated for 5 weeks with three subconvulsant doses of picrotoxin (PTX) and pentylenetetrazol (PTZ) per week to induce a persistent reduction of the GABAA receptor function which results in chemical kindling. Fifteen days after termination of this treatment schedule, the effect of desipramine (DMI) and alprazolam (ALP) on immobility time in the forced swim test (FST) was evaluated. Chronic PTX and PTZ did not alter the immobility time. Acute PTX and PTZ reduced the immobility of rats chronically treated with vehicle but not of those exposed chronically to PTX and PTZ. Chronic PTX did not influence the anti-immobility effect of DMI, but blocked that of ALP. Chronic PTZ markedly potentiated the anti-immobility effect of DMI but blocked that of ALP. Concomitant administration of chlordiazepoxide prevented the effects of chronic PTX and PTZ. These findings suggest that a long-lasting reduction in GABAA receptor function, unlike acute reduction, does not play an important role in the mobility of rats in the FST and in the anti-immobility effect of DMI while it blocks that of ALP.

Effects of carboxyamidotriazole on in vitro models of imatinib-resistant chronic myeloid leukemia.

Although imatinib mesylate (IM) has revolutionized the treatment of chronic myeloid leukemia (CML), some patients develop resistance with progression of leukemia. Alternative or additional targeting of signaling pathways deregulated in bcr‐abl‐driven CML cells may provide a feasible option for improving clinical response and overcoming resistance. In this study, we show that carboxyamidotriazole (CAI), an orally bioavailable calcium influx and signal transduction inhibitor, is equally effective in inhibiting the proliferation and bcr‐abl dependent‐ and independent‐signaling pathways in imatinib‐resistant CML cells. CAI inhibits phosphorylation of cellular proteins including STAT5 and CrkL at concentrations that induce apoptosis in IM‐resistant CML cells. The combination of imatinib and CAI also down‐regulated bcr‐abl protein levels. Since CAI is already available for clinical use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of CML. J. Cell. Physiol. 215: 111–121, 2008.

Role of calcium in E-selectin induced phenotype of T84 colon carcinoma cells.

The adhesion of cancer cells to the endothelium during the metastatic process involves the interaction of specific cell-cell adhesion receptors on the cell surface. E-selectin on endothelial cells and sialyl Lewis X carbohydrate component on tumor cells are mainly implicated in the adhesion of colon carcinoma cells to the endothelium of target organ. In this paper we show that binding of E-selectin to T84 colon tumor cells causes approximately a twofold increase in intracellular calcium concentration. In particular, using two inhibitors of receptor operated calcium channels, CAI and SK&F 96365, we present evidences that the augmentation in cytoplasmic calcium originates from ionic influx from extracellular sources. Furthermore, we demonstrated that modulation of [Ca2+]i by engagement of E-selectin receptor starts signal transduction pathways that affect cell spreading, tyrosine phosphorylation signaling, and cancer cell motility.

Role of S128R polymorphism of E-selectin in colon metastasis formation

The extravasation of cancer cells is a key step of the metastatic cascade. Polymorphisms in genes encoding adhesion molecules can facilitate metastasis by increasing the strength of interaction between tumor and endothelial cells as well as impacting other properties of cancer cells. We investigated the Ser128Arg (a561c at the nucleotide level) polymorphism in the E‐selectin gene in patients with metastatic colon cancer and its functional significance. Genotyping for a561c polymorphism was performed on 172 cancer patients and on an age‐matched control population. The colon cancer group was divided into groups with (M+) and without observable metastasis (M−). For in vitro functional assays, Huvec transfected cells expressing wild‐type (WT) or the S128R variant of E‐selectin were established to study in vitro binding ability and signal transduction processes of T84 colon cancer cell line. Our results demonstrated that the Arginine128 allele was more prevalent in the M+ group than in the M− group or normal controls (p < 0.005; odds ratio, 1.56; 95% confidence interval (CI) 1.16–1.92; p < 0.001, odds ratio = 1.65; CI = 1.24–1.99, respectively). In vitro, S128R E‐selectin transfected Huvec cells, supported increased adhesion as well as increased cellular signaling of T84 cancer cells compared to WT E‐selectin and mock‐transfected Huvec cells. These findings suggest that the E‐selectin S128R polymorphism can functionally affect tumor‐endothelial interactions as well as motility and signaling properties of neoplastic cells that may modulate the metastatic phenotype.

Identification and phenotypic characterization of a subpopulation of T84 human colon cancer cells, after selection on activated endothelial cells

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of the cells in the host connective tissue. The differences in metastatic ability could be attributed to properties intrinsic of the various primary tumor types. Thus, the clonal selection of neoplastic cells during cancer progression results in cells better equipped for survival and formation of colonies in secondary sites. A cell line (T84SF) exhibiting an altered phenotypic appearance was selected from a colon cancer cell line (T84) by repetitive plating on TNFα‐activated human endothelial cells and subsequent selection for adherent cells. Cell growth, motility, chemoinvasive abilities, tyrosine phosphorylation signaling, and the metastasis formation in nude mice of the two cell lines was compared. T84SF cells displayed in vitro an higher proliferation rate and a more invasive behavior compared to the parental cells while formed in vivo a greater number of metastatic colonies in nude mice. As concerns the signaling underlying the phenotypes of the selected cells, we examined the general tyrosine phosphorylation levels in both cell lines. Our results indicate that T84SF have an increased basal tyrosine phosphorylation of several proteins among which src kinase was identified. Treatment of cells with a specific inhibitor of src activity caused a greater in vitro inhibition of proliferation and invasive properties of T84 parental cells with respect to T84SF cells and diminished metastasis formation in vivo. Altogether, these data provide evidences that this new cell line may be valuable for identifying molecular mechanisms involved in the metastatic progression of colon cancer.