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.

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.

Carboxyamidotriazole inhibits cell growth of imatinib-resistant chronic myeloid leukaemia cells including T315I Bcr-Abl mutant by a redox-mediated mechanism.

Mutation of the Bcr-Abl oncoprotein is one of most frequent mechanisms by which chronic myelogenous leukemia (CML) cells become resistant to imatinib. Here, we show that treatment of cell lines harbouring wild type or mutant BCR-ABL with carboxyamidotriazole (CAI), a calcium influx and signal transduction inhibitor, inhibits cell growth, the expression of Bcr-Abl and its downstream signalling, and induces apoptosis. Moreover, we show that CAI acts by increasing intracellular ROS. Clinically significant, CAI has also inhibitory effects on T315I Bcr-Abl mutant, a mutation that causes CML cells to become insensitive to imatinib and second generation abl kinase inhibitors.

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.

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 Arginine(128) 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.

Comparative study of T84 and T84SF human colon carcinoma cells: in vitro and in vivo ultrastructural and functional characterization of cell culture and metastasis.

To better understand the relationship between tumor heterogeneity, differentiation, and metastasis, suitable experimental models permitting in vitro and in vivo studies are necessary. A new variant cell line (T84SF) exhibiting an altered phenotype was recently selected from a colon cancer cell line (T84) by repetitive plating on TNF-alpha treated human endothelial cells and subsequent selection for adherent cells. The matched pair of cell lines provides a useful system to investigate the extravasation step of the metastatic cascade. Since analysis of morphological differences can be instructive to the understanding of metastatic potential of tumor cells, we compared the ultrastructural and functional phenotype of T84 and T84SF cells in vitro and in vivo. The reported ultrastructural features evidence differences between the two cell lines; selected cells showed a marked pleomorphism of cell size and nuclei, shape, and greater surface complexity. These morphological differences were also coupled with biochemical data showing a distinct tyrosine phosphorylation-based signaling, an altered localization of beta-catenin, MAPK, and AKT activation, as well as an increased expression in T84SF cells of Bcl-X(L), a major regulator of apoptosis. Therefore, these cell lines represent a step forward in the development of appropriate models in vitro and in vivo to investigate colon cancer progression.