CarboxyAmido-Triazole Orotate inhibits the growth of Imatinib-resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

Chronic myelogenous leukemia is a myeloproliferative disorder characterized by the t(9:22) (q34:q11) reciprocal chromosomal translocation, resulting in the expression of the chimeric Bcr–Abl oncoprotein with constitutive tyrosine kinase activity. Deregulated Bcr–Abl induces the hyperactivation of various signalling pathways that promote cell growth, suppress apoptosis and alter cell adhesion. Bcr-Abl has also been involved in VEGF-mediated angiogenesis in CML and evidence indicates that the formation of new vessels plays an important role in the development and progression of CML. Imatinib mesylate (IM) is a selective well tolerated inhibitor of the Bcr–Abl tyrosine kinase that has significantly improved the prognosis of patients with chronic phase CML. Despite this remarkable progress, a major problem associated with the administration of imatinib is acquired resistance. Bcr-Abl gene amplification, increased expression of Bcr–Abl protein, point mutations in the Bcr–Abl tyrosine kinase domain have been reported as mechanisms of resistance to imatinib. Therefore, there is an urgent need for new anticancer agents and combinations that could improve responses and survival rates for CML. Recent studies from our laboratory have shown that addition of carboxyamidotriazole (CAI), an inhibitor of calcium-mediated signal transduction, to imatinib resistant human CML cells induces a marked decrease in cell viability and augmented apoptosis, events associated with downregulation of Bcr–Abl protein and inhibition of tyrosine phosphorylation of Bcr–Abl, STAT5, CrkL. Carboxyamidotriazole Orotate (CTO), is a derivate of CAI that has been developed at Tactical Therapeutics. CTO has a higher bioavailability and efficacy with respect to the parental compound. Exosomes are small vesicles of 40-100 nm diameter that are initially formed within the endosomal compartment and are secreted when a multivesicular body (MVB) fuses with the plasma membrane. These vesicles are released by many cell types including cancer cells and are considered messengers in intercellular communication. The exact function of exosomes in malignant cells has yet to be elucidated, but investigation has suggested roles in cell-to-cell communication, tumor-stroma interaction, and antigen presentation, thus potentially affecting cancer progression at different steps. Recent studies from our laboratory suggest that exosomes released from IM-sensitive CML cells directly affect endothelial cells modulating the process of neovascularization. Our data show that CTO is able to inhibit both in vitro and in vivo the growth of imatinib-resistant CML cells and to affect tumor microenvironment by modulating exosome-stimulated angiogenesis. CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility in the treatment of leukemia patients

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

Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis

The present 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

Crosstalk between chronic myelogenous leukemia (CML) and bone marrow stromal cells: role of interleukin 8 and CML derived- exosomes

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by the t(9:22) (q34:q11) reciprocal translocation, resulting in the expression of the chimeric Bcr–Abl oncoprotein with constitutive tyrosine kinase activity. Exosomes (Exo) are small vesicles of endosomal origin and of 40-100 nm diameter released by many cell types including cancer cells. Several data indicate that Exo play an important role in cell-to-cell communication and tumor-stroma interaction, thus potentially affecting cancer progression. It is well known that stromal microenvironment contributes to disease progression through the establishment of a bi-directional crosstalk with cancer cells. In the bone marrow (BM), stromal cells are able to sustain the growth and survival of leukemic cells by protecting malignant cells from chemotherapy- induced death; on the other hand, leukaemia cells induce changes in the bone marrow stroma composition. Our hypothesis is that CML exosomes could have a functional role in this crosstalk. We demonstrate that treatment of BM-derived-HS5 cells with LAMA84-released Exo induced a significant increase of Interleukin 8 (IL8), as well as an augmented LAMA84 cell adhesion to stromal monolayer and LAMA84 migration towards HS5 conditioned medium. To better investigate the possible role of IL8 in the modulation of leukemia phenotype, we treated CML cells with recombinant IL8 (rIL8). Addition of rIL8 to LAMA84 cells increases the adhesion of leukemic cells to stromal cells and triggers survival pathways, as demonstrated by colony formation assay in methocult and activation of signal transduction pathways by western blot. Inhibition of IL8 receptors, CXCR1 and CXCR2, with SB225002 on LAMA84 cells reverts the effects described previously, confirming a role of IL8 in this crosstalk. In conclusions our data show that LAMA84-derived Exo modulate bone marrow microenvironment, increasing the production of the IL8 by stromal cells; moreover IL8 is able to affect leukemia cell proliferation and survival in a paracrine fashion

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