Study of novel natural compounds as potential drugs in the treatment of acute leukemias

Introduction: Among all cancer types leukemia represents the leading cause of cancer death in man younger than 40 years. Single-target drug therapy has generally been highly ineffective in treating complex diseases such as cancer. A growing interest has been directed toward multi-target drugs able to hit multiple targets. In this context, plant products, based on their intrinsic complexity, could represent an interesting and promising approach. Aim of the research followed during my PhD was to indentify and study novel natural compounds for the treatment of acute leukemias. Two potential multi-target drugs were identified in Hemidesmus indicus and piperlongumine. Methodology/Principal Findings: A variety of cellular assays and flow cytometry were performed on different cell lines. We demonstrated that Hemidesmus modulates many components of intracellular signaling pathways involved in cell viability and proliferation and alters gene and protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential, raise of [Ca2+]i, inhibition of Mcl-1, increasing Bax/Bcl-2 ratio, and ROS formation. Moreover, we proved that the decoction causes differentiation of HL-60 and regulates angiogenesis of HUVECs in hypoxia and normoxia, by the inhibition of new vessel formation and the processes of migration/invasion. Clinically relevant observations are that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemia (AML) patients. Moreover, both Hemidesmus and piperlongumine showed a selective action toward leukemic stem cell (LSC). Conclusions: Our results indicate the molecular basis of the anti-leukemic effects of Hemidesmus indicus and indentify the mitochondrial pathways, [Ca2+]i, cytodifferentiation and angiogenesis inhibition as crucial actors in its anticancer activity. The ability to selectively hit LSC showed by Hemidesmus and piperlongumine enriched the knowledge of their anti-leukemic activity. On these bases, we conclude that Hemidesmus and piperlongumine can represent a valuable strategy in the anticancer pharmacology.

Shared monocyte subset phenotypes in HIV-1 infection and in uninfected subjects with acute coronary syndrome.

The mechanisms responsible for increased cardiovascular risk associated with HIV-1 infection are incompletely defined. Using flow cytometry, in the present study, we examined activation phenotypes of monocyte subpopulations in patients with HIV-1 infection or acute coronary syndrome to find common cellular profiles. Nonclassic (CD14(+)CD16(++)) and intermediate (CD14(++)CD16(+)) monocytes are proportionally increased and express high levels of tissue factor and CD62P in HIV-1 infection. These proportions are related to viremia, T-cell activation, and plasma levels of IL-6. In vitro exposure of whole blood samples from uninfected control donors to lipopolysaccharide increased surface tissue factor expression on all monocyte subsets, but exposure to HIV-1 resulted in activation only of nonclassic monocytes. Remarkably, the profile of monocyte activation in uncontrolled HIV-1 disease mirrors that of acute coronary syndrome in uninfected persons. Therefore, drivers of immune activation and inflammation in HIV-1 disease may alter monocyte subpopulations and activation phenotype, contributing to a pro-atherothrombotic state that may drive cardiovascular risk in HIV-1 infection.

Rôle du gène de fusion MLL-ENL dans le développement et le maintien du phénotype leucémique

Les translocations chromosomiques du gène MLL sont connues pour mener au développement de leucémies aiguës. La translocation avec un de ses partenaires de fusion les plus communs, ENL, peut engendrer des leucémies aiguës de plusieurs types différents pour cette même translocation. Une fois la leucémogenèse initiée par la fusion MLL-ENL, son rôle quant au maintien du phénotype leucémique n’est pas encore bien connu à ce jour. Pour mieux comprendre l’importance de MLL-ENL après la leucémogenèse, des cellules souches/progénitrices de sang de cordon ombilical humain purifiées ont ainsi été transduites par un virus exprimant le gène de fusion MLL-ENL bordé par des sites LoxP ainsi que le marqueur eGFP. Ces cellules infectées ont ensuite été injectées dans notre modèle de souris immunodéficientes irradiées et placées sous observation pendant 24 semaines pour voir le développement de leucémies aiguës. Elles ont alors été sacrifiées et les cellules la moelle osseuse et de la rate ont ensuite été analysées par cytométrie en flux pour déterminer si la xénogreffe a engendré une leucémie dans notre modèle ainsi que le phénotype de celle-ci. Les souris injectées par les cellules infectées par le MLL-ENL ont généré uniquement des leucémies lymphoïdes aiguës de type B. Les cellules de ces leucémies primaires isolées ont été par la suite infectées par un lentivirus exprimant la cre-recombinase et le marqueur BFP afin d’exciser le gène MLL-ENL des cellules leucémiques grâce aux sites LoxP. Les cellules ont ensuite été triées pour le marqueur BFP et injectées dans des souris secondaires pour de voir si les cellules leucémiques souches pouvaient toujours régénérer la leucémie. Les conséquences de l’absence de MLL-ENL dans le maintien du phénotype leucémique n’ont cependant pas pu être vérifiées à cause d’une erreur dans la séquence de la cre-recombinase, mais nous avons observé la régénération des leucémies secondaires.

An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis. (C) 2010 Elsevier Ltd. All rights reserved.

Invasive Aspergillus flavus sinusitis: case report in a patient with biphenotypic acute leukemia

Here we report a case of invasive pansinusitis with proptosis of the right eye caused by Aspergillus flavus in an immunocompromised patient with acute biphenotypic leukemia without aggressive therapy response.

An in vitro iron superoxide dismutase inhibitor decreases the parasitemia levels of Trypanosoma cruzi in BALB/c mouse model during acute phase.

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), two hydroxyphthalazine derivative compounds were prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels, by (1)H NMR and TEM studies. Finally, as these compounds are potentially capable of causing oxidative damage in the parasites, the study was completed, by assessing their activity as potential iron superoxide dismutase (Fe-SOD) inhibitors. High-selectivity indices observed in vitro were the basis of promoting one of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Compound 2 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression. Compound 2 turned out to be a great inhibitor of Fe-SOD. The high antiparasitic activity and low toxicity together with the modest costs for the starting materials render this compound an appropriate molecule for the development of an affordable anti-Chagas agent.

Cytotoxic T cells deficient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease.

Graft-versus-host disease (GVHD) is the main complication after allogeneic bone marrow transplantation. Although the tissue damage and subsequent patient mortality are clearly dependent on T lymphocytes present in the grafted inoculum, the lethal effector molecules are unknown. Here, we show that acute lethal GVHD, induced by the transfer of splenocytes from C57BL/6 mice into sensitive BALB/c recipients, is dependent on both perforin and Fas ligand (FasL)-mediated lytic pathways. When spleen cells from mutant mice lacking both effector molecules were transferred to sublethally irradiated allogeneic recipients, mice survived. Delayed mortality was observed with grafted cells deficient in only one lytic mediator. In contrast, protection from lethal acute GVHD in resistant mice was exclusively perforin dependent. Perforin-FasL-deficient T cells failed to lyse most target cells in vitro. However, they still efficiently killed tumor necrosis factor alpha-sensitive fibroblasts, demonstrating that cytotoxic T cells possess a third lytic pathway.

Hierarchal utilization of different T-cell receptor Vbeta gene segments in the CD8(+)-T-cell response to an immunodominant Moloney leukemia virus-encoded epitope in vivo.

The CD8(+)-T-cell response to Moloney murine leukemia virus (M-MuLV)-associated antigens in C57BL/6 mice is directed against an immunodominant gag-encoded epitope (CCLCLTVFL) presented in the context of H-2D(b) and is restricted primarily to cytotoxic T lymphocytes (CTL) expressing the Valpha3.2 and Vbeta5.2 gene segments. We decided to examine the M-MuLV response in congenic C57BL/6 Vbeta(a) mice which are unable to express the dominant Valpha3.2(+) Vbeta5.2(+) T-cell receptor (TCR) due to a large deletion at the TCR locus that includes the Vbeta5.2 gene segment. Interestingly, M-MuLV-immune C57BL/6 Vbeta(a) mice were still able to reject M-MuLV-infected tumor cells and direct ex vivo analysis of peripheral blood lymphocytes from these immune mice revealed a dramatic increase in CD8(+) cells utilizing the same Valpha3.2 gene segment in association with two different Vbeta segments (Vbeta3 and Vbeta17). Surprisingly, all these CTL recognized the same immunodominant M-MuLV gag epitope. Analysis of the TCR repertoire of individual M-MuLV-immune (C57BL/6 x C57BL/6 Vbeta(a))F(1) mice revealed a clear hierarchy in Vbeta utilization, with a preferential usage of the Vbeta17 gene segment, whereas Vbeta3 and especially Vbeta5.2 were used to much lesser extents. Sequencing of TCRalpha- and -beta-chain junctional regions of CTL clones specific for the M-MuLV gag epitope revealed a diverse repertoire of TCRbeta chains in Vbeta(a) mice and a highly restricted TCRbeta-chain repertoire in Vbeta(b) mice, whereas TCRalpha-chain sequences were highly conserved in both cases. Collectively, our data indicate that the H-2D(b)-restricted M-MuLV gag epitope can be recognized in a hierarchal fashion by different Vbeta domains and that the degree of beta-chain diversity varies according to Vbeta utilization.

Targeting PI3KC2β Impairs Proliferation and Survival in Acute Leukemia, Brain Tumours and Neuroendocrine Tumours.

BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2β isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2β was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2β or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2β also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2β contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.

Monoclonal antibody against a "Pan-T-cell" antigen expressed by thymocytes, peripheral T-lymphocytes and T-cell leukemias.

A monoclonal antibody, LAU-A1, which selectively reacts with all cells of the T-lineage, was derived from a fusion between spleen cells of a mouse immunized with paediatric thymocytes and mouse myeloma P X 63/Ag8 cells. As shown by an antibody-binding radioimmunoassay and analysis by flow microfluorometry of cells labelled by indirect immunofluorescence, the LAU-A1 antibody reacted with all six T-cell lines but not with any of the B-cell lines or myeloid cell lines tested from a panel of 17 human hematopoietic cell lines. The LAU-A1 antibody was also shown to react with the majority of thymocytes and E-rosette-enriched peripheral blood lymphocytes. Among the malignant cell populations tested, the blasts from all 20 patients with acute T-cell lymphoblastic leukemia (T-ALL) were found to react with the LAU-A1 antibody, whereas blasts from 85 patients with common ALL and 63 patients with acute myeloid leukemias were entirely negative. Examination of frozen tissue sections from fetal and adult thymuses stained by an indirect immunoperoxidase method revealed that cells expressing the LAU-A1 antigen were localized in both the cortex and the medulla. From the very broad reactivity spectrum of LAU-A1 antibody, we conclude that this antibody is directed against a T-cell antigen expressed throughout the T-cell differentiation lineage. SDS-PAGE analysis of immunoprecipitates formed by LAU-A1 antibody with detergent lysates of radiolabeled T-cells showed that the LAU-A1 antigen had an apparent mol. wt of 76,000 under non-reducing conditions. Under reducing conditions a single band with an apparent mol. wt of 40,000 was observed. Two-dimensional SDS-PAGE analysis confirmed that the 76,000 mol. wt component consisted of an S-S-linked dimeric complex. The surface membrane expression of LAU-A1 antigen on HSB-2 T-cells was modulated when these cells were cultured in the presence of LAU-A1 antibody. Re-expression of LAU-A1 antigen occurred within 24 hr after transfer of the modulated cells into antibody-free medium.

Immunological effects of donor lymphocyte infusion in patients with chronic myelogenous leukemia relapsing after bone marrow transplantation

Allogeneic bone marrow transplantation (alloBMT) is the only curative therapy for chronic myelogenous leukemia (CML). This success is explained by the delivery of high doses of antineoplastic agents followed by the rescue of marrow function and the induction of graft-versus-leukemia reaction mediated by allogeneic lymphocytes against host tumor cells. This reaction can also be induced by donor lymphocyte infusion (DLI) producing remission in most patients with CML who relapse after alloBMT. The immunological mechanisms involved in DLI therapy are poorly understood. We studied five CML patients in the chronic phase, who received DLI after relapsing from an HLA-identical BMT. Using flow cytometry we evaluated cellular activation and apoptosis, NK cytotoxicity, lymphocytes producing cytokines (IL-2, IL-4 and IFN-gamma), and unstimulated (in vivo) lymphocyte proliferation. In three CML patients who achieved hematological and/or cytogenetic remission after DLI we observed an increase of the percent of activation markers on T and NK cells (CD3/DR, CD3/CD25 and CD56/DR), of lymphocytes producing IL-2 and IFN-gamma, of NK activity, and of in vivo lymphocyte proliferation. These changes were not observed consistently in two of the five patients who did not achieve complete remission with DLI. The percent of apoptotic markers (Fas, FasL and Bcl-2) on lymphocytes and CD34-positive cells did not change after DLI throughout the different study periods. Taken together, these preliminary results suggest that the therapeutic effect of DLI in the chronic phase of CML is mediated by classic cytotoxic and proliferative events involving T and NK cells but not by the Fas pathway of apoptosis.

Asynchronous expression of myeloid antigens in leukemic cells in a PML/RARalpha transgenic mouse model

Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor a (RARa) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARa fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARa develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARa TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 ± 16.68, 10.83 ± 8.11, 7.4 ± 5.46%, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARa TM present a specific immunophenotype.

Mesenchymal stem cells from patients with chronic myeloid leukemia do not express BCR-ABL and have absence of chimerism after allogeneic bone marrow transplant

Bone marrow is a heterogeneous cell population which includes hematopoietic and mesenchymal progenitor cells. Dysregulated hematopoiesis occurs in chronic myelogenous leukemia (CML), being caused at least in part by abnormalities in the hematopoietic progenitors. However, the role of mesenchymal stem cells (MSCs) in CML has not been well characterized. The objectives of the present study were to observe the biological characteristics of MSCs from CML patients and to determine if MSCs originate in part from donors in CML patients after bone marrow transplantation (BMT). We analyzed MSCs from 5 untreated patients and from 3 CML patients after sex-mismatched allogeneic BMT. Flow cytometry analysis revealed the typical MSC phenotype and in vitro assays showed ability to differentiate into adipocytes and osteoblasts. Moreover, although some RT-PCR data were contradictory, combined fluorescence in situ hybridization analysis showed that MSCs from CML patients do not express the bcr-abl gene. Regarding MSCs of donor origin, although it is possible to detect Y target sequence by nested PCR, the low frequency (0.14 and 0.34%) of XY cells in 2 MSC CML patients by fluorescence in situ hybridization analysis suggests the presence of contaminant hematopoietic cells and the absence of host-derived MSCs in CML patients. Therefore, we conclude that MSCs from CML patients express the typical MSC phenotype, can differentiate into osteogenic and adipogenic lineages and do not express the bcr-abl gene. MSCs cannot be found in recipients 12 to 20 months after BMT. The influence of MSCs on the dysregulation of hematopoiesis in CML patients deserves further investigation.