Maja Matulić

Molecular mechanisms involved in the antiproliferative action of protein tyrosine phosphatase inhibitor potassium bisperoxo(1,10-phenanthroline)oxovanadate

Potassium bisperoxo(1,10-phenanthroline)oxovanadate, bpV(phen), a powerful protein phosphotyrosine phosphatase inhibitor and a potent insulinomimetic, influenced three fundamental cellular processes in HL-60 human leukemic cells: 1) inhibition of proliferation, 2) induction of differentiation and 3) apoptotic cell death. In the presence of micromolar concentrations of bpV(phen) cell number and DNA synthesis decreased progressively with time of incubation. A single treatment with bpV(phen) (3 microM) activated a differentiation program; after 6 days of incubation 82% of cells were differentiated, but differentiation started already within the first 24 h. Concentrations of 5-10 microM bpV(phen) caused the characteristic DNA ladder pattern, starting after 4.5 h. Differentiation in HL-60 cells appear to be associated with activation of extracellular signal-regulated kinase while apoptosis is connected with phosphorylation and activation of both extracellular signal-regulated kinase and c-Jun N-terminal kinase in a concentration and time-dependent manner. The antiproliferative and apoptotic action of bpV(phen) could be exploited in combination chemotherapy in leukemia.

Ikaros family transcription factors in chronic and acute leukemia

lkaros, Aiolos, and Helios are transcription factors important in lymphocyte differentiation and development [1-6]. One of the most interesting aspects of the Ikaros family members is that they are expressed as multiple splice variants and proteins arising from alternatively spliced transcripts may act as dominant negatives. Although expression of these short isoforms has been linked to lymphoid and myeloid leukemia [7-11] the comparison of Ikaros members mRNA expression level has not been reported. Here we show, by means of real time qRT-PCR method, a quantitative distribution of Ikaros, Aiolos, and Helios mRNA in hemopoietic cells of patients with lymphocytic leukemia, represented by the most frequent B cell chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (T cell ALL and common ALL), and compared with the acute leukemia of the myeloid lineage, acute myeloid leukemia (AML). We found that the relative quantity of Ikaros and Helios mRNA was similar in all subgroups. However, analysis of Aiolos showed a clear difference among groups because of its lower expression in all types of acute leukemia investigated. Our results associating a lower Aiolos expression with acute leukemia point to Aioloss role in human lymphocyte development, as previously shown in mice.

Sodium Salicylate Inhibits Urokinase Activity in MDA MB-231 Breast Cancer Cells

Micro‐Abstract We investigated the effect of the anti‐inflammatory drug, sodium salicylate, on urokinase plasminogen activator, a protease involved in metastasis and tumor invasion. Our results revealed concomitant urokinase activity inhibition and upregulation of its inhibitors, as well as cell‐specific activation of the transforming growth factor‐&bgr; pathway in triple‐negative breast cancer cells. Sodium salicylate‐induced downstream effectors of the transforming growth factor‐&bgr; pathway could regulate the processes of breast cancer progression. Introduction: Sodium salicylate (NaS) is a derivate of acetylsalicylic acid or aspirin, used as a nonsteroidal anti‐inflammatory drug for centuries, for its analgesic and anti‐inflammatory effects. It was found to modulate different signaling pathways, in a cell‐specific way. Here, we explore the effect of NaS on cell growth and urokinase activity in MDA MB‐231 breast cancer cells. Materials and Methods: We analyzed the effect of NaS treatment on cell growth by flow cytometry and viability test. The transwell migration assay was used to study the migratory response of the cells. The gene expression was analyzed by qRT‐PCR on RNA level and by Western blot analysis on protein level. Urokinase activity was assessed by caseinolysis. Results: Sublethal concentrations of NaS decreased cell growth and inhibited urokinase activity. The latter was a consequence of decrease in urokinase expression and increase in expression of its inhibitors. Analysis of signaling molecules revealed activation of transforming growth factor‐&bgr; signaling, increase in master transcription factors for epithelial‐mesenchymal transition and changes in integrin expression. Conclusions: We propose that NaS causes partial cellular reprogramming through transforming growth factor‐&bgr; signaling which, together with direct NaS influence, causes changes in expression in a set of genes involved in extracellular proteolysis. These data could be beneficial for the development of new therapeutic approaches in invasive breast cancer treatment.

Evaluation of the cytotoxic activity of Hypericum spp. on human glioblastoma A1235 and breast cancer MDA MB-231 cells

ABSTRACT Cytotoxic activity of 16 Hypericum ethanolic extracts was evaluated by MTT assay on two human cancer cell lines: glioblastoma A1235 and breast cancer MDA MB-231. Morphology and the type of induced cell death were determined using light and fluorescence microscopy. The majority of Hypericum extracts had no significant cytotoxic effect on MDA MB-231 cells. Eight extracts exhibited mild cytotoxic effect on A1235 cells after 24 h incubation, ranging from 8.0% (H. patulum) to 21.7% (H. oblongifolium). After 72 h of treatment, the strongest inhibition of A1235 viability was observed for extracts of H. androsaemum (26.4–43.9%), H. balearicum (25.8–36.3%), H. delphicum (14.8–27.4%) and H. densiflorum (11.2–24.1%). Micro-scopic examination of cells showed apoptosis as the dominant type of cell death. Due to observed high viability of treated cells, we propose that cytotoxic effects of Hypericum extracts could be related to alternations/interruptions in the cell cycle.

Modulation of urokinase plasminogen activator system by poly(ADP-ribose)polymerase-1 inhibition

The urokinase plasminogen activator (uPA) system is a complex regulator of extracellular proteolysis which is involved in various physiological and pathological processes. The major components of this system are the serine protease uPA, two inhibitors PAI-1 and PAI-2, and the receptor uPAR. It has been previously shown by several groups that the uPA system has an important role in cancer progression and therefore its possible prognostic and therapeutic value has been evaluated. The aim of this study is to tackle the role of poly(ADP-ribosyl)ation in the induction of uPA activity in a glioblastoma cell line, A1235. This cell line is sensitive to alkylation damage and is a model for drug treatment. The components of the uPA system and the level of DNA damage were analyzed after alkylation agent treatment in combination with poly(ADP-ribose)polymerase-1 (PARP-1) inhibition. Here we show that the increase in uPA activity results from the net balance change between uPA and its inhibitor at mRNA level. Further, PARP-1 inhibition exerts its influence on uPA activity through DNA damage increase. Involvement of several signaling pathways, as well as cell specific regulation influencing the uPA system are discussed.

Notch pathway connections in primary leukaemia samples of limited size

BackgroundThe Notch pathway combined with other signalling molecules acts specifically for the development of each blood cell type and differentiation stage. A causative role of Notch dysfunction in leukaemia development has been found in many studies so, initially only for T- acute lymphoblastic leukaemia (T-ALL) but more recently also for B cell and myeloid leukaemia. The aim of our study is to introduce a method for multiple direct analysis of the Notch pathway partners in a population of only 500 or fewer cells. The notion of this method consists in gaining insight into gene expression at the level of the malignant clone population. A small number of cells is a significant limitation when working on primary cells either when freshly isolated or when analysed after several days in cocultures.MethodsThe primers were designed to avoid genomic amplification through the selection of 3′ and 5′ primers that hybridise with different exons. Cell lines and primary cells were collected and multiplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) performed on a descending number of cells, ranging from 2,500 cells up to 50 cells per sample, for the Notch pathway genes and other transcription factors important for cell differentiation. ImageJ program, STATISTICA 13.1 software package and Student’s t-test were used for statistical evaluation. We checked protein expression by western blot.ResultsWe characterised the gene expression levels of Notch, Ikaros and Parp genes in leukaemia cell lines of B and T origin and in primary leukaemia samples of limited size. We further compared our results to the cDNA analysis obtained by total RNA isolation from a large number of cells as routinely performed in clinical laboratories, and finally tested the method described on primary cells from leukaemia patients.ConclusionsThis rapid multiple gene expression analysis of a small population of cells provides efficient cell classification determining malignant changes as an important additional information for clinical leukaemia diagnostics as well as for in vitro studies of primary cells.

Effect of Notch and PARP Pathways’ Inhibition in Leukemic Cells

Differentiation of blood cells is one of the most complex processes in the body. It is regulated by the action of transcription factors in time and space which creates a specific signaling network. In the hematopoietic signaling system, Notch is one of the main regulators of lymphocyte development. The aim of this study was to get insight into the regulation of Notch signalization and the influence of poly(ADP-ribose)polymerase (PARP) activity on this process in three leukemia cell lines obtained from B and T cells. PARP1 is an enzyme involved in posttranslational protein modification and chromatin structure changes. B and T leukemia cells were treated with Notch and PARP inhibitors, alone or in combination, for a prolonged period. The cells did not show cell proliferation arrest or apoptosis. Analysis of gene and protein expression set involved in Notch and PARP pathways revealed increase in JAGGED1 expression after PARP1 inhibition in B cell lines and changes in Ikaros family members in both B and T cell lines after γ-secretase inhibition. These data indicate that Notch and PARP inhibition, although not inducing differentiation in leukemia cells, induce changes in signaling circuits and chromatin modelling factors.

Notch affects the prodifferentiating effect of retinoic acid and PMA on leukemic cells.

Notch proteins determine cell fate decisions in the development of diverse tissues. Notch has been initially found in T‐ALL but its role has been also studied in myelopoiesis and myeloid leukemias. Studies in different model systems have led to a widespread controversy as to whether Notch promotes or blocks myeloid differentiation. In this work, we evaluated the influence of Notch activation on leukemic cell differentiation along the monocytic and myelocytic pathway induced by phorbol 12‐myristate 13‐acetate (PMA) or all‐trans retinoic acid (ATRA). We observed that differentiation of the human myeloblastic cell line HL‐60 can be retarded or blocked by Delta/Notch interaction. ATRA induces complete remission in patients with acute promyelocytic leukemia, but it cannot completely eliminate the leukemic clone and to be effective it should be combined with chemotherapy. Our findings suggest that Notch signaling may contribute to the incomplete elimination of the leukemic cells after PMA or ATRA treatment and the blockage of Notch pathway may be beneficial in the treatment of myeloid leukemia.