CYTOTOXIC ACTIVITY OF KAEMPFEROL GLYCOSIDES AGAINST HUMAN LEUKAEMIC CELL LINES IN VITRO.

Two kaempferol coumaroyl glycosides (i.e. platanoside and tiliroside) isolated from the methanolic extract of Platanus orientalis L. buds, were examined for their in vitro cytotoxic activity against a panel of human leukaemic cell lines. Platanoside (1) exhibited cytotoxic activity against most of the cell lines tested, while tiliroside (2) was active against two of the nine tested cell lines. Compound 1, was examined for its effect on the uptake of [(3)H]thymidine as a marker of DNA synthesis. Kaempferol was used as a control. 2000 Academic Press@p$hr Copyright 2000 Academic Press.

Cytotoxic activity of kaempferol glycosides against human leukaemic cell lines in vitro.

Two kaempferol coumaroyl glycosides (i.e. platanoside and tiliroside) isolated from the methanolic extract of Platanus orientalis L. buds, were examined for their in vitro cytotoxic activity against a panel of human leukaemic cell lines. Platanoside (1) exhibited cytotoxic activity against most of the cell lines tested, while tiliroside (2) was active against two of the nine tested cell lines. Compound 1, was examined for its effect on the uptake of [(3)H]thymidine as a marker of DNA synthesis. Kaempferol was used as a control.

Cytotoxic and antiproliferative effects of heptaacetyltiliroside on human leukemic cell lines.

The peracetylated derivative of kaempferol-3-O-beta-D-(6''-E-p-coumaroyl) glycopyranoside (tiliroside) (1a) was tested for its cytotoxic and cytostatic activity against several human leukemic cell lines. The significant cytotoxic activity of this derivative, prompted to an additional examination on some of the cell lines used. The effect on the uptake of [3H]thymidine as a marker of DNA synthesis and on the cell proliferation, was investigated as well as the morphology of the cells and the kind of death induced, using the Wright-Giemsa dye and horizontal agarose-gel electrophoresis. Flow cytometric experiments of 1a on some leukemic cell lines was also performed. Compound 1a showed a significant antiproliferative effect as soon as 1 h of continuous incubation at all cell lines tested. Cells were killed, through the process of apoptosis and the appearance of the apoptotic signs was time and dose-dependent, while from the flow cytometric experiments, a synchronisation (through a delay probably in the G(0/1) phase) of the cells seems to take place.

The effect of sclareol on growth and cell cycle progression of human leukemic cell lines.

Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 microg/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 microg/ml the compound showed a significant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using flow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the flow cytometry experiments, a delay of the cell population on G0/1 seems to take place. This is the first report, that a labdane type diterpene kills tumor cells via a phase specific mechanism which induces apoptosis.

Cytotoxic activity and antiproliferative effects of a new semi-synthetic derivative of Ent-3 beta-hydroxy-13-epi-manoyl oxide on human leukemic cell lines.

Ent-3 beta-hydroxy-13-epi-manoyl oxide (1), was converted to its thiomidazolide derivative (2) which was tested for its cytotoxic activity against a panel of established human leukemic cell lines. Compound 2, exhibited cytotoxic activity against 13 of the cell lines tested. Additionally, compound 2 was examined for its effect on the uptake of [3H]-thymidine as a marker of DNA synthesis and on cell proliferation. The morphology of the cells and the kind of death induced, was investigated. Flow cytometry experiments on a leukemic cell line was also performed. The results show that the semi-synthetic compound, showed a significant antiproliferative effect and kills cells through the process of apoptosis. The appearance of the apoptotic signs was time and dose dependent. From the flow cytometry experiments, a synchronisation through a delay of the cells in G0/1, phase seems to take place.

Cytotoxic activity of labdane type diterpenes against human leukemic cell lines in vitro.

Nine labdane type diterpenes isolated from the plant Cistus creticus subsp. creticus and from the resin "Ladano" which is excreted on the surface of the leaves and stems of this plant, were examined for their in vitro cytotoxic activity against 14 human leukemic cell lines. Compound 1, (13E)-labd-13-ene-8 alpha,15-diol, exhibited cytotoxic activity against 13 of the cell lines tested, while compound 2, (13E)-labd-7,13-dienol, was active only against HL60 cells. Further compound 1 was examined for its effect on the uptake of [3H]-thymidine as a marker of DNA synthesis.

Biotransformation of the flavonoid tiliroside to 7-methylether tiliroside: bioactivity of this metabolite and of its acetylated derivative.

Incubation of kaempferol-3-O-beta-D-(6"-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) with Aspergillus nidulans gives the 7-methyl ether of tiliroside (2) which is a new compound. Its structure is determined by spectroscopic methods. Cytotoxic studies of 2 and of its acetylated derivative 2a were carried out in vitro against fourteen human leukemic cell lines. Results clearly show that compound 2 is ineffective against all leukemic cell lines tested. On the contrary, compound 2a exhibited cytotoxic activity against four of the cell lines (HL60, DAUDI, HUT78 and MOLT3) and additionally, a dose- and time-dependent effect on DNA synthesis.

delta IK17: an antigen expressed on human lymphocytes.

Anti delta IK17 monoclonal antibody was produced by fusing SP2/0/Ag 14 myeloma with spleen cells of BALB/c mice immunized with normal human thymocytes. a delta IK17 antibody recognizes a 44kD cell surface protein detected on human lymphocytes. delta IK17 is expressed on human thymocytes, CD4+ and CD8+ T cell subsets, B, NK cells, as well as on activated cells. The antigen is detected on cells during the early, intermediate and late stages of lymphocyte maturation. In addition, the expression of the antigen is correlated with ontogenesis. A T+ delta IK17+ subpopulation responded poorly to TPA stimulation and provided a better helper signal for PWM-induced IgM synthesis than T+ delta IK17- cells. In addition, different levels of delta IK17 expression were detected in several hematological diseases tested.

delta IK17 antigen: a possible early marker of cancer development.

delta IK17 is a 44 kD molecule located on the surface of T, B and NK cells in normal peripheral blood mononuclear cells (PBMC) (1). The portion of PBMC expressing delta IK17 was determined in 52 patients with benign breast diseases, 182 patients with breast malignancies and 132 healthy individuals. The percentage of delta IK17-positive cells was significantly lower in the early stages (I-IIA) of malignancy compared to that of healthy donors. However, the percentage of PBMC expressing delta IK17 tended to increase as the stage of the disease advanced. delta IK17 seems to be the only antigen among the other cellular markers tested (CD2, CD4, CD8, HLA-DR) with a statistically significant correlation between a low percentage of positive cells and the early stages of malignancy and between a high expression and advanced disease. Its potential use as a tumor marker in breast cancer is discussed here.

Bioactive compounds from the buds of Platanus orientalis and isolation of a new kaempferol glycoside.

A new compound kaempferol 3-O-alpha-L-(2"-E-p-coumaroyl)-rhamnopyranoside, as well as the known flavonoids, kaempferol 3-O-beta-D-(6"-E-p-coumaroyl)-glucopyranoside, kaempferol 3-O-alpha-L-(2",3"-di-E-p-coumaroyl)-rhamnopyranoside, and caffeic acid were obtained from the methanolic extract of Platanus orientalis L. buds. All the compounds were isolated by column chromatography and identified using 1H-NMR, 2D-1H-NMR (COSY), 1H-13C-NMR, and CIDMS techniques. Cytotoxic and antimicrobial studies were carried out in vitro against human cell lines and against Gram-positive and Gram-negative organisms.

The use of nylon wool for the isolation of T lymphocyte subpopulations.

A simple method for the affinity isolation of lymphocyte subpopulations using nylon wool as a solid phase matrix is described. This matrix was coated with an acid-treated IgG fraction of polyclonal antibodies against mouse immunoglobulins. Using this approach, a simple isolation procedure for lymphocyte subpopulations, yielding purities higher than 95% was developed. The method can be used on a large scale for positive and negative cell selection, with very little non-specific binding. The isolated populations are fully functional.

A simple hemagglutination method for the detection of cell surface antigens.

A rapid, easy and reproducible hemagglutination method is described for the detection of cell surface antigens. This method can be used for the determination of cell subpopulations and the screening of monoclonal antibody-secreting hybridomas. Fresh or paraformaldehyde-fixed cells, which have previously been labeled with an appropriate monoclonal antibody (of known or unknown specificity), are reacted with human erythrocytes coupled with the second antibody (rabbit anti-mouse immunoglobulins), in a round bottomed 96-well microtiter plate. The presence of specific agglutination and the end-point of serial dilutions of the labeled cells with unlabeled ones give an approximate estimation of the percentage of cells reacting with the monoclonal antibody under examination. The hemagglutination results correlate well with the results obtained using a conventional immunofluorescence method.

Patterns of adenosine deaminase, ecto-5'-nucleotidase, poly(A)polymerase and surface light chain expression in chronic lymphocytic leukemias.

The levels of activity of three enzymes have been measured in the circulating malignant lymphocytes of 47 patients with B chronic lymphocytic leukemia (CLL). These were the purine degradative enzymes, adenosine deaminase (ADA) and ecto-5'-nucleotidase (5'NT) and the enzyme responsible for the polyadenylation of mRNA, poly(A) polymerase. The patterns of activity of the above enzymes and the expression of surface immunoglobulin light chains were examined. A heterogeneity in the specific activity of the enzymes was observed which could not be attributed to variations of the percentage of B lymphocytes. A positive correlation was found between ADA and poly(A)polymerase activity (r = 0.383, p less than 0.01). Furthermore, the expression of immunoglobulin light chain phenotype was inversely related to 5'NT specific activity; CLL cases in which less than 20% of the cells expressed lambda chain phenotype, presented 5'NT specific activity of 16.7 +/- 3.3 (S.E.) nmol/h/10(6) cells, whereas in CLL cases with more than 20% of the cells expressing this phenotype the enzyme specific activity was 4.8 +/- 1.6 (S.E.) nmol/h/10(6) cells (p less than 0.02). These findings suggest that the simultaneous determination of enzymatic activities and immunological markers, might be useful in defining subsets in CLL and the subsequent clinical treatment.

A colorimetric assay for the determination of 5'-nucleotidase activity.

A rapid, simple, quantitative and sensitive assay for the determination of 5'-nucleotidase has been developed. The method can be applied to both soluble and membrane bound forms of the leukocyte enzyme. Enzyme activity is determined by colorimetric estimation of NH3 released from adenosine, the product of 5'-nucleotidase activity in the presence of adenosine deaminase. The assay may be performed in microtitre plates and read with an automatic multiscan spectrophotometer. Thus it can be applied to a large number of samples for routine medical and research purposes.

Immunoregulatory effects of fraction 5 thymus peptides. I. Thymosin alpha 1 enhances while thymosin beta 4 suppresses the human autologous and allogeneic mixed lymphocyte reaction.

Thymosin alpha 1 and thymosin beta 4, two peptides isolated from preparations of calf thymus fraction 5, were tested in the human mixed lymphocyte reaction (MLR). Thymosin alpha 1 was found capable of enhancing both the allogeneic and autologous MLR. On the contrary, thymosin beta 4 suppressed MLR proliferative responses. Study of the responses of the T cell subpopulations revealed that T4+ (helper/inducer) cells but not T8+ (suppressor/cytotoxic) are responsible for the enhanced, proliferative response to allo- and autoantigens in the presence of thymosin alpha 1. Both the autologous and the allogeneic proliferative responses of either T4+ cells or T8+ cells were not influenced by the addition of thymosin beta 4 in the cultures. However, when T4+ and T8+ subsets were cocultured, thymosin beta 4 was capable of activating T8+ cells to suppress the allogeneic and the autologous proliferative response of T4+ cells. These studies show that thymosin fraction 5 peptides exert immunoregulatory effects on the human MLR proliferative responses in vitro.

Polyadenylic acid metabolizing enzyme levels during induction of differentiation in a human leukemia T-cell line with phorbol ester.

Induction of differentiation of MOLT3 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to affect the activity levels of the polyadenylic acid [poly(A)] metabolizing enzymes. TPA administration at a concentration of 16 nM resulted in an increase of poly(A) exonuclease and poly(A) polymerase activities after 2 and 2.5 hours, respectively, and in a decline to control levels thereafter. Cordycepin, an inhibitor of poly(A) polymerization, prevented the increasing binding of OKT11A monoclonal antibody induced by TPA treatment, whereas TPA-induced reduction in OKT6 monoclonal antibody binding persisted. The alterations in the poly(A) metabolizing enzyme activities following treatment of the cells with TPA, as well as the inhibitory effect of cordycepin, may suggest that these enzymes may be involved in the regulation of the differentiation process.

Effect of two thymosin fraction 5 polypeptides on human peripheral blood lymphocytes.

Thymosin fraction 5 polypeptides beta 4 and alpha 1 were tested for their ability to affect certain immunological parameters of human peripheral blood lymphocytes (PBL). PBL were cultured with various concentrations of the peptides for 24 hours. Thymosin beta 4 was found to induce a significant decrease in the expression of the Fc alpha receptors of PBL, as well as in their ability to express antibody dependent cellular cytotoxic (ADCC) activity. In addition, this peptide had the ability to increase the percentage of T4 lymphocytes in normal and immunosuppressed donors and to decrease the percentage of T8 positive cells in normal donors. Finally, beta 4 peptide caused a small increase in the capacity of peripheral blood lymphocytes to form sheep red blood cell (SRBC) rosettes (ER). In parallel experiments thymosin alpha 1 was found inactive. The results presented here indicate that thymosin beta 4 may be used as an immunoregulatory molecule in patients with immunodeficiencies.

Dexamethasone caused alterations in poly(A) polymerase levels of a human leukemic cell line.

The effect of glucocorticosteroids upon the poly(A) synthetic and degrading activity has been studied in steroid sensitive and resistant human leukemic cell lines. At least a two-fold increase in the levels of poly(A) polymerase activity was found in soluble, cytoplasmic extracts from the steroid sensitive human malignant T-cells of the MOLT3 line after 24-h treatment with dexamethasone. Longer exposure time of the cultured cells to the steroid resulted in a gradual decrease of the poly(A) polymerase activity level. Pretreatment of the cells with progesterone, a competitive inhibitor of dexamethasone, prevented the dexamethasone induced increase in the level of poly(A) polymerase activity, while progesterone alone had no effect on the enzyme level. In contrast, the levels of poly(A) nucleases activity remained constant following steroid treatment. In the steroid resistant human leukemic B-cell line Daudi no alterations in the poly(A) metabolizing enzymes could be measured in response to dexamethasone. Thus we suggest that poly(A) polymerase levels may be used to predict sensitivity of leukemic cells to glucocorticosteroid treatment.

Thymosin beta 4 induced phenotypic changes in Molt-4 leukemic cell line.

Thymosin beta 4 was tested for its ability to induce phenotypic changes in the human T-cell line Molt-4. Cells were cultured with nanogram concentrations of thymosin beta 4 for up to 16 days and were analyzed with a panel of monoclonal antibodies, sheep erythrocyte rosetting, peanut agglutinin binding (PNA) and an antibody to the enzyme, terminal deoxynucleotidyl transferase (TdT). Thymosin beta 4 induced Molt-4 cells to reduce the expression of a T-cell lineage specific antigen, with preferential expression on T blast-cells, detected by WT 1 monoclonal antibody. Thymosin beta 4 also induced an increase in sheep erythrocyte rosettes and PNA binding as well as an increased expression of OKT 11 A and OKT 8 in Molt-4 cells. TdT was found to be unchanged, however. Analysis of thymosin beta 4-treated cells with other monoclonal antibodies (OKT 3, OKT 6, OKT 9) showed no change when compared to controls. These results showed that thymosin beta 4 is capable of inducing phenotypic changes in Molt-4 cells. Such changes may represent a differentiation process of these cells through the early stages of the maturation process of thymus-dependent lymphocytes, albeit not to the stage of mature T cells.