Irene Herbacek

Multifactorial anticancer effects of digalloyl-resveratrol encompass apoptosis, cell-cycle arrest, and inhibition of lymphendothelial gap formation in vitro

Background:Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups.Methods:Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by 14C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers.Results:In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21Cip/Waf and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroid-secreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread.Conclusion:These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.

Daily Intake of Probiotic as well as Conventional Yogurt Has a Stimulating Effect on Cellular Immunity in Young Healthy Women

Background/Aims: The aim of this work was to study the effects of daily yogurt consumption on the cellular immunity of young healthy women and to compare a conventional with a probiotic product. Methods: 33 young healthy women (22–29 years) consumed 100 g/day of either probiotic or conventional commercially available yogurt for 2 weeks and 200 g/day for another 2 weeks followed by a 2-week washout period with no fermented food at all. Before the intervention and after each phase, a complete white blood count was done, the percentage of activated CD69+ T lymphocytes after stimulation of whole blood with pokeweed mitogen was determined as well as the natural cytotoxicity of peripheral blood mononuclear cells against a human erythroleukemic target cell line (K562). All analyses were done by flow cytometry. Results: In the probiotic group only, the numbers of cytotoxic T lymphocytes (CD3+CD16+CD56+) increased significantly (+30.8% with p = 0.001, +22.1 and +32.7% with p = 0.002, for T2, T3 and T4 compared to T1). There were no major changes for other cell populations, and all remained within the physiological range. In both groups, the expression of CD69 on T lymphocytes increased after yogurt consumption, especially on CD8+ (conventional: T2 +23%, T3 +27.2%, probiotic: T2 +15.7%; T3 +10.8% compared to T1) and to a lesser extent on CD4+ (conventional: T2 +7.7%, T3 +14.9%, probiotic: T2 +4% compared to T1. The cytotoxic activity also augmented following the intake, this effect persisting after cessation of consumption. However, there were no significant differences between the probiotic and the conventional yogurt group. Conclusion: Daily yogurt intake has a stimulating effect on cellular immune functions, but in this study the probiotic product did not perform better than the traditional one.

Hydrogen peroxide mediates EGCG-induced antioxidant protection in human keratinocytes

The beneficial health effects of (-)-epigallocatechin-3-gallate (EGCG), the main catechin of green tea, have been attributed to complex interactions with a focus on antioxidative properties. Susceptibility to autoxidation and production of cytotoxic reactive oxygen species (ROS), mostly H(2)O(2), have been suggested to occur in vitro but also in vivo. In this study, we address whether autoxidation-derived H(2)O(2) may be involved in the cytoprotective effects of EGCG. To that end we investigated keratinocyte-derived HaCat and HL-60 promyelocytic leukemia cells with significantly different sensitivities to H(2)O(2) (IC(50) 117.3 versus 58.3 μM, respectively) and EGCG (134.1 versus 84.1 μM). HaCat cells significantly resisted cytotoxicity and DNA damage based on enhanced H(2)O(2) clearance, improved DNA repair, and reduced intracellular ROS generation. Cumulative versus bolus EGCG and H(2)O(2) treatment and H(2)O(2) pretreatment before subsequent high-dose EGCG and vice versa significantly reduced DNA damage and cytotoxicity in HaCat cells only. Addition of catalase abolished the protective activities of low-dose H(2)O(2) and EGCG. In summary, our data suggest that autoxidative generation of low-dose H(2)O(2) is a significant player in the cell-type-specific cytoprotection mediated by EGCG and support the hypothesis that regular green tea consumption can contribute as a pro-oxidant to increased resistance against high-dose oxidative stressors.

Immunologic and Biochemical Effects of the Fermented Wheat Germ Extract Avemar

Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to have antitumor effects. Avemar has the potential to significantly improve the survival rate in patients suffering from malignant colon tumors. We studied its effects in the HT-29 human colon carcinoma cell line. Avemar had an inhibiting effect on colonies of HT-29 cells with an IC50 value of 118 μg/ml (7 days of incubation); this value could be decreased to 100 and 75 μg/ml in the presence of vitamin C. In the cell line examined, Avemar induced both necrosis and apoptosis, as demonstrated by Hoechst/propldium iodide staining. The incubation of cells with 3200 μg/ml Avemar for 24 hrs caused necrosis in 28% and the induction of apoptosis in 22% of the cells. Avemar inhibited the cell-cycle progression of HT-29 cells in the G1 phase of the cell cycle. In addition, Avemar inhibited the activity of the key enzyme of de novo DNA synthesis, ribonucleotide reductase. In addition, we determined the effects of Avemar on the activity of cyclooxygenase-1 and -2. Both enzymes were significantly inhibited by Avemar with IC50 values of 100 and 300 μg/ml, respectively. We outline new explanations for its antitumor activity, which might serve as the basis for further studies using Avemar.

Inducible expression of EVI1 in human myeloid cells causes phenotypes consistent with its role in myelodysplastic syndromes

The oncogene EVI1 has been implicated in the etiology of AML and MDS. Although AML cells are characterized by accelerated proliferation and differentiation arrest, MDS cells hyperproliferate when immature but fail to differentiate later and die instead. In agreement with its roles in AML and in immature MDS cells, EVI1 was found to stimulate cell proliferation and inhibit differentiation in several experimental systems. In contrast, the variant protein MDS1/EVI1 caused the opposite effect in some of these assays. In the present study, we expressed EVI1 and MDS1/EVI1 in a tetracycline‐regulable manner in the human myeloid cell line U937. Induction of either of these proteins caused cells to accumulate in the G0/G1‐phase of the cell cycle and moderately increased the rate of spontaneous apoptosis. However, when EVI1‐ or MDS1/EVI1‐expressing cells were induced to differentiate, they massively succumbed to apoptosis, as reflected by the accumulation of phosphatidylserine in the outer leaflet of the plasma membrane and increased rates of DNA fragmentation. In summary, these data show that inducible expression of EVI1 in U937 cells causes phenotypes that may be relevant for its role in MDS and provides a basis for further investigation of its contribution to this fatal disease.

Pro- and anticarcinogenic mechanisms of piceatannol are activated dose dependently in MCF-7 breast cancer cells

Estrogenic procarcinogenic effects of piceatannol (PIC) contrast reports about anticarcinogenic activities of PIC. To explain this contradiction, we investigated PIC in estrogen-dependent MCF-7 breast cancer cells and elucidated those cellular mechanisms that correlated with the observed cell effects induced by PIC. Low PIC concentrations (50 nM) induced c-Myc that depended on progesterone receptor (PR) and estrogen receptor (ER). PR-mediated c-Myc induction by PIC was independent of nuclear PR activity but depended on mitogen-activated protein kinase (MAPK) signaling and was associated with an acceleration of cancer cell proliferation. In contrast, 25 μM PIC inhibited deoxynucleotide triphosphate synthesis, activated Chk2 and p38-MAPK and this was accompanied by an attenuation of cancer cell growth. Apoptosis was most probably inhibited due to activation of Akt; however, high PIC concentrations (>100 μM) permitted apoptosis-like cell death in consequence to disruption of orchestrated mitotic signaling. The presented results show for the first time that nanomolar PIC concentrations signal through PR and Erk1/2 and provide a mechanistic explanation why moderate wine consumption-but not other alcoholic beverages-increases the breast cancer risk in women. In contrast, higher PIC concentrations in the micromolar range are considered for adjuvant anticancer therapeutic concepts.

EVI1 inhibits apoptosis induced by antileukemic drugs via upregulation of CDKN1A/p21/WAF in human myeloid cells.

Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec). Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs.

Overexpression of Hsp27 in a human melanoma cell line: regulation of E-cadherin, MUC18/MCAM, and plasminogen activator (PA) system

Abstract Hsp27 is considered a potential marker for cell differentiation in diverse tissues. Several aspects linked to the differentiation process and to the transition from high to low metastatic potential were analyzed in melanoma cells transfected with Hsp27. E-cadherin plays a central role in cell differentiation, migration, and normal development. Loss of expression or function of E-cadherin has been documented in a variety of human malignancies. We observed by fluorescence-activated cell sorter (FACS) as well as immunofluorescence (IF) analysis a pronounced expression of E-cadherin in Hsp27-transfected A375 melanoma cells compared with control melanoma cells. The expression of the adhesion molecule MUC18/MCAM correlates directly with the metastatic potential of melanoma cells. In contrast to wild-type and neotransfected melanoma cells, in Hsp27-transfected cells the expression of MUC18/MCAM could not be detected by FACS and IF analysis. The plasminogen activator (PA) system plays a central role in mediating extracellular proteolysis and also in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. Hsp27 transfectants revealed elevated messenger ribonucleic acid expression of the urokinase-type PA (uPA) and its inhibitor, PA inhibitor type 1, which might indicate a neutralization effect of the proteolytic activity of uPA. Control cells failed to express both these molecules. The influence of Hsp27 expression on uPA activity and the involvement of E-cadherin could be demonstrated by use of anti–E-cadherin–blocking antibody. Our data provide evidence for an inhibitory-regulatory role of Hsp27 in tumor progression as found in our system.

A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells.

Baculoviruses are widely used for protein production in insect cells, and their potential for gene transfer to mammalian cells is increasingly being recognized. Here we describe a baculovirus vector with a bicistronic mammalian expression cassette and demonstrate its suitability for efficient transient and stable protein expression in human glioblastoma cells. Bicistronic baculovirus vectors are safe, cost efficient, and easy to produce; thus, they represent an excellent gene transfer system for mammalian cells.

An apolar extract of Critonia morifolia inhibits c-Myc, cyclin D1, Cdc25A, Cdc25B, Cdc25C and Akt and induces apoptosis

Investigating the bioactivity of traditional medical remedies under the controlled conditions of a laboratory is an option to find additional applications, novel formulations or lead structures for the development of new drugs. The present work analysed the anti‑neoplastic activity of increasing polar extracts of the rainforest plant Critonia morifolia (Asteraceae) that has been successfully used as traditional remedy to treat various inflammatory conditions in the long-lasting medical tradition of the Central American Maya, which was here also confirmed in vitro. The apolar petroleum ether extract exhibited the most potent anti‑proliferative and pro‑apoptotic effects in HL‑60 cells and triggered down-regulation of Cdc25C and cyclin D1 within 30 min followed by the inhibition of c-Myc expression and the onset of caspase-3 activation within 2 h. Subsequent to these very rapid molecular responses Chk2 and H2AX became phosphorylated (γ‑H2AX) after 4 h. Analysis of the cell cycle distribution showed an accumulation of cells in the G2-M phase within 8 h and after 24 h in S-phase. This was temporally paralleled by the down-regulation of Cdc25A, Cdc25B, Wee1 and Akt. Therefore, the attenuation of cell cycle progression in the G2-M phase was consistent with the known role of Chk2 for G2-M arrest and with the role of Cdc25B in S-phase progression. These findings suggest the presence of two distinct active principles in the petroleum ether extract of C. moriflia. These facilitated the strong apoptotic response evidenced by the rapid activation of caspase-3 that was later enforced by the inhibition of the survival kinase Akt. Importantly, the efficient down-regulation of Akt, which is successfully tested in current clinical trials, is a unique property of C. morifolia.