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The fermented wheat germ extract (code name: MSC, trade name: Avemar), with standardized benzoquinone content has been shown to inhibit tumor propagation and metastases formation in vivo. The aim of this study was to understand the molecular and cellular mechanisms of the anti-tumor effect of MSC. Therefore, we have designed in vitro model experiments using T and B tumor lymphocytic cell lines. Tyrosine phosphorylation of intracellular proteins and elevation of the intracellular Ca 2+ concentration were examined using immunoblotting with anti-phosphotyrosine antibody and cytofluorimetry by means of Ca 2+ sensitive fluorescence dyes, Fluo-3AM and FuraRed-AM, respectively. Apoptosis was measured with cytofluorimetry by staining the DNA with propidium iodide and detecting the 'sub-G 1 ' cell population. The level of the cell surface MHC class I molecules was analysed with indirect immunofluorescence on cytofluorimeter using a monoclonal antibody to the non-polymorphic region of the human MHC class I. MSC stimulated tyrosine phosphorylation of intracellular proteins and the influx of extracellular Ca 2+ resulted in elevation of intracellular Ca 2+ concentration. Prominent apoptosis of 20-40% was detected upon 24 h of MSC treatment of the cell lines. As a result of the MSC treatment, the amount of the cell surface MHC class I proteins was downregulated by 70-85% compared to the non-stimulated control. MSC did not induce a similar degree of apoptosis in healthy peripheral blood mononuclear cells. Inhibition of the cellular tyrosine phosphatase activity or Ca 2+ influx resulted in the opposite effect increasing or diminishing the Avemar induced apoptosis as well as the MHC class I downregulation, respectively. A benzoquinone component (2,6-dimethoxi-pbenzoquinone) in MSC induced similar apoptosis and downregulation of the MHC class I molecules in the tumor T and B cell lines to that of MSC. These results suggest that MSC acts on lymphoid tumor cells by reducing MHC class I expression and selectively promoting apoptosis of tumor cells on a tyrosine phosphorylation and Ca 2+ influx dependent way. One of the components in MSC, 2,6-dimethoxi-pbenzoquinone was shown to be an important factor in MSC mediated cell response.

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Galectin-1 is a member of the family of β-galactoside binding animal lectins, galectins. Its presence in the bone marrow has been detected; however, its role in the regulation of hematopoiesis is unknown. In the present study, we have evaluated the effect of recombinant human galectin-1 on the proliferation and survival of murine and human hematopoietic stem and progenitor cells. We show that low amount of galectin-1 (10 ng/ml) increases the formation of granulocyte-macrophage and erythroid colonies and the frequencies of day-7 cobblestone area-forming cells on a lactose-inhibitable fashion. In contrast, high amount of galectin-1 (10 μg/ml) dramatically reduces the growth of the committed blood-forming progenitor cells as well as the much younger, lineage-negative hematopoietic cells (day-28 to -35 cobblestone area-forming cells). This inhibition is not blocked by lactose and, therefore, is largely independent of the β-galactoside-binding site of the lectin. Furthermore, assays to detect apoptosis render it likely that the high amount of galectin-1 acts as a classical proapoptotic factor for the premature hematopoietic cells.

Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

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Secreted, extracellular galectin-1 (exGal-1) but not intracellular Gal-1 (inGal-1) has been described as a strong immunosuppressive protein due to its major activity of inducing apoptosis of activated T-cells. It has previously been reported that T-cells express Gal-1 upon activation, however its participation in T-cell functions has remained largely elusive. To determine function of Gal-1 expressed by activated Tcells we have carried out a series of experiments. We have shown that Gal-1, expressed in Gal-1-transgenic Jurkat cells or in activated T-cells, remained intracellularly indicating that Gal-1-induced T-cell death was not a result of an autocrine effect of the de novo expressed Gal-1. Rather, a particular consequence of the inGal-1 expression was that T-cells became more sensitive to exGal-1 added either as a soluble protein or bound to the surface of a Gal-1-secreting effector cell. This was also verified when the susceptibility of activated T-cells from wild type or Gal-1 knockout mice to Gal-1-induced apoptosis were compared. Murine T-cells expressing Gal-1 were more sensitive to the cytotoxicity of the exGal-1 than their Gal-1 knockout counterparts. We also conducted a study with activated T-cells from patients with systemic lupus erythematosus (SLE), a disease in which dysregulated T-cell apoptosis has been well described. SLE T-cells expressed lower amounts of Gal-1 than healthy T-cells and were less sensitive to exGal-1. These results suggested a novel role of inGal-1 in T-cells as a regulator of T-cell response to exGal-1, and its likely contribution to the mechanism in T-cell apoptosis deficiency in lupus.