Dietary isoflavones: biological effects and relevance to human health.
Substantial evidence indicates that diets high in plant-based foods may explain the epidemiologic variance of many hormone-dependent diseases that are a major cause of mortality and morbidity in Western populations. There is now an increased awareness that plants contain many phytoprotectants. Lignans and isoflavones represent two of the main classes of phytoestrogens of current interest in clinical nutrition. Although ubiquitous in their occurrence in the plant kingdom, these bioactive nonnutrients are found in particularly high concentrations in flaxseeds and soybeans and have been found to have a wide range of hormonal and nonhormonal activities that serve to provide plausible mechanisms for the potential health benefits of diets rich in phytoestrogens. Data from animal and in vitro studies provide convincing evidence for the potential of phytoestrogens in influencing hormone-dependent states; although the clinical application of diets rich in these estrogen mimics is in its infancy, data from preliminary studies suggest beneficial effects of importance to health. This review focuses on the more recent studies pertinent to this field and includes, where appropriate, the landmark and historical literature that has led to the exponential increase in interest in phytoestrogens from a clinical nutrition perspective. (+info)
Selective killing of CD8+ cells with a 'memory' phenotype (CD62Llo) by the N-acetyl-D-galactosamine-specific lectin from Viscum album L.
As reported previously by our group, among the toxic proteins from Viscum album L. only the mistletoe lectins (MLs) induce the apoptotic killing pathway in human lymphocytes. Although one may expect a homogenous distribution of carbohydrate domains on cell surface receptors for the carbohydrate binding B chains of the toxic protein, the sensitivity of cells to these B chains obviously differ. Here we report a selective killing of CD8+ CD62Llo cells from healthy individuals by the galNAc-specific ML III (and RCA60, which binds to gal and galNAc), while the gal-specific ML I was less effective. This selective killing is not sufficiently explained by protein synthesis inhibition alone, since this subset was not affected by other ribosome inhibiting proteins such as the lectin from Ricinus communis (RCA120), lectin from Abrus precatorus (APA), abrin A, and inhibitors of RNA, DNA and/or protein synthesis such as actinomycin D, mitomycin C, and cycloheximide. We conclude that CD8+ cells with 'memory' phenotype (CD62Llo) are more sensitive to the ML III-mediated killing than their CD8+ CD62Lhi counterparts, CD4+ T cells, and CD19+ B cells. These cells probably express a distinct receptor with galNAc domains that is missing or not active on CD8+ cells with a 'naive' phenotype. (+info)
Mistletoe lectin activates caspase-8/FLICE independently of death receptor signaling and enhances anticancer drug-induced apoptosis.
Mistletoe lectin I (ML-I) is a major active component in plant extracts of Viscum album that is increasingly used in adjuvant cancer therapy. ML-I exerts potent immunomodulating and cytotoxic effects, although its mechanism of action is largely unknown. We show that treatment of leukemic T- and B-cell lines with ML-I induced apoptosis, which required the prior activation of proteases of the caspase family. The involvement of caspases is demonstrated because (a) a peptide caspase inhibitor almost completely prevented ML-I-induced cell death and (b) proteolytic activation of caspase-8, caspase-9, and caspase-3 was observed. Because caspase-8 has been implicated as a regulator of apoptosis mediated by death receptors, we further investigated a potential receptor involvement in ML-I-induced effects. Cell death triggered by ML-I was neither attenuated in cell clones resistant to CD95 nor in cells that were rendered refractory to other death receptors by overexpressing a dominant-negative FADD mutant. In contrast, ML-I triggered a receptor-independent mitochondria-controlled apoptotic pathway because it rapidly induced the release of cytochrome c into the cytosol. Because ML-I was also observed to enhance the cytotoxic effect of chemotherapeutic drugs, these data may provide a molecular basis for clinical trials using MLs in anticancer therapy. (+info)
Effects of soy or rye supplementation of high-fat diets on colon tumour development in azoxymethane-treated rats.
Evidence is accumulating that a diet high in plant-derived foods may be protective against cancer. One class of plant component under increasing investigation is the phytoestrogens of which there are two main groups: the isoflavones, found mainly in soy products, and the lignans, which are more ubiquitous and are found in fruit, vegetables and cereals with high levels being found in flaxseed. In this study, we have used carefully balanced high-fat (40% energy) diets: a control diet (containing low isoflavone soy protein as the sole protein source), a rye diet (the control diet supplemented with rye bran) and a soy diet (containing as protein source a high isoflavone soy protein). The effect of these diets on the development of colonic cancer was studied in F-344 rats treated with the carcinogen, azoxymethane (two doses of 15 mg/kg given 1 week apart). Colons from treated animals were examined for aberrant crypt foci (ACF) and tumours after 12 and 31 weeks. Results after 12 weeks showed no differences in the total number of ACF in the control, soy or rye bran groups. However, the soy group had increased numbers of small ACF (less than four crypts/focus) while the rye group had decreased numbers of large ACF (greater than six crypts/focus). Examination of colons after 31 weeks gave similar low numbers of ACF in each group with no differences in multiplicity. There were no differences in the number of tumours between the control (1.36 tumours/rat) and soy (1.38 tumours/rat) groups. However, there was a significant decrease in the number of tumours in the rye group (0.17 tumours/rat). These results suggest that soy isoflavones have no effect on the frequency of colonic tumours in this model while rye bran supplementation decreases the frequency of colon cancer. This effect is due not to a decrease in early lesions but in their progression to larger multi-crypt ACF. The study also supports the hypothesis that larger ACF are more predictive of subsequent tumorigenicity. (+info)
Incorporation of esterified soybean isoflavones with antioxidant activity into low density lipoprotein.
We have recently reported that dietary intake of soybean isoflavone phytoestrogens resulted in increased oxidation resistance of isolated low density lipoprotein (LDL). In order to explore the underlying mechanisms we designed two types of in vitro experiments. First, we prepared several different isoflavone fatty acid esters to increase their lipid solubility and studied their incorporation into LDL. Second, the oxidation resistance of the isoflavone-containing LDLs was investigated with Esterbauer's 'conjugated diene' method using Cu2+ as prooxidant. Unesterified daidzein and genistein as well as genistein stearic acid esters were incorporated into LDL to a relatively small extent (0.33 molecules per LDL particle, or less) and they did not significantly influence oxidation resistance. The oleic acid esters of isoflavones were incorporated more effectively, reaching a level of 2.19 molecules per LDL particle or more, and the 4',7-O-dioleates of daidzein and genistein exhibited prolongations of lag times by 46% (P<0.05) and 202% (P<0.01), respectively. A smaller but significant increase in lag time (20.5%, P<0.01) was caused by daidzein 7-mono-oleate. In summary, esterification of soybean isoflavones daidzein and genistein with fatty acids at different hydroxyl groups provided lipophilicity needed for incorporation into LDL. Some isoflavone oleic acid esters increased oxidation resistance of LDL following their incorporation. (+info)
Mistletoe lectin dissociates into catalytic and binding subunits before translocation across the membrane to the cytoplasm.
Hybridomas producing monoclonal antibodies (mAbs) against the mistletoe lectin A-chain (MLA) were obtained to investigate the intracellular routing and translocation of ribosome-inactivating proteins. Anti-MLA mAb MNA5 did not bind the holotoxin but interacted with isolated MLA. This epitope was not recognized upon MLA denaturation or conjugation of MLA with the ricin binding subunit (RTB). Furthermore, the mAbs did not appreciably react with a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. A study of the cytotoxicity of mistletoe lectin, ricin, and chimeric toxin MLA/RTB for the hybridomas revealed that interchain disulfide bond reduction and subunit dissociation are required for cytotoxic activity of mistletoe lectin. (+info)
Cloning of the mistletoe lectin gene and characterization of the recombinant A-chain.
Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodimeric disulphide-linked glycoprotein is classified as type II ribosome-inactivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two-chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post-translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full-length 1923 nucleotide DNA sequence coding for the prepro-protein was obtained showing the existence of a single intron-free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20-30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular mass of 27 kDa and a pI value of 6.4. The ribosome-inactivating activity of the unglycosylated recombinant A-chain (IC50 20.5 pM) protein was in the same range as that of the glycosylated plant-derived ML A-chain (IC50 3.7 pM), which was very similar to that of ricin A-chain (IC50 4.9 pM). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in the enzymatic activities of the type II RIP A-chains. (+info)
trans-Resveratrol inhibits calcium influx in thrombin-stimulated human platelets.
1. The phytoestrogenic compound trans-resveratrol (trans-3,5, 4'-trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans-resveratrol and pure trans-resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. 2. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans-resveratrol had on the Ca2+ channels in thrombin stimulated platelets. 3. In this study we showed that trans-resveratrol immediately inhibited Ca2+ influx in thrombin-stimulated platelets with an IC50 of 0.5 microM. trans-Resveratrol at 0.1, 1.0 and 10.0 microM produced 20+/-6, 37+/-6 and 57+/-4% inhibition respectively of the effect of thrombin (0.01 u ml(-1)) to increase [Ca2+]i. 4. trans-Resveratrol also inhibited spontaneous Ba2+ entry into Fura-2 loaded platelets, with 0.1, 1.0 and 10.0 microM trans-resveratrol producing 10+/-5, 30+/-5 and 50+/-7% inhibition respectively. This indicated that trans-resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation. 5. trans-Resveratrol also inhibited thapsigargin-mediated Ca2+ influx into platelets. This suggests that the store-operated Ca2+ channels are one of the possible targets of trans-resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell. 6. The phytoestrogens genistein, daidzein, apigenin and genistein-glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans-resveratrol. 7. We conclude that trans-resveratrol is an inhibitor of store-operated Ca2+ channels in human platelets. This accounts for the ability of trans-resveratrol to inhibit platelet aggregation induced by thrombin. (+info)