Suppression of growth of tumour cell lines in vitro and tumours in vivo by mistletoe lectins.

A variety of studies have shown that incubation of different tumour cell lines with mistletoe lectins (MLs) in vitro has a marked cytotoxic effect. In the concentration range of low cytotoxicity cell death induced by ML-I is quantitatively due to apoptotic processes. The first events observed being membrane perforation and protusions. Simultaneous treatment of certain tumour cells with MLs rendered them more sensitive to induction of apoptosis by TNFalpha. The immunomodulatory activity of ML-I was investigated by measuring cytokine release and the results confirmed that cytokine induction by the lectin is regulated at the transcriptional level. ML-I has been shown to potentiate the effect of chemotherapeutic drugs. In addition to an in vitro effect a number of workers have demonstrated that MLs suppress tumour growth in vivo. Mistletoe lectins have been administered to animals locally to the tumour, systemic, subcutaneously or by the oral route via the diet. In many cases apoptosis was observed in the tumour and instances where complete tumour ablation has occurred have been reported. It has been hypothesized that the anticancer efficacy of tumour necrosis factor-alpha (TNFalpha) is potentiated by MLs isolated from both European and Korean mistletoe. There is accumulating evidence that both types of MLs are able to induce an anti-angiogenic response in the host suggesting that the anti-metastatic effect observed on a series of tumour cell lines in mice is in part due to an inhibition of tumour-induced angiogenesis and in part due to an induction of apoptosis.

Dietary mistletoe lectin supplementation and reduced growth of a murine non-Hodgkin lymphoma.

The growth of a murine non-Hodgkin lymphoma (NHL) tumour has been shown to be reduced by incorporating mistletoe lectin (ML-1) into the diet. The morphological characteristics of NHL tumours in mice fed ML-1-supplemented diets were different from those in LA (control)-fed mice. The degree of mitotic activity was lower and nuclear area reduced. The degree of lymphocyte infiltration was increased in tumours from ML-1 fed mice and this was accompanied by a high incidence of apoptotic bodies. Visual observation of NHL tumours from individuals fed ML-1 diet showed a poorly developed blood supply in contrast to control-fed mice. A major reduction in number of blood capillaries in NHL tumours was confirmed by microscopic evaluation of tumour sections. The results suggested an anti-angiogenic response in ML-1-fed mice. The feeding of ML-1 compared to control diet thus provided several identifiable changes in the morphology of NHL tumours which were consistent with the observed reduction in tumour weight. There was no longer histological evidence of viable tumour in 25% mice fed the ML-1 diet for 11 days. Morphological studies of the small bowel indicated (a) that the lectin induces hyperplasia, and (b) that the lectin binds avidly to lymphoid tissue of Peyer's patches. There was evidence of limited endocytosis of the lectin. An experiment where ML-3 was added to the diet of mice three days after inoculation of tumour cells showed that the lectin was able to slow down further growth of an established tumour. The results show that ML lectins induce powerful anti-cancer effects when provided by the oral route.

Modulation of Salmonella infection by the lectins of Canavalia ensiformis (Con A) and Galanthus nivalis (GNA) in a rat model in vivo.

The plant lectins, Concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA) have been prefed to rats for 3 d pre- and 6 d postinfection with Salmonella typhimurium S986 or Salm. enteritidis 857. Con A significantly increased numbers of Salm. typhimurium S986 in the large intestine and in faeces, and severely impaired growth of the rats, more severely than is the case of infection with Salmonella typhimurium alone. Con A had much less effect on rats infected with Salm. enteritidis 857 only showing a significant increase in numbers in the colon, accompanied by intermittent increases of Salmonella in the faeces during the study. GNA significantly reduced pathogen numbers in the lower part of the small bowel and the large intestine of rats infected with Salm. typhimurium S986 and significantly improved rat growth. GNA had little effect on infection by Salm. enteritidis 857 with slight decreases in Salmonella numbers in the small intestine and large intestine and transient increases in the faeces.

Polyamine oxidase and tissue transglutaminase activation in rat small intestine by polyamines.

Polyamine degradation was studied in the small intestine from rats fed on a polyamine-supplemented diet. Lactalbumin diet was given to Hooded-Lister rats, with or without 5 mg rat(-1) day(-1) of putrescine or spermidine for 5 days. Polyamine oxidase activity increased with putrescine and spermidine in the diet, whereas spermidine/spermine N(1)-acetyltransferase and diamine oxidase activities were unchanged. We also studied the calcium-dependent and -independent tissue transglutaminase activities, since they can modulate intestinal polyamine levels. Both types of enzymes increased in the cytosolic fraction after putrescine (about 65%) or spermidine (80-100%). Our results indicate that exogenous polyamines stimulate intestinal polyamine oxidase and tissue transglutaminase activities, probably to prevent polyamine accumulation, when other pathways of polyamine catabolism (acetylation and terminal catabolism) are not activated.

Nutritional utilization by rats of chickpea (Cicer arietinum) meal and its isolated globulin proteins is poorer than that of defatted soybean or lactalbumin.

The effects on performance, digestibility, N utilization and plasma amino acid concentrations of dietary chickpea (Cicer arietinum, var. Kabuli) seed meal, globulin proteins or buffer-insoluble residue [starch + non-starch polysaccharides (NSP) + lignin] were studied in growing rats. Chickpea meal, defatted soybean meal, chickpea globulins and lactalbumin were each incorporated into diets as the sole source of dietary protein (100 g/kg). In addition, chickpea insoluble residue was included in a control diet in the same proportion found in the chickpea meal. Rats were killed while under halothane anesthesia after 10 d of consuming the diets, and ileal contents were washed out and freeze-dried for digestibility measurements. Weight gains and gain:feed ratios of rats fed chickpea diets for 10 d did not differ from those of rats fed defatted soybean but were significantly lower than those of rats given the control (lactalbumin) diet. However, ileal and fecal N digestibilities and N retention by rats fed the chickpea diet were significantly lower than those obtained with the lactalbumin or soybean diet. The inclusion of both chickpea meal or its globulin proteins in the diet significantly increased the amount of N excreted, primarily as urea, through the urine. However, although ileal N digestibility values for chickpea meal were significantly lower, those for its constituent globulins did not differ from control values. Urea levels in plasma in rats fed diets containing chickpea meal, globulins or soybean meal were significantly higher than in those fed lactalbumin. Furthermore, the concentrations of glycine, phenylalanine, histidine, arginine and ornithine in the plasma of rats fed chickpea meal, its globulins or defatted soybean were significantly higher, whereas those of threonine, leucine, lysine and tryptophan were significantly lower than lactalbumin-fed controls. The chickpea insoluble residue had no adverse effects on performance or N utilization by rats. We conclude that the low nutritional value of chickpea meal is likely to be due mainly to adverse effects of its globulin proteins on growth and N metabolism rather than to the action of any known antinutritional factor present in the diet.

Lipid accumulation in obese Zucker rats is reduced by inclusion of raw kidney bean (Phaseolus vulgaris) in the diet.

The effects of inclusion of different levels of raw kidney bean (Phaseolus vulgaris) of high lectin content (27 g/kg meal) in a high-quality (lactalbumin) control diet were tested in nutritional trials on the growth and metabolism of obese Zucker (fafa) rats and their lean littermates in comparison with pair-fed controls. All diets contained 100 g total protein/kg and either 50 g lipids/kg (low fat) or 150 g lipids/kg (moderate fat). The growth of both obese and lean rats on bean diets was retarded by the daily bean intake in a dose-dependent manner. However, most of this was because bean-fed rats contained less body fat than the controls after 10 d. Thus, after feeding low-fat diets containing up to 130 g kidney bean/kg (lectin intake < or = 0.2 g/kg body weight (BW) per d) in both 10 d and 70 d trials, the bodies of obese rats contained less fat but not protein than their pair-fed controls. Moreover, by increasing the lipid content of the diet to 150 g/kg, the level of bean inclusion could be increased to 280 g/kg (lectin intake > or = 0.4 g/kg BW per d) without loss of body protein and skeletal muscle. Although these rats contained more body fat than those which were fed on low-fat diets, their weight reduction could be accounted for exclusively by reduced lipid content. In contrast, significant body protein loss occurred when the same diet of high lectin content was fed to lean littermates. Plasma insulin levels were significantly depressed in the obese Zucker rats on bean diets but the pancreas was not significantly enlarged nor its insulin content changed in 10 d trials. However, significant pancreatic growth occurred on long-term (70 d) bean feeding compared with pair-fed controls. The results suggest that, in addition to animal nutrition, it may also be possible to use the bean lectin as a dietary adjunct or therapeutic agent to stimulate gut function and ameliorate obesity if a safe and effective dose-range can be established for human subjects.

The utilization of lupin (Lupinus angustifolius) and faba bean globulins by rats is poorer than of soybean globulins or lactalbumin but the nutritional value of lupin seed meal is lower only than that of lactalbumin.

The effects of dietary sweet lupin (Lupinus angustifolius, Unicrop) seed meal or its insoluble fiber (nonstarch polysaccharides + lignin) on performance, digestibility and nitrogen utilization in growing rats were studied in four experiments. Globulin proteins isolated from lupin, faba bean (Vicia faba L. minor) or soybean (Glycine max) were also incorporated into purified diets as replacements for lactalbumin (control) and the nutritional effects were evaluated. Isocaloric, legume-based diets supplemented with amino acids were used. Final weight gain, gain:feed ratios, nitrogen retention and net protein utilization of the animals fed whole lupin meal-based diets for 10 d were inferior to those of controls. In contrast, adding lupin insoluble fiber to a control diet produced no adverse effects. Ileal starch and apparent nitrogen digestibilities, and fecal digestibility of starch in lupin-fed rats were higher than those of controls, but fecal true nitrogen digestibility was lower. Replacement of lactalbumin with globulin proteins from lupin or faba bean depressed food intake and protein utilization, but only performance was affected by consumption of soybean globulins. Rats consuming lupin or faba bean globulins excreted significantly more nitrogen, particularly as urea through the urine. This did not occur in rats fed soybean globulins. Urea concentration in plasma was higher in rats fed diets containing lupin meal or legume globulins. The concentrations of urea, arginine and ornithine in plasma increased significantly compared with control values after 3 to 9 h of a lupin diet. After 9 h, plasma lysine was also decreased. We concluded that the main reasons for the low nutritional value of sweet lupin seed meal are likely to be related to the chemical structure of the globulin proteins and their adverse effects on growth and nitrogen metabolism, and not to any known antinutritional factor or poor digestibility.

Inhibition of starch digestion by alpha-amylase inhibitor reduces the efficiency of utilization of dietary proteins and lipids and retards the growth of rats.

Digestion/absorption and nutritional utilization of starch, protein and lipids were studied in rats fed diets containing purified kidney bean alpha-amylase inhibitor at levels of 0, 1.6, 3.3 and 6.6 g/kg diet. At the two higher levels, the growth rate of rats and the apparent digestibilities and utilization of dietary starch and protein were significantly less than in control rats, and losses of nitrogen, lipids and carbohydrate resulted in a significant reduction in dry body weight. Some organs of the body were also affected: the relative dry weights of the intestines and the pancreas were higher, whereas liver and thymus weights were lower than in control rats. As starch digestion in the small intestine was negligible at higher inhibitor concentrations, the cecum was practically blocked by solidified digesta. This effect and the ensuing bacterial fermentation stimulated the growth of this tissue by hyperplasia and hypertrophy. However, as the distension was not always sufficient, the organ was occasionally ruptured and the rats had to be killed. Inhibitor doses in this work were comparable to those in clinical studies, implying that the use of the inhibitor is not without health risks. Moreover, diets rich in alpha-amylase inhibitor such as those containing transgenic plants with high levels of inhibitor gene expression cannot be recommended in intensive animal production.

Lectins as markers of rumen epithelial cell differentiation.

Lectins of different carbohydrate specificities (GNA (Galanthus nivalis), con A (Canavalia ensiformis), VFL (Vicia faba), PSL (Pisum sativum), LCA (Lens culinaris), PNA (Arachis hypogaea; with or without prior neuraminidase treatment), WGA (Triticum vulgare), SBA (Glycine max), UEA-I (Ulex europaeus), LPA (Limulus polyphemus), BS-I B4 (Bandeiraea simplicifolia, isolectin B4)) were explored for use as differentiation markers of rumen epithelial cells in vivo and in vitro. Lectins specific for mannose (GNA), mannose/glucose (con A, VFL, PSL and LCA), N-acetylglucosamine (WGA) or for N-acetylneuraminic acid (LPA) reacted generally with all types of rumen epithelial cell from both rumen tissue and cell culture. They were, therefore, not suitable markers of epithelial differentiation. SBA was unsuitable because, although it reacted with both tissue and cultured rumen epithelial cells, it was also bound to non-stratified areas of primary rumen epithelial cell cultures. Both BS-I B4 and PNA (after neuraminidase treatment) had to be ruled out because they did not react with differentiated rumen tissue epithelial cells, although they did bind to both stratified and non-stratified cultured cells. In contrast, UEA-I reacted strongly with differentiated rumen epithelial cells both from rumen tissue and cell cultures and therefore appears to be a good general marker for rumen epithelial cell differentiation.

Kidney bean lectin-induced Escherichia coli overgrowth in the small intestine is blocked by GNA, a mannose-specific lectin.

The reversible and dose-dependent hyperplastic growth of the small intestine and accelerated epithelial cell turnover caused by feeding rats with diets containing kidney bean lectin (PHA) increased the proportion of immature cells on the villi whose membrane and/or cytoplasm contained mainly simple, polymannosylated glycans. These new alpha-linked mannosyl terminals, particularly of the damaged epithelium, facilitated the preferential adherence of opportunistic Escherichia coli with mannose-sensitive Type 1 fimbriae, and other coliforms, to the glycocalyx. Accordingly, the growth of the gut was accompanied by a reversible and PHA dose-dependent overgrowth with E. coli. As expected from their common carbohydrate specificity, the inclusion in the diet of the mannose-specific agglutinin from snowdrop (Galanthus nivalis) bulbs (GNA) significantly reduced the extent of E. coli overgrowth, but abolished neither the growth nor the damage caused by PHA to the small intestine. Thus, GNA and perhaps other mannose-specific lectins, especially when used in a preventive mode, can be used to specifically block the proliferation of Type 1 E. coli in the small intestine.

Nutritional evaluation of the trypsin (EC 3.4.21.4) inhibitor from cowpea (Vigna unguiculata Walp.).

The effect of feeding rats purified cowpea (Vigna unguiculata Walp.) trypsin (EC 3.4.21.4) inhibitor in a semi-synthetic high-quality diet based on lactalbumin (10 g inhibitor/kg) for 10 d was a moderate reduction in the weight gain of rats in comparison with controls, despite an identical food intake in the two groups. The reduction in the growth rate was about 20% on a live weight basis. However, the corresponding value calculated from the weight of dry carcasses was less, only about 7%, probably because the water content of the body of the two groups of rats was different. Although most of the cowpea trypsin inhibitor (CpTI) was rapidly broken down in the digestive tract, its inclusion in the diet led to a slight, though significant, increase in the nitrogen content of faeces but not of urine. Accordingly, the net protein utilization of rats fed on inhibitor-containing diets was also slightly depressed while their energy expenditure was elevated. In agreement with results obtained for the protease inhibitors of soya bean, the slight anti-nutritional effects of CpTI were probably due mainly to the stimulation of the growth and metabolism of the pancreas. Thus, the nutritional penalty for increased insect-resistance after the transfer of the cowpea trypsin inhibitor gene into food plants is slight in the short-term.

Alterations in the level of insulin receptor and GLUT-4 mRNA in skeletal muscle from rats fed a kidney bean (Phaseolus vulgaris) diet.

1. A decline in the level of circulating insulin was observed in rats fed a diet containing kidney bean. 2. Consumption of a diet containing kidney bean caused an increase in the level of mRNAs for the insulin receptor (327%) and GLUT-4 (185%) in the gastrocnemius muscle. In contrast there was only a small increase in the amount of actin mRNA (125%). Since the kidney bean-fed rats are euglycaemic the results suggest that insulin receptor and GLUT-4 mRNA levels are regulated in response to circulating insulin concentrations rather than glucose. 3. No increases in the level of insulin receptor and actin mRNA were evident in the soleus muscle of rats fed the diet containing kidney bean; however a decline was observed in the level of GLUT-4 mRNA. 4. It is proposed that a component of kidney beans, most likely the lectin phytohaemagglutinin, has systemic effects which lead to changes in expression of the insulin receptor and GLUT-4 genes and to the sensitivity of muscle to insulin.

Mineral excretion of rats fed on diets containing faba beans (Vicia faba L.) or faba bean fractions.

The effects on faecal mineral excretion of two commercial varieties (local cultivar and Troy cultivar) of raw faba beans (Vicia faba L., minor) meal (VFM) and its fractions have been studied in growing rats. Diets contained local-VFM (dark seed coat) and Troy-VFM (light seed coat) at 474-500 g/kg diet, hull (VFH) from both varieties at 65 g/kg diet, and the insoluble cotyledon residue (VFCR) obtained from the Troy variety at 237 g/kg diet. Rats were pair-fed on diets which had been supplemented with amino acids to target requirements and contained similar amounts of zinc, manganese, iron and copper. With VFM diets the apparent absorption of Zn and Mn was significantly reduced. On the other hand, with hulls the apparent absorption of Fe was reduced while that of Cu slightly increased. As the amounts of calcium, phosphorus and magnesium in VFM and VFH diets were higher than in the controls, the increased intake resulted in a significant increase in both the apparent absorption and the faecal excretion of these minerals. The inclusion of VFCR in the diet had no significant effect on the mineral content of faeces. The relatively low concentrations of phytate in the bean seeds of 7.8 and 6.7 g/kg for the local and Troy cultivars respectively, could not adequately account for the increased mineral excretion. The results suggest that other seed constituents, possibly the soluble non-starch polysaccharides, may be involved in the elevated loss of Zn and Mn in rats fed on diets containing faba bean for extended periods, while some insoluble structural hull components may interfere with the absorption of Fe from the gut.

Nutritional response of growing rats to faba beans (Vicia faba L., minor) and faba bean fractions.

The effects of raw faba bean (Vicia faba L., minor) meal (VFM) and its fractions on the growth and nitrogen utilization of rats have been determined in two experiments. Two commercial varieties of VFM were tested, local VFM (409-439 g/kg diet) and Troy VFM (439 g/kg diet). The bean fractions tested were V. faba lectin-depleted protein (VFDP), V. faba lectin (VFL) and V. faba cotyledon residue (VFCR). All diets were supplemented with amino acids to target requirements. Body-weight, body N and lipid contents of rats fed on VFM were reduced significantly in comparison with control rats fed on lactalbumin. This was due, in part, to the lower digestibility of the protein, lipid and dry matter (DM) of VFM diets. As a result, net protein utilization (NPU) and biological value (BV) of faba bean proteins were less than expected. Urine and urea-N outputs of the VFM-fed rats were also elevated in both experiments. Increasing the energy content of local VFM diets led to significantly higher dry body-weight, body N and lipid contents, with the result that the NPU and BV values of the protein also increased. However, the NPU values for VFM-fed rats were still significantly lower than those for the controls in both experiments. In contrast, true N, lipid and DM digestibilities in rats given local VFM were not significantly affected by the difference in the energy content of the diets. The replacement of two-thirds of the lactalbumin in the diet with VFDP (65 g/kg) reduced dry body-weight, N and lipid contents, NPU and BV compared with the control rats, even though N, lipid and DM digestibilities were not significantly different. The nutritional performance of rats fed on lactalbumin-based diets containing 7 g VFL/kg was similar to that of the controls. Similarly, the inclusion of the cotyledon residue (237 g VFCR/kg diet) had no appreciable effect on any of the variables studied. As VFL and VFCR had no antinutritional effects in these rats, it appears that the low nutritional value of VFM for rats (NPU 0.66) results not only from the low digestibility of the bean proteins, but also from disturbances in N metabolism.

Luminal and basolateral polyamine uptake by rat small intestine stimulated to grow by Phaseolus vulgaris lectin phytohaemagglutinin in vivo.

Luminal and basolateral uptake of polyamines by the rat small intestine was studied in vivo. In the concentration range studied (0.1-5 mg per rat) 23-47% of the individual polyamines given intragastrically were found in the body after 1 h, with the small intestine retaining 4-12% of the dose. With spermidine or spermine, labelled polyamines accounted for 85-96% of the counts in the small intestine and between 72-82% were in the form given. However, with putrescine only 29-39% of the label found in the tissue remained in polyamine form and even less, 11-15%, as putrescine. Luminal uptake of polyamines was linear, non-saturable and was not stimulated when small intestinal growth was stimulated by phytohaemagglutinin (PHA). On the basolateral side of the gut, polyamine uptake was stimulated by PHA in a time-dependent way in advance of detectable growth. Overall polyamine recoveries were high (89-99%) with intraperitoneally administered spermidine and spermine. Moreover, a large proportion of the counts in the tissue (63-89%) were still in the original form. Even with putrescine, total recoveries of polyamines (72-88%) and putrescine (24-33%) were elevated in comparison with those from the lumen. Treatment of rats with alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, reduced tissue polyamine content, although it had slight effects only on basolateral polyamine transport. The PHA-stimulated increase of polyamine uptake was not abolished in the presence of DFMO.

Relationship between survival and binding of plant lectins during small intestinal passage and their effectiveness as growth factors.

The effects on the small intestine and the growth of rats of six pure plant lectins: PHA (Phaseolus vulgaris); SBL (Glycine maxima); SNA-I and SNA-II (Sambucus nigra); GNA (Galanthus nivalis) and VFL (Vicia faba), covering most sugar specificities found in nature, were studied in vivo. Variable amounts, 25% (VFL) to 100% (PHA, GNA) of the lectins administered intragastrically, remained in immunochemically intact form in the small intestine after 1 h. All lectins, except GNA, showed binding to the brush border on first exposure, although this was slight with VFL. Thus, binding to the gut wall was not obligatory for resistance to proteolysis. Exposure of rats to lectins, except VFL, for 10 days, retarded their growth but induced hyperplastic growth of their small intestine. The two activities were directly related. PHA and SNA-II, whose intestinal binding and endocytosis was appreciable after 10 days of feeding the rats with diets containing these lectins and similar to that found on acute (1 h) exposure, were powerful growth factors for the small intestine. GNA, which did not bind at the start but was reactive after 10 days, and SNA-I, which behaved in the opposite way, induced changes in receptor expression in the gut. As they were bound to the brush border transiently, they were less effective growth factors. VFL was not bound or endocytosed, was non-toxic and did not promote gut growth.