Microbial carcinogenic toxins and dietary anti-cancer protectants

Several toxins are known which account for the ability of some bacteria to initiate or promote carcinogenesis. These ideas are summarised and evidence is discussed for more specific mechanisms involving chymotrypsin and the bacterial chymotryptic enzyme subtilisin. Subtilisin and Bacillus subtilis are present in the gut and environment and both are used commercially in agriculture, livestock rearing and meat processing. The enzymes deplete cells of tumour suppressors such as deleted in colorectal cancer (DCC) and neogenin, so their potential presence in the food chain might represent an important link between diet and cancer. Over-eating increases secretion of chymotrypsin which is absorbed from the gut and could contribute to several forms of cancer linked to obesity. Inhibition of these serine proteases by Bowman–Birk inhibitors in fruit and vegetables could account for some of the protective effects of a plant-rich diet. These interactions represent previously unknown non-genetic mechanisms for the modification of tumour suppressor proteins and provide a plausible explanation contributing to both the pro-oncogenic effects of meat products and the protective activity of a plant-rich diet. The data suggest that changes to farming husbandry and food processing methods to remove these sources of extrinsic proteases might significantly reduce the incidence of several cancers

An expanding range of targets for kynurenine metabolites of tryptophan

The kynurenine pathway of tryptophan metabolism accounts for most of the tryptophan that is not committed to protein synthesis and includes compounds active in the nervous and immune systems. Kynurenine acts on the aryl hydrocarbon receptor, affecting the metabolism of xenobiotics and promoting carcinogenesis. Quinolinic acid is an agonist at N-methyl-D-aspartate receptors (NMDARs), but is also pro-oxidant, has immunomodulatory actions, and promotes the formation of hyperphosphorylated tau proteins. Kynurenic acid blocks NMDARs and α7-homomeric nicotinic cholinoceptors and is also an agonist at the orphan G-protein-coupled receptor GPR35. 3-Hydroxykynurenine and 3-hydroxyanthranilic acid have pronounced redox activity and regulate T cell function. Cinnabarinic acid can activate metabotropic glutamate receptors. This review highlights the increasing range of molecular targets for components of the kynurenine pathway in both the nervous and immune systems in relation to their relevance to disease and drug development

Obesity and cancer: existing and new hypotheses for a causal connection

Existing explanations of obesity-associated cancer emphasise direct mutagenic effects of dietary components or hormonal imbalance. Some of these hypotheses are reviewed briefly, but recent evidence suggests a major role for chronic inflammation in cancer risk, possibly involving dietary content. These ideas include the inflammation-induced activation of the kynurenine pathway and its role in feeding and metabolism by activation of the aryl hydrocarbon receptor (AHR) and by modulating synaptic transmission in the brain. Evidence for a role of the kynurenine pathway in carcinogenesis then provides a potentially major link between obesity and cancer. A second new hypothesis is based on evidence that serine proteases can deplete cells of the tumour suppressors Deleted in Colorectal Cancer (DCC) and neogenin. These enzymes include mammalian chymotryptic proteases released by pro-inflammatory neutrophils and macrophages. Blood levels of chymotrypsin itself increase in parallel with food intake. The mechanistically similar bacterial enzyme subtilisin is widespread in the environment, animal probiotics, meat processing and cleaning products. Simple public health schemes in these areas, with selective serine protease inhibitors and AHR antagonists and could prevent a range of intestinal and other cancers

Dependence receptor involvement in subtilisin-induced long-term depression and in long-term potentiation

The serine protease subtilisin induces a form of long-term depression (LTD) which is accompanied by a reduced expression of the axo-dendritic guidance molecule Unco-ordinated-5C (Unc-5C). One objective of the present work was to determine whether a loss of Unc-5C function contributed to subtilisin-induced LTD by using Unc-5C antibodies in combination with the pore-forming agents Triton X-100 (0.005%) or streptolysin O in rat hippocampal slices. In addition we have assessed the effect of subtilisin on the related dependence receptor Deleted in Colorectal Cancer (DCC) and used antibodies to this protein for functional studies. Field excitatory postsynaptic potentials (fEPSPs) were analysed in rat hippocampal slices and protein extracts were used for Western blotting. Subtilisin produced a greater loss of DCC than of Unc-5C, but the antibodies had no effect on resting excitability or fEPSPs and did not modify subtilisin-induced LTD. However, antibodies to DCC but not Unc-5C did reduce the amplitude of theta-burst long-term potentiation (LTP). In addition, two inhibitors of endocytosis – dynasore and tat-gluR2(3Y) – were tested and, although the former compound had no effect on neurophysiological responses, tat-gluR2(3Y) did reduce the amplitude of subtilisin-induced LTD without affecting the expression of DCC or Unc-5C but with some loss of PostSynaptic Density Protein-95. The results support the view that the dependence receptor DCC may be involved in LTP and suggest that the endocytotic removal of a membrane protein or proteins may contribute to subtilisin-induced LTD, although it appears that neither Unc-5C nor DCC are involved in this process. (220)

Prenatal inhibition of the tryptophan–kynurenine pathway alters synaptic plasticity and protein expression in the rat hippocampus

Glutamate receptors sensitive to N-methyl-d-aspartate (NMDA) are important in early brain development, influencing cell proliferation and migration, neuritogenesis, axon guidance and synapse formation. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at these receptors. Rats were treated in late gestation with 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]-benzene-sulphonamide (Ro61-8048), an inhibitor of kynurenine-3-monoxygenase which diverts kynurenine metabolism to kynurenic acid. Within 5 h of drug administration, there was a significant decrease in GluN2A expression and increased GluN2B in the embryo brains, with changes in sonic hedgehog at 24 h. When injected dams were allowed to litter normally, the brains of offspring were removed at postnatal day 21 (P21). Recordings of hippocampal field excitatory synaptic potentials (fEPSPs) showed that prenatal exposure to Ro61-8048 increased neuronal excitability and paired-pulse facilitation. Long-term potentiation was also increased, with no change in long-term depression. At this time, levels of GluN2A, GluN2B and postsynaptic density protein PSD-95 were all increased. Among several neurodevelopmental proteins, the expression of sonic hedgehog was increased, but DISC1 and dependence receptors were unaffected, while raised levels of doublecortin and Proliferating Cell Nuclear Antigen (PCNA) suggested increased neurogenesis. The results reveal that inhibiting the kynurenine pathway in utero leads to molecular and functional synaptic changes in the embryos and offspring, indicating that the pathway is active during gestation and plays a significant role in the normal early development of the embryonic and neonatal nervous system

Selective depletion of tumour suppressors Deleted in Colorectal Cancer (DCC) and neogenin by environmental and endogenous serine proteases: linking diet, obesity and cancer

Background: The related tumour suppressor proteins Deleted in Colorectal Cancer (DCC) and neogenin are absent or weakly expressed in many cancers, whereas their insertion into cells suppresses oncogenic behaviour. Serine proteases influence the initiation and progression of cancers although the mechanisms are unknown. Methods: The effects of environmental (bacterial subtilisin) and endogenous mammalian (chymotrypsin) serine proteases were examined on protein expression in fresh, normal tissue and human neuroblastoma and mammary adenocarcinoma lines. Cell proliferation and migration assays (chemoattraction and wound closure) were used to examine cell function. Cells lacking DCC were transfected with an ectopic dcc plasmid. Results: Subtilisin and chymotrypsin selectively depleted DCC and neogenin from cells at nanomolar concentrations without affecting related proteins. Cells showed reduced adherence and increased migration, but after washing they re-attached within 24 h, with recovery of protein expression. These effects are induced by chymotryptic activity as they are prevented by chymostatin and the soybean Bowman-Birk inhibitor typical of many plant protease inhibitors. Conclusions: Bacillus subtilis, which secretes subtilisin is widely present in soil, the environment and the intestinal contents, while subtilisin itself is used in meat processing, animal feed probiotics and many household cleaning agents. With chymotrypsin present in chyme, blood and tissues, these proteases may contribute to cancer development by depleting DCC and neogenin. Blocking their activity by Bowman-Birk inhibitors may explain the protective effects of a plant diet. Our findings identify a potential non-genetic contribution to cancer cell behaviour which may explain both the association of processed meats and other factors with cancer incidence and the protection afforded by plant-rich diets, with significant implications for cancer prevention

On the Biological Importance of the 3-hydroxyanthranilic Acid: Anthranilic Acid Ratio

Of the major components of the kynurenine pathway for the oxidative metabolism of tryptophan, most attention has focussed on the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid, and the glutamate receptor blocker kynurenic acid. However, there is increasing evidence that the redox-active compound 3-hydroxyanthranilic acid may also have potent actions on cell function in the nervous and immune systems, and recent clinical data show marked changes in the levels of this compound, associated with changes in anthranilic acid levels, in patients with a range of neurological and other disorders including osteoporosis, chronic brain injury, Huntington’s disease, coronary heart disease, thoracic disease, stroke and depression. In most cases, there is a decrease in 3-hydroxyanthranilic acid levels and an increase in anthranilic acid levels. In this paper, we summarise the range of data obtained to date, and hypothesise that the levels of 3-hydroxyanthranilic acid or the ratio of 3-hydroxyanthranilic acid to anthranilic acid levels, may contribute to disorders with an inflammatory component, and may represent a novel marker for the assessment of inflammation and its progression. Data are presented which suggest that the ratio between these two compounds is not a simple determinant of neuronal viability. Finally, a hypothesis is presented to account for the development of the observed changes in 3-hydroxyanthranilic acid and anthranilate levels in inflammation and it is suggested that the change of the 3HAA:AA ratio, particularly in the brain, could possibly be a protective response to limit primary and secondary damage

Kynurenine pathway metabolism following prenatal KMO inhibition and in Mecp2+/- mice, using liquid chromatography-tandem mass spectrometry

To quantify the full range of tryptophan metabolites along the kynurenine pathway, a liquid chromatography – tandem mass spectrometry method was developed and used to analyse brain extracts of rodents treated with the kynurenine-3-mono-oxygenase (KMO) inhibitor Ro61-8048 during pregnancy. There were significant increases in the levels of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (3-HK) in the maternal brain after 5 h but not 24 h, while the embryos exhibited high levels of kynurenine, kynurenic acid and anthranilic acid after 5 h which were maintained at 24 h post-treatment. At 24 h there was also a strong trend to an increase in quinolinic acid levels (P = 0.055). No significant changes were observed in any of the other kynurenine metabolites. The results confirm the marked increase in the accumulation of some neuroactive kynurenines when KMO is inhibited, and re-emphasise the potential importance of changes in anthranilic acid. The prolonged duration of metabolite accumulation in the embryo brains indicates a trapping of compounds within the embryonic CNS independently of maternal levels. When brains were examined from young mice heterozygous for the meCP2 gene – a potential model for Rett syndrome - no differences were noted from control mice, suggesting that the proposed roles for kynurenines in autism spectrum disorder are not relevant to Rett syndrome, supporting its recognition as a distinct, independent, condition